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Summary: Impaired mitochondrial respiratory activity contributes to the development of insulin resistance in type 2 diabetes. Metformin, a first-line antidiabetic drug, functions mainly by improving patients’ hyperglycemia and insulin resistance. However, its mechanism of action is still not well understood. We show here that pharmacological metformin concentration increases mitochondrial respiration, membrane potential, and ATP levels in hepatocytes and a clinically relevant metformin dose increases liver mitochondrial density and complex 1 activity along with improved hyperglycemia in high-fat- diet (HFD)-fed mice. Metformin, functioning through 5′ AMP-activated protein kinase (AMPK), promotes mitochondrial fission to improve mitochondrial respiration and restore the mitochondrial life cycle. Furthermore, HFD-fed-mice with liver-specific knockout of AMPKα1/2 subunits exhibit higher blood glucose levels when treated with metformin. Our results demonstrate that activation of AMPK by metformin improves mitochondrial respiration and hyperglycemia in obesity. We also found that supra-pharmacological metformin concentrations reduce adenine nucleotides, resulting in the halt of mitochondrial respiration. These findings suggest a mechanism for metformin’s anti-tumor effects. : The mechanism of metformin action still remains controversial, in particular on mitochondrial activity and the involvement of AMPK. Wang et al. show that pharmacological metformin concentration or dose improves mitochondrial respiration by increasing mitochondrial fission through AMPK-Mff signaling; in contrast, supra-pharmacological metformin concentrations reduce mitochondrial respiration through decreasing adenine nucleotide levels. Keywords: metformin, diabetes, insulin resistance, mitochondrial respiration/fission, membrane potential, adenine nucleotides, AMPK, Drp1

Carney complex (CNC) is a rare disease associated with multiple neoplasias, including a predisposition to pancreatic tumors; it is caused most frequently by the inactivation of thePRKAR1Agene, a regulator of the cyclic AMP (cAMP)-dependent kinase (PKA). The method used was to create null alleles ofprkar1ain mouse cells expressingpdx1(Δ-Prkar1a). We found that these mice developed endocrine or mixed endocrine/acinar cell carcinomas with 100% penetrance by the age of 4–5 months. Malignant behavior of the tumors was seen as evidenced by stromal invasion and metastasis to locoregional lymph nodes. Histologically, most tumors exhibited an organoid pattern as seen in the islet-cell tumors. Biochemically, the lesions exhibited high PKA activity, as one would expect from deletingprkar1a. The primary neuroendocrine nature of these tumor cells was confirmed by immunohistochemical staining and electron microscopy, the latter revealing the characteristic granules. Although the Δ-Prkar1amice developed hypoglycemia after overnight fasting, insulin and glucagon levels in the plasma were normal. Negative immunohistochemical staining for the most commonly produced peptides (insulin, c-peptide, glucagon, gastrin and somatostatin) suggested that these tumors were non-functioning. We hypothesize that the recently identified multipotentpdx1+/insulin−cell in adult pancreas, gives rise to endocrine or mixed endocrine/acinar pancreatic malignancies with completeprkar1adeficiency. In conclusion, this mouse model supports the role ofprkar1aas a tumor suppressor gene in the pancreas and points to the PKA pathway as a possible therapeutic target for these lesions.

Kisspeptin was initially identified as a metastasis suppressor. Shortly after the initial discovery, a key physiologic role for kisspeptin emerged in the regulation of fertility, with kisspeptin acting as a neurotransmitter via the kisspeptin receptor, its cognate receptor, to regulate hypothalamic GnRH neurons, thereby affecting pituitary–gonadal function. Recent work has demonstrated a more expansive role for kisspeptin signaling in a variety of organ systems. Kisspeptin has been revealed as a significant player in regulating glucose homeostasis, feeding behavior, body composition as well as cardiac function. The direct impact of kisspeptin on peripheral metabolic tissues has only recently been recognized. Here, we review the emerging endocrine role of kisspeptin in regulating metabolic function. Controversies and current limitations in the field as well as areas of future studies toward kisspeptin’s diverse array of functions will be highlighted.

Type 2 diabetes mellitus (T2DM) is caused by relative insulin deficiency, subsequent to both reduced β-cell mass and insufficient insulin secretion, and both augmenting β-cell mass and β-cell function are therapeutic strategies for treating T2DM. However, the relative significance of increasing β-cell mass vs improving β-cell stimulus secretion coupling remains unclear. We have developed a mouse model that allows proliferation of β-cells in adult mice without affecting β-cell function by inducible expression of the positive cell cycle regulator cyclin A2 specifically in β-cells. In these mice, when kept on a standard diet, doubling of β-cell mass does not result in altered glucose tolerance or glucose-stimulated circulating insulin levels. Notably, a doubling of β-cell mass also does not confer improved glycemic control and ability of β-cells to respond to diabetogenic high-fat diet-induced glucose intolerance. However, in high-fat diet-exposed mice, an increase in endogenous β-cell mass confers increased potentiation of in vivo glucose-stimulated rise in circulating insulin in response to acute pharmacologic treatment with the incretin glucagon-like peptide-1 receptor agonist exendin-4. These observations indicate that increasing endogenous β-cell mass may not be sufficient to improve glycemic control in T2DM without additional strategies to increase β-cell stimulus secretion coupling.

Impaired estrogens action is associated with features of the metabolic syndrome in animal models and humans. We sought to determine whether disruption of hepatic estrogens action in adult male mice could recapitulate aspects of the metabolic syndrome to understand the mechanistic basis for the phenotype. We found 17β-estradiol (E2) inhibited hepatic gluconeogenic genes such as phosphoenolpyruvate carboxykinase 1 (Pck-1) and glucose 6-phosphatase (G6Pase) and this effect was absent in mice lacking liver estrogen receptor α (Esr1) (LERKO mice). Male LERKO mice displayed elevated hepatic gluconeogenic activity and fasting hyperglycemia. We also observed increased liver lipid deposits and triglyceride levels in male LERKO mice, resulting from increased hepatic lipogenesis as reflected by increased mRNA levels of fatty acid synthase (Fas) and acetyl-CoA carboxylase (Acc1). ChIP assay demonstrated estradiol (E2) induced ESR1 binding to Pck-1, G6Pase, Fas and Acc1 promoters. Metabolic phenotyping demonstrated both basal metabolic rate and feeding were lower for the LERKO mice as compared to Controls. Furthermore, the respiratory exchange rate was significantly lower in LERKO mice than in Controls, suggesting an increase in lipid oxidation. Our data indicate that hepatic E2/ESR1 signaling plays a key role in the maintenance of gluconeogenesis and lipid metabolism in males.

