Control of glycaemia: from molecules to men. Minkowski Lecture 2003
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Regulation of glycaemia represents a fundamental biological principle, and its failure underlies Type 2 diabetes. The complex aetiology of Type 2 diabetes, which probably involves a medley of molecular mechanisms, requires dissection out of diabetes-associated subphenotypes, such as the non-obese with increased liver fat or the obese with low plasma adiponectin. The concepts of the hyperbolic relationship of insulin secretion and insulin sensitivity with glucose allostasis help us to establish the pathophysiological framework within which such mechanisms must operate. The translation of burgeoning new basic science findings into a physiological and clinical context calls for novel and imaginative clinical experimental tools. For the purpose of this review, four molecules (adiponectin [APM1], stearoyl CoA desaturase-1 [SCD1], insulin receptor substrate-1 [IRS1], peroxisome proliferator-activated receptor-γ [PPARG]), each with a plausible role in the disease process, have been selected to illustrate the use of such techniques in humans. These include procedures as diverse as isotope dilution for turnover studies (e.g. glycerol turnover as a proxy for lipolysis), conventional and modified clamp procedures, association studies of functionally relevant single nucleotide polymorphisms in candidate genes (e.g. IRS-1 and PPARγ), multivariate correlational analyses (as with plasma adiponectin), magnetic resonance spectroscopy to quantify intra-tissue lipid deposition and regional fat distribution, and gas chromatography to determine fatty acid patterns in selected lipid fractions as proxy for intrahepatic enzyme activity. A concerted effort by scientists from many disciplines (genetics and cell biology, physiology and epidemiology) will be required to bridge the growing gap between basic scientific concepts of biological modifiers of glycaemia and concepts that are truly relevant for human Type 2 diabetes.
KeywordsAdipose tissue Insulin resistance Obesity Type 2 diabetes
peroxisome proliferator-activated receptor
single nucleotide polymorphism
The regulation of glucose levels is a fundamental biological process that becomes deranged in diabetes mellitus, a condition that affects millions of people around the world  and, via its associated complications, imposes enormous strain on health care systems . Some 90% of affected individuals have Type 2 diabetes mellitus, and within this category no more than 10% can be accounted for by monogenic forms such as maturity-onset diabetes of the young , by mitochondrial diabetes , or by late-onset autoimmune diabetes . The common form of Type 2 diabetes is, in itself, likely to represent a melting pot of disease entities that differ in terms of aetiology and pathogenesis. By definition, whatever the primary aetiology, all of these mechanisms culminate in the inability to regulate hyperglycaemia.
Traditionally, little effort has been made to distinguish between one person with Type 2 diabetes and another, except perhaps for the distinction between the lean and the obese. Our knowledge about the pathogenesis of this condition has, however, advanced to a stage where pathophysiological substratification into categories such as beta cell dysfunction, increased endogenous glucose production or impaired insulin-stimulated glycogen storage has become possible. Having said this, such characterisation is virtually impossible after the onset of overt hyperglycaemia and associated metabolic derangements, because hyperglycaemia can, in itself, induce all of these abnormalities. Nevertheless, an understanding of the molecular mechanisms underlying the loss of normoglycaemia will form the essential basis for our ultimate success in predicting, preventing and treating Type 2 diabetes. This requires us to have a clear idea about the general principles of control of glycaemia in humans, especially in the pre-diabetic state.
This review will identify the principal physiological constraints within which any mechanism affecting glycaemia must operate. The aim will be to provide a framework within which the flow of new knowledge from disciplines ranging from genetics and cell biology to physiology and epidemiology can be channelled into the identification of molecules truly relevant for human Type 2 diabetes. Within this framework each and every advance in basic science will need to be scrutinised carefully before its relevance for human disease can be accepted.
The hyperbolic law of glycaemia
The concept of glucose allostasis
In 1988, Sterling and Eyer expanded the general concept of homeostasis  by introducing the term “allostasis” as an essential component of maintaining health in response to stress . McEwen and others applied the concept of allostasis to help explain adaptive neuroendocrine responses to chronic stress, which ensure day-to-day survival but take a cumulative toll on the individual’s health [11, 12, 13].
