Insulin Action and Its Disturbances in Disease - part 5 doc

White adipose tissue Although obesity is robustly associated with impaired insulin sensitivity, the severe insulin resistance of both humans with lipodystrophy and of mice with... This i

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132 Tomlinson, J W., Moore, J., Cooper, M S., Bujalska, I., Shahmanesh, M., Burt, C.,Strain, A., Hewison, M and Stewart, P M (2001) Regulation of expression of 11beta-hydroxysteroid dehydrogenase type 1 in adipose tissue: tissue-specific induction by

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Homeostasis

Robert K Semple and Stephen O’Rahilly

It has long been known that various xenobiotic compounds, when tered to mice, give rise to exuberant proliferation of hepatic peroxisomes, and ultimately to tumour development In 1990 the mediator of this response was cloned and identified as a nuclear hormone receptor subsequently called per-

developments, large scale chemical screening in the 1980s identified lidinediones as potent agents for lowering blood glucose and improving lipid

two lines of investigation with the realization that the molecular target of the

interplay between lipid and glucose metabolism This occurred at a time in the early 1990s when the ‘glucocentric’ view of type 2 diabetes as a disease princi-

usurped by the resurgent appreciation that it is a complex metabolic disease in which abnormal lipid and glucose homeostasis are intimately and inextricably linked In the decade since then, a wealth of experimental data has confirmed the

insulin-sensitive tissues, and thiazolidinediones have proved beneficial tically as the first new class of insulin-sensitizing agents for several decades.

pathophysiology of this most prevalent condition, many questions remain about

Insulin Resistance. Edited by Sudhesh Kumar and Stephen O’Rahilly

 2005 John Wiley & Sons, Ltd ISBN: 0-470-85008-6

its biology Here the progress of investigation to date and the outstanding issues will briefly be reviewed.

9.1 Evidence from cell and rodent models

the retinoic acid receptor (RXR) In the presence of ligand it recruits activator molecules, which target chromatin-decondensing complexes to the promoter region and render it accessible for the initiation of transcription Con- versely, in the presence of an antagonist, and perhaps in the unliganded state,

chro-matin and sequestration of promoter elements In addition, there is an evolving

usu-ally negatively, through competition with other transcription factors for such accessory molecules Although thiazolidinediones have been identified as potent

potent endogenous ligands exist The most widely studied candidate has been 15

but a more recent reanalysis has suggested not only that in vivo concentrations

are too low for it to be a relevant ligand, but also that levels fail to correlate

eicosanoids and prostaglandins have also been shown in vitro to activate the

receptor The binding affinities of these agents tend to be rather low, leading

to the suggestion that, instead of conforming to the paradigm of receptors with

of fatty acid flux, a property which might help subserve a role as a nutritional sensor and co-ordinator of metabolic responses Further complexity is attested

to by the ability of RXR ligands, too, to stimulate the transactivational activity

where around 30 per cent of its protein expression is accounted for by a splice

expressed at high levels in large intestine and white blood cells of both the lymphoid and myeloid lineages, and at lower levels in kidney, liver, skeletal

is incompletely understood, and will form the remainder of this discussion.

White adipose tissue

Although obesity is robustly associated with impaired insulin sensitivity, the severe insulin resistance of both humans with lipodystrophy and of mice with

EVIDENCE FROM CELL AND RODENT MODELS 239

genetically ablated white adipose tissue bears witness to the importance of

Importantly, transplantation of white adipose tissue into these mice dramatically

absence of fat per se that leads to the abnormal metabolic phenotype Human

subjects with lipodystrophy exhibit a similar pattern of severe insulin resistance and dyslipidaemia, and are discussed in detail in Section 17.5.

characterized in vitro models of adipogenesis, as detailed in Figure 9.1 Mouse

embryo-derived preadipocyte cell lines such as 3T3-L1 have been key tools in establishing the transcriptional cascade of adipogenesis Comparison of this data

C/EBP β

C/EBPδ PPAR γ

C/EBPα dexamethasone

10% serum Insulin

10% serum (a)

(b)

ERK

C/EBPα PPARγ

with adipose phenotypes of genetically modified animals suggests that they do,

at least in part, model the in vivo situation, although it is also clear that many more influences on in vivo adipogenesis remain to be discovered Figure 9.1(a)

shows a typical pattern of expression of some of the key genes implicated in L1 differentiation, showing details of the artificial differentiation medium used Figure 9.1(b) shows a simplified model of the transcriptional cascade, showing

3T3-a complex series of kindling re3T3-actions le3T3-ading to 3T3-a robust mutu3T3-ally sust3T3-aining

adipocyte gene expression.

