Type 2 diabetes mellitus is caused by a combination of varying degrees of

insulin resistance and relative insulin deficiency. Its occurrence most likely
represents a complex interaction among many genes and environmental
factors, which are different among different populations and individuals.
●The search for plausible candidate genes has focused upon genes
coding for proteins that might be involved in pancreatic development,
insulin synthesis, secretion, or action. ●The most striking environmental
risk factors in most patients who develop type 2 diabetes are increased
weight gain and decreased physical activity, each of which increases the
risk of diabetes. ●The mechanism by which obesity induces insulin
resistance is poorly understood. Inflammation may be the common
mediator linking obesity to the pathogenesis of diabetes.

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INTRODUCTION — Type 2 diabetes mellitus is characterized by hyperglycemia,
insulin resistance, and relative impairment in insulin secretion.
PATHOPHYSIOLOGY — Understanding the pathogenesis of type 2 diabetes is
complicated by several factors. Patients present with a combination of varying
degrees of insulin resistance and relative insulin deficiency, and it is likely that
both contribute to type 2 diabetes. Furthermore, each of the clinical features can
arise through genetic or environmental influences, making it difficult to determine
the exact cause in an individual patient. Moreover, hyperglycemia itself can
impair pancreatic beta-cell function and exacerbate insulin resistance, leading to
a vicious cycle of hyperglycemia causing a worsening metabolic state. Type 2
diabetes is often accompanied by other conditions, including hypertension, high
serum low-density lipoprotein (LDL) cholesterol concentrations, and low serum
high-density lipoprotein (HDL) cholesterol concentrations that, like type 2
diabetes, increase cardiovascular risk. This constellation of clinical conditions is
referred to as the metabolic syndrome. Hyperinsulinemia occurring in response to
insulin resistance may play an important role in the genesis of these
abnormalities. Increased free fatty acid levels, inflammatory cytokines from fat,
and oxidative factors have all been implicated in the pathogenesis of metabolic
syndrome, type 2 diabetes, and their cardiovascular complications.

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It is a common disorder with a prevalence that rises markedly with increasing
degrees of obesity. The prevalence of type 2 diabetes has risen alarmingly in the
past decade, in large part linked to the trends in obesity and sedentary lifestyle

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Observations that demonstrate a genetic influence on the development of type 2
diabetes include:
●The prevalence of type 2 diabetes varies remarkably among ethnic groups living
in the same environment. Type 2 diabetes is two to six times more prevalent in
African Americans, Native Americans, Pima Indians, and Hispanic Americans in
the United States than in whites.
●Thirty-nine percent of patients with type 2 diabetes have at least one parent with
the disease.
●Among monozygotic twin pairs with one affected twin, approximately 90 percent
of unaffected twins eventually develop the disease.
●First-degree relatives of patients with type 2 diabetes frequently have impaired
nonoxidative glucose metabolism (indicative of insulin resistance) long before
they develop type 2 diabetes. In addition, they may have beta-cell dysfunction, as
evidenced by decreases in insulin and amylin release in response to glucose
stimulation.
●The lifetime risk for a first-degree relative of a patient with type 2 diabetes is 5 to
10 times higher than that of age- and weight-matched subjects without a family
history of diabetes .
Even among groups with increased genetic risk for diabetes, however,
environmental factors play a major role in the development of diabetes. As an
example, the prevalence of diabetes among Pima Indians in Mexico is less than
one-fifth that in United States Pima Indians (6.9 versus 38 percent).

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Type 2 diabetes most likely represents a complex interaction among many genes
and environmental factors. Monogenic causes of type 2 diabetes represent only a
small fraction of cases and commonly inherited polymorphisms individually
contribute only small degrees of risk for, or protection from, diabetes (figure 3).
Most of the genetic risk for type 2 diabetes results from complex polygenic risk
factors.

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The search for plausible candidate genes has focused upon genes coding for
proteins that might be involved in pancreatic development, insulin synthesis,
secretion, or action (table 1). Un intrón es una región del ADN que forma parte
de la transcripción primaria de ARN, pero a diferencia de los exones, son
eliminados del transcrito maduro, previamente a su traducción.
Pancreatic development and beta-cell function — Genome-wide association
analysis has played an important role in identifying several new diabetes
susceptibility loci. Some of these loci are in genes involved in pancreatic
development and insulin synthesis. As an example, a genome-wide association
study to find genetic loci associated with type 2 diabetes in a French population
confirmed the known association with the transcription factor 7-like 2 gene
(TCF7L2) and identified four new loci associated with an increased risk of type 2
diabetes. These loci (SLC30A8, HHEX/IDE, and KCNJ11) are involved in beta-
cell development and insulin synthesis and appear to contribute substantially to
type 2 diabetes risk. The population attributable risk for the four new loci
plus TCF7L2 was 70 percent. The results of the original genome-wide
association study were quickly confirmed by four independent studies in diverse
populations. A meta-analysis of three genome-wide association studies found six
new loci associated with diabetes. Some of the loci (NOTCH 2, JAZF1) may be
involved in pancreatic beta-cell growth and development.
In subsequent genome-wide association studies, the following findings were
noted:
●A susceptibility locus in the KCNQ1 gene was noted in Japanese individuals.

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The association was subsequently confirmed in samples from Singaporean,
Danish, and Mexican populations. The KCNQ1 gene encodes the alpha subunit of
the slowly acting component of the outward-rectifying potassium channel
(KvLQT1). Mutations in KCNQ1 are responsible for a subset of long QT-interval
syndromes. KCNQ1 is also expressed in pancreatic islet cells and KCNQ1 risk
alleles disrupt beta-cell function. Twelve new loci were noted in individuals of
European descent, including a second independent signal at the KCNQ1 locus.
The identified loci affected both beta-cell function and insulin action.
●Common variants in WFS1, a gene involved in beta-cell survival, were
associated with susceptibility to type 2 diabetes. Mutations in this gene also cause
a rare syndrome, called Wolfram syndrome, characterized by diabetes insipidus,
nonautoimmune diabetes mellitus, optic atrophy, and deafness.
Insulin release
Transcription factor genes — One gene variant, representing single nucleotide
polymorphisms at one of two loci (rs7903146 and rs12255372) in
the TCF7L2 gene, was found to significantly increase risk for type 2 diabetes in a
case control study of a population in Iceland. It was also shown to increase
diabetic risk in two other populations, including in the United States where the
gene variant is widely prevalent: 38 percent of the United States cohort studied
were heterozygous and 7 percent homozygous for the variant allele. Compared
with non-carriers, the relative risk for type 2 diabetes was 1.45 for heterozygotes
and 2.41 for homozygotes. The population-attributable risk for diabetes for this
gene is estimated at 21 percent. Subsequent analysis for this variant gene in
samples from participants (n = 3548) in the Diabetes Prevention Program (DPP
trial) found that homozygotes for the variant TCF7L2 gene were more likely to
progress from impaired glucose tolerance to diabetes over three years than those
without the variant gene (HR 1.55, 95% CI 1.20-2.01). The impact was strongest
in the placebo DPP group where the incidence of diabetes per 100 person-years
for the homozygous variant genotype at rs7903146 compared with heterozygous
and nonvariant genotypes was 18.5, 10.7, and 10.8, respectively. This variant
genotype predisposing to type 2 diabetes is associated with decreased insulin
secretion from the beta cell in response to oral and intravenous glucose.
MODY2 and MODY4 — Maturity onset diabetes of the young (MODY) is a rare
cause of type 2 diabetes that has autosomal dominant transmission and features
of both impaired insulin secretion and insulin resistance. One form, MODY2,
appears to be due to mutations in the glucokinase gene on chromosome 7.
Markers in this region have been linked to type 2 diabetes in American blacks and
some other ethnic groups but not in whites. Glucokinase, which phosphorylates
glucose to glucose-6-phosphate, probably acts as the glucose sensor within
pancreatic beta cells, and therefore, glucokinase defects likely result in decreased
insulin secretion. Another form of MODY (MODY4) is associated with mutations in
insulin promoter factor-1 (IPF-1/PDX-1), a pancreatic beta-cell transcription factor.
These mutations result in decreased insulin secretion in response to glucose due
to reduced binding of the protein to the insulin gene promoter and perhaps by
altering fibroblast growth factor signaling in beta cells. Less severe mutations
in IPF-1/PDX-1 may predispose to late-onset type 2 diabetes.

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Other — Several other genes that might affect insulin secretion, including the
insulin gene and the genes for amylin and glucose transporters, have not been
found to have any relationship to type 2 diabetes. On the other hand, a
polymorphism in the human alpha adrenergic receptor gene (ADRA2A) was
associated with reduced insulin secretion. In addition, a mutation in mitochondrial
DNA has been associated with a rare subtype of type 2 diabetes that has been
called maternally inherited diabetes and deafness; insulin secretion is impaired via
an uncertain mechanism in this disorder.
Insulin action — Insulin exerts its effects by first binding to a specific insulin
receptor that is present on many cells throughout the body. The insulin receptor is
a large transmembrane protein composed of two extracellular alpha subunits and
two transmembrane and intracellular beta subunits that have intrinsic tyrosine
kinase activity. When insulin binds to the extracellular portion of the receptor, the
tyrosine kinase is activated, initiating a sequence of intracellular responses
mediated in part by insulin receptor substrates. Several genetic syndromes of
severe insulin resistance have been identified, many of which are associated with
point mutations of the insulin receptor gene. These patients have marked
hyperinsulinemia and sometimes other abnormalities, such as acanthosis
nigricans and hyperandrogenism. However, as noted above, impaired glucose
tolerance or overt diabetes only occurs in humans or in animals if compensatory
increases in insulin secretion are inadequate. None of the syndromes associated
with an insulin receptor defect plays an important role in the common forms of
type 2 diabetes. Thus, the decrease in insulin responsiveness in type 2 diabetes is
probably due to a postreceptor defect, presumably affecting one of the intracellular
enzymes involved in glucose metabolism. In animals, for example, the gene for
glycogen synthase (which promotes the conversion of glucose-6-phosphate to
glycogen) is involved in the susceptibility to diet-induced hyperglycemia. This
observation may be relevant to humans because impaired glycogen synthesis is
responsible for the early insulin resistance in nondiabetic, first-degree relatives of
patients with type 2 diabetes, and an association has been noted between a
polymorphism of the glycogen synthase gene and the presence of diabetes in a
subgroup of patients with a strong family history of type 2 diabetes, hypertension,
and marked insulin resistance. However, a relationship between type 2 diabetes
and the promoter or coding regions of the glycogen synthase gene has not been
confirmed. Furthermore, there is evidence that impaired insulin-stimulated glucose
transport is responsible for the reduced muscle glycogen synthesis in patients with
this disorder.
Hepatocyte nuclear factors — Three of the recognized forms of what was
formerly called maturity onset diabetes of the young (MODY1, MODY3, and
MODY5) are due to mutations in the genes for hepatocyte nuclear factors 4-alpha,
1-alpha, and 1-beta, respectively, indicating that genes not directly related to
insulin can be responsible for the development of diabetes. Genes in
the MODY1 region of chromosome 20 and the MODY3 region of chromosome 12
also contribute to the development of type 2 diabetes in whites.
Other genes — Other genes that may affect susceptibility to type 2 diabetes are
the genes for insulin-receptor substrates, the beta-3-adrenergic receptor, and
peroxisome-proliferator-activated receptor (PPAR) gamma-2.

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●Insulin-receptor substrates are a common substrate for insulin receptor tyrosine
kinases. Disruption of the IRS-2 gene in mice results in insulin resistance in the
liver and skeletal muscle and hyperglycemia because of an inadequate
compensatory increase in insulin secretion. In another mouse study, upregulation
of IRS-2 in pancreatic beta cells could prevent the onset of diabetes caused
by IRS-2 disruption or diet-induced obesity. Other tissue-specific, IRS-2 knockout
(KO) mouse models suggest that IRS-2 signaling may play an important role in
hypothalamic regulation of leptin, peripheral insulin sensitivity, and, possibly,
regeneration of beta cells. In contrast, disruption of the IRS-1 gene does not result
in diabetes, because the insulin resistance is mild, and therefore, less of an
increase in insulin secretion is needed to overcome it.
●The beta-3-adrenergic receptor regulates lipolysis in visceral fat (the possible
human component of brown fat in animals) and increases thermogenesis in this
tissue. Initial observations in humans suggest that a mutation in the gene for the
beta-3-adrenergic receptor is associated with a low metabolic rate, high risk of
obesity, and the early onset of type 2 diabetes.
●PPAR gamma-2 is a transcription factor that has a key role in adipocyte
differentiation. Polymorphisms in this gene may contribute to the variability in body
mass index (BMI) and insulin sensitivity in the general population. The common
Pro12Ala polymorphism of PPAR gamma has been associated with a modestly
decreased risk for type 2 diabetes. PPAR gamma is also the receptor for the
thiazolidinediones, which lower the blood glucose in type 2 diabetes by increasing
insulin sensitivity.
●It is possible that impaired action and secretion of insulin in type 2 diabetes
share a common pathogenesis. In mice, a gain of function mutation in the Foxo1
gene (which encodes a transcription factor) targeted to the liver and pancreatic
beta cells resulted in diabetes (due to an increase in hepatic glucose production
and impaired beta cell compensation). Espadas y corazones Daniel Balmaceda
●Calpain-10 locus on chromosome 2A (called NIDDM1) may confer major
susceptibility to type 2 diabetes in Mexican Americans. It may also play a role in
other populations, but its contribution appears less than in Mexican Americans.
The involved gene encodes calpain-10, a cysteine protease. At least three
polymorphisms within this gene can act in concert with a gene on chromosome 15
to increase the predisposition to the development of type 2 diabetes. How this
occurs is not clear, but decreased insulin responsiveness plays at least a
contributory role. Human immunodeficiency virus (HIV) protease inhibitors also
are associated with the development of diabetes, suggesting that other proteases
may be involved in diabetes susceptibility.
●SLC16A11 was also identified as a candidate gene for type 2 diabetes in
Mexican and Latin American individuals. Although common in Mexican
populations (50 percent frequency in Native American samples), it is rare in
European and African samples. Analysis of archaic genome sequence suggests
that it is derived from Neanderthal introgression. It may play a role in lipid
metabolism in the liver.
Animal models — Additional insights into the genetic influences on the
development of type 2 diabetes and on the requirement for both insulin resistance

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and a relative decrease in insulin secretion are provided by the phenotypes of
double transgenic animals:
●Double-KO mice homozygous for null alleles of genes for beta-cell glucokinase
(which would impair insulin secretion) and IRS-1 (which would impair insulin
responsiveness) have overt diabetes. By comparison, single-KO mice lacking
either one of these genes do not have diabetes.
●In another model, 40 percent of mice doubly heterozygous for null alleles of the
insulin receptor and insulin receptor substrate-1 genes develop diabetes (with
marked hyperinsulinemia and beta-cell hyperplasia) at the age of four to six
months. In contrast, mice heterozygous for only one of these null alleles do not
develop diabetes.
These findings and the animal and human studies cited previously indicate that
insulin resistance alone is insufficient to cause diabetes in humans. This
requirement for multiple abnormalities in the expression and interaction of genes
controlling insulin secretion and action may explain the non-Mendelian inheritance
and the variable penetrance of type 2 diabetes.