Based on our observations and the presented concepts, we propose to make an analogous distinction between homeostasis and allostasis in the context of glucose metabolism. The widely used term, glucose homeostasis, describes a system that is essential for life and becomes operative when an acute and transient stressor (e.g. an oral glucose load) is introduced. In sequence, glucose levels rise, insulin secretion increases, glucose disposal is stimulated, and finally, glucose levels return to where they started. A chronic glucotoxic state is prevented. When the stressor is chronic, however, (i.e. insulin resistance due to obesity) beta cell function improves and insulin secretion increases, but this usually fails to bring glucose concentration back to where it was before insulin resistance was introduced. This increment may be minute, but over time it accumulates and takes its toll. Such a situation fulfils the original criteria for the term allostasis. Thus, with chronic insulin resistance, the regulatory process becomes allostatic while continuing to maintain homeostatic regulation during the next acute challenge. It appears that this higher glycaemia (among other signals like non-esterified fatty acids, sympathetic nerve activity or glucagon-like peptide-1) is necessary to keep the beta cell continuously informed that insulin resistance is present.
Though not appreciated in such terms, an allostatic increase in glycaemia despite normal beta cell regulation has been previously documented. In a longitudinal study in 60 children going through insulin resistance of puberty (from Tanner I to Tanner III), fasting glucose increased by an average of 0.2 mmol/l with each stage, a difference that was statistically significant . It can be safely assumed that beta cell compensation at that age is as good as it gets. Thus, despite perfect compensation, the glucoregulatory system is not designed to maintain perfectly constant glycaemia as implied by the term glucose homeostasis. It follows logically that any mechanism that induces insulin resistance has the potential to lead ultimately to Type 2 diabetes.
A significant portion of this variability is accounted for by factors such as measurement error (due to the limited reproducibility of the in vivo procedure, for example) or incompleteness of the artificial experimental condition to quantify every biologically important nuance of the parameter of interest. For example, how precisely can a euglycaemic-hyperinsulinaemic clamp at insulin concentrations of ~400 pmol/l assess insulin sensitivity of glucose uptake at say 100 pmol/l, or insulin sensitivity of the vessel wall to vasodilate? The answer is probably not at all, and as with any experiment, the data obtained by such an in vivo procedure are only valid under the defined experimental conditions unless proven otherwise. In any case, measurement technique and understanding of the disease mechanism are inseparable.
A good example to show that the two go hand in hand is the issue of fat distribution. It has been well established that measuring the absolute or relative fat mass is insufficient to capture fat-related associations. With the advances of molecular tools (for expression studies, for example) and imaging techniques (CT scanning) it has become clear that regional differences have to be taken into consideration. Visceral adipose tissue is metabolically more detrimental than subcutaneous adipose tissue . Further advancements in imaging techniques (magnetic resonance spectroscopy) shed light on the metabolic importance of fat (usually small quantities in absolute terms) at atypical sites such as intrahepatic  or intramyocellular lipids . Thus, developing and advancing measurement techniques to the best possible level, and controlling for the measured parameter (either by matching or mathematically) should progressively reduce interindividual variability and help us to understand why one person develops diabetes and another does not. This concept must be taken all the way to the molecular level by comparing the genetic make up of one individual with that of another. Theoretically, measurement of as many relevant parameters as possible, including those that can be influenced (e.g. physical fitness due to regular exercise) and those that cannot be influenced (genetic factors), should permit near-perfect assessment of disease risk.
Genetic factors in control of glycaemia
The most compelling evidence for a genetic component of Type 2 diabetes is the excess concordance rate in monozygotic versus dizygotic twins. The heritability of abnormal glucose tolerance has been estimated as about 60% [18, 19]. Moreover, excess concordance rates in monozygotic versus dizygotic twins also suggest a contribution of genetic factors to insulin resistance and beta cell dysfunction . There can be no doubt, therefore, that in the control of glycaemia and in the pathogenesis of Type 2 diabetes, genetic factors play a role. But what exactly is meant by genetic factors?