Insulin signaling in osteoblasts regulates global energy balance by stimulating the production of osteocalcin, a bone-derived protein that promotes insulin production and action. To identify the signaling pathways in osteoblasts that mediate insulin's effects on bone and energy metabolism, we examined the function of the tuberous sclerosis 2 (Tsc2) protein, a key target important in coordinating nutrient signaling. Here, we show that loss of Tsc2 in osteoblasts constitutively activates mTOR and destabilizes Irs1, causing osteoblasts to differentiate poorly and become resistant to insulin. Young Tsc2 mutant mice demonstrate hypoglycemia with increased levels of insulin and undercarboxylated osteocalcin. However, with age, Tsc2 mutants develop metabolic features similar to mice lacking the insulin receptor in the osteoblast, including peripheral adiposity, hyperglycemia, and decreased pancreatic β cell mass. These metabolic abnormalities appear to result from chronic elevations in undercarboxylated osteocalcin that lead to downregulation of the osteocalcin receptor and desensitization of the β cell to this hormone. Removal of a single mTOR allele from the Tsc2 mutant mice largely normalizes the bone and metabolic abnormalities. Together, these findings suggest that Tsc2 serves as a key checkpoint in the osteoblast that is required for proper insulin signaling and acts to ensure normal bone acquisition and energy homeostasis.

SummaryEarly in the pathogenesis of type 2 diabetes mellitus (T2DM), dysregulated glucagon secretion from pancreatic α cells occurs prior to impaired glucose-stimulated insulin secretion (GSIS) from β cells. However, whether hyperglucagonemia is causally linked to β cell dysfunction remains unclear. Here we show that glucagon stimulates via cAMP-PKA-CREB signaling hepatic production of the neuropeptide kisspeptin1, which acts on β cells to suppress GSIS. Synthetic kisspeptin suppresses GSIS in vivo in mice and from isolated islets in a kisspeptin1 receptor-dependent manner. Kisspeptin1 is increased in livers and in serum from humans with T2DM and from mouse models of diabetes mellitus. Importantly, liver Kiss1 knockdown in hyperglucagonemic, glucose-intolerant, high-fat-diet fed, and Leprdb/db mice augments GSIS and improves glucose tolerance. These observations indicate a hormonal circuit between the liver and the endocrine pancreas in glycemia regulation and suggest in T2DM a sequential link between hyperglucagonemia via hepatic kisspeptin1 to impaired insulin secretion.

A hallmark of type 2 diabetes (T2DM) is the reduction in functional β-cell mass, which is considered at least in part to result from an imbalance of β-cell renewal and apoptosis, with the latter being accelerated during metabolic stress. More recent studies, however, suggest that the loss of functional β-cell mass is not as much due to β-cell death but rather to de-differentiation of β-cells when these cells are exposed to metabolic stressors, opening the possibility to re-differentiate and restore functional β-cell mass by therapeutic intervention. In parallel, clinical observations suggest that temporary intensive insulin therapy in early diagnosed humans with T2DM, so as to "rest" endogenous β-cells, allows these patients to regain adequate insulin secretion and to maintain euglycemia for prolonged periods free of continued pharmacotherapy. Whether observations made in (mostly rodent) models of diabetes mellitus and in clinical trials are revealing identical mechanisms and therapeutic opportunities remains a tantalizing possibility. Our intention is for this review to serve as an overview of the field and commentary of this particularly exciting field of research.

Incretin hormone action on β-cells stimulates in parallel two different intracellular cyclic AMP-dependent signaling branches mediated by protein kinase A and exchange protein activated by cAMP islet/brain isoform 2A (EPAC2A). Both pathways contribute toward potentiation of glucose-stimulated insulin secretion (GSIS). However, the overall functional role of EPAC2A in β-cells as it relates to in vivo glucose homeostasis remains incompletely understood. Therefore, we have examined in vivo GSIS in global EPAC2A knockout mice. Additionally, we have conducted in vitro studies of GSIS and calcium dynamics in isolated EPAC2A-deficient islets. EPAC2A deficiency does not impact GSIS in mice under basal conditions. However, when mice are exposed to diet-induced insulin resistance, pharmacologic secretagogue stimulation of β-cells with an incretin hormone glucagon-like peptide-1 analog or with a fatty acid receptor 1/G protein–coupled receptor 40 selective activator, EPAC2A is required for the increased β-cell response to secretory demand. Under these circumstances, EPAC2A is required for potentiating the early dynamic increase in islet calcium levels after glucose stimulation, which is reflected in potentiated first-phase insulin secretion. These studies broaden our understanding of EPAC2A function and highlight its significance during increased secretory demand or drive on β-cells. Our findings advance the rationale for developing EPAC2A-selective pharmacologic activators for β-cell–targeted pharmacotherapy in type 2 diabetes.

The incidence of type 2 diabetes mellitus (T2DM) is rapidly increasing worldwide with significant consequences on individual quality of life as well as economic burden on states’ healthcare costs. While origins of the pathogenesis of T2DM are poorly understood, an early defect in glucose-stimulated insulin secretion (GSIS) from pancreatic β-cells is considered a hallmark of T2DM 1 . Upon a glucose stimulus, insulin is secreted in a biphasic manner with an early first-phase burst of insulin, which is followed by a second, more sustained phase of insulin output 2 . First phase insulin secretion is diminished early in T2DM as well is in subjects who are at risk of developing T2DM 3 4 5 6 . An effective treatment of T2DM with incretin hormone glucagon-like peptide-1 (GLP-1) or its long acting peptide analogue exendin-4 (E4), restores first-phase and augments second-phase glucose stimulated insulin secretion. This effect of incretin action occurs within minutes of GLP-1/E4 infusion in T2DM humans. An additional important consideration is that incretin hormones augment GSIS only above a certain glucose threshold, which is slightly above the normal glucose range. This ensures that incretin hormones stimulate GSIS only when glucose levels are high, while they are ineffective when insulin levels are below a certain threshold 7 8 . Activation of the GLP-1 receptor, which is highly expressed on pancreatic β-cells, stimulates 2 ­distinct intracellular signaling pathways: a) the cAMP-protein kinase A branch and b) the cAMP-EPAC2 (EPAC=exchange protein activated by cAMP) branch. While the EPAC2 branch is considered to mediate GLP-1 effects on first-phase GSIS, the PKA branch is necessary for the former branch to be active 9 10 . However, how these 2 branches interplay and converge and how their effects on insulin secretion and insulin vesicle exocytosis are coordinated is poorly understood. Thus, at the outset of our studies we have a poorly understood intracellular interplay of cAMP-dependent signaling pathways, which – when stimulated – restore glucose-dependent first phase and augment second phase insulin secretion in the ailing β-cells of T2DM.

SummaryImpaired insulin secretion contributes to the pathogenesis of type 2 diabetes mellitus (T2DM). Treatment with the incretin hormone glucagon-like peptide-1 (GLP-1) potentiates insulin secretion and improves metabolic control in humans with T2DM. GLP-1 receptor-mediated signaling leading to insulin secretion occurs via cyclic AMP stimulated protein kinase A (PKA)- as well as guanine nucleotide exchange factor-mediated pathways. However, how these two pathways integrate and coordinate insulin secretion remains poorly understood. Here we show that these incretin-stimulated pathways converge at the level of snapin, and that PKA-dependent phosphorylation of snapin increases interaction among insulin secretory vesicle-associated proteins, thereby potentiating glucose-stimulated insulin secretion (GSIS). In diabetic islets with impaired GSIS, snapin phosphorylation is reduced, and expression of a snapin mutant, which mimics site-specific phosphorylation, restores GSIS. Thus, snapin is a critical node in GSIS regulation and provides a potential therapeutic target to improve β cell function in T2DM.