In view of the physiological importance of adipose tissue, the simplest

beneficial effects of its activation derive solely from its ability to promote the expansion of adipose tissue However, thiazolidinediones are not used in clinical practice principally as a means of inducing adipogenesis in lipodys- trophic subjects, but rather are used effectively in patients of normal or more commonly increased adiposity to enhance insulin sensitivity Thus, apparently paradoxically, a pro-adipogenic agent is used to treat a condition that is often precipitated by the development of excessive adipose tissue This paradox is at least partly resolved by consideration of the complex biology of adipose tis-

sue in vivo, which cannot be replicated fully in vitro: far from the historical

perception of adipose tissue as a relatively inert reservoir for excess dietary fat, it is now understood that white adipose tissue is a complex ‘organ’, which plays a key role in orchestrating numerous metabolic processes It is constantly sensing the nutritional status of the whole organism, is in continuous commu- nication with other tissues such as liver and muscle and is moreover spatially heterogeneous, with fat depots at different anatomical sites exhibiting markedly different patterns of gene expression, presumably reflecting distinct metabolic functions Thus, modifying the hypothesis by invoking depot-selective responses

by pharmacological studies in mice: administration of potent and selective azolidinediones results in a preferential expansion of inguinal fat, analogous to human subcutaneous adipose tissue, at the expense of retroperitoneal and other

depots that are less hormonally sensitive, and that do not have direct access

to the portal circulation and hence the liver, insulin sensitivity is enhanced However, the increased mass of inguinal fat pads is not simply due to accu- mulation of more tissue of the same morphology: analysis of the distribution

of adipocyte size reveals that, while the total number of cells does increase, these cells are of smaller size due to a combination of hyperplasia of precursor

in different genetic and dietary models of obesity have consistently revealed a

EVIDENCE FROM CELL AND RODENT MODELS 241

adipose tissue, but also in regulating the function of that mature tissue.

sen-sitivity has been to manipulate mice genetically in order to look at the effects of

het-erozygous knockout mice has been instructive, and has revealed some surprising

knockout mice are more insulin sensitive than their wild type counterparts at

underlying this showed that, as in thiazolidinedione-treated wild type animals, the mean size of the adipocytes decreased, though in this case they also declined

in number, so that body weight and fat mass of the heterozygotes was reduced However, when these heterozygous knockout mice were treated with antagonists

found no difference in adipocyte hypertrophy and insulin resistance between erozygous knockout and wild type animals, but did find the heterozygous animals

A second, and complementary, genetic approach involved generation of mice

in vitro to reduce PPAR γ transactivational activity, and so loss of the

at least intermittently The homozygous mice had no more adipose tissue than wild type counterparts, and were protected from high-fat-diet-induced insulin resistance and adipocyte hypertrophy.

sensi-tivity is more complex than first imagined, with either stimulation or a moderate reduction in its action apparently leading to metabolic benefits These metabolic

thresh-old It appears that the unifying feature of the two situations is a change in adipocyte morphology, such that the cells are predominantly smaller and less lipid laden The possible functional connections between these adipocyte mor- phological changes and enhanced insulin sensitivity may broadly be classified

effects on the insulin sensitivity of the adipocytes, thus augmenting the rate of glucose disposal in adipose tissue Second, the trapping of fatty acids in adipose tissue in the fed state may be rendered more efficient, and finally the change in adipocyte phenotype may result in an altered profile of secretory factors, which have remote effects on other insulin-sensitive tissues.

