Decreased fetal size is associated with beta-cell hyperfunction in early life and failure with age

OBJECTIVE—Low birth weight is associated with diabetes in adult life. Accelerated or “catch-up” postnatal growth in response to small birth size is thought to presage disease years later. Whether adult disease is caused by intrauterine β-cell–specific programming or by altered metabolism associated with catch-up growth is unknown.RESEARCH DESIGN AND METHODS—We generated a new model of intrauterine growth restriction due to fatty acid synthase (FAS) haploinsufficiency (FAS deletion [FASDEL]). Developmental programming of diabetes in these mice was assessed from in utero to 1 year of age.RESULTS—FASDEL mice did not manifest catch-up growth or insulin resistance. β-Cell mass and insulin secretion were strikingly increased in young FASDEL mice, but β-cell failure and diabetes occurred with age. FASDEL β-cells had altered proliferative and apoptotic responses to the common stress of a high-fat diet. This sequence appeared to be developmentally entrained because β-cell mass was increased in utero in FASDEL mice and in another model of intrauterine growth restriction caused by ectopic expression of uncoupling protein-1. Increasing intrauterine growth in FASDEL mice by supplementing caloric intake of pregnant dams normalized β-cell mass in utero.CONCLUSIONS—Decreased intrauterine body size, independent of postnatal growth and insulin resistance, appears to regulate β-cell mass, suggesting that developing body size might represent a physiological signal that is integrated through the pancreatic β-cell to establish a template for hyperfunction in early life and β-cell failure with age.Low birth weight predisposes to type 2 diabetes, cardiovascular disease, and premature death (1,2), prompting the hypothesis (3) that impairing growth in early life programs metabolic disease in adulthood. Much of this programming is attributed to postnatal catch-up growth, which is linked to insulin resistance and cardiovascular disease later in life (4). Modeling impaired growth in utero using calorie restriction (5), protein malnutrition (6), prenatal glucocorticoid administration (7), or ligation of the uterine arteries (8) produces catch-up growth and glucose intolerance. Catch-up growth is associated with changes in food intake, metabolism, and insulin resistance that confound the search for mechanisms linking low birth weight and adult disease. In particular, insulin resistance increases β-cell mass (9) and makes it difficult to determine whether adult disease is caused by in utero β-cell–specific programming instead of altered body composition and feeding behavior associated with accelerated postnatal growth.Insulin and its downstream signals are critical for growth and development in species ranging from worms and insects to mammals (10–15). In Drosophila, body size is sensed in the fat body (equivalent to the vertebrate liver) to antagonize insulin-induced growth by ecdysone (16). If an analogous process occurs in mammals, β-cells are likely involved because they are affected by mediators of body size, including insulin, amino acids, and other signals (17). These factors also regulate fatty acid synthase (FAS), which catalyzes the first committed step in fatty acid biosynthesis (18). FAS is regulated by nutrients independent of insulin, suggesting that it could be important for nutrient-dependent growth. Its global loss results in early embryonic lethality (19). Tissue-specific inactivation of FAS is possible (20), and, surprisingly, the loss of FAS in pancreatic β-cells has no effect on β-cell mass or the capacity to secrete insulin (21). Thus, FAS, unlike glucokinase (22) and insulin receptor substrate (IRS)-2 (23), is not required for normal β-cell function.Here, we report that FAS heterozygous mice are born small yet have expanded β-cell mass and increased insulin secretion without insulin resistance. This hyperplastic β-cell phenotype was reversed by promoting growth in utero, and increased β-cell mass was confirmed in another model of intrauterine growth restriction (IUGR). Despite the absence of catch-up postnatal growth, FAS-deficient mice develop diet-induced diabetes and β-cell failure with age.     