While Kisspeptin was initially found to function as a metastasis suppressor, after identification of its receptor KissR1 and their expression profile in tissues like the hypothalamus and adrenals, kisspeptin and Kiss1R were predominantly assigned endocrine functions, including regulating puberty and fertility through their actions on hypothalamic gonadotropin releasing hormone production. More recently, an alter ego for kisspeptin has emerged, with a significant role in regulating glucose homeostasis, insulin secretion, as well as food intake and body composition, with lack of kisspeptin signaling resulting in reduced locomotor activity and increased adiposity. This review will highlight these recent observations of kisspeptin's role in metabolism and the several key questions that have emerged and that need to be addressed in the future.

Kisspeptin, encoded by the Kiss1 gene, binds to a specific G protein-coupled receptor (kisspeptin1 receptor) to regulate the central reproductive axis. Kisspeptin has also been reported to be expressed in peripheral tissues, including the testes. However, factors regulating testicular kisspeptin and its role in reproduction are unknown. Our objective herein was to begin to address kisspeptin function in the testis. In particular, we sought to determine the level of kisspeptin in the testis in comparison with the brain and other tissues, how these levels change from the prepubertal period through sexual maturation, and the factors involved in kisspeptin regulation in the testis. Immunohistochemical analysis of testis sections using a validated kisspeptin antibody localized kisspeptin to the Leydig cells. Kisspeptin was not detected in germ cells or Sertoli cells within the seminiferous tubules at any developmental time period studied, from prepuberty to sexual maturation. A developmental time course of testicular kisspeptin revealed that its mRNA and protein levels increased during development, reaching robust levels at postnatal day 28, correlating with pubertal onset. In vitro studies of primary mouse Leydig cells, as well as in vivo studies, indicated clearly that LH is involved in regulating levels of Leydig cell kisspeptin. Interestingly, gonadectomy resulted in elevated LH but reduced serum kisspeptin levels, suggesting that testicular kisspeptin may be secreted. These data document kisspeptin expression in mouse Leydig cells, its secretion into peripheral serum, and its regulation by changes in reproductive neuroendocrine function.

Coherent management of thyroid nodules that grow after being identified as benign will vary depending on their biology. Some are thyroid-stimulating hormone (TSH) regulated, so a dose of levothyroxine that reduces TSH to the lower limits of normal (subsuppression) may prevent or reverse enlargement; others are controlled by poorly understood mechanisms that must be elucidated before effective medical treatment can be designed; a few are autonomous requiring obliteration, and a very few are "missed" malignancies that should be excised.

Signal transduction properties of exendin-4 (Ex-4) underlying its ability to stimulate rat insulin I gene promoter (RIP1) activity were assessed in the pancreatic beta-cell line INS-1. Ex-4 acted via glucagon-like peptide-1 receptors to stimulate RIP1 in a glucose-dependent manner, as measured in cells transfected with a -410-bp RIP1-luciferase construct (RIP1-Luc). The action of Ex-4 was independent of cAMP and PKA because it was not blocked by cotransfection with dominant-negative G alpha(s), was unaffected by pretreatment with the membrane-permeant cAMP antagonist 8-Br-Rp-cAMPS, and remained apparent after treatment with PKA inhibitors H-89 or KT 5720. Similarly, cotransfection with a dominant-negative isoform of the type-2 cAMP-regulated guanine nucleotide exchange factor (Epac2) failed to alter the response to Ex-4. Ro 31-8220, a serine/threonine protein kinase inhibitor that targets PKC as as well as the 90-kDa ribosomal S6 kinase (RSK) and mitogen- and stress-activated protein kinase (MSK) family of cAMP response element-binding protein (CREB) kinases, blocked the stimulatory action of Ex-4 at RIP1-Luc. However, selective inhibition of PKC using K-252c, prolonged exposure to phorbol 1,2-myristate-13-acetate, or cotransfection with dominant-negative atypical PKC-zeta, was without effect. A-CREB, a dominant-negative inhibitor of basic region-leucine zipper transcription factors (bZIPs) related in structure to CREB, inhibited the action of Ex-4 at RIP1-Luc, whereas A-ATF-2 was ineffective. Similarly, introduction of deletions at the RIP1 cAMP response element (CRE), or truncation of RIP1 to remove the CRE, nearly abolished the action of Ex-4. Inactivating mutations introduced at the A4/A3 elements, binding sites for the glucose-regulated homeodomain transcription factor PDX-1, did not diminish the response to Ex-4, although a marked reduction of basal promoter activity was observed. The glucose-dependent stimulation of RIP1-Luc by Ex-4 was reproduced using a synthetic reporter (RIP1-CRE-Luc) incorporating multimerized CREs of the RIP1 nonpalindromic sequence 5'-TGACGTCC-3'. It is concluded that the bZIP and CRE-mediated stimulation of RIP1 by Ex-4 explains, at least in part, how this insulinotropic hormone facilitates transcriptional activity of the rat insulin I gene.

Recent advances in conditional gene targeting and cyclic nucleotide research further our understanding of how the incretin hormone GLP-1 exerts a therapeutically important action to restore pancreatic insulin secretion in patients with type 2 diabetes mellitus (T2DM). These studies demonstrate that the pancreatic β-cell GLP-1 receptor has the capacity to signal through two distinct branches of the adenosine 30,50-cyclic monophosphate (cAMP) signal transduction network; one branch activates protein kinase A (PKA), and the second engages a cAMPregulated guanine nucleotide exchange factor designated as Epac2. Under normal dietary conditions, specific activation of the cAMP-PKA branch in mice dramatically augments glucose-stimulated insulin secretion (GSIS). However, under conditions of diet-induced insulin resistance, cAMP-Epac2 signaling in the control of GSIS becomes prominent. This chapter provides an update on GLP-1 receptor signaling in the islets of Langerhans, with special emphasis on key molecular events that confer “plasticity” in the β-cell cAMP signal transduction network. The reader is reminded that an excellent review of β-cell cAMP signaling can also be found in the prior first edition of this book.