Direct effects on adipose tissue insulin sensitivity

To the extent that preadipocyte differentiation involves the expression of many genes that confer insulin sensitivity upon adipose tissue, it is to be expected

glu-cose uptake of adipose tissue However, besides effects on the total number of adipocytes and on the population distribution of differentiated and undifferen- tiated cells, there is also evidence that enhanced glucose uptake in response to

the 3T3-L1 murine embryo fibroblast model of adipogenesis, it has been shown that in cells differentiated for 15 days, and regarded as equivalent to mature

reduc-tion in cell size and triglyceride content are seen in the presence of a dominant

The induction of Glut4 expression and glucose transport in response to insulin

is known to depend in part on activation of phosphatidyl inositol-3-kinase stream from IRS1 and 2 However, a second pathway, involving interaction of the tyrosine kinase cCbl with the insulin receptor, is also thought to be involved.

adipocytes to insulin: cCbl interacts with the insulin receptor only via the tor protein CAP, or cCbl-associated protein, and expression of CAP appears to

analysis of the CAP gene has confirmed the presence therein of a functional

phosphory-lated by the receptor, and the CAP–cCbl complex then translocates to specific

complex in these rafts further recruits the CrkII–C3G complex to the rafts, which phosphorylates the small GTP-binding protein TC10 This step has been shown to be necessary for normal translocation of Glut4-containing vesicles to

arms of the signalling network that links insulin binding to Glut4 translocation and increased glucose uptake in adipose tissue However, as glucose uptake into adipose tissue accounts for only a small proportion of whole body glucose disposal, this is likely to account at most for only part of the enhanced insulin

Effects on dietary lipid handling

The central metabolic role played by adipose tissue is in the storage of excess caloric intake as triglycerides in the postprandial state, the controlled release

of this stored energy under fasting conditions and above all the tight coupling

EVIDENCE FROM CELL AND RODENT MODELS 243

of these processes to the prevailing nutritional conditions Consonant with this,

sig-nificantly lower levels of mRNA found in adipose tissue in the fasting state and

in experimental states of insulin deficiency, and higher levels seen after high

are indeed a constellation of polyunsaturated fatty acids and their derivatives,

is likely to be of great functional significance that the transcriptional response

mediate uptake of the resulting free fatty acids into the adipocyte, fatty acid

stored in the adipocyte as triglyceride, glycerol is also required This may be provided by uptake of circulating glycerol, mediated by the adipocyte homo-

The rate-limiting step in this last process is catalysed by phosphoenolpyruvate

of glycerol prior to esterification with fatty acids is undertaken by glycerol kinase Until recently, the dogma has been that this enzyme is not present in adipocytes, preventing a futile cycle between triglyceride hydrolysis and resyn- thesis being established However, one report has now shown not only that this

in adipose tissue in situations where ligand and receptor are plentiful This is

upregulates the transcription of almost all stages of fatty acid trapping, from fatty acid release from lipoproteins and uptake into adipocytes, to esterifica- tion to glycerol The futile glycerol cycle recently proposed to be stimulated by

These actions of PPARγ form the basis of the ‘lipid steal hypothesis’, which provides at present the best supported and most widely held explanation for the insulin-sensitizing action of thiazolidinediones According to this hypothesis,

effi-ciently in adipose tissue, prevents the exposure of other insulin-sensitive tissues such as liver and skeletal muscle to these molecules It is well established that there is a strong correlation between ectopic accumulation of lipid at these sites

EVIDENCE FROM CELL AND RODENT MODELS 245

In type 2 diabetes, the habitually tight coupling between fatty acid trapping in adipose tissue and nutritional state is dysregulated, rendering lipid metabolism relatively inflexible in the face of fluctuating nutritional states Because of this, mean circulating levels of free fatty acids are high Pharmacological activation

sequestered in adipocytes, ‘stealing’ them from the other insulin-sensitive tissues such as skeletal muscle Indeed, the level of improvement of insulin sensitiv-

insulin-stimulated free fatty acid clearance, and also the rate of fasting free fatty acid

acid levels alone are likely to be only crude indicators of subtly dysregulated coupling of free fatty acid flux to nutritional status, and perhaps explain some

of the inconsistent findings in human studies.