Blockade of alpha4 integrin signaling ameliorates the metabolic consequences of high-fat diet-induced obesity

OBJECTIVE—Many prevalent diseases of advanced societies, such as obesity-induced type 2 diabetes, are linked to indolent mononuclear cell–dependent inflammation. We previously proposed that blockade of α4 integrin signaling can inhibit inflammation while limiting mechanism-based toxicities of loss of α4 function. Thus, we hypothesized that mice bearing an α4(Y991A) mutation, which blocks signaling, would be protected from development of high-fat diet–induced insulin resistance.RESEARCH DESIGN AND METHODS—Six- to eight-week-old wild-type and α4(Y991A) C57Bl/6 male mice were placed on either a high-fat diet that derived 60% calories from lipids or a chow diet. Metabolic testing was performed after 16–22 weeks of diet.RESULTS—α4(Y991A) mice were protected from development of high-fat diet–induced insulin resistance. This protection was conferred on wild-type mice by α4(Y991A) bone marrow transplantation. In the reverse experiment, wild-type bone marrow renders high-fat diet–fed α4(Y991A) acceptor animals insulin resistant. Furthermore, fat-fed α4(Y991A) mice showed a dramatic reduction of monocyte/macrophages in adipose tissue. This reduction was due to reduced monocyte/macrophage migration rather than reduced monocyte chemoattractant protein-1 production.CONCLUSIONS—α4 integrins contribute to the development of HFD-induced insulin resistance by mediating the trafficking of monocytes into adipose tissue; hence, blockade of α4 integrin signaling can prevent the development of obesity-induced insulin resistance.Obesity leads to insulin resistance that results in type 2 diabetes (1) and that contributes to hypertension and cardiovascular disease (2). Mononuclear cell–mediated inflammation in obese adipose tissue plays a pathogenetic role in insulin resistance (3,4). Thus, there is great interest in the possibility of using anti-inflammatory strategies to ameliorate obesity-induced insulin resistance.Blockade of leukocyte adhesion is a proven therapeutic strategy for a wide variety of inflammatory diseases (5). In particular, inhibiting α4 integrins or their counter-receptors (vascular cell adhesion molecule-1 [VCAM-1] and mucosal adressin cell adhesion molecule-1 [MadCAM-1]) blocks inflammatory responses mediated by mononuclear leukocytes (6). α4 integrin antagonists are of proven benefit in several human inflammatory diseases (7,8). These antagonists, such as the monoclonal antibody natalizumab, block ligand binding function, thus producing a complete loss of α4 integrin function. Lack of α4 integrins is embryonic lethal and results in defective placentation, heart development, and hematopoiesis (9–11). Furthermore, natalizumab therapy has been associated with fatal progressive multifocal leukoencephalopathy in humans, possibly because of defective T-cell trafficking to the brain (12,13). Thus, currently available α4 integrin antagonists are of proven value in mononuclear cell–mediated diseases; however, complete loss of α4 integrin function is associated with developmental defects and abnormal hematopoiesis.As noted above, whereas α4 integrin antagonists show promise for several autoimmune and inflammatory diseases, mechanism-based toxicities may limit their use, particularly in low-grade chronic inflammatory conditions, such as obesity-induced insulin resistance. We recently proposed an alternative strategy—blockade of α4 integrin signaling—to perturb functions involved in inflammation, while limiting mechanism-based adverse effects (14). α4 integrin signaling involves the binding of paxillin to the α4 integrin tail, and a point mutation (α4Y991A) that selectively blocks this interaction reduces α4-mediated leukocyte migration (15) and adhesion strengthening in flowing blood (16) while sparing α4-mediated static cell adhesion (17). Furthermore, mice bearing an α4(Y991A) mutation are viable and fertile and have intact lympho-hematopoiesis and humoral immune responses; however, they exhibit defective recruitment of mononuclear leukocytes in experimental inflammation (18). Here, we report that the α4(Y991A) mutation reduces mononuclear leukocyte infiltration of white adipose tissue (WAT) in high-fat diet–induced obese mice and hence reduce high-fat diet–induced insulin resistance. Thus, we establish that blocking α4 integrin signaling can ameliorate the metabolic consequences of high-fat diet–induced obesity.     

Small interfering RNA-mediated suppression of proislet amyloid polypeptide expression inhibits islet amyloid formation and enhances survival of human islets in culture