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Type 2 diabetes mellitus results from the interaction of environmental factors with a
combination of genetic variants, most of which were unknown. A systematic search for
these variants was recently made possible by the development of high-density arrays
that permit the genotyping of hundreds of thousands of polymorphisms. We tested
392,935 single-nucleotide polymorphisms in a French case–control cohort. Markers with
the most significant difference in genotype frequencies between cases of type 2 diabetes
and controls were fast-tracked for testing in a second cohort. This identified four loci
containing variants that confer type 2 diabetes risk, in addition to confirming the known
association with the TCF7L2 gene. These loci include a non-synonymous polymorphism
in the zinc transporter SLC30A8, which is expressed exclusively in insulin-producing b-
cells, and two linkage disequilibrium blocks that contain genes potentially involved in b-
cell development or function (IDE–KIF11–HHEX and EXT2–ALX4). These associations
explain a substantial portion of disease risk and constitute proof of principle for the
genome-wide approach to the elucidation of complex genetic traits. The rapidly
increasing prevalence of type 2 diabetes mellitus (T2DM) is thought to be due to
environmental factors, such as increased availability of food and decreased opportunity
and motivation for physical activity, acting on genetically susceptible individuals. The
heritability of T2DM is one of the best established among common diseases and,
consequently, genetic risk factors for T2DM have been the subject of intense research.
Although the genetic causes of many monogenic forms of diabetes (maturity onset
diabetes in the young, neonatal mitochondrial and other syndromic types of diabetes
mellitus) have been elucidated, few variants leading to common T2DM have been clearly
identified and individually confer only a small risk (odds ratio 1.1–1.25) of developing
T2DM. Linkage studies have reported many T2DM-linked chromosomal regions and
have identified putative, causative genetic variants in CAPN10, ENPP1 ,HNF4A and
ACDC (also called ADIPOQ). In parallel, candidate-gene studies have
reportedmanyT2DM-associated loci,with coding variants in the nuclear receptor PPARG
(P12A) and the potassium channel KCNJ11 (E23K) being among the very few that have
been convincingly replicated. The strongest known (odds ratio 1.7) T2DM association

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was recently mapped to the transcription factor TCF7L2 and has been consistently
replicated in multiple populations

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Algunos ejemplos para dimensionar la complejidad: Polimorfismo TCF7L2:
común en Dinamarca y USA donde el 38% de la población es heterocigota y el
7% homocigota, con riesgo de D2 de 1,4 y 2,4 respectivamente.TCF7L2 actuaría
regulando la expresión del proglucagón y podría afectar la producción del GLP-1.
Polimorfismo KCNJ11 codifica los canales de K ATP dependientes, críticos para
la secreción de insulina acoplada a nutrientes
Common polymorphisms of the transcription factor 7–like 2 gene (TCF7L2) have
recently been associated with type 2 diabetes. We examined whether the two
most strongly associated variants (rs12255372 and rs7903146) predict the
progression to diabetes in persons with impaired glucose tolerance who were
enrolled in the Diabetes Prevention Program, in which lifestyle intervention or
treatment with metformin was compared with placebo. Methods We genotyped
these variants in 3548 participants and performed Cox regression analysis using
genotype, intervention, and their interactions as predictors. We assessed the
effect of genotype on measures of insulin secretion and insulin sensitivity at
baseline and at one year. Results Over an average period of three years,
participants with the risk-conferring TT genotype at rs7903146 were more likely to
have progression from impaired glucose tolerance to diabetes than were CC
homozygotes (hazard ratio, 1.55; 95 percent confidence interval, 1.20 to 2.01;
P<0.001). The effect of genotype was stronger in the placebo group (hazard
ratio, 1.81; 95 percent confidence interval, 1.21 to 2.70; P = 0.004) than in the
metformin and lifestyle-intervention groups (hazard ratios, 1.62 and 1.15,
respectively; P for the interaction between genotype and intervention not
significant). The TT genotype was associated with decreased insulin secretion
but not increased insulin resistance at baseline. Similar results were obtained for
rs12255372. Conclusions Common variants in TCF7L2 seem to be associated
with an increased risk of diabetes among persons with impaired glucose
tolerance. The risk-conferring genotypes in TCF7L2 are associated with impaired
beta-cell function but not with insulin resistance.

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A large body of evidence indicates that the risk of developing chronic diabetic
complications is under the control of genetic factors. Previous studies using a
candidate gene approach have uncovered a number of genetic loci that may
shape this risk, such as the VEGF gene for retinopathy, the ELMO1 gene for
nephropathy, and the ADIPOQ gene for coronary artery disease. Recently, a new
window has opened on identifying these genes through genome-wide association
studies. Such systematic approach has already led to the identification of a major
locus for coronary artery disease on 9p21 as well three potential genes for
nephropathy on 7p, 11p, and 13q. Further insights are expected from a broader
application of this strategy. It is anticipated that the identification of these genes
will provide novel insights on the etiology of diabetic complications, with crucial
implications for the development of new drugs to prevent the adverse effects of
diabetes.

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ROLE OF INTRAUTERINE DEVELOPMENT
Low birth weight — The presence of insulin resistance in obesity and type 2
diabetes led to the theory of the "thrifty" genotype in which insulin resistance
might improve survival during states of caloric deprivation but would lead to
diabetes in states of caloric excess or even adequacy. However, other
observations have suggested a different hypothesis: the thrifty genotype might be
induced by malnutrition during fetal and early life. In particular, intrauterine growth
restriction leading to low birth weight may be associated with an increased risk in
adulthood of insulin resistance, glucose intolerance, type 2 diabetes,
dyslipidemia, and hypertension. The inverse relationship between birth weight
and diabetes mellitus was illustrated in an analysis from the Nurses' Health Study
of over 69,000 women. The relative risk of type 2 diabetes compared with a
reference group by ascending birth weight categories decreased progressively
from 1.8 for a birth weight less than 2.3 kg to 0.8 for a birth weight greater than
4.5 kg. Adjustment for ethnicity, childhood socioeconomic status, and adult
lifestyle factors did not substantially alter this association.

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Contrasting relationship between ponderal index at birth (kg/m3) and body mass
index (kg/m2) in adulthood on mean insulin resistance. Insulin resistance varies
directly with adult BMI and inversely with ponderal index at birth. Thus, the
degree of insulin resistance is greatest with lower ponderal index at birth and
higher BMI in adulthood (first panel, orange column). Insulin resistance is
measured as the half-life (t1/2) of the fall in blood glucose during an intravenous
insulin tolerance test. Phillips DI, Barker DJ, Hales CN, et al. Thinness at birth
and insulin resistance in adult life. Diabetologia 1994; 37:150.
A meta-analysis of 30 studies, including the Nurses' Health Study, confirmed the
inverse association between birth weight and type 2 diabetes. Thus, thinness at
birth and in adult life have opposing effects on insulin resistance, such that
subjects who were underweight at birth but who become overweight in middle
age have the most severe insulin resistance and the greatest risk for type 2
diabetes (figure 7). Even among infants with a normal birth weight (≥3.5 kg),
those who had slow growth in length in the first three months after birth were
more likely to develop diabetes later in life, suggesting that the critical period for
pancreatic beta-cell development extends beyond the intrauterine period.
High birth weight — Higher birth weight (>4.0 kg) may also be associated with
an increased risk of diabetes. A meta-analysis of 14 studies (involving 132,180
individuals) of birth weight and subsequent risk of type 2 diabetes demonstrated
a U-shaped relationship between birth weight and diabetes risk. High birth weight
was associated with increased risk of diabetes in later life to the same extent as
low birth weight (ORs 1.36 versus 1.47). The association between high birth
weight and risk of type 2 diabetes may be related to maternal hyperglycemia

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during pregnancy. Prenatal exposure to hyperglycemia may increase the risk of
type 2 diabetes, independent of genetic predisposition. This was demonstrated in
a study of 31 nondiabetic adults, 15 of whom were exposed to a diabetic
environment in utero (mothers with type 1 diabetes) and 15 who had not been
exposed but whose fathers had type 1 diabetes (controls). The exposed subjects
had an increased risk of impaired glucose tolerance (5 of 15 versus 0 of 16), and a
defective insulin secretory response when compared with control subjects.
Prematurity — Children born prematurely, whether they were appropriate or small
for gestational age, may also be at increased risk for type 2 diabetes and other
diseases of adulthood associated with insulin resistance. This was illustrated in a
study of 50 healthy children ages 4 to 10 years who had been born prematurely
(<32 weeks gestation; 38 with a birth weight that was appropriate for gestational
age and 12 who were small for gestational age) and 22 control subjects (≥37
weeks gestation with a normal birth weight). A similar reduction in insulin
sensitivity (as measured by paired insulin and glucose values on an intravenous
glucose tolerance tests) was seen in both groups of children born prematurely
(normal or small for gestational age) when compared with the control group. The
alteration in insulin dynamics appears to be present at birth. However,
measurements in neonates/infants are limited. In a prospective birth cohort of
1358 children (418 born preterm), there was an inverse association between
gestational age (regardless of birth weight for gestational age) and elevated
plasma insulin levels at birth. Plasma insulin levels in early childhood were also
inversely associated with gestational age, but the association was attenuated after
adjustment for rapid weight gain in the first year of life. In another study, the
reduction in insulin sensitivity associated with preterm birth persisted into
adulthood. The implications for future development of type 2 diabetes require
further study.

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Fixed genomic variation explains only a small proportion of the risk of adiposity. In animal models, maternal
diet alters offspring body composition, accompanied by epigenetic changes in metabolic control genes. Little
is known about whether such processes operate in humans. RESEARCH DESIGN AND METHODS—Using
Sequenom MassARRAY we measured the methylation status of from five candidate genes in umbilical cord
tissue DNA from healthy neonates. Methylation varied greatly; we related methylation status to maternal
pregnancy diet and to child’s adiposity at age 9 years. Retinoid X receptor-a (RXRA) chr9 and endothelial
nitric oxide synthase (eNOS) chr7 methylation had independent associations with sex-adjusted childhood fat
mass. Higher methylation of RXRA chr9, but not of eNOS chr7, was associated with lower maternal
carbohydrate intake in early pregnancy, previously linked with higher neonatal adiposity in this population.
CONCLUSIONS—Our findings suggest a substantial component of metabolic disease risk has a prenatal
developmental basis. Perinatal epigenetic analysis may have utility in identifying individual vulnerability to
later obesity and metabolic disease.

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Fitness cardiorrespiratorio (FCR) es una condición fisiológica medible con
precisión a través de la VO2Máx durante un test de ejercicio máximo y se
expresa en mLO2/min/kg o en METs (equivalente metabólico) 1 MET = 3,5
mLO2/min/kg.
La VO2Máx refleja respiración, output cardíaco, función autonómica,
función endotelial, flujo muscular y extracción de O2 por el músculo
esquelético
Existe clara relación entre insulinorresistencia y ↓ del FCR
La distribución poblacional del FCR va de 10 a 90 mLO2/min/kg (3 a 25
METs)
Cada aumento de 1 MET del FCR se asocia con una ↓ mortalidad de 13%
The hunter-gatherer lifestyle A physiological benchmark Since the late
Paleolithic era, huntergatherers have had to explore large areas in order to find
high-quality food, traveling approxi mately 20 km per day. A high level of cardio
respiratory fitness is, therefore, vital for an individual with a hunter- gatherer
lifestyle, and endurance performance in humans is exceptional among
mammals. Humans have adapted to conditions that require us to supply our
large brains with nutrients during long fasting periods, which include sleep and
heavy physical activity, such as hunting. Since insulin is the main hormone that
regulates blood glucose levels in feeding and fasting periods, insulin blood
levels, secretion patterns and tissue activ ities are tightly linked to physical
activity

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Regulation of fatty acid oxidation by AMPK. The AMPK pathway has
profound effects on the regulation of lipid metabolism. Fatty acid oxidation
in skeletal muscle involves a rate-controlling step that is regulated by
carnitine palmitoyltransferase 1 (CPT1). CPT1 transfers long-chain acyl-
CoA into the mitochondria, and this process is inhibited allosterically by
malonyl-CoA, synthesized by acetyl CoA carboxylase (ACC). The activity
of ACC is regulated by reversible phosphorylation, and AMPK directly
phosphorylates and inactivates this downstream target. During exercise
and skeletal muscle contraction, activated AMPK inhibits ACC to reduce
malonyl-CoA concentration, thereby driving the entry of long-chain acyl-
CoA into the mitochondria for -oxidation to restore energy balance. The
ability of AMPK to induce lipid oxidation and thus lower skeletal muscle
and liver lipid deposition is considered an important feature for the
insulinsensitizing effect of AMPK activation. Indeed, when an activating
form of AMPK 3(R225Q) subunit is expressed in skeletal muscle via
genetic manipulation, the transgenic mice are protected against the
development of diet-induced skeletal muscle insulin resistance. This effect
is associated with lower skeletal muscle triglyceride stores as a result of
increased fatty acid oxidation.

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22
Insulin resistance — Insulin resistance may be the best predictor of type 2
diabetes. The vast majority of patients appear to have a genetic risk for type 2
diabetes. It is possible, for example, that insulin resistance becomes more severe
with increasing age and weight, thereby unmasking a concurrent defect in insulin
secretion in susceptible subjects to cause impaired glucose tolerance and
eventually overt hyperglycemia. In normal-weight, nondiabetic subjects at high
risk for type 2 diabetes, both fasting and post-glucose hyperinsulinemia predict
future weight gain, which in turn predisposes to hyperglycemia. Hyperglycemia
itself may contribute to further progression by a toxic effect on beta cells, possibly
by decreasing insulin gene expression. Insulin resistance may, at least in part, be
related to substances secreted by adipocytes ("adipokines," including leptin,
adiponectin, tumor necrosis factor-alpha [TNFa], and resistin). The importance of
genetic factors in the pathogenesis of type 2 diabetes is suggested by the
observation that lean, normoglycemic offspring of parents with type 2 diabetes
have reduced nonoxidative glucose metabolism associated with reduced muscle
glycogen synthesis. Thus, insulin resistance is present years before the onset of
hyperglycemia. An increase in intracellular lipid content in muscle has been
identified in these insulin-resistant offspring, suggesting that dysregulation of fatty
acid metabolism may mediate the insulin resistance in these individuals. In one
study, this dysregulation appeared to be due to an inherited defect in
mitochondrial function.

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The importance of the combination of genetic and environmental factors is
suggested by another study of nondiabetic offspring of two parents with type 2
diabetes. Their insulin sensitivity was similar to that of normal subjects with no
first-degree relatives with type 2 diabetes at near ideal body weight; with
increasing degrees of obesity, however, the progressive decrease in insulin
sensitivity was much more pronounced in those with a family history of type 2
diabetes.