Approximately 35 000 genes, organised in 46 chromosomes, encode for an even greater number of proteins (due to alternate splicing and post-translational modification, for example), which regulate the structure and function of the human organism. What makes humans different from one another—not only in terms of eye colour but also in terms of disease susceptibility—is the presence or absence of genetic polymorphisms such as single nucleotide polymorphisms (SNPs). An SNP with biological significance is expected to alter the function (when located in an exon) or the expression (when located in the promotor region or in an intron) of the resulting protein. This could lead to a decrease or increase in activity of an enzyme, to altered signal transmission for signalling proteins, or to modified expression of dependent genes in the case of a transcription factor.
Among the 3 billion base pairs of the human genome there is a common site of variation every 300 base pairs. The crucial question is which of these SNPs accounts for the fact that one person develops diabetes, say with a given degree of obesity, while another does not? The enormous heterogeneity of the diabetic phenotype has impeded the identification of significant SNPs, and very few genetic variants have been identified to account for even a minor proportion of common Type 2 diabetes. The most widely used experimental techniques for dissecting complex traits in general are the candidate gene approach and genome-wide random mapping (linkage analysis).
The candidate gene approach targets naturally occuring variants in genes that encode for proteins critical for insulin secretion, insulin action or obesity. The variants or polymorphisms in these genes are subsequently examined for significant association with Type 2 diabetes. Ideally, evidence should also be available from in vitro experiments, demonstrating functional significance of a variant, such as impaired binding of a docking protein, altered catalytic activity, or reduced expression as a result of an amino acid exchange or of a promoter polymorphism in a transcription factor binding site. Classical historical examples include the insulin receptor as a candidate gene for insulin resistance and the beta-cell-specific potassium channel KIR6.2 as a candidate gene for beta cell dysfunction. More recent candidates (IRS1 and PPARG) will be discussed below.
The genome-wide random mapping approach, in contrast, is not based on assumptions about the biological involvement of any gene product. It locates genes through their genomic position alone and is based on the following rationale: family members who share a specific phenotype (e.g. Type 2 diabetes) will also share chromosomal regions surrounding the gene, which contribute to that phenotype in excess of what is expected by chance alone . Allele sharing is assessed after typing several hundred polymorphic chromosomal markers across the genome. The first (and to date only) successful example of a positionally cloned “common Type 2 diabetes gene” is CAPN10 in the NIDDM1 region of chromosome 2 [22, 23]. This gene encodes for calpain-10, a cysteine protease that is ubiquitously expressed . Although an association with insulin-stimulated glucose disposal was reported in Pima Indians , the biological role of this protein in the control of glycaemia is far from understood.
Successful identification of genes through association with Type 2 diabetes is, however, only the beginning. To take full advantage of a “diabetes gene” for prevention and therapy it is necessary to understand the mechanism by which it modifies disease risk or progression. This is the interface between basic and clinical science. The basic scientist develops the biological concept, according to which the gene or protein in question might affect relevant pathways. For example, peroxisome proliferator-activated receptor-γ (PPARγ) interferes with development of obesity and insulin resistance by regulating transcription of genes important in adipogenic differentiation and lipid metabolism. Can this concept become relevant for humans? The clinical scientist’s task is to devise an experimental approach to test a plausible hypothesis, for example whether variability in transcriptional activity of PPARγ affects the risk of developing obesity in humans. The presence of the Pro12Ala polymorphism, leading to proline–alanine exchange, alters transcriptional activity in vitro. Comparing carriers and non-carriers of this polymorphism provides one opportunity to test the above hypothesis.
In the following section I shall present human data obtained in vivo to illustrate the potential role of four proteins that have been implicated in pathways or mechanisms related to insulin secretion or insulin sensitivity and thus to control of glycaemia. This will serve as a platform to describe different clinical experimental approaches to questions such as those discussed above.
Selected molecules involved in control of glycaemia
The striking association between insulin resistance and obesity justifies the study of factors originating from adipose tissue that have insulin-sensitising or -desensitising effects. Conceivably, greater release of an insulin desensitising factor for a given fat mass should make an individual more insulin-resistant. Adipose tissue releases not only fatty acids but also peptide hormones (adipocytokines or adipokines) with distant sites of action, making it an endocrine organ. While the role of leptin appears to be most prominent in the context of lipoatrophy , and that of TNF-α is confined to rodents, or in humans is only detectable at a local but probably not systemic (i.e. circulating) level, the role of adiponectin as an endogenous insulin sensitiser is progressively emerging.