Effects on adipocytokines

Fatty acids, as metabolic substrates themselves, provide an appealing link between adipose tissue and other insulin-sensitive tissues, and provide perhaps the simplest explanation of thiazolidinedione action However, in the last decade it has become apparent that adipose tissue also has the capacity to elaborate a wide variety of small molecules with autocrine, paracrine or endocrine activity, and many of these

coined for some of these molecules, and they have been grouped into those that enhance insulin sensitivity (such as leptin and adiponectin) and those that blunt

cytokine-like peptide hormone is secreted in proportion to total body fat mass, and is best characterized as a centrally acting suppressor of appetite and food intake Acting principally through the autonomic nervous system, it also induces

in addition some evidence in rodents that it has direct local actions on skeletal

for the importance of leptin in glucose homeostasis is most compelling in less or lipodystrophic mice, where insulin sensitivity is significantly enhanced

degree of metabolic improvement is contingent upon the particular fatless model used, genetic background and details of the leptin regimen Further to these observations, it has been demonstrated in A-ZIP/F1 fatless mice that the marked

metabolic benefit seen with transplantation of adipose tissue is not observed with

least in the context of this model of complete lipodystrophy, leptin seems to be pre-eminent as the mediator of the beneficial effects of fat Disappointingly, it appears that the dramatically beneficial effects of leptin may only be relevant to complete lipodystrophy, or genetic deficiency of the hormone, as the vast major- ity of models of obesity feature high leptin levels, often posited as evidence of

‘leptin resistance’ Such differences between the situation of dramatically low levels of leptin and that of normal or increased fat mass and high leptin perhaps

suppresses expression of leptin This is apparent in the decreased circulating leptin in mice treated with thiazolidinediones, and the higher levels of leptin

response to administration of leptin appears to be enhanced in mice with only

for normal glucose homeostasis, its beneficial metabolic effects are not tional to its concentration over the higher part of its range, and in cases of insulin resistance in animals that have normal or increased adipose stores other

decreasing leptin levels, enhances insulin sensitivity.

Unlike leptin, adiponectin, an adipocyte-derived multimeric hormone with homology to complement factor 1q, circulates at levels that are inversely related

to the amount of white adipose tissue Also unlike leptin, circulating levels of adiponectin have been shown to correlate with insulin sensitivity in both genetic

has been shown that in a murine model of lipoatrophy (this time heterozygous

alone substantially improved insulin sensitivity, while co-administration of leptin

analy-sis suggested that these effects of adiponectin were mediated by an enhanced capacity to oxidize fatty acids in muscle and liver, possibly via AMP-activated

at these sites Interestingly, this protein kinase has also been suggested recently

produc-tion both in vitro and in vivo, these findings render adiponectin a more appealing

action However, the picture has been clouded by the contradictory findings from different workers who have produced adiponectin knockout mice: while one report suggested this resulted in moderate insulin resistance without body

EVIDENCE FROM CELL AND RODENT MODELS 247

however, was the observation of a marked reduction in neointimal proliferation

Compared with wild type animals, these mice are mildly resistant to obesity and insulin resistance induced by high fat feeding or gold thioglucose lesion-

the double-knockout mice were protected from insulin resistance and

hypergly-caemia when put on a ob/ob background, the same mice, exposed to high fat

diets, were equally or even more insulin resistant in comparison to wild type

Plasma resistin levels are approximately in proportion to adipose tissue mass, and in some reports correlate with insulin resistance in both dietary and genetic

hepatic insulin resistance in mice, but had no effect on the insulin sensitivity of

in vivo in several different models of obesity and insulin resistance.84

sensitization lies in its effects on nitric oxide (NO) production: in diet-induced obesity and insulin resistance it is known that NO is overproduced in adipose

been shown to impair insulin-stimulated glucose uptake in L6 myotubes and

this has already been shown in other cell types, and so NO may potentially

activation.

Thus, tantalizing clues from these studies of adipocytokines suggest that they

but the many confusing and contradictory findings from different experimental protocols and animal models mean that consensus has yet to be reached.