The sections in this article are: 1 History 1.1 Glucagon 1.2 Glucagon-like Peptides 2 The Glucagon Superfamily of Peptide Hormones 3 Tissue Distribution of Proglucagon Expression 3.1 Pancreas 3.2 Intestine 3.3 Brain 4 Proglucagon Biosynthesis 4.1 Organization and Structure of the Proglucagon Gene 4.2 Regulation of Glucagon Gene Expression 4.3 Posttranslational Processing of Proglucagon 4.4 Chemistry and Structure 5 Regulation of Glucagon Secretion 5.1 Overview 5.2 Intracellular Signals 5.3 Nutrients 5.4 Endocrine/Paracrine 5.5 Neural 5.6 Pulsatility 6 Regulation of Glucagon-like Peptide-1 Secretion 6.1 Overview 6.2 Intracellular Signals 6.3 Nutrients 6.4 Endocrine 6.5 Neural 7 Metabolism and Degradation 7.1 Overview 7.2 Renal Clearance 7.3 Hepatic Clearance 7.4 Degradation in the Circulation 7.5 Biologically Active Fragments 8 Physiological Actions 8.1 Glucagon 8.2 Glucagon-like Peptide-1 8.3 Glucagon-like Peptide-2 9 Mechanisms of Action 9.1 Glucagon 9.2 Glucagon-like Peptide-1 9.3 Glucagon-like Peptide-2 10 Human Disease 10.1 Glucagon 10.2 Glucagon-like Peptide-1 10.3 Glucagon-like Peptide-2

Diabetes is caused by a failure of the pancreas to produce insulin in amounts sufficient to meet the body's needs. A hallmark of diabetes is an absolute (type 1) or relative (type 2) reduction in the mass of pancreatic beta-cells that produce insulin. Mature beta-cells have a lifespan of approximately 48-56 days (rat) and are replaced by the replication of preexisting beta-cells and by the differentiation and proliferation of new beta-cells (neogenesis) derived from the pancreatic ducts. Here, we show that the insulinotropic hormone glucagon-like peptide (GLP)-1, which is produced by the intestine, enhances the pancreatic expression of the homeodomain transcription factor IDX-1 that is critical for pancreas development and the transcriptional regulation of the insulin gene. Concomitantly, GLP-1 administered to diabetic mice stimulates insulin secretion and effectively lowers their blood sugar levels. GLP-1 also enhances beta-cell neogenesis and islet size. Thus, in addition to stimulating insulin secretion, GLP-1 stimulates the expression of the transcription factor IDX-1 while stimulating beta-cell neogenesis and may thereby be an effective treatment for diabetes.

Metabolism Without Modification Obesity-associated metabolic disease has rapidly become a public health priority in the developed world and is being addressed through prevention strategies aimed at lifestyle changes and through pharmacological approaches. Barnett et al. (p. 1689 , published online 18 November) designed a drug that inhibits the action of ghrelin, a circulating peptide hormone that increases fat mass and food intake. The drug, a bisubstrate analog called GO-CoA-Tat, is a selective antagonist of ghrelin O-acyltransferase (GOAT), an enzyme that catalyzes a posttranslational modification that is essential for ghrelin activity. Injection of GO-CoA-Tat into wild-type mice on a high-fat diet improved glucose tolerance and reduced weight gain, probably through changes in metabolic activity. Because GO-CoA-Tat is a peptide-based drug that requires repeated injection, it is unsuitable for clinical use, but GOAT does represent a potentially valuable target for future drug development efforts in metabolic disease.

Zeus looked down from the top of Mount Olympus and laid his eyes on Thurma, the beautiful princess of the small island kingdom of Styconos in the Aegean Sea. Smitten with desire, he instantly transformed himself into a white dove and flew down to win her affections. Thurma, delighted and utterly charmed by the sweet bird, easily succumbed to the god and soon thereafter found herself pregnant with a child. Hera, Zeus’ sister and first wife had been watching, and, in her jealousy, had turned the son into a dragon while he was still in the womb of his mother. In her rage, Hera devised that the monster would grow evil and turn against the people of the kingdom. Meanwhile the King had received an oracle prophesying that his kingdom would be devastated by a dragon born from his daughter. Upon finding her pregnant, he immediately banished her to an isolated cave at the edge of the island, forbidding her to return until she had given birth and all was declared safe. Though she was visited daily by a royal attendant, Thurma was quite lonely in the dim light of the grotto, and spent countless hours singing to herself and her child. She sang of the pleasantries of the outside world, which she missed, as well as of the follies of famous heroes, and so, when her son was finally born among the shadows, while still dragon in form, his mind and spirit were gentle. Such was the King’s despair at birth of the first dragon prince ‐ seeing the fulfilment of the oracle ‐ he abandoned Styconos and left with the disheartened Queen on a rough sea never to return. But Thurma loved her childdragon Hesperon (whose name means the son of a god and a beautiful princess), who at birth was so beautiful and gentle that the people of the island took him into their hearts. Even jealous Hera, who had come to see the fate of the island, was taken by Hesperon’s looks. She regretted what she had done and took the evil spirit from the dragon so that now he would not harm the people of the island but protect them from intruders. Hesperon would catch an intruder but do him no further harm unless the people of the island signalled that they did not recognize the intruder as friendly. Then only would Hesperon devour the intruder. We find the myth of the dragon Hesperon reflected in our present understanding of how the immune system distinguishes self (native) from non-self (intruder). Knowledge of how the immune system is kept from turning against itself has allowed the induction of specific tolerance even towards transplanted tissue. This Highlight summarizes the pertinent molecular mechanisms involved in the discrimination of self and non-self by the immune system and outlines exciting new studies in transplant tolerance. Until the mid-1960s it was thought that the immune system discriminated between self molecules and those belonging to pathogens by deleting all self-reactive