With a concentration of around 5 µg/ml in human plasma, adiponectin represents one of the most abundant circulating proteins. In cases of obesity, plasma adiponectin levels are significantly decreased [27, 28], and after weight reduction, levels increase . Moreover, two independent nested case-control studies, one in Pima Indians and one in a German cohort, demonstrated that high plasma adiponectin concentrations predicted lower risk of developing Type 2 diabetes in the future, independently of any known covariate [30, 31].
In conclusion, variability in adiponectin release may contribute to the variation in insulin sensitivity for any given degree of adiposity. It is still unclear what influences the variability in adiponectin release independently of the determinants such as age, sex and percentage body fat. Although the data are not convincing, SNPs in the adiponectin (APM1) gene itself, such as the T-G polymorphism in exon 2 , or any of the host of proteins involved in the regulation of adiponectin production and degradation, could play a role. Nevertheless, the emerging role of adiponectin as an endogenous insulin sensitiser might lead to novel therapeutic modalities for Type 2 diabetes, conceivably also in a preventive context. Although recent observations of exophthalmos and increased interscapular fat pads in transgenic mice with elevated circulating adiponectin  have somewhat dampened earlier enthusiasm, cloning of the adiponectin receptor  might lead the way to safe and effective adiponectin analogues for human use.
Stearoyl-CoA desaturase (SCD1)
Human obesity involves accumulation of excess triglycerides not only at classical sites (subcutaneous, visceral) but also at intramuscular and intrahepatic locations. More so than intramyocellular lipids, intrahepatic lipids are associated with insulin resistance . A step crucial for hepatic VLDL and triglyceride synthesis is the enzyme stearoyl-CoA desaturase-1 (SCD1), which catalyses the conversion of saturated fatty acids (palmitate and stearate) to monounsaturated fatty acids (palmitoleic and oleate). Ob/ob mice are characterised not only by excessive whole-body adiposity (as a result of hyperphagia) but also by a markedly increased hepatic triglyceride content. Interestingly, transgenic reduction of SCD1 activity almost completely prevented hepatic triglyceride accumulation in ob/ob mice .
We sought to develop a method to probe SCD1 activity non-invasively in humans in vivo. Oleate incorporated into secretory VLDL is the product of stearate desaturated by SCD1. The oleate:stearate ratio in VLDL particles, the main secretory triglyceride originating in the liver, should therefore semiquantitavely reflect intrahepatic SCD1 activity. Since plasma triglycerides are the predominant content of VLDL particles, we measured the oleate:stearate ratio in fatty acids contained in plasma triglycerides. Stearate (18:0) and oleate (18:1 ω9) relative to total fatty acids in plasma triglycerides were determined in 98 healthy individuals using gas chromatography and flame ionisation detection.
Insulin receptor substrate-1 (IRS1, Gly972Arg polymorphism)
IRS-1 is a classical insulin signalling molecule and therefore a prime candidate gene for insulin resistance. The Gly972Arg polymorphism in IRS-1 therefore represented a prime candidate for genetic causes of impaired insulin signalling. Over the years, however, conflicting data have accumulated [40, 41, 42, 43, 44, 45, 46] and this polymorphism has not held up to expectations as an insulin resistance gene. More recently, it was shown that insulin-secreting cells overexpressing the IRS-1 Gly972Arg variant had decreased sulphonylurea and glucose-stimulated insulin secretion compared with cells overexpressing the wild-type IRS-1 . Moreover, cell apoptosis in isolated human islets obtained from organ donors heterozygous for Arg972 was increased two-fold compared with that of wild-type IRS-1 carriers , and the mature to immature insulin-containing granules were unfavourably affected by this polymorphism . It was consequently suggested that this polymorphism might represent a genetic variant unifying insulin resistance and beta cell dysfunction.