Skeletal muscle

on adipose tissue, it is also expressed in other insulin-sensitive tissues Indeed,

mass of muscle and its importance as the site of most insulin-mediated glucose

tissue and elsewhere has been attempted by administration of thiazolidinediones

to lipoatrophic mice, but once again results have been inconsistent between ferent models: while rosiglitazone and troglitazone failed to have any effect on glucose or insulin levels in A-ZIP/F1 fatless mice (although circulating triglyc-

in mice that had had 90 per cent of their adipose tissue ablated by coupling of

even the tiny residual amount of adipose tissue in the second model may have permitted a beneficial effect of troglitazone to have been expressed through its effects on those remaining adipocytes.

com-plicated by the potential for phenomena that are secondary to improved

prob-lem has been circumvented experimentally in two ways: first, isolated cells

in culture have been examined, and positive effects of thiazolidinediones on insulin-stimulated glucose uptake have been reported in both rat-derived L6 myotubes and in cultured human skeletal muscle cells, mediated by enhance-

modest whole body insulin resistance, but surprisingly the glucose disposal into muscle is normal, with the effect attributable instead to hepatic and perhaps adipose insulin resistance Moreover, the knockout animals accumulate adipose tissue at a faster rate than wild type controls despite reduced food consumption This evidence of metabolic cross-talk between different insulin-sensitive tissues

is a recurring theme of different tissue-specific genetic manipulations, including

Glut4, but the mechanisms are at best only partly understood In this case no excess of intracellular lipid was seen in the livers of the knockout animals, and

no increase in whole animal fatty acid oxidation was observed However, when knockout animals were treated with thiazolidinediones, their response was as

EVIDENCE FROM CELL AND RODENT MODELS 249

have complex roles in normal physiology, the principal site of the therapeutic action of potent agonists is adipose tissue This genetic model failed to pro- vide support for the previous suggestion, based on gene expression profiling in

phosphorylates and inhibits pyruvate dehydrogenase, downregulating glucose oxidation and reciprocally promoting fatty acid oxidation, and was found to be

Pancreatic β-cells

some evidence that its activation can both enhance insulin secretion and protect β-cells from the apoptosis thought to be triggered by excessive accumulation

pancreatic islets, treatment of insulin-resistant animals with thiazolidinediones

Levels of intracellular triglyceride may not only affect cell survival, but are also likely to interfere with insulin secretion A second, more direct mode of enhancement of insulin secretion has also been suggested, based on the finding of

PPARγ-mediated stimulation of GLUT2 expression would increase glucose uptake and

be an important player in systemic insulin resistance, but also to play a role

pathogenesis of type 2 diabetes.

Liver

The data relating to effects of chronic thiazolidinedione administration on

in adipose tissue) they suggest that the liver is not a physiologically important

diabetes and insulin resistance, encompassing both lipoatrophy and hyperphagic

these models feature hepatic steatosis, and patterns of gene expression suggest

indeed stimulate lipid accumulation in hepatocytes in vivo has recently been

hep-atic steatosis is the same, and further that fat accumulation per se is insufficient

circumstances, has a significant role in the liver comes from two tary approaches First, detailed analysis of tissue-specific insulin sensitivity in A-ZIP/F-1 fatless mice treated with rosiglitazone demonstrated improvement of muscle insulin sensitivity at the expense of increased intracellular triglyceride

animals, of the A-ZIP/F1 fatless model, and ob/ob genetic obesity models

decreased triglyceride accumulation in the liver, with concomitantly enhanced hepatic insulin sensitivity However circulating levels of free fatty acids and triglycerides were increased, and insulin sensitivity in muscle and adipose tissue

still resulted in marked improvement in these indices, further supporting the view

worsened muscle insulin sensitivity, but this time the effect of rosiglitazone on

hyperlip-idaemia and insulin resistance compared with controls in the face of a high fat diet.

signif-icance in lean animals, in those with obesity and insulin resistance it undergoes compensatory upregulation to accommodate excess lipid in the liver Further-

that there is a hierarchy of triglyceride storage: by far the largest site of storage, and probably the only one of relevance in lean animals, is the white adipose tissue However, when presented with chronic caloric excess, the liver also has a significant capacity for fat storage Only when both these depots are overwhelmed does skeletal muscle experience the adverse effects of lipid accu- mulation and insulin resistance In this regard it is worth reflecting that the diet

of large parts of Western industrialized society most closely resembles the types

of high fat diet used in rodent studies.

Figure 9.3 As well reducing adipocyte size, improving the profile of secreted

INSIGHTS FROM HUMAN STUDIES 251

Adiponectin

Resistin

LeptinNO

liver

Figure 9.3 Potential mechanisms of the insulin-sensitizing effect of PPARγ activation

permit excess triglyceride to be stored in the liver, giving further buffering

to have deleterious consequences for the insulin sensitivity (or secretion) of the others.