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Muscle. Using the euglycemic insulin clamp technique in combination with tritiated glucose to
measure total body glucose disposal, we and others conclusively have demonstrated that lean type 2
diabetic individuals are severely resistant to insulin compared with age-, weight-, and sex-matched
control subjects. Employing femoral arterial and venous catheterization in combination with the insulin
clamp, we further demonstrated that muscle insulin resistance could account for over 85–90% of the
impairment in total body glucose disposal in type 2 diabetic subjects. Even though the insulin
clamp was extended for an additional hour in diabetic subjects to account for the delay in onset of
insulin action, the rate of insulin-stimulated glucose disposal remained 50% less than in control
subjects. A similar defect in insulin-stimulated muscle glucose uptake in type 2 diabetic subjects has
been demonstrated by others. In type 2 diabetic subjects we, as well as others, have documented the
presence of multiple intramyocellular defects in insulin action, including impaired glucose
transport and phosphorylation, reduced glycogen synthesis, and decreased glucose oxidation.
However, more proximal defects in the insulin signal transduction system play a paramount
role in the muscle insulin resistance. Insulin signal transduction. For insulin to work, it
must first bind to and then activate the insulin receptor by phosphorylating key tyrosine residues on
the chain. This results in the translocation of insulin receptor substrate (IRS)-1 to the plasma
membrane, where it interacts with the insulin receptor and also undergoes tyrosine phosphorylation.
This leads to the activation of PI 3-kinase and Akt, resulting in glucose transport into the cell,
activation of nitric oxide synthase with arterial vasodilation, and stimulation of multiple intracellular
metabolic processes. Studies from our laboratory were the first to demonstrate in humans that the
ability of insulin to tyrosine phosphorylate IRS-1 was severely impaired in lean type 2 diabetic
individuals, in obese normal glucose tolerant individuals, and in the insulin-resistant, normal glucose
tolerant offspring of two type 2 diabetic parents. Similar defects have been demonstrated by others in
human muscle. This defect in insulin signaling leads to decreased glucose transport, impaired release
of nitric oxide with endothelial dysfunction, and multiple defects in intramyocellular glucose
metabolism. In contrast to the severe defect in IRS-1 activation, we have shown that the mitogen-
activated protein (MAP) kinase pathway, which can be activated by Shc, is normally responsive to
insulin. The MAP kinase pathway, when stimulated, leads to the activation of a number of intracellular
pathways involved in inflammation, cellular proliferation, and atherosclerosis Thus, the block at the
level of IRS-1 impairs glucose transport into the cell and the resultant hyperglycemia stimulates insulin
secretion. Because the MAP kinase pathway retains its sensitivity to insulin, this causes excessive
stimulation of this pathway and activation of multiple intracellular pathways involved in inflammation
and atherogenesis. This, in part, explains the strong association between insulin resistance and
atherosclerotic cardiovascular disease in nondiabetic, as well as in type 2 diabetic, individuals. As
shown by Miyazaki et al. in our laboratory, there is only one class of oral antidiabetic drugs—the
TZDs—that simultaneously augment insulin signaling through IRS-1 and inhibit the MAP kinase
pathways. These molecular observations help to explain the recent results from the CHICAGO
(Carotid Intima-Media Thickness in Atherosclerosis Using Pioglitazone) and PERISCOPE
(Pioglitazone Effect on Regression of Intravascular Sonographic Coronary Obstruction Prospective
Evaluation) studies, in which pioglitazone was shown to halt the progression of carotid intima-media
thickness and coronary atherosclerosis, respectively, in type 2 diabetic patients. Consistent with these
anatomical studies, pioglitazone in the PROactive study was shown to decrease (P 0.027) the second
principal end point of death, myocardial infarction, and stroke by 16%. The primary composite end

25
point was reduced by 10% but did not reach statistical significance because of an increase in leg
revascularization, which is not an end point in most cardiovascular studies. This is not surprising since
gravity, not lipids or blood pressure, is the most important risk for peripheral vascular disease. Route of
glucose administration: oral vs. intravenous. The euglycemic insulin clamp, by maintaining plasma
glucose and insulin levels constant, has become the gold standard for quantitating insulin sensitivity.
However, the normal route of glucose administration in every day life is via the gastrointestinal tract. Using a
double tracer technique (1-14C-glucose orally and 3-3H-glucose intravenously) in combination with hepatic
vein catheterization, we set out to examine the disposal of oral versus intravenous glucose in healthy, normal
glucose tolerant and type 2 diabetic subjects. Under basal conditions, with fasting plasma glucose and insulin
concentrations of 90 mg/dl and 11 mU/ml, respectively, the splanchnic tissues, which primarily reflect the
liver, take up glucose at the rate of 0.5 mg/kg per min. When insulin was administered intravenously to raise
the plasma insulin concentration to 1,189 U/ml, while maintaining euglycemia, in subjects with NGT, no
stimulation of hepatic glucose uptake was observed. When insulin was infused with glucose to elevate both
glucose and insulin levels, hepatic glucose uptake increased, but only in proportion to the increase in plasma
glucose concentration, despite plasma insulin concentrations in excess of 1,000 U/ml. In contrast, when
glucose was administered orally, hepatic glucose uptake increased 4.5-fold, despite plasma insulin and
glucose concentrations that were much lower than with intravenous glucose plus insulin administration (Fig.
10). When the same oral glucose load was administered to type 2 diabetic individuals, despite higher plasma
glucose and insulin concentrations than in nondiabetic subjects, hepatic glucose uptake was reduced by 50%.
Thus, individuals with type 2 diabetes lack the gut factor that is responsible for augmenting hepatic glucose
uptake following glucose ingestion.

25
26
1. Este fue un estudio de entrecruzamiento en el que participaron sujetos sanos.
2. Se administraron cargas de glucosa oral o infusiones intravenosas de glucosa
isoglicémica de 25, 50, o 100 g a seis sujetos jóvenes sanos. Arriba se exponen los
datos correspondientes a 50 g
3. Es posible que el péptido C constituya una mejor medida de secreción insulínica que la
insulina plasmática, dado que los niveles del péptido C no se ven afectados por la
extracción hepática de insulina
4. Esta diferencia en los niveles de péptido C en respuesta a la glucosa oral vs la glucosa
intravenosa sugiere que otros factores (incretinas), y no sólo las acciones directas de la
glucosa plasmática, afectan la respuesta secretora de insulina

27
GLP-1 Secretion, Metabolism, and Clearance
1. GLP-1 is secreted from intestinal endocrine Lcells, which are located mainly in the distal ileum
and colon. In contrast, GIP is released from intestinal K-cells that are localized to more
proximal regions (duodenum and jejunum) of the small intestine. However, endocrine cells
that produce GLP-1 or GIP, as well as cells that produce both peptides, can be found
throughout all regions.
2. The L-cell is an open-type intestinal epithelial endocrine cell that directly contacts luminal
nutrients through its apical surface and neural and vascular tissue through its basolateral
surface. Accordingly, GLP-1 secretion from intestinal L-cells is stimulated by a variety of
nutrient, neural, and endocrine factors. Meal ingestion, particularly one rich in fats and
carbohydrates, is the primary physiologic stimulus for GLP-1 secretion. GLP-1 release can be
stimulated by mixed meals or individual nutrients including glucose and other sugars, fatty
acids, essential amino acids, and dietary fiber. Oral, but not intravenous, glucose
administration stimulates GLP-1 secretion in humans.
3. GLP-1 is released rapidly into the circulation after oral nutrient ingestion, and its secretion
occurs in a biphasic pattern starting with an early (within 10–15 min) phase that is followed by
a longer (30 –60 min) second phase.
4. Because the majority of GLP-1–secreting L-cells are located in the distal small intestine, it is
unlikely that the early phase of GLP-1 secretion can be mediated by direct nutrient contact
with the L-cell. Indeed, several studies have shown that the autonomic nervous system, the
neurotransmitters GRP and acetylcholine, and the peptide hormone GIP all can contribute to
the rapid release of GLP-1 after nutrient ingestion. The role of the vagus nerve as an
important mediator of nutrient-induced GLP-1 secretion has been established by studies in
rats in which it was shown that bilateral subdiaphragmatic vagotomy completely blocks fat-
induced GLP-1 secretion, whereas direct electrical stimulation of the celiac branches of the
vagus (that innervate the jejunum, ileum, and colon) increases GLP-1 secretion. In humans,
administration of atropine, a nonspecific muscarinic- receptor antagonist, diminishes oral
glucose-stimulated first-phase GLP-1 secretion. In similar studies, either atropine or the M1
muscarinic-receptor antagonist pirenzepine could completely inhibit fat-induced GLP-1
secretion in rats. GIP-induced GLP-1 secretion has been demonstrated in vitro in canine L-
cells and in vivo inrodents, and may be mediated by GRP. However, GIP has no effect on
GLP-1 secretion in humans.
5. In contrast to the indirect mechanisms that mediate early GLP-1 release, the second or late
phase of GLP-1 secretion likely is caused by direct stimulation of intestinal L-cells by digested
nutrients. Therefore, nutrient generated stimulatory signals can be transmitted to Lcells either
indirectly, through neural or endocrine mediators, or via direct contact, to produce the early
and late phases of GLP-1 secretion, respectively. However, because L-cells seem to be
present throughout the entire length of the small intestine, it is possible that early GLP-1
secretion also can occur by direct association of nutrients with L-cells located in more

28
 proximal regions of the small intestine.
6. Leptin receptors are expressed in rodent and human intestinal L-cells, and leptin stimulates GLP-1
secretion from fetal rat, mouse, and human endocrine L-cell cultures in vitro, as well as in vivo in rats
and leptin-deficient ob/ob mice. Moreover, high-fat diet–induced obesity in mice is associated with leptin
resistance and decreased basal and oral glucose-stimulated GLP-1 levels.
7. GLP-1 secretion is stimulated by activation of a number of intracellular signals including PKA, PKC,
calcium, and MAPK. Studies using a mouse intestinal L-cell line that expresses the adenosine
triphosphate (ATP)-sensitive potassium channel (KATP) subunits sulfonylurea receptor 1 and inward
rectifying potassium channel 6.2, as well as glucokinase, and sodium-glucose cotransporters 1 and 3
suggest that glucose stimulates GLP-1 secretion via glucose metabolism and KATP channel closure.
8. Unsaturated long-chain free fatty acids stimulate GLP-1 secretion via GPR120, a G-protein– coupled
receptor that is expressed abundantly in the intestine.
9. In comparison with the stimulation of GLP-1 secretion, relatively few studies have examined the factors
responsible for inhibition of GLP-1 release. However, limited studies have shown that insulin,
somatostatin, and the neuropeptide galanin can inhibit GLP-1 secretion from intestinal L-cells in vitro and
in vivo.

28
29
30
31
1. Multiple forms of GLP-1 are secreted in vivo, including GLP-1(1-37)
and GLP-1(1-36)NH2, which are thought to be inactive, and GLP-1(7-
37) and GLP-1(7-36)NH2, which are biologically active. GLP-1(7-37)
and GLP-1(7-36)NH2 appear to be equipotent in their ability to
stimulate insulin secretion.
2. The addition of an amide group to GLP-1(1-36)NH2 and GLP-1(7-
36)NH2 likely is mediated by the enzyme peptidylglycine-amidating
monooxygenase and may enhance the survival of GLP-1 in plasma.
3. In humans, the majority of GLP-1 in the circulation is GLP-1(7-36)NH2.
The half-life of bioactive GLP-1 in the circulation is less than 2
minutes owing to rapid inactivation by the ubiquitous proteolytic
enzyme dipeptidyl peptidase-4 (DPP-4). DPP-4, also known as CD26,
is a serine protease that specifically cleaves dipeptides from the amino
terminus of oligopeptides or proteins that contain an alanine or proline
residue in position 2, thereby modifying or inhibiting their activity. GLP-
1, which contains a penultimate alanine residue and thus is a
substrate for DPP-4, is metabolized rapidly to GLP-1 (9-37) or GLP-1
(9-36)NH2.
4. DPP-4 also binds collagen and adenosine deaminase, and plays a role
in T-cell costimulation and proliferation. DPP-4 is widely expressed
and can be found in multiple tissues and cell types including the
kidney, lung, adrenal gland, liver, intestine, spleen, testis, pancreas,
and CNS, as well as on the surface of lymphocytes and macrophages.

32
 Notably, DPP-4 also is found on the surface of endothelial cells, including
those lining blood vessels that drain the intestinal mucosa, which are
positioned directly adjacent to the sites of GLP-1 secretion. Consequently,
more than half of the GLP-1 that enters the portal circulation already has been
inactivated by DPP-4 before entry into the systemic circulation.
5. In addition to a cell-surface membrane-bound form, DPP-4 also exists as a
soluble protein in the circulation. In healthy or diabetic humans, intravenous or
subcutaneous GLP-1 is metabolized rapidly (within 30 min) to GLP-1 (9-
36)NH2, which accounts for more than 75% of the immunodetectable
circulating GLP-1 in these individuals. Numerous studies in both animals and
humans have demonstrated that inhibition of DPP-4 activity prolongs the half-
life of intact, biologically active GLP-1.
6. Neutral endopeptidase 24.11 (NEP-24.11), a membrane-bound zinc
metallopeptidase, has been shown to have endoproteolytic activity on GLP-1
in vitro and up to 50% of GLP-1 entering the circulation may undergo C
terminal cleavage by NEP-24.11.
7. The plasma half-life of intact GLP-1 is approximately 2 minutes, whereas that
of its metabolite has been estimated to be approximately 5 minutes as a result
of renal clearance. The major route of GLP-1 elimination is through the kidney
and involves mechanisms that include glomerular filtration and tubular uptake
and catabolism. Concentrations of GLP-1 metabolites are increased in patients
with renal failure, whereas levels of intact bioactive GLP-1 are similar to those
of healthy individuals. These studies indicate that the kidneys are important for
elimination of GLP-1 and its metabolites.
8. Fasting plasma levels of bioactive GLP-1 typically range between 5 and 10
pmol/L in humans and increase approximately 2- to 3-fold after a meal, with
the absolute peak values being dependent on both the size and nutrient
composition of the meal. Postprandial levels of intact, biologically active GLP-1
are reduced in obese and type 2 diabetic individuals. Because the elimination
rates of GLP-1 are similar in healthy, obese, and type 2 diabetic individuals the
decrease in GLP-1 levels observed in obese and type 2 diabetic humans likely
is caused by reductions in GLP-1 secretion. Although leptin can stimulate
GLP-1 secretion, obese individuals often exhibit leptin resistance. Hence, it
has been proposed that leptin resistance may be responsible for the
decreased GLP-1 levels in obese humans. The factors responsible for
decreased meal-stimulated GLP-1 secretion in type 2 diabetic patients are not
known.

9. Plasma levels of GLP-1 are low in the fasted state, in the

range of 5–10 pmol/L, and increase rapidly after eating,
reaching 15–50 pmol/L. The circulating levels of intact
GLP-1 and GIP decrease rapidly because of enzymatic
inactivation, mainly dipeptidyl peptidase-4 (DPP-4), and
renal clearance. Both GIP and GLP-1 contain alanine at
position 2, and hence are excellent substrates for DPP-4.