The pathogenesis of type 2 diabetes is characterized by insulin resistance, primarily in skeletal muscle, fat and liver, and a relative failure of the pancreatic b-cell (1, 2). Considerable controversy surrounds the issue of which of these deficiencies is the primary cause of diabetes. In some studies, the earliest observed defect is dysfunctional secretion and in others insulin resistance appears to be the first detectable problem. Some important new studies open a new perspective to these questions. Glucose is the principal regulator of insulin secretion from pancreatic b-cells. Insulin is secreted immediately in response to elevated glucose concentrations. After insulin secretion, an immediate response takes place within the b-cell to replenish insulin stores through activation of insulin biosynthesis at the transcriptional (3) as well as translational levels (4, 5). The signals that govern the signal transduction pathway that link the glucose stimulus to the initiation of insulin gene transcription have been largely unclear. Intraislet interactions as well as extrapancreatic hormones and neural inputs exert an important level of control over insulin synthesis and secretion and ultimately glucose homeostasis. Several lines of evidence support the possibility of an autocrine action of insulin on b-cells. Insulin binds to the surface of b-cells (6, 7), and functional insulin receptors and insulin receptor substrates (IRSs) identical to those found in peripheral tissues have been identified in both clonal and primary b-cells (6, 8, 9). Glucose stimulation of b-cell lines activates the b-cell insulin receptor much in the same way as application of exogenous insulin, suggesting that insulin secreted from b-cells binds to the insulin receptor eliciting a physiological response (10). The complete physiological consequences of insulin receptor activation of the b-cell have yet to be completely elucidated, but at least one effect is initiation of protein synthesis at both transcriptional and translational levels (11). However, there remains controversy on the effects of insulin on b-cell secretion. Several reports have shown that glucose-stimulated insulin or C-peptide secretion from islets or perfused pancreas is suppressed in the presence of exogenous insulin, leading to the concept of a negative feedback of insulin secretion by insulin (12–18). Under similar conditions, however, other investigators have found little or no effect of insulin on glucose-stimulated insulin secretion (19– 21). Furthermore, these data have been difficult to interpret because of neuronal and intraislet hormonal regulatory mechanisms that could interact with exogenous insulin, and the use of high glucose levels in these studies could per se evoke the effects of substantial stimulation of the b-cell insulin receptors masking the effect of exogenous insulin. Experiments with purified b-cells have also generated conflicting evidence for insulin feedback. Glucosestimulated insulin secretion from purified b-cell lines is inhibited by exogenous insulin levels (1 mmol/l) (21). In contrast, measurements of the effect of insulin on C-peptide secretion in bTC3 cells failed to show direct evidence of secretory regulation by insulin. Furthermore, transfected bTC6-F7 cells in which the insulin receptor was overexpressed showed enhanced basal and glucose-stimulated insulin secretion, but fractional secretory levels remained unchanged at all glucose concentrations whereas cells expressing kinase negative (inactive) insulin receptors showed decreased glucosestimulated insulin secretion (11). These results suggest an autocrine pathway regulating one or more of the following: insulin secretion, insulin synthesis, and glucose sensing/utilization. Several studies have revisited the question on whether insulin may have an autocrine effect on insulin synthesis and secretion. Leibiger et al. (22) studied the effects on insulin gene transcription of depolarization of b-cells by adding (i) glucose, (ii) KCl, or (iii) sulfonylureas to the culture medium. The depolarization, which takes place in all three cases and induces insulin secretion, was also accompanied by an increase in insulin gene transcription as assessed by reverse transcription PCR and by transfected insulin-promoter–green fluorescent protein reporter constructs. By adding various pharmacological inhibitors of the signal transduction pathway, the investigators delineated two pathways leading to an increased insulin gene transcription rate: the PI-3 kinase/p70 s6 and the CaM kinase II. Furthermore, stimulation of hamster insulinoma (HIT) cells with glucose or insulin led to increased PI-3 kinase activity in immunoprecipitates obtained with anti-IRS-2 antibodies. These same pathways are also involved in insulin receptor signal transduction. Thus the investigators studied the effect of overexpression of type A and B insulin receptors in HIT cells and found that the overexpression of type A led to an enhanced insulin European Journal of Endocrinology (1999) 141 95–97 ISSN 0804-4643 H IG H L IG H T

The cloning of the glucagon gene from anglerfish, rodents and humans ((1) and references therein) in the early 1980s yielded the surprising finding that the glucagon gene not only encoded the known peptide hormone glucagon, but also contained sequences for two additional peptides, which, because of their homology to glucagon, were named glucagon-like peptide (GLP)-1 and -2 (Fig. 1). The protein product of the gene, preproglucagon, is processed in a tissuespecific manner to glucagon and the major proglucagon fragment in pancreatic endocrine a-cells or to glicentin, oxyntomodulin, GLP-1, intervening peptide 2 (IP-2), and GLP-2 in intestinal L-cells and certain neurons of the brain (Fig. 1). GLP-1 has a high sequence homology to the incretin hormone, gastric inhibitory polypeptide (GIP), and thus, at the time of its identification, was already hypothesized to represent a second incretin hormone (1). Subsequent studies did, indeed, reveal an incretin effect of GLP-1 (2). Furthermore, GLP-1 has been shown to be involved in satiety control and gastric emptying (3, 4). GLP-2 is co-secreted with GLP-1. Fasting levels in human plasma are 151 6 14 pmol/l and these increase to 225 6 15 pmol/l by 2 h after a mixed meal (5). In contrast, GLP-2, a 33-amino acid peptide, has only recently been ascribed a biological function, elucidated in a pioneering effort by Dr Drucker’s group in Toronto. The background of their studies were earlier observations that over-expression of the glucagon gene in rodents was associated with bowel growth and regeneration (6, 7). Furthermore, two reports of patients presenting with proglucagon-expressing tumors in the kidney, increased circulating concentrations of the proglucagon-derived peptides (PGDP), and small-bowel hypertrophy that regressed after tumor resection, suggested a link between peptide products derived from proglucagon and the control of small-bowel epithelium proliferation (8, 9). Small-bowel epithelial proliferation can also be reproduced in glucagon promoter‐SV40 T antigen transgenic mice, which develop proglucagon-producing tumors and increased plasma concentrations of PGDP (10‐12). When nude mice are injected subcutaneously with proglucagonproducing tumors, circulating PGDP are increased and the small bowel appears enlarged (13). To identify further which PGDP was leading to the intestinal enlargement, each PGDP was injected separately to normal mice. Whereas GLP-1 and IP2 had no effect, glicentin treatment led to a small increase in bowel weight. Remarkably, GLP-2 treatment resulted in a 50% increase in total small-bowel wet and dry weight. GLP-2 increased mucosal thickness, which was attributable to an increase in villus height; crypt depth and muscle thickness did not change. Markers for cell proliferation were increased in epithelial cells of the crypts and also extended to the lower portions of the villus surfaces. Remarkably, the effects of GLP-2 were confined to the crypt and villus epithelium, and no changes were detectable in other layers of the intestine or in other organs. Changes in intestinal epithelial proliferation markers were already evident after a single dose of GLP-2, and morphologic changes were detectable after a treatment period of as little as 4 days (13). Food intake and body weight were similar between control and GLP-2 treated animals, excluding the possibility that increased enteral nutrient ingestion attributable to GLP-2 may