OBJECTIVE—Islet amyloid, formed by aggregation of the β-cell peptide islet amyloid polypeptide (IAPP; amylin), is a pathological characteristic of pancreatic islets in type 2 diabetes. Toxic IAPP aggregates likely contribute to the progressive loss of β-cells in this disease. We used cultured human islets as an ex vivo model of amyloid formation to investigate whether suppression of proIAPP expression would inhibit islet amyloid formation and enhance β-cell survival and function.RESEARCH DESIGN AND METHODS—Islets from cadaveric organ donors were transduced with a recombinant adenovirus expressing a short interfering RNA (siRNA) designed to suppress human proIAPP (Ad-hProIAPP-siRNA), cultured for 10 days, and then assessed for the presence of islet amyloid, β-cell apoptosis, and β-cell function.RESULTS—Thioflavine S–positive amyloid deposits were clearly present after 10 days of culture. Transduction with Ad-hProIAPP-siRNA reduced proIAPP expression by 75% compared with nontransduced islets as assessed by Western blot analysis of islet lysates 4 days after transduction. siRNA-mediated inhibition of IAPP expression decreased islet amyloid area by 63% compared with nontransduced cultured islets. Cell death assessed by transferase-mediated dUTP nick-end labeling staining was decreased by 50% in transduced cultured human islets, associated with a significant increase in islet insulin content (control, 100 ± 4 vs. +Ad-siRNA, 153 ± 22%, P < 0.01) and glucose-stimulated insulin secretion (control, 222 ± 33 vs. +Ad-siRNA, 285 ± 21 percent basal, P < 0.05).CONCLUSIONS—These findings demonstrate that inhibition of IAPP synthesis prevents amyloid formation and β-cell death in cultured human islets. Inhibitors of IAPP synthesis may have therapeutic value in type 2 diabetes.Islet amyloid deposits are a pathological lesion of the pancreas in type 2 diabetes (1–3) formed by aggregation of islet amyloid polypeptide (IAPP; amylin) (4,5), a 37–amino acid hormone that is colocalized with insulin in β-cell granules and secreted along with insulin in response to β-cell secretagogues (6–8). Aggregates of IAPP, including small oligomeric species, are toxic to β-cells (9–14) and likely play an important role in the progressive loss of β-cells in this disease. Despite considerable study during the past decade, it is still not well understood why soluble IAPP molecules form toxic IAPP aggregates in type 2 diabetes. The presence of an amyloidogenic amino acid sequence in the human IAPP molecule (Gly-Ala-Iso-Leu-Ser [GAILS]) appears to be important but not sufficient for amyloid formation (2,3,15,16). Because production and secretion of IAPP closely mimic that of insulin, IAPP levels are elevated in conditions associated with insulin resistance and hyperinsulinemia, such as the early stages of type 2 diabetes (17). Elevated IAPP production and secretion because of increased demand for insulin along with defective trafficking and/or processing of proIAPP associated with β-cell dysfunction have been implicated as two possible factors contributing to aggregation of (pro)IAPP (i.e., proIAPP and its intermediate forms) in type 2 diabetes (2,3,16,18–23).Studies performed using transgenic rodent models have demonstrated a strong association between β-cell expression of human IAPP and development of hyperglycemia associated with loss of β-cells (24–29). β-Cell loss in these models was attributed to the formation of toxic IAPP aggregates as either small oligomeric species or amyloid fibrils. Studies on human and nonhuman primates have also demonstrated development of islet amyloid associated with β-cell loss in type 2 diabetes (30,31). It is not clear from these studies, however, whether IAPP aggregation and amyloid formation is the cause or a consequence of β-cell dysfunction and death. In the present study, we tested the hypothesis that suppression of human IAPP expression would inhibit amyloid formation and enhance survival and/or function of human islets. We and others have found that islet amyloid forms rapidly in islets from transgenic mice with β-cell expression of human IAPP, and its formation is potentiated by elevated glucose concentrations (23,32–35). We used cultured human islets as an ex vivo model of amyloid formation to investigate whether short interfering RNA (siRNA)-mediated suppression of endogenously expressed human proIAPP will enhance β-cell survival in human islets.     

Combination therapy with glucagon-like peptide-1 and gastrin restores normoglycemia in diabetic NOD mice