We studied subjects with and without the polymorphism using a modified hyperglycaemic clamp method . This consisted of a sequential and additive combination of square-wave hyperglycaemia (at 10 mmol/l glucose concentration), glucagon-like peptide-1 and arginine. To increase statistical power, we also analysed OGTTs of 212 subjects (31 with and 181 without the mutation), using a number of validated indices to estimate beta cell function from insulin (and C-peptide) concentrations obtained during an OGTT. During the modified hyperglycaemic clamp insulin secretion rates were significantly lower in Gly/Arg than in Gly/Gly during first-phase secretion and after maximal stimulation with arginine. During second-phase insulin secretion the differences reached statistical significance, while during the GLP-1 phases they were not significant. Several validated indices of beta cell function from the OGTT were significantly lower in X/Arg than in Gly/Gly (p values 0.002–0.05). Insulin sensitivity as determined by the euglycaemic-hyperinsulinaemic clamp was not different . The most striking difference in absolute terms was seen during maximal stimulation with glucose plus GLP-1 plus arginine (5340±639 vs 9075±722 pmol/min), compatible with the finding of reduced mature granules in beta cells of arginine carriers . Although no association with impaired beta cell function was observed in an older Dutch cohort , it is possible that the Gly972Arg polymorphism in IRS1 contributes to the variation in insulin secretion in people with normal glucose tolerance.
Peroxisome proliferator-activated receptor-γ (PPARG, Pro12Ala polymorphism)
The PPARγ is a transcription factor that belongs to the same family of nuclear receptors as steroid and thyroid hormone receptors . It is activated by certain fatty acids, prostanoids and thiazolidinediones [53, 54, 55, 56] and promotes transcription of numerous target genes . While the isoform PPARγ1 is expressed in most tissues, PPARγ2 is specific for adipose tissue, where it plays a key role in regulating adipogenic differentiation . The PPARG gene is located on chromosome 3 , and the specific isoforms are a result of alternative mRNA splicing. The highly prevalent Pro12Ala polymorphism in PPARγ2 is the result of a CCA to GCA missense mutation in codon 12 of exon B of the PPARG gene. This exon encodes the N-terminal residue that defines the adipocyte-specific PPARγ2 isoform. The Pro12Ala polymorphism in PPARγ2 was first identified in 1997  and studied as a plausible obesity candidate variant.
When16 candidate gene variants with previously published evidence for association with Type 2 diabetes (or related disorders) were tested using transmission disequilibrium testing (a powerful variant of association studies) in 333 Scandinavian parent–offspring trios with abnormal glucose tolerance, only the Pro12Ala polymorphism in the PPARγ2 gene was found to be significant . An additional meta-analysis demonstrated a signficant risk reduction of 21% and a population-attributable risk of approximately 25% . In other words, if the entire population carried the Ala allele, the prevalence for Type 2 diabetes would be 25% lower. In a recent prospective analysis (n>1000) from the Nurses’ Health Study, carriers of the Ala variant had 27% reduced risk of Type 2 diabetes compared with carriers of the Pro/Pro genotype . This underlines the importance of alleles with weak individual effect but high population prevalence, such as the Pro/Pro wild type of PPARγ (75% prevalence in Caucasians).
The mechanism by which the Ala allele of the polymorphism protects from Type 2 diabetes probably involves improved insulin sensitivity [63, 64, 65]. It is worth noting that the heterozygous PPARγ knockout mouse is also more insulin-resistant. In subgroups with obesity, the difference in insulin sensitivity is more pronounced [66, 67], suggesting an interaction of the polymorphism with factors originating from adipose tissue. Most investigators observed lower fasting insulin concentrations in carriers of the Ala allele [64, 68, 69]. While this observation is generally compatible with the suggestion of greater insulin sensitivity, it could also indicate greater insulin clearance.
In a recent series of analyses from our laboratory we therefore addressed the question of whether carriers of the Pro12Ala polymorphism have greater insulin clearance than wild-type controls. We used insulin concentrations during the steady state of a standard euglycaemic-hyperinsulinaemic clamp produced by a constant insulin infusion based on kg body weight. We also used C-peptide:insulin ratio and insulin concentrations adjusted for C-peptide concentrations during an OGTT and a hyperglycaemic clamp, taking advantage of endogenous hyperinsulinaemia.