32
 Indeed, DPP-4 is essential for incretin inactivation. As a
result of DPP-4 activity, intact, biologically active GLP-1
represents only 10–20% of total plasma GLP-1.
10. The observation that GLP-1 is rapidly degraded by DPP-4 has fostered the
development of specific protease inhibitors that prevent the rapid fall of GLP-1
in circulating plasma after eating. DPP-4 is a ubiquitous membrane-spanning
cell-surface aminopeptidase widely expressed in many tissues, such as liver,
lung, kidney, intestinal brush-border membranes, lymphocytes, and endothelial
cells.The extracellular domain of DPP-4 can also be cleaved from its
membrane-anchored form and circulate in plasma, where it retains its full
enzymatic activity. DPP-4 preferentially cleaves peptides with a proline or
alanine residue in the second aminoterminal position. Many gastrointestinal
hormones, neuropeptides, cytokines, and chemokines are substrates for DPP-
4 among them both GIP and GLP-1.
11. DPP-IV is a 766 amino acid cell-surface, serine-type protease belonging to the
prolyloligopeptidase family. It has a characteristic Asp-His-Ser motif at the
active site and is widely distributed in various tissues, for example, kidney,
intestine, liver, placenta, uterus, prostate, skin and in capillary endothelium. A
soluble form of DPP-IV is also present in plasma and other body fluids. DPP-
IV selectively cleaves two amino acids from the N-terminal end of peptides
which have proline or alanine in the second position. In the case of GLP-1 and
GIP, proline and alanine are important for incretin-receptor activation, so a
truncated molecule devoid of either of these amino acids is not biologically
active. The presence of DPP-IV in the capillary bed of the gut mucosa,
adjacent to GLP-1-secreting L cells, facilitates rapid inactivation. Fifty per cent
of newly synthesized GLP-1 is degraded and inactivated even before it leaves
the local capillary bed, but DPP-IV-mediated inactivation also occurs during
the passage of GLP-1 and GIP across the hepatic endothelial surface. The
inactive incretin metabolites are then eliminated via the kidney.
12. Incretin hormones are not the only substrates for DPPIV. Other substrates
include various neuropeptides, for example, pituitary adenylate cyclase–
activating polypeptide (PACAP), vasoactive intestinal polypeptide, gastrin
releasing peptide (GRP), neuropeptide Y (NPY), growth hormone–releasing
hormone, GLP-2 and peptide YY. It remains unclear, however, if these
peptides are important physiologically as endogenous substrates for DPP-IV,
and the extent to which they are also metabolized by other enzymes.
13. A second enzyme involved in incretin hormone metabolism has been
identified. GLP-1 is partially and slowly degraded by the enzyme neutral
endopeptidase 24.11 (NEP24.11) with at least six potential cleavage sites.
NEP24.11 has widespread tissue distribution and is abundantly expressed in
the kidneys, but the physiological significance of NEP24.11 in vivo is still under
investigation.
14. Apart from its catalytic properties, DPP-IV has also been implicated in the
cellular uptake of human immunodeficiency virus-1, malignant transformation
and tumour invasion, and is under investigation as a diagnostic marker in solid

32
tumours, haematological malignancies and immunological disorders. DPP-IV
is also expressed on the surface of T lymphocytes (where it is known as
CD26) and plays an important role in the immune system. It interacts with
several membrane-expressed antigens, for example, the protein tyrosine
phosphates CD45 and adenosine deaminase (ADA), to activate T cells and
modulate chemotaxis. Given the importance of DPP-IV in immunoregulation, it
is reassuring to note that mice with tissue-targeted deletion of the gene
encoding for CD26 suffer no adverse effects. Furthermore, no immunological
side effects were reported after chronic DPP-IV inhibition in animals. Given the
large number of potential substrates inactivated by DPP-IV, and the functional
importance of DPP-IV in the immune, haematological and endocrine systems,
there will be keen interest in the long-term safety and tolerability profile of
DPP-IV inhibitors.

32
1. The acute insulinotropic effects of GLP-1 raised interest in the use of this peptide for diabetes
treatment. Moreover, the peptide possesses a number of additional effects which in the
context of diabetes treatment, must be considered favorable.
2. Effects on the islets GLP-1’s insulinotropic activity, which is strictly glucose dependent, is, at
least partly, exerted via interaction with the GLP-1 receptor located on the cell membrane of
the beta cells. Binding of GLP-1 to the receptor causes activation – via a stimulatory G-protein
– of adenylate cyclase resulting in the formation of cAMP. Most of the actions of GLP-1 are
secondary to the formation of cAMP.
3. Subsequent activation of protein kinase A and the cAMP-regulated guanine nucleotide
exchange factor II (cAMP-GEFII, also known as Epac2) leads to a plethora of events including
altered ion channel activity, intracellular calcium handling and enhanced exocytosis of insulin
containing granules. The effects of glucose and GLP-1 may converge at the level of the KATP
channels of the beta cells. These channels are sensitive to the intracellular ATP levels and,
thereby, to glucose metabolism of the beta cells, but may also be affected by protein kinase A
activated by GLP-1.
4. There is also evidence that GLP-1 acts as a glucose-sensitizer. Thus, GLP-1 has been found
to facilitate glucose-dependent mitochondrial ATP production.
5. Cyclic AMP generated by activation of the GLP-1 receptor may also influence the exocytotic
process directly, and this process has been estimated to account for up to 70% of the entire
secretory response. Also ATP may directly influence the exocytotic process, and may,
therefore, represent another site of convergence for the glucose and GLP-1 mediated signals.
6. The transcription factor PDX-1, a key regulator of islet growth and insulin gene transcription,
appears to be essential for most of the glucoregulatory, proliferative and cytoprotective
actions of GLP-1.
7. In addition, GLP-1 up regulates the genes for the cellular machinery involved in insulin
secretion, such as the glucokinase and GLUT-2 genes.
8. GLP-1 also has trophic effects on beta cells. Not only does it stimulate beta cell proliferation, it
also enhances the differentiation of new beta cells from progenitor cells in the pancreatic duct
epithelium. Thus, GLP-1 has been demonstrated to be capable of differentiating the
pancreatic duct cell line, AR42J, into endocrine insulin secreting cells. Most recently, GLP-1
has been shown to be capable of inhibiting apoptosis of beta cells including human beta cells.
Since the normal number of beta cells is maintained in a balance between apoptosis and
proliferation, this observation is of considerable interest, and also raises the possibility that
GLP-1 could be useful in conditions with increased beta cell apoptosis. A most striking
demonstration of the beta cell protective/ proliferative effects of GLP-1 receptor activation was
provided by Stoffers et al. (2003), who studied the diabetes developing in rats subjected to
intrauterine growth retardation. Treatment with exendin 4 in the neonatal period completely
prevented development of diabetes and restored beta cell mass, which otherwise is strongly

33
 reduced in these animals.
9. Reflex activation of the beta cell As will be discussed further below, GLP-1 is
extensively degraded by the enzyme dipeptidyl-peptidase-4 and it has been
demonstrated that only about 1/4 of what leaves the gut (the endocrine organ)
is still in the intact, active form, and only about 8% of what is secreted actually
reaches the pancreas as the intact peptide. This has given rise to speculations
that the actions of GLP-1 on the beta cells might be exerted indirectly via
activation of long vago-vagal reflexes. Indeed, the GLP-1 receptor is
expressed in cell bodies in the nodose ganglion from which the sensory
afferents from the GI-tract emanate, and it has been demonstrated that
blockade of the autonomic ganglia also blocked the effects of intraportally
injected GLP-1 on insulin secretion. Thus it may be that under physiological
conditions the majority of the effects of GLP-1 on insulin secretion is
transmitted via autonomic nerves.

33
1. Effects on glucagon secretion GLP-1 also strongly inhibits glucagon
secretion. Since in patients with T2DM there is fasting
hyperglucagonemia as well as exaggerated glucagon responses to
meal ingestion, and since it has been demonstrated that the
hyperglucagonaemia contributes to the hyperglycaemia of the
patients, this effect may be as important as the insulinotropic effects.
2. The mechanism whereby GLP-1 inhibits glucagon secretion has been
suggested to be indirect relative to its effects on the beta cell (insulin,
zinc, glutamate).
3. However, GLP-1 also suppressed glucagon secretion in subjects with
type 1 diabetes and no residual beta cell function, and recent studies
in isolated perfused rat pancreas demonstrated inhibition of glucagon
secretion by GLP-1 at glucose levels too low to cause measurable
insulin secretion.
4. A specific antagonist of the somatostatin receptors (subtype 2)
completely abolished the GLP-1 effect and actually increased the
secretion of glucagon, suggesting that the somatostatin-producing D-
cells of the islets transmit the effects of GLP-1 by paracrine inhibition
of the alpha cells and keep them under tonic suppression.
5. Relación entre el núcleo y el manto del islote que indica potenciales
interacciones paracrinas y flujo portal dentro del islote. Estas
relaciones sugieren que las células beta, al estar torrente arriba,
tienen pocas posibilidades de verse influenciadas por el glucagon y la

34
somatostatina producidas por las células alfa y delta del manto. Por el
contrario las alfa que están torrente abajo pueden ser fuertemente afectadas
por la insulina de las células beta, la cual tiene una acción supresora sobre la
secreción de glucagon. Esto explica por qué la liberación de dicha hormona
no es suprimida por la hiperglucemia de la diabetes. Este patrón vascular se
conoce como circulación portal islote-acino, lo que implica que las hormonas
insulares son liberadas torrente abajo directamente sobre las células
exocrinas, se cree que particularmente la insulina tiene un efecto trófico en el
páncreas exocrino

34
35
1. It is now well established that T2DM is characterized not only by
insulin resistance, but also by a beta cell defect which renders the beta
cells incapable of responding adequately to the insulin resistance. It is,
therefore, relevant to ask how the incretin effect functions in these
patients. Careful studies by Nauck et al. (1986b) indicated that the
incretin effect is severely reduced or lost in relatively lean type 2
diabetic patients.
2. In a similar study carried out in our own laboratory in obese subjects
with T2DM (BMI 37 kg/m2), we could confirm the loss of the incretin
effects, which was actually more extensive than in lean patients.
3. And also observed that the amount of intravenous glucose required for
copying of the oral glucose response was similar to the oral dose
(about 50 g for each), another indication that in these patients, the
route of administration did not result in different handling of the
glucose. Thus, there is little doubt that a defective incretin effect
contributes to the glucose intolerance of these patients.
4. Given that GLP-1 and GIP are the most important incretin hormones, it
is possible also to dissect their contribution to the defective incretin
action in diabetic patients. Such contributions could consist of defects
with respect to secretion, action or metabolism. Detailed studies of the
secretion of GIP and GLP-1 in response to mixed meals in patients
with T2DM revealed a slightly impaired secretion of GIP but a more
pronounced impairment with respect to the secretion of GLP-1. In fact,

36
 the GLP-1 response expressed as the incremental area under the curve was
reduced to approximately 50% in the patients compared to healthy glucose
tolerant controls. The reduction was particularly prominent during the second
and third hour of the meal test, while the initial response was unimpaired. The
decreased response was related to both BMI (lower the higher the BMI) and to
the actual diabetic state, but was independent of, e.g. the presence of
neuropathy. A decreased GLP-1 secretion in obese subjects has been
observed repeatedly and is related to an impaired incretin effect. How obesity
affects GLP-1 secretion is not known, but part of the effect may be related to
the insulin resistance of obesity. However insulin resistance is independently
related to a decreased GLP-1 meal response. It should be noted though, that
GLP-1 secretion is not decreased in all obese or insulin resistant subjects and
the impairment may be missed in small cohorts of subjects. But, if present,
impaired secretion of GLP-1 is likely to contribute to the failing incretin effect.
5. The metabolism of GLP-1 and GIP was compared by Vilsboll et al. in diabetic
patients and controls, but both hormones were metabolized at similar rates, so
that differences in their elimination do not explain the reduced plasma
concentrations when present.
6. With respect to the actions of the incretin hormones, it was discovered in 1993
by Nauck that infusion of rather large amounts of GLP-1 resulted in near
normal insulin responses in patients with T2DM, whereas GIP had no
significant effect. Similar observations were made by Elahi (1994). In
subsequent studies involving infusion of various doses of GLP-1 during
stepwise increases in plasma glucose, it was possible to analyse the influence
of GLP-1 on the beta cell sensitivity to glucose. It was found that although
GLP-1 at supraphysiological infusion rates was capable of restoring the beta
cell sensitivity to glucose to completely normal values, the sensitivity of the
diabetic islets to GLP-1 was, nevertheless, severely decreased. In further
studies in patients with T2DM, infusions of GIP and GLP-1 at rates resulting in
physiological elevations of the incretin levels (as observed after mixed meal
ingestion) while glucose was clamped at 15mmol/L, failed to affect insulin
secretion at all, although these infusion rates greatly (and equally) increased
insulin secretion in healthy subjects. These findings illustrate the dramatic loss
of potency of GLP-1 in T2DM, and when combined with the finding that the
secretion of GLP-1 is often reduced, one may conclude that also the
insulinotropic effects of endogenous GLP-1 may be severely compromised in
these patients. Thus a severely decreased efficacy characterizes the incretin
action of both GLP-1 and GIP in T2DM. Whereas supraphysiological amounts
of GLP-1 retain the capability to enhance glucose-induced secretion, GIP
remains ineffective regardless of dose. The mechanisms whereby GLP-1, but
not GIP, stimulate insulin secretion in T2DM remain unknown, but the
observation raises the possibility that GLP-1 in pharmacological doses could
be used clinically to enhance insulin secretion in T2DM.

36
1. One may ask whether the incretin defect is a primary event, perhaps a
major etiological contributor to the beta cell failure that characterizes
T2DM. Several observations, however, suggest that this is not the
case. Thus, the impaired secretion of GLP-1 seems to be a
consequence of diabetes. In identical twins that were discordant for
type T2DM, meal-induced GLP-1 secretion was reduced only in the
diabetic twin, and in first degree relatives of patients with T2DM, 24-h
incretin hormone profiles were normal. A reduced insulinotropic action
of GIP to almost diabetic levels was observed in about 50% of first
degree relatives of patients with T2DM, suggesting that this might
represent a primary, genetic defect (Meier et al., 2001). However,
subsequent observations have questioned this interpretation.
2. Vilsboll et al. studied insulin responses to GIP in patients with diabetes
of different etiologies, including diabetes secondary to pancreatitis. In
these patients, there was a similar loss of insulinotropic effects of GIP
as observed in the classical type 2 diabetic subjects. These findings
suggest that the lost effect of GIP is also secondary to the diabetic
condition. In further studies, Knop et al. (2007) studied healthy
controls, lean patients with T2DM and patients with chronic
pancreatitis with normal or diabetic glucose tolerance, and found that
the incretin effect was lost only in the diabetic patients, suggesting that
the loss of incretin effect develops as glucose intolerance progresses.
Interestingly, in these subjects incretin hormone secretion was
comparable to that of healthy controls.

37
3. Thus, although a therapeutic strategy based on incretin hormones may restore
beta cell responsiveness to glucose in T2DM, the incretin defect is not a
primary cause of diabetes.
4. In agreement with this notion, there is now data to suggest that intensified
treatment resulting in near normal glucose levels may lead to a partial
restoration of incretin action of GLP-1 and GIP (Hojbjerg et al., 2007; Hojberg
et al., 2008).
5. Abnormalities in the incretin axis have been shown to play an important role in
the progressive –cell failure of type 2 diabetes. GLP-1 and glucose-dependent
insulinotrophic polypeptide (also called gastric inhibitory polypeptide [GIP])
account for 90% of the incretin effect. In type 2 diabetes, there is a deficiency
of GLP-1 and resistance to the action of GIP. The deficiency of GLP-1 can be
observed in individuals with IGT and worsens progressively with progression
to type 2 diabetes. In addition to deficiency of GLP-1, there is resistance to the
stimulatory effect of GLP-1 on insulin secretion. In contrast to GLP-1, plasma
levels of GIP are elevated in type 2 diabetes, yet circulating plasma insulin
levels are reduced. This suggests that there is -cell resistance to the
stimulatory effect of GIP on insulin secretion, and this, in fact, has been
demonstrated. Of note, recent studies have shown that tight glycemic control
can restore the -cells’ insulin secretory response to GIP. Thus, -cell resistance
to GIP is another manifestation of glucotoxicity. Because GLP-1 deficiency
occurs early in the natural history of type 2 diabetes, it follows that GLP-1
replacement therapy is a logical choice to restore the deficient insulin
response that is characteristic of the diabetic condition.