OBJECTIVE—Glucagon-like peptide-1 (GLP-1) and gastrin promote pancreatic β-cell function, survival, and growth. Here, we investigated whether GLP-1 and gastrin can restore the β-cell mass and reverse hyperglycemia in NOD mice with autoimmune diabetes.RESEARCH DESIGN AND METHODS—Acutely diabetic NOD mice were treated with GLP-1 and gastrin, separately or together, twice daily for 3 weeks. Blood glucose was measured weekly and for a further 5 weeks after treatments, after which pancreatic insulin content and β-cell mass, proliferation, neogenesis, and apoptosis were measured. Insulin autoantibodies were measured, and adoptive transfer of diabetes and syngeneic islet transplant studies were done to evaluate the effects of GLP-1 and gastrin treatment on autoimmunity.RESULTS—Combination therapy with GLP-1 and gastrin, but not with GLP-1 or gastrin alone, restored normoglycemia in diabetic NOD mice. The GLP-1 and gastrin combination increased pancreatic insulin content, β-cell mass, and insulin-positive cells in pancreatic ducts, and β-cell apoptosis was decreased. Insulin autoantibodies were reduced in GLP-1–and gastrin-treated NOD mice, and splenocytes from these mice delayed adoptive transfer of diabetes in NOD-scid mice. Syngeneic islet grafts in GLP-1–and gastrin-treated NOD mice were infiltrated by leukocytes with a shift in cytokine expression from interferon-γ to transforming growth factor-β1, and β-cells were protected from apoptosis.CONCLUSIONS—Combination therapy with GLP-1 and gastrin restores normoglycemia in diabetic NOD mice by increasing the pancreatic β-cell mass and downregulating the autoimmune response.Pancreatic β-cells can regenerate in response to experimental injury in adult animals (1–3) and can increase in humans in response to conditions such as pregnancy (4) and obesity (5). In addition, there is histological evidence of attempts at β-cell regeneration in humans with type 1 diabetes (6,7). Similarly, β-cell proliferation is increased before diabetes onset in NOD mice, an animal model for human type 1 diabetes, but not sufficiently to keep up with the ongoing autoimmune response that decreases the β-cell mass (8). Therefore, therapies directed at stimulating β-cell regeneration in addition to arresting autoimmunity may restore the β-cell mass and reverse type 1 diabetes.Many putative β-cell growth factors have been identified, one of the most promising being glucagon-like peptide-1 (GLP-1), a peptide secreted from intestinal L-cells in response to nutrient ingestion (9). The actions of GLP-1 to stimulate glucose-dependent insulin secretion and inhibit glucagon release, gastric emptying, and food intake (10) have led to its application as a therapy for type 2 diabetes (11). GLP-1 has additional actions that suggest a therapeutic role in conditions with a deficit in β-cell mass. GLP-1 and long-acting GLP-1 receptor agonists, such as exendin-4, increase the β-cell mass in rodents with surgically or chemically induced diabetes through stimulation of β-cell proliferation and islet neogenesis and inhibition of β-cell apoptosis (12–15). Also, GLP-1 (16) and exendin-4 (17) reduce insulitis and protect β-cells in NOD mice when given before diabetes onset. Exendin-4 has also been reported to reverse diabetes in NOD mice; however, this required combination of exendin-4 with immunosuppressive therapy using antilymphocyte serum (18).Gastrin is a gastrointestinal peptide reported to induce β-cell neogenesis from pancreatic exocrine duct cells in rodents (19,20). Combined gastrin and epidermal growth factor (EGF) treatment induces islet regeneration and restores normoglycemia in alloxan-treated mice (21) and ameliorates hyperglycemia after diabetes onset in NOD mice (22). Here, we report that addition of gastrin to GLP-1 treatment restored normoglycemia in acutely diabetic NOD mice by increasing the pancreatic β-cell mass and downregulating the autoimmune response.     

Plasma Ceramides Are Elevated in Obese Subjects With Type 2 Diabetes and Correlate With the Severity of Insulin Resistance