Hardly a day passes without news of stem cells. Stem cell therapy implies the replacement of diseased or lost cells from the progeny of stem cells that differentiate into the required phenotypes. Stem cells have the capacity to divide asymmetrically, that is, yielding one identical ‘daughter’ stem cell and a second daughter cell that proceeds towards terminal differentiation. Thus, stem cells are theroretically endlessly self-renewing and from thence derives their hoped-for uses. There are two large groups of stem cells: embryonic stem cells (stem cells derived from early embryonic stages) and adult stem cells (stem cells found in the postnatal organism). Reconstitution of bone marrow by hematopoietic stem cell infusion is an established practice in the treatment of certain cancers and of certain disorders of hematopoiesis. Recently, bone marrow and other hematopoietic organs (liver and spleen) have been shown to harbor cells that possess the capacity to enter extramedullary tissues and to differentiate into functional parenchymal cells of the respective tissue, such as the liver, intestinal, skin and bronchial epithelia (1, 2), skeletal (3, 4), heart (5, 6) muscle and cells of the central nervous system (7, 8). These observations are not limited to experimental rodent models but are also made in biopsy material of human recipients of bone marrow transplantation (9, 10). Several recent reports have uncovered the possibility of using adult stem cells from hematopoietic organs for the replacement of pancreatic endocrine cells and the treatment of type 1 diabetes mellitus (11 –15). We summarize these findings as they represent different, albeit similar, paradigms of cellular regeneration in adult diabetic organisms. In a first report, Ianus et al. (11) demonstrated that in mice, cells from transplanted bone marrow engraft into the pancreatic islets. A genetic mouse model allowed identification of transplanted bone marrowderived cells by virtue of a fluorescent protein that is expected to be expressed in cells if they transcribe insulin. Fluorescent cells were found in pancreatic islets of recipient mice approximately 1 –2 months after bone marrow transplantation. Importantly, fluorescent cells were also positive for the Y chromosome that came from the male donor in the female bone marrow recipient (Fig. 1A). These donor-derived engrafted cells express insulin and other genetic markers typical of pancreatic beta cells. When tested in isolated culture conditions, the bone marrow-derived cells secrete insulin in response to a physiological glucose stimulus, and show intracellular calcium fluctuations reminiscent of pancreatic beta cells. However, 1–2 months after bone marrow transplantation, only about 1–3% of the islet cells originate from the transplanted bone marrow. This rate of engraftment does not account for the normal estimated turnover of 30 days of the endocrine cells in an islet (16). Additional studies demonstrated that cell fusion is not a likely explanation for the observed phenomenon of differentiation of bone marrow-derived cells into pancreatic endocrine cells. In a second report by Hess et al. (12), streptozotocindiabetic mice were lethally irradiated and transplanted with bone marrow from donors that ubiquitously express green fluorescent protein (GFP). Whereas control mice remained hyperglycemic and showed increased mortality, bone marrow-transplanted mice were normoglycemic, had normal insulin production after 17 days and had an increased survival rate at day 42 (75 – 85% vs 0–50%). Increases in serum insulin correlated with reduction in blood glucose. Furthermore, the investigators showed that bone marrow cells that express c-kit – one of the ‘stem cell markers’ – as a surface marker could, when transplanted, lead to reduction in glycemia, whereas c-kit-negative cells had no effect. Comparison of engraftment into liver and pancreas revealed that bone marrow-derived cells engrafted preferentially into the streptozotocin-damaged pancreas. In the pancreas, bone marrow-derived cells were found in two regions: the ducts and the islets. In the islets, most bone marrow-derived cells did not contain insulin, although a small number of bone marrowderived cells also contained insulin protein. These insulin-positive cells were found only in streptozotocin-diabetic bone marrow recipients. However, the insulin-positive cells did not coexpress the beta-cellspecific transcription factor PDX-1. The authors speculate that the insulin-positive cells may have been incorporating insulin from their surrounding environment rather than producing insulin themselves. Because of the small number of donor-derived, insulin-positive cells in the face of normoglycemia in bone H IG H L IG H T European Journal of Endocrinology (2004) 150 415–419 ISSN 0804-4643

1. An Historical and Phylogenetic Perspective of Islet-Cell Development R.C. Heller, J. Jensen, O.D. Madsen, H.V. Petersen, P. Serup. 2. Glucose Signalling to Transcription Factors of the Insulin Gene D. Melloul. 3. beta-Cell Dysfunction and Chronic Hyperglycaemia J.L. Leahy. 4. Glucose Toxicity of the Pancreatic beta-cell C. Gleason, J. Harmon, V. Poitout, R.P. Robertson, G. Sacchi, Y. Tanaka, Phuong Oanh T. Tran. 5. The alpha-Cell and Regulation of Glucagon Gene Transcription W. Knepel. 6. The KATP Channel and the Sulfonylurea Receptor T. Miki, S. Seino, H. Yano. 7. Glucagon-Like Peptide-1: An Insulinotropic Hormone with Potent Growth Factor Actions at the Pancreatic Islets of Langerhans G.G. Holz, C.A. Leech. 8. Communication of Islet Cells: Molecules and Functions D. Bosco, P. Meda. 9. Soluble Factors Important for Pancreas Development P. Czernichow, R. Scharfmann. 10. Role of Mesenchymal-Epithelial Interactions in Pancreas Development C.A. Crisera, G.K. Gittes, A.S. Kadison, M.T. Longaker, T.S. Maldonado. 11. Homeodomain Proteins in Pancreas Development U. Ahlgren, H. Edlund. 12. bHLH Factors and Notch in Pancreatic Development L.E. Flores, R.S. Heller, J. Jensen, O.D. Madsen, P. Serup. 13. Identification, Biological Functions, and Contribution to Human Diabetes of Islet-Brain 1 C.Bonny, G. Waeber. 14. Pax4 and Pax6 in Islet Differentiation P. Gruss, Xunlei Zhou. 15. The Role of the Hepatocyte Nuclear Factor Network in Glucose Homeostasis M. Stoffel. 16. Regulation of PDX-1 Gene Expression M.A. Cissell, K. Gerrish, S. Samaras, R. Stein, C.V.E. Wright. 17. Mechanisms of Postnatal b-Cell Mass Regulation S. Bonner-Weir, G.C. Weir. 18. Genetic Models of Insulin Resistance: Alterations in b-Cell Biology C.R. Kahn, R.N. Kulkarni. 19. Clinical Consequences of Genetic Defects in b-Cell Genes J.C. Evans, T.M. Frayling, A.T. Hattersley. 20. Pathophysiology of Glut2 in Diabetes Mellitus B. Thorens. 21. Use of a Cre-LoxP Strategy in Mice to Determine the Cell-Specific Roles of Glucokinase in MODY-2 M.A. Magnuson, C. Postic. 22. Development of b-Cell Lines for Transplantation in Type 1 Diabetes Mellitus S. Efrat. 23. Gene Therapeutic Approaches for b-Cell Replacement G.M. Beattie, A. Hayek, F. Levine. Colour Figures. Index.