37
38
The last, and perhaps most important, player to be implicated in the
pathogenesis of type 2 diabetes is the brain, which, along with his seven
companions, forms the ominous octet. It is abundantly clear that the current
epidemic of diabetes is being driven by the epidemic of obesity. Porte and
colleagues were among the first to demonstrate that, in rodents, insulin was a
powerful appetite suppressant. Obese individuals, both diabetic and nondiabetic,
are characterized by insulin resistance and compensatory hyperinsulinemia.
Nonetheless, food intake is increased in obese subjects despite the presence of
hyperinsulinemia, and one could postulate that the insulin resistance in peripheral
tissues also extends to the brain. Our laboratory has attempted to address the
issue of impaired appetite regulation by insulin in obese subjects using functional
magnetic resonance imaging (MRI) to examine the cerebral response to an
ingested glucose load. After glucose ingestion, two hypothalamic areas with
consistent inhibition were noted: the lower posterior hypothalamus, which
contains the ventromedial nuclei, and the upper posterior hypothalamus, which
contains the paraventricular nuclei. In both of these hypothalamic areas, which
are key centers for appetite regulation, the magnitude of the inhibitory response
following glucose ingestion was reduced in obese, insulin-resistant, normal
glucose tolerant subjects, and there was a delay in the time taken to reach the
maximum inhibitory response, even though the plasma insulin response was
markedly increased in the obese group. Whether the impaired functional MRI
response in obese subjects contributes to or is a consequence of the insulin
resistance and weight gain remains to be determined. Nonetheless, these results
suggest that the brain, like other organs (liver, muscle, and fat) in the body, may

39
be resistant to insulin. Studies by Obici et al.in rodents have also provided
evidence for cerebral insulin resistance leading to increased HGP and reduced
muscle glucose uptake.

39
Peptide signals from the pancreatic islets and the gastrointestinal tract influence the regulation of
energy homeostasis by the brain, and the brain in turn influences the secretions of both the islets
and the gut. This article focuses on how insulin interacts with the brain to influence food intake,
blood glucose, and cognitive behavior. Insulin is secreted in response to changes of ambient
glucose, and the levels achieved are directly proportional to body adiposity.
Hence, insulin, like leptin, is an adiposity signal. An increased insulin signal in the mediobasal
hypothalamus indicates that ample or excess energy is available in the body and elicits responses
that limit food intake and reduce hepatic glucose secretion. Increased insulin (and leptin as well)
locally within the brain complements other signals that indicate a surfeit of energy in the body,
including satiety signals generated by the gut during meals, glucose, and some fatty acids. There is
compelling evidence that overlapping intracellular signaling pathways within the mediobasal
hypothalamus mediate the overall catabolic response to these diverse metabolic signals. Insulin
receptors are also densely expressed in the hippocampus, and insulin acts there to facilitate
learning and memory. The function of insulin receptors in other brain areas is poorly understood.
Obesity and/or the consumption of diets high
in fat render the brain as well as the body insulin resistant. In the hypothalamus, this is manifest as a
reduced ability of insulin to reduce food intake and body weight, and in the hippocampus, it is
manifest as a reduced ability of insulin to improve learning and/or memory. Diabetes 55 (Suppl.
2):S114–S121, 2006

40
41
INSULIN RESISTANCE Both the liver and muscle are severely resistant to insulin in
individuals with type 2 diabetes. However, when discussing insulin resistance, it is
important to distinguish what is responsible for the insulin resistance in the basal or
fasting state and what is responsible for the insulin resistance in the insulin-stimulated
state. Liver. The brain has an obligate need for glucose and is responsible for 50% of
glucose utilization under basal or fasting conditions. This glucose demand is met
primarily by glucose production by the liver and to a smaller extent the kidneys.
Following an overnight fast, the liver of nondiabetic individuals produces glucose at the
rate of 2 mg/kg per min. In type 2 diabetic individuals, the rate of basal HGP is
increased, averaging 2.5 mg/kg per min. In an average 80-kg person, this amounts to
the addition of an extra 25–30 g of glucose to the systemic circulation every night. As
shown in Fig control subjects cluster with a fasting plasma glucose concentration of 85–
90 mg/dl, and their rate of HGP averages 2 mg/kg per min. In type 2 diabetic subjects,
as the rate of basal HGP rises, so also does the fasting plasma glucose concentration,
and these two variables are strongly correlated with an R value of 0.847 (P 0.001). This
overproduction of glucose by the liver occurs in the presence of fasting plasma insulin
levels that are increased 2.5- to 3-fold, indicating severe resistance to the suppressive
effect of insulin on HGP. Similar observations have been made by others. The increase
in basal HGP is explained entirely by an increase in hepatic gluconeogenesis. In
addition to hepatic insulinresistance, multiple other factors contribute to accelerated rate
of HGP including: 1) increased circulating glucagon levels and enhanced hepatic
sensitivity to glucagon; 2) lipotoxicity leading to increased expression and activity of
phosphoenolpyruvate carboxykinase and pyruvate carboxylase, the rate-limiting
enzymes for gluconeogenesis; and 3) glucotoxicity, leading to increased expression and
activity of glucose-6-phosphatase, the rate-limiting enzyme for glucose escape from the
liver

42
Since phosphorylation of glucose by glucokinase is the rate limiting step in the
uptake of extracellular glucose by the liver (18,19), these data strongly suggest a
defect in hepatic glucokinase activity. The increase in basal HGP is explained
entirely by an increase in hepatic gluconeogenesis. In addition to hepatic
insulinresistance, multiple other factors contribute to accelerated rate of HGP
including: 1) increased circulating glucagon levels and enhanced hepatic
sensitivity to glucagon; 2) lipotoxicity leading to increased expression and activity
of phosphoenolpyruvate carboxykinase and pyruvate carboxylase, the rate-
limiting enzymes for gluconeogenesis; and 3) glucotoxicity, leading to increased
expression and activity of glucose-6-phosphatase, the rate-limiting enzyme for
glucose escape from the liver

43
44
45
46
SETACEOUS SEXTET The sixth member, who establishes the setaceous sextet,
is the pancreatic -cell. Many groups, dating back to the 1970s, have
demonstrated that the basal plasma glucagon concentration is elevated in type 2
diabetic individuals. The important contribution of elevated fasting plasma
glucagon levels to the increased basal rate of HGP in type 2 diabetic individuals
was provided by Baron et al. with control subjects, diabetic individuals had a
markedly elevated rate of basal HGP, which correlated closely with the increase
in fasting plasma glucagon concentration. Following somatostatin infusion,
plasma glucagon levels declined by 44% in association with a 58% decrease in
basal HGP. These results conclusively demonstrate the pivotal role of
hyperglucagonemia in the pathogenesis of fasting hyperglycemia in type 2
diabetes. There also is evidence that the liver may be hypersensitive to the
stimulatory effect of glucagon in hepatic gluconeogenesis. In summary, drugs that
inhibit glucagon secretion or block the glucagon receptor are likely to be effective
in treating patients with type 2 diabetes. One such example is exenatide, but
glucagon receptor antagonists also have been shown to be effective
.

47
JCEM, en el que se demuestra que el GLP-1 endógeno regula la glucosa
postprandial en humanos y que la supresión de la liberación del glucagon
inducida por esta incretina juega un papel fundamental en ese sentido. Para
completar el cuadro, también les envío un estudio que demuestra de manera
fehaciente que el GLP-1:
1) Sí tiene receptores en las células alfa de los islotes
2) Inhibe de manera DIRECTA la secreción de glucagon por células alfa
Se había planteado previamente que el efecto supresor de la secreción del
glucagon del GLP-1 ocurría de manera parácrina, ya que esta incretina aumenta
la secreción de somatostatina por las células delta, hormona que inhibe la
secreción de glucagon. Este estudio que les envío demuestra que el GLP-1 tiene
una acción glucagonostática independiente de su efecto sobre la secreción de
somatostatina.

48
OBJECTIVE— To determine the time course of changes in glucagon and
insulin secretion in children with recently diagnosed type 1 diabetes.
RESEARCH DESIGN AND METHODS— Glucagon and C-peptide
concentrations were determined in response to standard mixed meals in
23 patients with type 1 diabetes aged 9.44.6 years, beginning within 6
weeks of diagnosis, and every 3 months thereafter for 1 year.
RESULTS— Glucagon secretion in response to a physiologic stimulus
(mixed meal) increased by 37% over 12 months, while C-peptide secretion
declined by 45%. Fasting glucagon concentrations remained within the
normal (nondiabetic) reference range. CONCLUSIONS— Postprandial
hyperglucagonemia worsens significantly during the first year after
diagnosis of type 1 diabetes and may represent a distinct therapeutic
target. Fasting glucagon values may underestimate the severity of
hyperglucagonemia. The opposing directions of abnormal glucagon and
C-peptide secretion over time support the link between dysregulated
glucagon secretion and declining -cell function.

49
Impaired insulin secretion and insulin resistance — The relative importance
of impaired insulin release and insulin resistance in the pathogenesis of type 2
diabetes has been evaluated in several studies. As an example, in a prospective
study of over 6500 British civil servants without diabetes at baseline, 505
subjects were diagnosed with diabetes during 9.7 years (median) of follow-up. In
those who developed diabetes compared with those who did not, there was a
marked decrease in insulin sensitivity during the five years prior to diagnosis.
Beta-cell function (insulin secretion) increased three to four years prior to
diagnosis and then decreased until diagnosis. In addition, a seven-year
prospective study of 714 nondiabetic Mexican-Americans suggested that
decreased insulin secretion and insulin resistance were independent risk factors
for type 2 diabetes. Among Pima Indians, in whom the frequency of diabetes is
very high, the transition from normal glucose tolerance to impaired glucose
tolerance to diabetes is characterized by concomitant decreases in insulin-
stimulated glucose disposal and glucose-stimulated insulin secretion.
Insulin secretion — Insulin secretion by beta cells requires glucose transport
into the cell, which is, at least in part, mediated by the glucose transporter 2
(GLUT-2). A mouse model with a genetic alteration affecting GLUT-2 expression
produced mice with glucose intolerance; similar changes in GLUT-2 could be
induced in normal mice fed a high-fat diet and suggests a possible mechanism
for the link between high-fat diet and the development of diabetes. Impaired
insulin secretion has also been demonstrated to occur in mice lacking Abca1, a
cellular cholesterol transporter. Mice with inactivation of Abca1 in beta cells have
defective insulin secretion, impaired glucose tolerance but normal insulin

50
sensitivity.
CELL FUNCTION Although the plasma insulin response to the development of
insulin resistance typically is increased during the natural history of type 2
diabetes , this does not mean that the -cell is functioning normally. To the contrary,
recent studies from our group have demonstrated that the onset of -cell failure
occurs much earlier and is more severe than previously appreciated. In the San
Antonio Metabolism (SAM) study and the Veterans Administration Genetic
Epidemiology Study (VAGES), we examined
a large number of subjects with NGT (n 318), IGT (n 259), and type 2 diabetes (n
201). All subjects had an OGTT with plasma glucose and insulin concentrations
measured every 15 min to evaluate overall glucose tolerance and -cell function
and a euglycemic insulin clamp to measure insulin sensitivity. It now is recognized
that simply measuring the plasma insulin response to a glucose challenge does
not provide a valid index of beta cell function. The beta cell responds to an
increment in glucose (G) with an increment in insulin (I). Thus, a better measure of
beta cell function is I/G. However, the beta cell also is keenly aware of the body’s
sensitivity to insulin and adjusts its secretion of insulin to maintain normoglycemia.
Thus, the gold standard for measuring beta cell function is the insulin
secretion/insulin resistance (I/G ÷ IR), or so called
disposition, index. Note that insulin resistance is the inverse of insulin sensitivity.
displays the glucose area under the curve (AUC) and insulin AUC in NGT, IGT,
and type 2 diabetic subjects who participated in VAGES and SAM. In the right
panel, the typical inverted U-shaped or Starling’s curve of the pancreas for the
plasma insulin response is evident. Although subjects with IGT have an increase
in the absolute plasma insulin concentration, this should not be interpreted to
mean that the beta cells in these
individuals are functioning normally. Figure 3 depicts the insulin secretion/insulin
resistance index (I/G ÷ IR) in NGT, IGT, and type 2 diabetic subjects as a function
of the 2-h plasma glucose concentration during the OGTT. If a 2-h plasma glucose
140 mg/dl is considered to represent “normal” glucose tolerance, subjects in the
upper tertile (2-h PG 120–139 mg/dl) have lost two-thirds of their beta cell
function. Most disturbingly, subjects in the upper tertile of IGT (2-h PG 180–199
mg/dl) have lost 80–85% of their -cell function. Although not commented upon,
similar conclusions can be reached from data in previous publications. The
therapeutic implications of these findings are readily evident. By the time that the
diagnosis of diabetes is made, the patient has lost over 80% of his/her beta cell
function, and it is essential that the physician intervene aggressively with therapies
known to correct known pathophysiological disturbances in beta cell function. In
biomedical phenomena, most reactions take place as a log function. Figure 4
depicts the natural log of the 2-h plasma glucose concentration during the OGTT
as a function of the natural log of the insulin secretion/insulin resistance (beta cell
function) index. These two variables are strongly and linearly related with an r
value of 0.91 (P 0.00001). There are no cut points that distinguish NGT from IGT
from type 2 diabetes. Rather, glucose intolerance is a continuum, and subjects
simply move up and down this curve as a function of the insulin secretion/insulin
resistance index. Therefore, the current diagnostic criteria for IGT and type 2

50
diabetes are quite arbitrary and, like plasma cholesterol, glucose tolerance should
be viewed as a continuum of risk. The higher the 2-h plasma glucose
concentration, even within the range of IGT, the greater is the risk for
microvascular complications.