Since PPARγ2 is exclusively expressed in adipose tissue, a primary mechanism in this tissue with an immediate impact on hepatic insulin sensitivity and insulin clearance can be invoked. Increased NEFA delivery to the liver could induce hepatic insulin resistance and decreased insulin clearance . In vitro, NEFAs reduced insulin binding, degradation and action . In vivo, elevation of NEFAs substantially decreased insulin clearance [73, 74]. Moreover, human obesity, a condition characterised by excessive NEFA turnover , is usually associated with reduced insulin clearance [76, 77, 78]. Finally, in dogs [79, 80, 81] and in humans , insulin-induced suppression of endogenous glucose production is prevented by experimental elevation of NEFA. It is therefore highly possible that the greater insulin clearance in carriers of the Ala allele mirrors reduced portal NEFA availability. Our observation of significantly lower insulin-suppressed NEFA concentrations in carriers of the polymorphism  (Fig. 7b) suggested that one effect of the Ala allele is the more efficient suppression of lipolysis.
Nevertheless, our results are also compatible with an alternative hypothesis. Although PPARγ is mainly expressed in adipose tissue, a direct effect of this polymorphism on hepatic insulin binding, action, internalisation and degradation cannot be excluded. In such a scenario, the Ala allele would primarily cause enhanced hepatic insulin clearance. That this could impact on insulin sensitivity and glucose tolerance derives support from a very recent report. Carcinoembryogenic-antigen-related cell adhesion molecule-1 (CEACAM1) is a protein with a key role in hepatic internalisation and degradation of receptor-bound insulin. Mutation of a functional serine residue resulted in hyperinsulinaemia, insulin resistance and impaired glucose tolerance in mice, while not affecting metabolic insulin signalling . This clearly demonstrated that a primary defect in insulin clearance can trigger the cascade of events leading to Type 2 diabetes. Thus, a mutation that leads to an increase in insulin clearance would secondarily produce an improvement in insulin sensitivity and glucose tolerance.
In conclusion, the diabetes-protective effect of the Ala allele in PPARγ is probably secondary to the association with greater insulin sensitivity. This in turn is probably secondary to decreased release of NEFAs and possibly other factors from (perhaps mainly visceral) adipose tissue, and may operate through greater hepatic insulin sensitivity and insulin clearance .
Summary and conclusions
Unravelling the pathogenesis of Type 2 diabetes requires continued efforts to understand the mechanisms relevant for the control of glycaemia, from the molecular to the epidemiological level. Translation of the ever-growing basic scientific findings into a physiological and clinical context calls for novel and imaginative clinical experimental tools. The scope of such techniques encompasses procedures as diverse as isotope dilution for turnover studies (such as glycerol turnover as proxy for lipolysis) in addition to conventional clamp procedures, association studies of functionally relevant SNPs in candidate genes (such as IRS-1 and PPARγ), intelligent multivariate correlational approaches (such as with plasma adiponectin), magnetic resonance spectroscopy to quantify intra-tissue lipid deposition in addition to regional fat distribution, and advanced analytical biochemistry to determine fatty acid patterns in selected lipid fractions as proxy for enzymatic activity. Whenever human cohorts are studied, it is crucial to control for possible confounders of the variable under investigation based on a profound insight into human physiology. This can range from simple control for age or sex to taking into account differences in fat distribution or even genetic background. Future generations of scientists from all backgrounds and disciplines will need to make an honest effort to bridge the growing gap between basic scientific concepts and their relevance for human disease in prediction, prevention, diagnosis and therapy.
I wish to thank the many volunteers who participated in our studies. I gratefully acknowledge the teaching, patience and continuous support of my mentors and their co-workers: John Gerich, University of Rochester, N.Y., USA, who raised my interest in clinical research; Hans Häring, University of Tübingen, Germany, who provided a wonderful platform for most of my studies; Bo Bogardus, National Institutes of Health, Phoenix, Ariz., USA, who put the idea, though not the word, of glucose allostasis in my mind. I also thank Bruce McEwen, The Rockefeller University, New York, N.Y., USA, for his discussions on the allostasis concept, and Hans-Günther Wahl for meticulously establishing the technique of measuring fatty acid patterns in Tübingen. This review was in large parts presented as the Minkowski Lecture at the 2003 IDF Meeting in Paris, at which time the author was still affiliated with the University of Tübingen, Department of Medicine.
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