In 1932, Harvey Cushing reviewed the medical history of Minnie G and 11 other patients with a rapid weight gain confined to the face, neck, and trunk, but sparing the extremities (1). This adiposity was associated with kyphosis, amenorrhea or impotence, hirsutism, purplish lineae atrophicae, hypertension, and extreme weakness. Cushing concluded ‘that the peculiar polyglandular syndrome may accompany basophil adenoma in the absence of any apparent alteration in the adrenal cortex other than a possible secondary hyperplasia.’ Cushing’s reasoning was correct. The majority of patients with the syndrome that now bears his name suffer from adrenal hypersecretion of cortisol due to excess of corticotropin (ACTH). The basophilic cells in Cushing’s series were most likely expansions of the population of pituitary corticotropes. But what is the pathogenesis of ACTH hypersecretion? Why do some ‘silent’ pituitary tumors show histochemical evidence for ACTH production but do not hypersecrete it? And why are not all ACTH-secreting tumors associated with atrophy of the neighboring normal corticotropes which should be suppressed? Current research has begun to elucidate the answers to some of these questions. The anterior pituitary corticotropes produce ACTH by processing products of the pro-opiomelanocortin (POMC) gene (see Fig. 1). Up to 10% of normal subjects may harbor small, POMC-containing pituitary adenoma (2). These adenoma are endocrinologically silent, presumably due to production of inactive POMC peptides or peptides that are not secreted. In patients with Cushing’s Disease, the frequency of identifiable pituitary tumors is much greater. However, the pathological findings are sometimes classified as corticotrope hyperplasia rather than adenoma. This has led to two major outlooks on the pathogenesis of Cushing’s disease, i.e. the hypothalamic theory and the pituitary theory (3). Evidence favoring the hypothalamic theory is that many histopathological specimens are classified as hyperplasia rather than adenoma. Of course, this may be due to difficulty in interpreting pathology of small tumor fragments. However, excess corticotropin-releasing hormone (CRH) has been shown to produce Cushing’s disease in patients with ectopic CRH production and in transgenic mice that overexpress the CRH gene (4). Evidence favoring the pituitary theory, which proposes that mutated corticotropes give rise to neoplastic clones that produce excess ACTH, is that permanent cure results from successful resection of pituitary tumors in the majority of patients. Successfully treated patients regain normal circadian rhythms for ACTH and other pituitary hormones. The factors that may lead to the development of corticotrope neoplasms, whether entirely of pituitary or perhaps of hypothalamic origin, are yet to be identified. Although most corticotrope neoplasms are believed to arise from the anterior pituitary, there have been patients with ACTH-secreting tumors whose origins seem to have been from the intermediate lobe (2). In humans, the intermediate lobe is a rudimentary structure; however, in other species it is well developed and functions to regulate skin pigmentation. Intermediate lobe melanotropes produce a-melanocyte-stimulating hormone (a-MSH), an alternative splice product of POMC, which is generated by tissue-specific enzyme cleavage. The enzyme prohormone convertase 2 (PC2), a member

Both glucose and glucagon-like peptide 1 (GLP-1) stimulate insulin gene transcription in endocrine pancreatic beta-cells within the islets of Langerhans. The effects of glucose are mediated by the homeodomain transcription factor islet duodenum homeobox -1 (IDX-1) that binds to two adenine thymidine-rich (A1 and A2/3) motifs within the rat insulin promoter. Glucose stimulates the activity of the transactivation domain of IDX-1 that lies within the first 80 amino acids of the IDX-1 protein. The effects of GLP-1 on insulin gene expression are primarily conferred by the cAMP responsive element (CRE) within the insulin promoter. GLP-1 stimulates glucose-dependent insulin release from beta-cells. We hypothesize that GLP-1 may augment the effects of glucose on insulin gene transcription. Here we show that GLP-1 stimulates insulin gene transcription independent of the CRE and is glucose-dependent. Furthermore, we show that GLP-1 stimulates the transactivational activity of IDX-1.

Background & Aims: The endoderm-specific homeodomain transcription factor IDX-1 is critical for pancreas development and for the regulation of islet cell–specific genes. During development, IDX-1 is expressed in the epithelial cells of the endoderm in the pancreatic anlage of the foregut. The aim of this study was to determine whether IDX-1 may have potential properties of a master homeotic determinant of pancreas and/or gut development. Methods: Transgenic mice were generated in which the expression of IDX-1 was misdirected by a promoter of the mesoderm-specific homeodomain protein Hoxa-4 known to express in the stomach and hindgut during development. The expectation was the formation of ectopic pancreatic tissue or alterations of gut patterning or morphology. Results: Although no ectopic induction of pancreatic markers was found in these transgenic mice, they manifested an altered midgut-hindgut union and agenesis of the cecum. Further, IDX-1 binds to the gut-specific homeodomain protein Cdx-2 and inhibits transactivation of the sucrase-isomaltase promoter by Cdx-2. Conclusions: These findings further support the emerging understanding that interactions among different classes of homeodomain proteins, expressed in a spatially and temporally restricted manner during development, determine the pattern of organogenesis. A possible mechanism for the dysmorphogenesis of the proximal colon may be an inhibition of Cdx-2 actions by IDX-1. GASTROENTEROLOGY 1998;115:381-387

The genetic factors that underlie the increasing incidence of diabetes with age are poorly understood. We examined whether telomere length, which is inherited and known to shorten with age, plays a role in the age-dependent increased incidence of diabetes. We show that in mice with short telomeres, insulin secretion is impaired and leads to glucose intolerance despite the presence of an intact β-cell mass. In ex vivo studies, short telomeres induced cell-autonomous defects in β-cells including reduced mitochondrial membrane hyperpolarization and Ca(2+) influx which limited insulin release. To examine the mechanism, we looked for evidence of apoptosis but found no baseline increase in β-cells with short telomeres. However, there was evidence of all the hallmarks of senescence including slower proliferation of β-cells and accumulation of p16(INK4a). Specifically, we identified gene expression changes in pathways which are essential for Ca(2+)-mediated exocytosis. We also show that telomere length is additive to the damaging effect of endoplasmic reticulum stress which occurs in the late stages of type 2 diabetes. This additive effect manifests as more severe hyperglycemia in Akita mice with short telomeres which had a profound loss of β-cell mass and increased β-cell apoptosis. Our data indicate that short telomeres can affect β-cell metabolism even in the presence of intact β-cell number, thus identifying a novel mechanism of telomere-mediated disease. They implicate telomere length as a determinant of β-cell function and diabetes pathogenesis.