50
Even more ominous are the observations of Butler et al. In a postmortem
analysis, these investigators quantitated relative beta cell volume and related it to
the fasting plasma glucose concentration. As individuals progressed from NGT to
impaired fasting glucose (IFG), there was a 50% decline in beta cell volume,
suggesting a significant loss of beta cell mass long before the onset of type 2
diabetes. With the progression to overt diabetes, there was a further and
significant loss of beta cell volume. Although beta cell volume should not be
viewed to be synonymous with beta cell mass, these results suggest that
significant loss of beta cell mass occurs long before the onset of type 2 diabetes,
according to current diagnostic criteria. In summary, our findings demonstrate
that, at the stage of IGT, individuals have lost over 80% of their beta cell function,
while the results of Butler et al. suggest that subjects with “pre-diabetes” have
lost approximately half of their beta cell volume. “PRE-DIABETES” The
recently published results of the Diabetes Prevention Program (DPP) have raised
further concern about the clinical implications of the term “pre-diabetes.” In the
DPP, individuals who entered with a diagnosis of IGT and still had IGT 3 years
later had a 7.9% incidence of background diabetic retinopathy at the time of
study end. Individuals who entered the DPP with IGT but who progressed to
diabetes after 3 years had a 12.6% incidence of diabetic retinopathy at the time
of study end. Moreover, these IGT individuals developed diabetic retinopathy with
an A1C of 5.9 and 6.1%, respectively, values much less than the current
American Diabetes Association (ADA) treatment goal of 7%. Peripheral
neuropathy also is a common finding in IGT, occurring in as many as 5–10%
individuals. In summary, individuals with IGT are maximally or near maximally

51
insulin resistant, they have lost 80% of their beta cell function, and they have an
approximate 10% incidence of diabetic retinopathy. By both pathophysiological
and clinical standpoints, these pre-diabetic individuals with IGT should be
considered to have type 2 diabetes. The clinical implications of these findings for
the treatment of type 2 diabetes are that the physician must intervene early, at the
stage of IGT or IFG, with interventions that target pathogenic mechanisms known
to promote beta cell failure.
PATHOGENESIS OF -CELL FAILURE (SUPPLEMENTAL) Age. Advancing age
plays an important role in the progressive beta cell failure that characterizes type 2
diabetes. Numerous studies have demonstrated a progressive age-related decline
in beta cell function. This is consistent with the well-established observation that
the
incidence of diabetes increases progressively with advancing age. Genes. -Cell
failure also clusters in families, and studies in first-degree relatives of type 2
diabetic parents and in twins have provided strong evidence for the genetic basis
of the -cell dysfunction. Impaired insulin secretion has been shown to be an
inherited trait in Finnish families with type 2 diabetes with evidence for a
susceptibility locus on chromosome 12. Most recently, a number of genes
associated with beta cell dysfunction in type 2 diabetic individuals have been
described. Of these genes, the transcription factor TCF7L2 is best established.
Studies by Groop and colleagues have shown that the T-allele of single nucleotide
polymorphism rs7903146 of the TCF7L2 gene is associated with impaired insulin
secretion in vivo and reduced responsiveness to glucagon-like peptide 1 (GLP-1).
Both the CT and TT genotypes predict type 2 diabetes in multiple ethnic groups. In
both the Malmo and Botnia studies, presence of either the CT or TT genotype was
associated with a significant reduction in the diabetes-free survival time, with odds
ratios of 1.58 and 1.61, respectively. TCF7L2 encodes for a transcription factor
involved in Wnt signaling, which plays a central role in the regulation of beta-cell
proliferation and insulin secretion. Unfortunately, at present there are no known
therapeutic interventions that can reverse either the age-related decline or
genetic-related factors responsible for impaired insulin secretion. However, there
are a number of causes of beta-cell failure that can be reversed or ameliorated.
Insulin resistance. Insulin resistance, by placing an increased demand on the
beta-cell to hypersecrete insulin, also plays an important role in the progressive
beta-cell failure of type 2 diabetes. Therefore, interventions aimed at enhancing
insulin sensitivity are of paramount importance. The precise mechanism(s) via
which insulin resistance leads to beta-cell failure remain(s) unknown. It commonly
is stated that the beta-cell, by being forced to continuously hypersecrete insulin,
eventually wears out. Although simplistic in nature, this explanation lacks a
mechanistic cause. An alternate hypothesis, for which considerable evidence
exists, is that the cause of the insulin resistance is also directly responsible for the
beta-cell failure. Thus, just as excess deposition of fat (LC-fatty acyl CoAs,
diacylglycerol, and ceramide) in liver and muscle has been shown to cause insulin
resistance in theseorgans, i.e., lipotoxicity, deposition of fat in the beta-cell leads
to impaired insulin secretion and beta-cell failure. Similarly, hypersecretion of islet
amyloid polypeptide (IAPP), which is co-secreted in a one-to-one ratio with insulin,
can lead to progressive –cell failure.

51
52
Lipotoxicity. Elevated plasma free fatty acid (FFA) levels impair insulin
secretion, and this has been referred to as lipotoxicity. Studies from our
laboratory have shown that a physiological elevation of the plasma FFA
concentration for as little as 48 h markedly impairs insulin secretion in genetically
predisposed individuals (Fig. 5). In this study, the normal glucose tolerant
offspring of two type 2 diabetic parents received a 48-h infusion of saline or
Intralipid to approximately double the plasma FFA concentration and then
received a 2-h hyperglycemic (125 mg/dl) clamp. Compared with saline infusion,
lipid infusion markedly impaired both the first and second phases of C-peptide
release. and reduced the insulin secretory rate, calculated by deconvolution of
the plasma C-peptide curve. Conversely, a sustained reduction in plasma FFA
concentration with acipimox in nondiabetic subjects with a strong family history of
type 2 diabetes improved insulin secretion. In vivo studies in rodents and in
humans, as well as in vitro studies, also support an important role for lipotoxicity.
Thus, when human pancreatic islets were incubated for 48 h in presence of 2
mmol/l FFA (oleate-to-palmitate ratio 2:1), insulin secretion, especially the acute
insulin response, was markedly reduced. Exposure to FFA caused a marked
inhibition of insulin mRNA expression, decreased glucose-stimulated insulin
release, and reduction of islet insulin content. Rosiglitazone, a peroxisome
proliferator–activated receptor (PPAR) agonist, prevented all of these deleterious
effects of FFA. Consistent with these in vitro observations, we have shown that
both rosiglitazone and pioglitazone markedly improve the insulin secretion/insulin
resistance index in vivo in type 2 diabetic humans. In summary, interventions—
such as weight loss and TZDs—that mobilize fat out of the -cell would be

53
expected to reverse lipotoxicity and preserve -cell function.

53
Glucotoxicity. Chronically elevated plasma glucose levels also impair -cell
function, and this has been referred to as glucotoxicity. Studies by Rossetti et al.
have provided definitive proof of this concept. Partially pancreatectomized
diabetic rats are characterized by severe defects in both first- and second-phase
insulin secretion compared with control rats. Following treatment with phlorizin,
an inhibitor of renal glucose transport, the plasma glucose profile was normalized
without changes in any other circulating metabolites. Normalization of the plasma
glucose profile was associated with restoration of both the first and second
phases of insulin secretion. In vitro studies with isolated human islets also have
demonstrated that chronic exposure to elevated plasma glucose levels impairs
insulin secretion. In rats, Leahy et al. showed that elevation of the mean day-long
plasma glucose concentration in vivo by as little as 16 mg/dl leads to a marked
inhibition of glucose-stimulated insulin secretion in the isolated perfused
pancreas. Thus, strict glycemic control is essential not only to prevent the
microvascular complications of diabetes but also to reverse the glucotoxic effect
of chronic hyperglycemia on the BETA cells, as well as on hepatic and muscle
insulin resistance.
IAPP. Hypersecretion of IAPP and amyloid deposition within the pancreas have
also been implicated in the progressive BETA cell failure of type 2 diabetes.
Although convincing evidence for a pathogenic role of IAPP exists in rodents, the
natural history of pancreatic amylin deposition in humans has yet to be defined.
To address this issue, Chavez and colleagues examined the relationship between
pancreatic amylin deposition and BETA cell function in 150 baboons spanning a
wide range of glucose tolerance. Since the baboon genome shares more than

54
98% homology with the human genome, results in baboons are likely to be
pertinent to those in humans. As the relative amyloid area of the pancreatic islets
increased from 5.5% to 51%, there was a progressive decline in the log of HOMA-
beta. The decline in beta-cell function was strongly correlated with the increase in
fasting plasma glucose concentration. Studies by Butler and colleagues have also
provided additional evidence for a beta-cell toxic effect for the soluble IAPP fibrils.
Because amylin is secreted in a one-to-one ratio with insulin and IAPP oligomers
are toxic, interventions that improve insulin sensitivity, i.e., TZDs/metformin/weight
loss, by leading to a reduction in insulin secretion, would be expected to preserve
beta-cell function on a long-term basis. Of note, rosiglitazone has been shown to
protect human islets against human IAPP toxicity by a phosphatidylinositol (PI) 3-
kinase–dependent pathway.
Incretins. Abnormalities in the incretin axis have been shown to play an important
role in the progressive beta-cell failure of type 2 diabetes. GLP-1 and glucose-
dependent
insulinotrophic polypeptide (also called gastric inhibitory polypeptide [GIP])
account for 90% of the incretin effect. In type 2 diabetes, there is a deficiency of
GLP-1 and resistance to the action of GIP. The deficiency of GLP-1 can be
observed in individuals with IGT and worsens progressively with progression to
type 2 diabetes. In addition to deficiency of GLP-1, there is resistance to the
stimulatory effect of GLP-1 on insulin secretion. In contrast to GLP-1, plasma
levels of GIP are elevated in type 2 diabetes, yet circulating plasma insulin levels
are reduced. This suggests that there is beta-cell resistance to the stimulatory
effect of GIP on insulin secretion, and this, in fact, has been demonstrated. Of
note, recent studies have shown that tight glycemic control can restore the beta-
cells’ insulin secretory response to GIP. Thus, beta-cell resistance to GIP is
another manifestation of glucotoxicity. Because GLP-1 deficiency occurs early in
the natural history of type 2 diabetes, it follows that GLP-1 replacement therapy is
a logical choice to restore the deficient insulin response that is characteristic of the
diabetic condition.
Summary: beta-cell dysfunction and development of type 2 diabetes. In
summary, although insulin resistance in liver and muscle are well established early
in the natural history of the disease, type 2 diabetes does not occur in the absence
of progressive beta-cell failure.

Up to date 2017
Impaired insulin processing — Insulin production in normal subjects involves
cleavage of insulin from proinsulin; 10 to 15 percent of secreted insulin is
proinsulin and its conversion intermediates. In contrast, the proportion of
immunoreactive insulin that is proinsulin in type 2 diabetes is increased
considerably in the basal state (>40 percent). The difference between normal and
diabetic subjects becomes even more pronounced after stimulation with arginine
or glucagon. The increase in proinsulin secretion persists after matching for
degree of obesity, suggesting that it represents beta-cell dysfunction and not
merely the response to the increased secretory demand imposed by the insulin

54
resistance of obesity. These findings suggest that the processing of proinsulin to
insulin in the beta cells is impaired in type 2 diabetes or that there is insufficient
time for granules to mature properly so that they release more proinsulin.
Role of islet amyloid polypeptide — Islet amyloid polypeptide (amylin) is stored
in insulin secretory granules in the pancreatic beta cells. It is cosecreted with
insulin, resulting in serum concentrations approximately one-tenth those of insulin,
and is present in increased amounts in the pancreas of many patients with type 2
diabetes. First-phase serum insulin and amylin concentrations are lower in
patients with impaired glucose tolerance compared with patients with normal
glucose tolerance, and the concentrations are very low in patients with type 2
diabetes. High concentrations of amylin decrease glucose uptake and inhibit
endogenous insulin secretion, suggesting that amylin may be directly involved in
the pathogenesis of type 2 diabetes. However, administration of physiologic
amounts of amylin has no acute effect on insulin secretion or insulin action in
humans. On the other hand, the administration of an amylin antagonist to rats
results in a fall in blood glucose and an increase in insulin secretion, suggesting
that amylin may tonically inhibit insulin secretion. Thus, it remains unclear whether
amylin has a causative role in type 2 diabetes or is merely present in increased
amounts as a consequence of the defect in insulin secretion. There is no apparent
association between the amylin gene and type 2 diabetes. Pramlintide is a
synthetic analog of human amylin that slows gastric emptying, reduces
postprandial rises in blood glucose concentrations, and improves glycated
hemoglobin (A1C) concentrations in patients with type 1 and type 2 diabetes when
given subcutaneously.

54
SEPTICIDAL SEPTET
The next, and most recent member, implicated in the pathogenesis of type 2
diabetes is the kidney who along with the muscle, liver, -cell, -cell, adipocyte, and
gut, forms the septicidal septet. The kidney filters 162 g ([glomerular filtration rate
180 l/day] [fasting plasma glucose 900 mg/l]) of glucose every day. Ninty
percent of the filtered glucose is reabsorbed by the high capacity SGLT2
transporter in theconvoluted segment of the proximal tubule, and the remaining
10% of the filtered glucose is reabsorbed by the SGLT1 transporter in the straight
segment of the descending proximal tubule (227). The result is that no glucose
appears in the urine. In animal models of both type 1 and type 2 diabetes, the
maximal renal tubular reabsorptive capacity, or Tm, for glucose is increased (228
–230). In humans with type 1 diabetes, Mogensen et al. (231) have shown that
the Tm for glucose is increased. In human type 2 diabetes, the Tm for glucose
has not been systematically examined. No studies in either type 1 or type 2
diabetic individuals have examined the splay in the glucose titration curve in
humans. However, cultured human proximal renal tubular cells from type 2
diabetic patients demonstrate markedly increased levels of SGLT2 mRNA and
protein and a fourfold increase in the uptake of -methyl-D-glucopyranoside
(AMG), a nonmetabolizeable glucose analog. These observations have important
clinical implications. Thus, an adaptive response by the kidney to conserve
glucose, which is essential to meet the energy demands of the body, especially
the brain and other neural tissues, which have an obligate need for glucose,
becomes maladaptive in the diabetic patient. Instead of dumping glucose in the
urine to correct the hyperglycemia, the kidney chooses to hold on to the glucose.

55
Even worse, the ability of the diabetic kidney to reabsorb glucose appears to be
augmented by an absolute increase in the renal reabsorptive capacity for glucose.
In summary, the development of medications that inhibit renal proximal tubular
glucose reabsorption provides a rational approach to the treatment of type 2
diabetes.