9.2 Insights from human studies

Informing and motivating these rodent and cellular studies has been the

resistance and type 2 diabetes in humans As ever, caution must be exercised in extrapolating the results in these model systems to human pathophysiology, but the evolving evidence suggests that this is, in large part, appropriate The prin-

will be considered in turn.

Pharmacological studies

Effects on insulin sensitivity

clinical practice The prototype, troglitazone, was unfortunately withdrawn by

the manufacturer due to the occurrence in a small proportion of patients of serious, and sometimes fatal, hepatotoxicity, but subsequently both rosiglitazone and pioglitazone have been used with no evidence of similar problems Extensive clinical trial data has accumulated for all three agents Results have been consistent: used as monotherapy in patients with type 2 diabetes,

and postprandial plasma glucose by around 2 mmol/l, and glycosylated haemoglobin A1 by between 1 and 1.5 per cent When combined with either a

beneficial results are seen When analysed in more detail by hyperinsulinaemic clamp studies, significant improvements in whole body insulin sensitivity have

increased glucose disposal rates, although a minor suppression of hepatic glucose

the effects of metformin and troglitazone in parallel suggested that their effects were complementary, with metformin principally suppressing hepatic glucose output, and troglitazone preferentially acting to increase the rate of glucose

with diabetes: when used in 18 obese patients with normal glucose tolerance, troglitazone still reduced insulin levels in the fasting state and after glucose challenge, concomitant with a significant increase in insulin sensitivity measured

Effects on adipose tissue

with thiazolidinediones promotes weight gain in humans and several studies have shown that the increase in body weight associated with thiazolidinedione treat- ment is accounted for principally by accumulation of subcutaneous fat (reviewed

These observations are in keeping with ex vivo studies in which preadipocytes

isolated from subcutaneous abdominal adipose tissue differentiated more ily in response to thiazolidinediones than cells from visceral depots of the same

fat mass in all body regions equally, a question that is of interest in light of the burgeoning evidence of important functional metabolic differences between upper body (abdominal) and lower body (including femoro-gluteal) subcuta-

Beyond gross effects on white adipose tissue distribution, whether TZD ment in humans induces apoptosis of hypertrophic adipocytes and increased

treat-INSIGHTS FROM HUMAN STUDIES 253

differentiation of preadipocytes, leading to alterations in average adipocyte size

as seen in rodents, remains unresolved Other aspects of thiazolidinedione action

on adipose tissue appear to correspond rather variably with data from animal models Thus, thiazolidinediones have generally been reported to lower free fatty acid (FFA) levels in clinical trials, consistent with the ‘lipid steal’ hypothesis of insulin sensitization It is likely that many of the mechanisms subserving this are the same as those outlined earlier in mice, although whether the induction

of glycerol kinase expression and activity in human adipocytes is significant

As in rodents, alterations of the profile of adipocyte-secreted proteins may play a role in the therapeutic actions of thiazolidinediones Of those adipocytokines discussed earlier, adiponectin appears to be the best candidate in

γ-mediated severe insulin resistance, suggesting a direct correlation between

determine the extent to which this contributes to the insulin-sensitizing effects

of PPARγ agonists.

It is not clear whether resistin is significantly expressed in mature human

be presented.

Studies of human genetic variants

Rare mutations

Recently, three groups have independently identified loss-of-function mutations

subjects, all of whom exhibit a stereotyped form of partial lipodystrophy and severe insulin resistance, with the insulin resistance being evident even in early

and gluteal region, with relative preservation of both the subcutaneous and visceral abdominal depots was uniformly reported, although some differences were observed in facial adipose tissue, said either to be reduced, preserved and increased in different kindreds carrying different point mutations Although these

adipogenesis, it is difficult to reconcile the pattern of selective partial

and the adipogenic response to receptor agonists in humans.