Non-insulin-dependent diabetes mellitus (NIDDM) is characterized by peripheral insulin resistance and insulin secretory dysfunction (1, 2). These abnormalities are either genetically defined or augmented by hyperglycemia itself, and may interact in a complex manner to cause and sustain hyperglycemia. Previous prospective studies have demonstrated that both insulin resistance/ hyperinsulinemia and low insulin secretory response to glucose are predictors of NIDDM in humans (3–7). The notion that either peripheral insulin resistance or an insulin secretory defect alone would be sufficient for the development of NIDDM has been tested in several knockout mouse models. Mice lacking the insulin receptor exhibit severe insulin resistance and die from ketoacidosis within 3–7 days after birth. Mice lacking insulin receptor substrate (IRS)-1 are growth retarded in addition to being insulin resistant, but are not diabetic, because of compensatory hyperinsulinemia. Glucokinase-deficient mice are diabetic as a result of defective glucose sensing in the pancreatic b cell and consequently disturbed insulin secretion, but do not present with peripheral insulin resistance, which is characteristic of NIDDM. Heterozygous animals of the first two models do not exhibit any obvious clinical phenotype. Heterozygous glucokinase-deficient mice have decreased glucose tolerance as a result of reduced insulin secretory capacity. These mouse models with single genetic defects represent rare diabetic syndromes found in humans, but do not serve as models for the most abundant form of diabetes: NIDDM. Thus the hypothesis that has emerged from these observations is that an accumulation of several genetic defects (and/or variants), each of which in isolation would not lead to frank NIDDM, together may be the basis for the development of NIDDM. The most plausible combination would be a defect leading to peripheral insulin resistance, plus an insulin secretory defect. Two important studies have now tested this hypothesis and have established polygenic models of NIDDM (8, 9). The first model involved the crossing of homozygous IRS1-deficient mice with heterozygous-glucokinase deficient mice. Thus peripheral insulin resistance, together with disturbed glucose sensing and insulin secretion in the pancreatic b cell, leads to an animal model having a close resemblance to NIDDM (8). In the second model (9), double heterozygotes of animals lacking one allele each of IRS-1 and the insulin receptor were developed. The mice presented with profound peripheral insulin resistance. These animals could be further divided into two distinct groups: those that would (approximately 40%) and those that would not develop NIDDM. It may be assumed that an as yet unknown genetic factor is the cause of the stratification, and, in conformity with the first model, this defect may be hypothesized to cause b cell dysfunction. Both mouse models have a remarkable resemblance to NIDDM in humans. First, they are polygenic and inheritable; secondly, they bear the major metabolic hallmarks of NIDDM: insulin resistance, fasting hyperglycemia and hyperinsulinemia, and b cell dysfunction; and thirdly, the development of NIDDM is age-dependent and appears in older (begins at 4 months of age) and not young animals. Why age is a factor in the development of NIDDM is not clear from these studies. The authors do not mention in detail the results of glucose tolerance tests in young animals. However, it may be speculated that insulin secretory dysfunction requires some time to develop (perhaps when b cell regenerative capacity is exhausted or b cells fail as a result of ‘glucose toxicity’). Notably, both studies have revealed increased b cell mass and altered islet architecture in the diabetic animals, suggesting that a proliferative stress has been placed on the islets. The results are consistent with the concept that increased insulin resistance and decreased insulin secretion are independent yet co-operative risk factors for the development of NIDDM. It must be stated, however, that knocking out an element in the pathway of intracellular glucose metabolism or insulin signaling is expected to interfere with glucose metabolism, therefore these new mouse models may reflect NIDDM at the metabolic, but not at the genetic, level. An inverse approach to elucidating the genetic basis of NIDDM – searching for trait loci in established NIDDM animal models – has also resulted in the observation that NIDDM is most probably attributable to the coincidence of several genetic traits. The (patho)physiologically well characterized spontaneously diabetic Goto-Kakiuaki (GK) rat model is one of the best animal models for studying genetic susceptibility to NIDDM. The GK rat manifests the main features of the metabolic, hormonal and vascular disorders described in NIDDM, having mild basal hyperglycemia, marked glucose intolerance and both hepatic and peripheral insulin resistance. Moreover, GK rats exhibit basal hyperinsulinemia and impaired insulin response to glucose. A major difference between the GK rat model (and the above mentioned mouse models) and human NIDDM is, however, the lack of obesity in the animals. Two recent articles reported on the combined use of physiological and genetic studies to identify quantitative trait loci responsible for the control of glucose homeostasis and insulin secretion (10, 11). The loci, which in European Journal of Endocrinology (1997) 137 453–454 ISSN 0804-4643 H IG H L IG H T

Hypoglycemia as a limiting factor in the treatment of diabetes mellitus with insulin was apparent even before the hormone was administered to patients. A passage in a biography of Banting notes: ‘The pace picked up as Banting and Best began adjusting extract . . . On December 2, 1921 . . . the injections threw the longevity dog, number 27, into repeated convulsions. . . . It finally died, killed by the extract.’ (1). It is acknowledged that the organism has an intricate control mechanism to regulate glycemic levels within a narrow range; insulin as the hypoglycemic agent, and an array of mechanisms as a control against potentially fatal hypoglycemia. The central nervous system (CNS) plays a major role in sensing glucopenia and triggering hormone release during imminent hypoglycemia (2–4). As previous studies have demonstrated, selective perfusion of the cranial arteries with glucose abrogate counterregulation elicited by insulin-induced hypoglycemia, but hepatic glucoreceptors have also been reported to sense hypoglycemia and to activate the sympathoadrenal system (5). This mechanism is regarded to be secondary to the dominant role of the CNS. Several lines of evidence localize the specific region which is responsible for activation of counterregulatory mechanisms to the ventromedial hypothalamus (VMH): focal lesioning of this region abolishes the hormonal response to systemic hypoglycemia (6) and production of local hypoglycemia by perfusion of 2-deoxy-glucose into the VMH is able to trigger the release of counterregulatory hormones despite systemic normoglycemia (7). Alternatively one may ask: when peripheral hypoglycemia is induced by insulin, does continued supply of glucose to the VMH prevent the peripheral neurohumoral counterregulatory response? This question has been studied in a recent and most remarkable piece of work in which the authors were able to demonstrate the central role of the VMH in sensing and responding to hypoglycemia (8). Indeed, they showed that in awake rats, local perfusion of glucose to the VMH by a microdialysis technique blocks counterregulation during systemic hypoglycemia (8). Most of the counterregulatory responses were abrogated. At this point it is important to note that counterregulatory mechanisms normally come into play well before hypoglycemia ensues (4), such that it may be assumed that the VMH is usually active in physiologic situations. Glucagon – an important counterregulatory hormone – is an exception since it appears to be regulated normally regardless of the state of the VMH, suggesting that ambient glucose levels directly or indirectly regulate glucagon secretion independent of the above mentioned mechanisms. Let us now turn to the pathophysiology of hypoglycemia when it arises from prolonged fasting or strenuous exercise. During these situations circulating ketone bodies and lactate levels increase severalfold. In both situations certain signs and symptoms of hypoglycemia are diminished. In experimental situations, hyperketonemia and hyperlactacidemia lowered responses to hypoglycemia in humans (9–11). One of these studies reported that lactate infusions protect cerebral function during hypoglycemia suggesting a potential therapeutic application of lactate during hypoglycemia in patients with insulin overdosage (11). Another important finding of these studies is that, during glucopenia, ketone bodies and lactate can serve as an immediate energy substitute to the CNS. Future studies will reveal whether – similar to glucose – local infusion of ketone bodies and lactate to the VMH will abrogate the hormonal response to hypoglycemia. Moreover, the sensing at the molecular level of hypoglycemia and/or adequate alternative energy supplies to the brain remain to be investigated.

Global energy balance in mammals is controlled by the actions of circulating hormones that coordinate fuel production and utilization in metabolically active tissues. Bone-derived osteocalcin, in its undercarboxylated, hormonal form, regulates fat deposition and is a potent insulin secretagogue. Here, we show that insulin receptor (IR) signaling in osteoblasts controls osteoblast development and osteocalcin expression by suppressing the Runx2 inhibitor Twist2. Mice lacking IR in osteoblasts have low circulating undercarboxylated osteocalcin and reduced bone acquisition due to decreased bone formation and deficient numbers of osteoblasts. With age, these mice develop marked peripheral adiposity and hyperglycemia accompanied by severe glucose intolerance and insulin resistance. The metabolic abnormalities in these mice are improved by infusion of exogenous undercarboxylated osteocalcin. These results indicate the existence of a bone-pancreas endocrine loop through which insulin signaling in the osteoblast ensures osteoblast differentiation and stimulates osteocalcin production, which in turn regulates insulin sensitivity and pancreatic insulin secretion to control glucose homeostasis.