55
Dapaglifl ozin, an SGLT2 inhibitor, for diabetes In the 2nd century, Aretaeus of Cappadocia considered
polyuria as a compensatory mechanism in patients with diabetes (diabaino in Ionian Greek, meaning “go
through”) and concluded that the disease was caused by a fault in the kidneys. However, the organ’s role in
normal glucose homoeostasis has received little attention in the treatment of type 2 diabetes in modern
times. The kidneys contribute to glucose homoeostasis in three ways: gluconeogenesis of 15–55 g per day,
utilisation of 25–35 g glucose, and reabsorption of glucose. In healthy people, around 180 g of glucose is fi
ltered by the kidneys in 24 h but nearly all of this is reabsorbed by sodium-glucose transporter 2 (SGLT2)
expressed in the proximal tubules. 1 Glucose reabsorption in the kidney should be the primary target for the
regulation of glucose homoeostasis in people with hyperglycaemia. This mechanism is independent from β-
cell capacity and insulin resistance, the two major pathogenetic determinants for progression of glucose
intolerance. Clinical investigations in patients with familial renal glycosuria, caused by a mutation of the
SLC5A2 gene
that encodes SGLT2, have shown that despite glycosuria of 50 g to more than 100 g per day, these patients
are asymptomatic without relevant loss of electrolytes and with no increased risk of urogenital infections. 2
On the basis of this clinical experience, a new class of SGTL2 inhibitors was developed at the beginning of
this century, and some of them are in phase 3 clinical trials. 3 A nonspecific SGLT1/2 inhibitor—phlorizin—
has already shown beneficial effects on diabetes in animal experiments, but because of serious
gastrointestinal side-eff ects resulting from additional inhibition of SGLT1, the drug could not be considered
for clinical use. 4 In The Lancet today, Clifford Bailey and colleagues present results from the first large-
scale clinical study investigating the efficacy of the SGLT2 inhibitor dapagliflozin. The trial included 534
patients with type 2 diabetes who had insufficient glycaemic control with preceding metformin treatment.
There was a dosedependent reduction in HbA1c in patients treated with dapagliflozin, with a reduction of 0
・84% in patients assigned to a maximum dose of 10 mg per day compared with a reduction of 0・3% in
the placebo group. Beyond glucose control, treatment with dapagliflozin reduced bodyweight and decreased
systolic and diastolic blood pressure. The clinical significance of these effects, attributed to mild osmotic
diuresis and renal elimination of glucose, remains to be clarified. By contrast with previous phase 1 and 2
studies, in which no increased risk for urinary tract infections was reported, in today’s study a slightly higher
incidence of genital infections was seen in patients assigned to dapagliflozin than in those assigned to
placebo. The increase in blood urea-nitrogen and packed-cell volume seen during treatment with
dapagliflozin might reflect haemoconcentration caused by osmotic diuresis and needs to be measured
carefully in continuing trials of SGLT2 inhibitors. Whether SGLT2 inhibitors promote sustained weight loss is
unclear, but 1 g of glucose excreted in the urine equates to energy depletion of about 4 kcal. By contrast
with patients who have benign familial glycosuria, patients with diabetes might also show anomalies in
immune response and impaired cellular defence against infection of the urinary tract and against genital
fungal infection. Many patients with diabetes have asymptomatic bacteriuria and pyelonephritis in
combination with dysfunction of the efferent urinary tract by comparison with the non-diabetic population.
Such latent infections might be activated by increased glycosuria. Even though, so far, no clinicallysignificant
increase in the risk of urinary tract infections has been seen with SGLT2 inhibitors, a final appraisal requires
long-term observational studies that directly measure colonisation with pathogens. One of the major goals in
the treatment of type 2 diabetes is to decrease the cardiovascular risk caused by the coincidence of obesity,

56
hyperglycaemia, dyslipidaemia, hypertension, and endothelial dysfunction in an individual patient. Therefore
the assessment of new therapeutic approaches should not be judged solely by their glucose-lowering
efficacy, but rather by considering the effect on the overall cardiovascular risk profile in patients with type 2
diabetes. Selective SGLT2 inhibitors represent an innovative new class of oral antidiabetic agents at a time
when disappointing results of large trials with established antidiabetic drugs, such as ACCORD or RECORD,
require a critical re-evaluation of antidiabetic treatment. Beyond reducing glucotoxicity, dapagliflozin might
improve cardiovascular outcome by reducing overweight and blood pressure. Glucose reabsorption in the
proximal tubules of the kidneys as a target for controlling hyperglycaemia by specifi c SGLT2 inhibitors brings
us back to the roots of diabetes. Dapagliflozin was only moderately effective as an add-on drug in reducing
glycaemic load in patients with type 2 diabetes who were insufficiently controlled with metformin according to
an internationally accepted treatment algorithm. The only relevant adverse effect was a minor increase in
genital infections. But the net balance of this novel group of oral antidiabetic drugs looks promising. Longterm
trials should be designed with careful monitoring of urogenital infections on the basis of comparative
investigations with established oral antidiabetic drugs. In the absence of randomised trials, SGLT2 inhibitors
are candidates for add-on therapy to metformin as shown in today’s study. Because of the role of glucotoxicity
in the pathophysiology of type 2 diabetes, and in view of weight loss and low risk of hypoglycaemia, SGLT2
inhibitors in the future also might be considered for the treatment of early-stage and late-stage type 2
diabetes.

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ROLE OF DIET, OBESITY, AND INFLAMMATION — The prevalence of
impaired glucose tolerance and type 2 diabetes has increased dramatically in the
United States population in the past two decades. The most striking features in
these groups and of most patients who develop type 2 diabetes are increased
weight gain and decreased physical activity, each of which increases the risk of
diabetes. Obesity, for example, causes peripheral resistance to insulin-mediated
glucose uptake and may also decrease the sensitivity of the beta cells to glucose.
These defects are largely reversed by weight loss, leading to a fall in blood
glucose concentrations toward normal. Although not as effective as weight loss,
an exercise regimen also may improve glucose tolerance and prevent the
development of overt diabetes. The mechanism by which obesity induces insulin
resistance is poorly understood. The pattern of fat distribution and perhaps a
genetic abnormality in the beta-3-adrenergic receptor, as described above,
appear to contribute. The c-Jun amino-terminal kinase (JNK) pathway may be an
important mediator of the relationship between obesity and insulin resistance as
JNK activity is increased in obesity, an effect that can interfere with insulin action.
In animal models of obesity, absence of JNK1 results in decreased adiposity and
enhanced insulin sensitivity.

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Pattern of fat distribution — Upper body or male-type obesity has a much
greater association with insulin resistance and impaired glucose tolerance than
lower body or female-type obesity (figure 6).
Serial changes in blood glucose concentrations during an oral glucose tolerance
test in nine patients with upper body obesity, 16 patients with lower body obesity,
and nine normal subjects. Both obese groups had impaired glucose tolerance,
but the abnormality was more pronounced in the group with upper body obesity.
The degree of glucose intolerance underestimated the degree of insulin
resistance, since peak serum insulin concentrations were also much higher in the
group with upper body obesity (225 µU/mL versus 115 and 65 µU/mL in the other
two groups). To convert blood glucose to mmol/L, divide by 18; to convert serum
insulin to pmol/L, multiply by 6.

58
59
Many studies have focused on the role of inflammation as a common mediator
linking obesity to both the pathogenesis of diabetes and atherosclerosis. The
incidence of type 2 diabetes has been correlated with increased levels of markers
of inflammation, including C-reactive protein, interleukin (IL)-6, plasminogen
activator inhibitor-1 (PAI-1), tumor necrosis factor-alpha (TNFa), and white cell
count. Adipokines (factors released from adipose tissue) stimulate inflammatory
activity, which correlates with insulin resistance, as demonstrated in mouse
models. Intensive lifestyle interventions have been shown to decrease markers of
inflammation. Anti-inflammatory properties of medications including
thiazolidinediones and statins may contribute therapeutic benefit beyond their
activity to lower glucose and cholesterol levels, respectively. Among patients with
rheumatoid arthritis or psoriasis, use of anti-inflammatory, disease-modifying
antirheumatic drugs, such as TNF inhibitors and hydroxychloroquine, is
associated with a lower incidence of diabetes than other agents.
Free fatty acids — Plasma free fatty acid (FFA) concentrations are high in obese
patients. A high plasma FFA concentration is a risk factor for type 2 diabetes
(relative risk 2.3), may inhibit insulin secretion, and can inhibit insulin-stimulated
glucose uptake in patients with type 2 diabetes. Elevated plasma free fatty acid
(FFA) levels impair insulin secretion, and this has been referred to as lipotoxicity.
Physiological elevation of the plasma FFA concentration for as little as 48 h
markedly impairs insulin secretion in genetically predisposed individuals. In this
study, the normal glucose tolerant offspring of two type 2 diabetic parents
received a 48-h infusion of saline or Intralipid to approximately double the plasma
FFA concentration and then received a 2-h hyperglycemic (125 mg/dl) clamp.

60
Compared with saline infusion, lipid infusion markedly impaired both the first and
second phases of C-peptide release and reduced the insulin secretory rate,
calculated by deconvolution of the plasma C-peptide curve.
Factors released from adipose tissue
Leptin — Leptin is produced by adipocytes and is secreted in proportion to
adipocyte mass. It signals the hypothalamus about the quantity of stored fat.
Studies in humans and animals have shown that leptin deficiency and leptin
resistance are associated with obesity and insulin resistance. In addition to its
hypothalamic actions, leptin also has biologic actions in peripheral tissues, such
as the pancreas. In a preliminary study of a pancreas-specific, leptin receptor-
knockout (KO) mouse model, the absence of leptin signaling was associated with
improved glucose tolerance in KO mice compared with control mice when the
mice were fed a standard diet. The administration of a high-fat diet resulted in
weight gain and insulin resistance in both KO and control mice. The control mice
developed islet-cell hyperplasia to compensate for insulin resistance. However,
deterioration in the acute insulin response to glucose and poor islet-cell growth
were seen in the KO mice. These results suggest conflicting roles for leptin,
depending upon diet and presence of insulin resistance. Additional studies are
required to determine if leptin is important for the regulation of beta-
cell mass/function, and whether abnormal leptin receptor signaling in islet cells
plays a role in the pathogenesis of obesity-related type 2 diabetes.
Adiponectin — Adiponectin, an adipocyte-derived cytokine, reduces levels of
blood FFAs and has been associated with improved lipid profiles, better glycemic
control, and reduced inflammation in diabetic patients. Adiponectin has also been
inversely associated with risk for diabetes in the nondiabetic population. A number
of observations suggest that deficiency of adiponectin, an adipocyte-derived
hormone, plays a role in the development of insulin resistance and subsequent
type 2 diabetes:
●Lower adiponectin levels are more closely related to the degree of insulin
resistance and hyperinsulinemia than to the degree of adiposity and glucose
intolerance. ●In a study of subjects with insulin resistance, administration of
thiazolidinediones, which has been shown to decrease the development of
diabetes, increased serum adiponectin concentrations without affecting body
weight. ●Adiponectin is down-regulated in obesity. In obese or lipoatrophic mice,
adiponectin administration decreases the degree of insulin resistance associated
with these conditions. ●In adiponectin-KO mice, plasma and adipocyte
concentrations of TNFa increased, resulting in severe diet-induced insulin
resistance. ●Two adiponectin receptors have been cloned: AdipoR1 and AdipoR2,
which are expressed primarily in skeletal muscle and the liver, respectively.
Adiponectin, and adiponectin receptors, may become an important target in the
management of diabetes. Two studies have suggested that dietary cereal fiber
and reduced glycemic load can increase adiponectin in diabetic men and women.
In addition to its strong association with type 2 diabetes risk, preliminary data
suggest that adiponectin may be moderately associated with cardiovascular
morbidity and mortality. High adiponectin concentrations are associated with a
favorable cardiovascular risk profile. However, the relationship is more complex.

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High adiponectin concentrations have also been associated with increased all-
cause and cardiovascular mortality. The discrepancy may be related to the patient
population studied (men versus women, older versus younger, prevalent
cardiovascular disease). In addition, adiponectin may not directly affect
cardiovascular risk, but may be a marker of other risks. Additional studies are
needed to clarify the relationship between adiponectin and cardiovascular disease.
Tumor necrosis factor-alpha — Studies in genetically obese animals suggest
that increased release of TNFa from adipose tissue may play a major role in the
impairment in insulin action. This suggestion is based on the following
observations: administration of anti-TNFa antibody led to marked improvement in
glucose utilization in obese rats, and obese mice genetically lacking TNFa have
more normal insulin sensitivity. In another study, weight reduction in obese
animals was associated with both improved insulin activity and decreased TNFa
gene expression. A fatty acid-binding protein in adipocytes, aP2, may provide the
link by which FFA in obesity leads to increased expression of TNFa in obesity.
Targeted mutations in the gene for this protein are associated with obesity but not
insulin resistance or increased TNFa expression. In addition, activation of Toll-like
receptor 4 (TLR4) by nutritional fatty acids has been reported to increase TNFa
and IL-6. Thus, there may be more than one mechanism by which obesity induces
insulin resistance. The applicability of these findings to humans is uncertain. A
preliminary study found a strong correlation among the degree of obesity,
hyperinsulinemia, and TNFa mRNA in adipose tissue. In addition, in a study of a
homogeneous Native Canadian population, plasma TNFa concentrations were
positively correlated with insulin resistance.
Chemokine molecules — Chemokines (chemotactic proinflammatory cytokines)
are a family of low molecular weight proteins that are potent chemo-attractants of
leukocytes and may modulate the formation of reactive oxygen species and
cytokines. Chemokines specific for neutrophils, called CXC chemokines, are
distinguished from other chemokines by a protein motif in which the first two
cysteines are separated by one amino acid. CXC chemokines are produced by
many different cell types, including endothelial cells, platelets, neutrophils, T
lymphocytes, and monocytes. The chemokine molecule CXCL5 (CXC ligand 5) is
expressed at high levels in the macrophage fraction of white adipose tissue. When
it binds to the chemokine receptor CXCR2, it reduces insulin-stimulated glucose
uptake in muscle, suggesting a potential role of CXCL5 in mediating insulin
resistance. In support of this hypothesis are the observations that serum levels of
CXCL5 are higher in obese compared with normal-weight individuals and in
insulin-resistant obese compared with noninsulin-resistant obese individuals.
CXCL5 concentrations decrease with weight loss. In addition, inhibition of CXCL5
via administration of a neutralizing antibody or a selective CXCR2 antagonist in
two mice models of insulin resistance resulted in an improvement in insulin
sensitivity. Thus, CXCL5 may be another link among obesity, inflammation, and
insulin resistance.
Plasminogen activator inhibitor — PAI-1, an inhibitor of fibrinolysis, is another
protein related to adipocytes. It is also secreted from endothelial cells,
mononuclear cells, hepatocytes, and fibroblasts and has been associated with an
increased risk for cardiovascular disease. A five-year prospective study of 2356

60
adults aged 70 to 79 years identified 143 new cases of diabetes. Elevated levels
of PAI-1 were an independent predictor of onset of diabetes OR 1.35 after
controlling for components of the metabolic syndrome (body mass index [BMI],
visceral fat, lipids, hypertension, and fasting plasma glucose [FPG]); other
adipocytokines, including adiponectin, TNFa, and leptin, did not independently
predict diabetes.
Resistin — In diet-induced or genetic obesity in mice, adipocytes secrete a
signaling molecule named resistin. Administration of resistin decreases while
neutralization of resistin increases insulin-mediated glucose uptake by adipocytes.
Hypothalamic administration of resistin also enhances glucose production,
independent of changes in glucoregulatory hormones. Thus, resistin may be a
hormone that links obesity to diabetes.
Retinol-binding protein 4 — Retinol-binding protein 4 (RBP4), another protein
released from adipocytes, correlates with the degree of insulin resistance in
patients with obesity, impaired glucose tolerance, or type 2 diabetes, as well as in
nonobese subjects with or without a strong family history of type 2 diabetes.
Exercise training reduced RBP4 levels in patients whose insulin resistance
improved with exercise. In a mouse model, mice lacking adipocyte glucose
transporter 4 (GLUT4) had increased levels of RBP4, and RBP4 was shown to
cause insulin resistance in mouse muscle and liver. An inverse relationship
between GLUT4 in adipocytes and serum RBP4 was demonstrated in the human
study, as well. Whether RBP4 in humans causes, or is correlated with, insulin
resistance has not been determined.
Other factors
Interleukin-1 beta — Another cytokine, interleukin-1 beta, a recognized inhibitor
of glucose-induced insulin secretion, has been reported to undergo increased
synthesis by the islet (presumably the beta cell itself) under conditions of high
glucose concentrations. This raises the possibility of a negative downward spiral:
chronic exposure to hyperglycemia leading to high concentrations of interleukin-1
beta within the islet, which in turn worsens beta-cell function. Interleukin-1
receptor antagonists, which decrease the activity of interleukin-1 beta, may
therefore improve beta-cell function and glycemic control.
Uncoupling protein 2 — Uncoupling protein 2 (UCP2) is an inhibitor of insulin
secretion. Ob/ob mice are extremely obese, insulin-resistant, and diabetic, with
increased levels of UCP2 in pancreatic islet cells. When UCP2-KO mice were
crossed with ob/ob mice to produce ob/ob mice, the obesity and insulin resistance
did not change, but the degree of hyperglycemia decreased as a result of
increased insulin secretion.
Animal studies suggest a possible additional genetic association between obesity
and diabetes. Leptin, a hormone released from adipocytes, modulates body
weight by regulating food intake and energy expenditure.
Obestatin — Obestatin, a hormone that was isolated from rat stomach, is
encoded by the ghrelin gene and opposes the effects of ghrelin on food intake.
Treatment of rats with obestatin suppresses food intake, inhibits jejunal
contraction, and decreases weight gain. Circulating obestatin concentrations are

60
decreased in individuals with diabetes and impaired glucose tolerance compared
with normal glucose tolerance. In addition, expression of the obestatin receptor in
adipose tissue is downregulated in obesity-associated type 2 diabetes, but not in
normoglycemic obese subjects, suggesting that obestatin may play a role in
glucose regulation and development of type 2 diabetes independent of obesity.