As both partial and generalized lipodystrophy have consistently been found to

dramatic diminution in peripheral limb and gluteal fat contributes to the severe insulin resistance of these rare patients Additionally, even the residual adipose tissue depots in the individuals studied so far is metabolically inflexible, probably exacerbating the exposure of skeletal muscle and liver to dysregulated fatty acid

with or without lipodystrophy, discussed earlier, permit the speculation that the

of these subjects further denies them the compensatory FFA-buffering capacity afforded in lipodystrophy by the liver in particular, and so abnormalities in these tissues also may contribute significantly to the severe systemic insulin resistance observed.

Although study of the metabolic cross-talk between insulin-sensitive tissues

in humans is hampered by the lack of tissue-specific genetic deletions, the recent report of a kindred with a digenic pattern of severe insulin resistance may offer

a premature stop mutation in the muscle-specific glycogen-targeting subunit of protein phosphatase 1 (PPP1R3A) cosegregated with severe insulin resistance, while either mutation alone appeared to have no effect on insulin sensitivity in

adipose tissue, while the PPP1R3A product is specific for cardiac and skeletal muscle, further investigation of these subjects, and of analogous animal models, may provide important insights into the factors mediating the metabolic dialogue between insulin-sensitive tissues, which has been a prominent but ill understood feature of many of the genetic models already described This kindred may also

be a first step away from rare monogenic insulin resistance towards the oligo- or polygenic patterns which account for most of the population burden of insulin resistance and diabetes.

antici-pated to increase body fat mass Indeed, four morbid subjects have been described who are heterozygous for a proline to glutamine substitution in the N-terminal

inter-feres with this phosphorylation, resulting in a receptor with constitutive

segregation studies were reported, and no more similar mutations were found in

a larger screen of morbidly obese subjects Furthermore, the observed severely obese phenotype is at odds with the recently reported mouse harbouring a mutation

at the phosphorylation site itself: even homozygous animals had no more tendency

to weight gain than wild type littermates, and moreover were protected from

INSIGHTS FROM HUMAN STUDIES 255

reported to have low insulin levels relative to their massive obesity, all three also had type 2 diabetes, making inferences about their insulin sensitivity impossible.

Pro12Ala polymorphism

The receptor mutations described hitherto are rare, and although they have

profound phenotypic effects in affected individuals, they make a negligible bution to the risk of insulin resistance or type 2 diabetes in the general population.

date is a polymorphism, replacing alanine for proline at codon 12 (Pro12Ala)

functional assays, the Pro12Ala variant exhibits reduced binding to DNA and modest impairment in transcriptional activation, and these functional properties have been correlated with the association of this polymorphism with reduced body mass index (BMI), although subsequent studies have failed to confirm this

was first reported in a Finnish population, in whom a lower BMI appeared to correlate with improved insulin sensitivity in those carrying the Ala allele, while

in a group of second generation Japanese Americans the Pro/Pro genotype was

poorly reproducible, with only one of five subsequent studies showing

of published association studies confirmed a modest (1.25-fold) but significant ( p = 0.002) increase in diabetes risk with the Pro allele,149the discrepancy being accounted for by the underpowering of many of the individual studies Thus, if an entire population carried the Ala allele, the global prevalence of type 2 diabetes

common ‘diabetogene’ thus far discovered Further reports have strengthened

favoured positive studies is a significant one.

If the association does stand the test of time, it raises the question of how the Ala genetic variant influences diabetes risk In the index study carriers of the Ala polymorphism had a significantly lower BMI, and after correcting for

in conjunction with the lower transcriptional activity of the Ala variant in vitro,

led to the hypothesis that improved insulin sensitivity might be accounted for entirely by changes in adiposity Although this would unify the observation that

• dominant negative mutations

• heterozygous knockout mice + PPARγ/RXR antagonist

Figure 9.4 Integrated model of the relationship between PPARγ activity and insulinsensitivity

that body fat mass is a strong determinant of insulin sensitivity, subsequent studies have failed to yield consistent findings, with some even demonstrating

likely to be lessons from rodent studies, where not only the amount of fat, but also the size and function of individual adipocytes within it, is crucial to the optimal physiological function of the adipose tissue Such issues have yet to be examined in human subjects Furthermore, gene–environment interactions are likely to be more complex in humans, as evidenced by a recent study indicating that variations in dietary polyunsaturated fat versus saturated fat intake can

and insulin sensitivity in humans, based on the above diverse observations, is represented in Figure 9.4.

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