60
Reinventing Type 2 Diabetes Pathogenesis, Treatment, and Prevention Roger H. Unger, MD THE
CONVENTIONAL GLUCOCENTRIC PERSPECTIVE OF type 2 diabetes views hyperglycemia as a primary
disease caused by an etiologically uncertain combination of obesity-associated insulin resistance and beta
cell loss (a disease of glucose metabolism to be treated with antihyperglycemic agents, including high-dose
insulin, if necessary). By contrast, the novel lipocentric view depicts the hyperglycemia of type 2 diabetes,
and the underlying insulin resistance and beta cell loss, as being secondary to the metabolic trauma caused
by ectopic lipid deposition or lipotoxicity. If this is in fact the case, hyperglycemia should be corrected by
eliminating the lipid overload. The study by Dixon et al provides support for this lipocentric hypothesis, by
demonstrating that weight loss that follows gastric banding is accompanied by remission of diabetes in 73%
of obese patients with type 2 diabetes. This finding supports 45 years of biochemical, physiological, and
clinical research pointing to lipid overload as the underlying cause of this disease and of the other coexisting
components of the metabolic syndrome.
History of the Lipocentric Concept During the past half century, US individuals have been exposed to 2
historically unprecedented changes in their caloric
environment that would predispose to lipid overload. First, meal preparation was increasingly outsourced
from the family kitchen to commercial processors and purveyors
of lipid-rich, calorie-dense foods, resulting in a 168-kcal/d and a 335-kcal/d increase in the caloric intake of
men and women, respectively, during the past 30 years.3 Second,
during the same period physical activities that had always been a part of normal life have been substantially
decreased or eliminated by a variety of immobilizing technologies, causing a substantial decrease in daily
caloric expenditure. Not surprisingly, this caloric surplus has dramatically altered the body habitus of most
US individuals,
more than two-thirds of whom are now overweight or obese. Not long after the start of this obesity epidemic,
Yalow and Berson reported the development of the radioimmunoassay for insulin, an achievement that was
to earn a Nobel Prize. This new technology led to the discovery that overweight individuals with normal
glucose levels have higher insulin levels than normal-weight individuals.The coexistence of hyperinsulinemia
and normoglycemia implied resistance to the action of insulin. This interpretation was soon confirmed with
the development of techniques for precise measurement of insulin action on glucose metabolism, and along
with other studies demonstrated a relationship between obesity and insulin resistance. However, the
mechanism of this relationship and its clinical consequences continued to be a matter of some controversy,
despite a relative consensus that insulin resistance is related to overt type 2 diabetes and the other
morbidities of the metabolic syndrome. During the same decade, the concept of a relationship between
glucose and lipid metabolism emerged. In 1963, Randle et al proposed a “glucose-fatty acid cycle,” which
provided a plausible biochemical explanation for lipid induced impairment of glucose metabolism that could
cause insulin resistance. In 1992, McGarry advanced the position that abnormal metabolism of lipids, not
glucose, might be the primary metabolic defect in type 2 diabetes. Indeed, there is now evidence that in
muscle, fatty acids inhibit insulin-mediated glucose uptake by interfering with the translocation of the glucose
transporter GLUT-4 to the plasma membrane, thus effectively blocking glucose uptake by myocytes;
whereas, in liver, fatty acids inhibit insulin-mediated suppression of glycogenolysis and gluconeogenesis.

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New insights into the mechanistic links between lipid metabolism and insulin resistance have been provided
by the use of isotopes and magnetic resonance spectroscopy. But whatever the precise molecular pathways
to insulin resistance, there seems to be broad consensus that ectopic accumulation of unoxidized fatty acids
is a major factor. In 1994, Lee et al demonstrated that ectopic lipids accumulate in pancreatic islets in parallel
with other tissues and can cause the subtotal lipotoxic destruction of beta cells that precipitates the
hyperglycemia, thereby providing the final evidence for the lipocentric theory of type 2 diabetes. The Insulin
Resistance Paradox If surplus lipids are in fact the link between obesity and insulin
resistance, the phrase “insulin resistance of obesity” becomes oxymoronic, in as much as the presence of
obesity constitutes evidence for robust activity of a major insulinmediated process, lipogenesis. Neither
obesity nor ectopic lipid overload with which it is so commonly associated could occur were there resistance
to insulin-mediated lipogenesis. Resistance to insulin action on glucose metabolism but sensitivity to insulin
action on lipogenesis is a paradox. A molecular explanation for the paradox was reported in 2000 when
insulin was shown to down-regulate insulin receptor substrate, while stimulating the production of sterol
response element binding protein 1c (SREBP-1c), a transcription factor that stimulates lipogenesis. This
dichotomous relationship explains how the liver continues to synthesize fatty acids while resisting insulin-
mediated suppression of hepatic glucose production. The Lipocentric Pathway to Hyperglycemia Based on
the epidemiological, clinical, metabolic, and molecular information now available, the following teleologically
plausible pathway emerges, with increased caloric balance as the primary perturbation: (1) caloric surplus →
(2) hyperinsulinemia → (3) increased expression of the lipogenic transcription factor SREBP-1c → (4)
increased lipogenesis → (5) increased adiposity → (6) ectopic lipid deposition → (7) insulin resistance→ (8)
beta cell lipotoxicity→ (9) hyperglycemia. Not only is this scheme historically and epidemiologically congruent
with the change in the caloric environment, but it fits physiologically with the known actions of overnutrition on
insulin secretion and the known actions of insulin on the disposition of unused calories—initially as fat in
adipocytes—but ultimately as ectopic fat in nonadipocytes, such as myocytes and hepatocytes. If this
formulation is correct, resistance to insulin-mediated uptake of glucose in tissues associated with increasing
ectopic lipid deposition may serve as a compensatory adaptation designed to limit further lipid accumulation
by keeping lipogenic substrate out of liver cells, even at the cost of an abnormal glucose tolerance test.
Clinical Implications of the Lipocentric Concept This lipocentric conceptual revision may have at least 2
important clinical implications. First, there has long been evidence that relatively modest weight loss by
caloric restriction, by exercise, or both can reduce insulin resistance and hyperglycemia, even if it fails to
achieve its desired cosmetic goal. In other words, overweight individuals should restrict calories to prevent
disease, whether or not optimal weight loss is achieved. Second, the lipocentric concept raises questions as
to the preferred therapy for obese patients with poorly controlled, insulin-resistant type 2 diabetes. The
availability of U500 insulin now makes it easier to administer the doses of insulin necessary to overpower
insulin resistance. But is it rational to overpower resistance to insulin without eliminating the caloric excess
that created the abnormalities? Or might the superimposition of exogenous hyperinsulinemia on preexisting
endogenous hyperinsulinemia worsen the ectopic lipid overload by providing yet more substrate for
lipogenesis from the continuing surplus of calories? If so, intensive insulin therapy would be relatively
contraindicated. For instance, the National Institutes of Health announced on February 8, 2008, that the
National Heart, Lung, and Blood Institute has halted a clinical trial of aggressive lowering of blood glucose
levels in patients with high-risk type 2 diabetes because of an increase in deaths from myocardial infarction or
stroke. In this study, apart of the ACCORD (Action to Control Cardiovascular Risk in Diabetes) trial, blood
glucose levels were maintained as close
to normal as possible and some patients reportedly received as many as 5 insulin injections per day. The
unfortunate outcome suggests that overpowering the insulin resistance may be harmful, quite possibly
because doing so forces lipogenesis and promotes ectopic deposition of lipids. This should in no way be
construed as minimizing the
importance of treating the hyperglycemia. It merely advocates a strategy that would eliminate hyperglycemia
without amplifying the underlying abnormality, the ectopic lipid overload. The most rational therapy would be
one that eliminates the caloric surplus and thus reduces the hyperinsulinemia and the lipogenesis. This will
slowly decrease lipogenesis and ectopic lipid deposition, and the hyperglycemia will gradually decline. If
hyperglycemia fails to decline, anti-diabetic drugs that reduce food intake, that reduce ectopic lipids, or that
do both can be added. Bariatric surgery may provide perhaps the most certain way to reverse the chronic
caloric surplus and the lipid overload. If hyperglycemia persists despite aggressive diet restriction and weight
loss, insulin therapy will be required as a last resort. The preferred strategy to correct hyperglycemia is to
eliminate its proximal cause, caloric surplus. This will gradually reduce the diet-driven hyperinsulinemia and
excessive lipogenesis that is responsible for the abnormal glucose metabolism.

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The antidiabetic drug metformin also activates hepatic AMPK. Treatment
of ob/ob mice with metformin markedly reduced hepatic steatosis, and its
administration to humans with NASH improved LFT numbers and
decreased liver size. A second class of antidiabetic drugs, the
thiazolidinediones, are principally recognized as drugs that activate PPAR-
γ; however, recent data suggest that they also can activate AMPK.
Insulin resistance in nonalcoholic steatohepatitis is frequently associated
with chronic hyperinsulinemia, hyperglycemia, and an excessive supply of
plasma free fatty acids to the liver; this metabolic milieu promotes hepatic
lipogenesis. Thiazolidinediones reverse these abnormalities by
ameliorating insulin resistance in adipose tissues, the liver, and muscles.
Patients with nonalcoholic steatohepatitis have low plasma adiponectin
levels and adiponectin receptor expression in the liver.Thiazolidinediones
increase plasma adiponectin levels, activate AMP-activated protein
kinase, stimulate fatty acid oxidation, and inhibit hepatic fatty acid
synthesis. In patients with nonalcoholic steatohepatitis, there is activation
of the intracellular proinflammatory signaling pathways, and
thiazolidinediones have antiinflammatory effects.
Estudios posteriores no demostraron tal aumento de G6F y los estudios
de Shulman sugiere lo que está en la diapo.

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Liver biopsy specimen showing fatty infiltration with so-called chicken-wire
appearance.
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67
Obesity is accompanied by resistance to insulin action in skeletal muscle,
liver, and blood vessels. Insulin resistance in muscle could be a
mechanism to protect muscle from excessive postprandial glucose uptake
in conditions of calorie excess. We have suggested that lowgrade
inflammation, dependent on production of proinflammatory cytokines by
adipose tissue, might underlie relations between insulin resistance and
vascular disease.1 We now propose that depots of fat around both large
and small vessels could be an important source of these cytokines, and
that periarteriolar fat plays a physiological role in regulating distribution of
blood flow, through outside-to-inside cellular cross-talk and through
regional vascular signalling—mechanisms we term vasocrine. Moreover,
perivascular fat might contribute both to insulin resistance and to
macrovascular disease.
We propose that increased adipose mass also generates cytokine signals
to blood vessels through the medium of perivascular fat, which is also
ectopic although within adipocytes. We suggest that the organism’s
perivascular adipocytes act as an integrated organ responsible for
paracrine and vasocrine signalling, which in turn contributes to skeletal
muscle insulin resistance. Substantial data from rats, and more limited
human studies, have shown insulin to have a striking and rapid effect in
increasing nutritive blood flow to skeletal muscle and skin, an effect
shared, in muscle, with exercise. We propose that this action of insulin
helps coordinate postprandial delivery of substrate and hormone to
insulin-sensitive tissues. Insulin’s ability to recruit capillaries is likely to

68
depend on its action in dilating precapillary arterioles. Our in-vitro studies in larger
(so-called .rst order) arterioles from rat cremaster muscle, however, show a dual
effect of insulin on arteriolar endothelium. In vessels from healthy rats, insulin has
no net effect on vessel diameter, because of a balance between stimulation of two
pathways—NO-mediated vasodilatation and endothelin-1-mediated
vasoconstriction. Insulin
stimulates activation of endothelial NO synthase: the signalling pathway is through
insulin receptor substrate-1, phosphoinositol-3-kinase, and Akt. However, if this
pathway is inhibited, the arteriole constricts, a response mediated by endothelin 1
through the extracellular signal-related kinase-1/2 pathway.13 In the cremaster
muscle arteriole from a Zucker fatty rat, our findings show that insulin induces
vasoconstriction because of unopposed action of endothelin 1. These
observations imply a dual insulin signalling
mechanism—one pathway stimulating synthesis of NO, the other endothelin-1
release. In obesity, the endothelin-1 pathway dominates, insulin resistance in
these arterioles being limited to the insulin receptor substrate-1, phosphoinositol-
3-kinase, and Akt pathway. We propose that this dual mechanism allows for local
regulation of insulin action.
We propose that, in situations of chronic calorie excess or inactivity, an organism
protects its muscle from substrate over-supply by creating a local fat pad at the
vessel origin with specialist vasoregulatory function. Adipocytokines from these
pads, such as TNF, inhibit the phosphoinositol-3-kinase signalling pathway of
endothelial NO synthase activation and NO production.14 In vivo, insulin-induced
vasodilatation and production nutritive capillary recruitment are inhibited by
TNF.15 In consequence, these vasoregulatory fat depots have the capacity locally
to modulate the systemic vasodilating effects of insulin, instead causing
vasoconstriction. We also suggest a novel mechanism to enhance the signalling
function of TNF. The effects of insulin to stimulate nutritive flow, and of TNF to
inhibit it, is likely to result from recruitment or drop-out of muscle capillary
networks depending on vasodilatation or constriction in smaller, precapillary
arterioles.

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Figure 2: Vasocrine signalling from perivascular fat
Adipocytokines secreted from perivascular adipocytes inhibit the PI3-K
pathway of insulin signalling, leaving unopposed vasoconstrictor effects of
endothelin 1. High concentrations of TNF access the vascular lumen,
resulting in inhibition of endothelial PI3-K pathway insulin signalling in
downstream vessels. Reduced insulinmediated
enhancement of muscle nutritive blood .ow will contribute to insulin
resistance. EC=endothelial cell. VSMC=vascular smooth muscle cell.
eNOS=endothelial nitric oxide synthase. PI3-K=phosphoinositol-3-kinase.
TNF=tumour necrosis factor . ERK=extracellular signal-related kinase.
NEFA=non-esteri.ed fatty acids.

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