OBJECTIVE—To quantitate plasma ceramide subspecies concentrations in obese subjects with type 2 diabetes and relate these plasma levels to the severity of insulin resistance. Ceramides are a putative mediator of insulin resistance and lipotoxicity, and accumulation of ceramides within tissues in obese and diabetic subjects has been well described.RESEARCH DESIGN AND METHODS—We analyzed fasting plasma ceramide subspecies by quantitative tandem mass spectrometry in 13 obese type 2 diabetic patients and 14 lean healthy control subjects. Results were related to insulin sensitivity measured with the hyperinsulinemic-euglycemic clamp technique and with plasma tumor necrosis factor-α (TNF-α) levels, a marker of inflammation. Ceramide species (C18:1, 18:0, 20:0, 24:1, and 24:0) were quantified using electrospray ionization tandem mass spectrometry after separation with high-performance liquid chromatography.RESULTS—Insulin sensitivity (mg · kg⁻¹ · min⁻¹) was lower in type 2 diabetic patients (4.90 ± 0.3) versus control subjects (9.6 ± 0.4) (P < 0.0001). Type 2 diabetic subjects had higher (P < 0.05) concentrations of C18:0, C20:0, C24:1, and total ceramide. Insulin sensitivity was inversely correlated with C18:0, C20:0, C24:1, C24:0, and total ceramide (all P < 0.01). Plasma TNF-α concentration was increased (P < 0.05) in type 2 diabetic subjects and correlated with increased C18:1 and C18:0 ceramide subspecies.CONCLUSIONS—Plasma ceramide levels are elevated in type 2 diabetic subjects and may contribute to insulin resistance through activation of inflammatory mediators, such as TNF-α.Type 2 diabetes is an insulin-resistant state characterized by impaired glucose tolerance (1) and inflammation (2). Much evidence has demonstrated the role of increased circulating free fatty acids and tissue fat accumulation in the development of muscle and liver insulin resistance (1,3,4). The disturbances in plasma and tissue lipid metabolism result from an oversupply of lipid substrates, both exogenously and endogenously (increased lipolysis secondary to adipocyte insulin resistance), and perturbations in fat oxidation and utilization by muscle and liver, resulting in the accumulation of ectopic fat (4). Ectopic fat is “lipotoxic” and has been linked to the severity of insulin resistance and pancreatic β-cell dysfunction, i.e., the core defects in type 2 diabetes (1,4). Ectopic fat comprises various lipid species, including long-chain fatty acyl CoAs, diacylglycerol, and ceramide. It is well documented that ceramide accumulates within insulin-resistant tissues of animals (5–7) and humans (8–10) and inhibits insulin action and subsequent glucose uptake through inactivation of Akt. Ceramide also induces inflammation through activation of the nuclear factor-κB–tumor necrosis factor-α (TNF-α) axis (5–7).TNF-α is released from adipocytes and circulating mononuclear cells (MNCs) in response to stimuli, such as lipid infusion, lipopolysaccharide, reactive oxygen species, and hyperglycemia, and elevated TNF-α concentrations have been shown to induce insulin resistance (11–14). TNF-α also activates the plasma membrane enzyme sphingomyelinase (SMase) that hydrolyzes sphingomyelin to ceramide, allowing ceramides to accumulate within the cell (5,6,15–17). This accumulation of ceramide within tissues is thought to initiate a positive feedback mechanism, leading to enhanced production of proinflammatory cytokines (5), resulting in further inhibition of insulin-stimulated glucose uptake. Both plasma TNF-α concentrations and intracellular lipid intermediates, such as ceramides, are elevated in subjects with type 2 diabetes (8,18). Thus, ceramide is a bioactive lipid and putative mediator of insulin resistance that could link nutrient (fat) oversupply and cytokine-induced inflammation in tissues (5–7).Plasma ceramide levels also have been shown to correlate with coronary artery disease, independent of the plasma cholesterol concentration (19,20). However, the role of circulating ceramides has received little attention with respect to the development of insulin resistance and type 2 diabetes. Conflicting reports exist as to whether total circulating ceramides are elevated in obese (21) and type 2 diabetic subjects (22). Subspecies of plasma ceramides have been demonstrated to be increased in patients with sepsis and atherosclerosis (23–25), but the relationship between plasma ceramide subspecies levels and insulin resistance has not been investigated in patients with type 2 diabetes.Given their central role in the induction of insulin resistance and inflammation, elevated plasma ceramide levels may serve as a biomarker or direct perpetuator of insulin resistance and lipid-induced inflammation. Elevated plasma ceramide concentrations also may serve to identify individuals who are at risk to develop type 2 diabetes. The objective of this study was to quantify the concentration of individual ceramide subspecies in the circulation of patients with type 2 diabetes and healthy control subjects and to examine the correlation between plasma levels of ceramide subspecies and insulin sensitivity, measured with the euglycemic-hyperinsulinemic clamp, and plasma TNF-α concentration, a marker of inflammation.     

Spontaneous diabetes in hemizygous human amylin transgenic mice that developed neither islet amyloid nor peripheral insulin resistance

OBJECTIVES—We sought to 1) Determine whether soluble-misfolded amylin or insoluble-fibrillar amylin may cause or result from diabetes in human amylin transgenic mice and 2) determine the role, if any, that insulin resistance might play in these processes.RESEARCH DESIGN AND METHODS—We characterized the phenotypes of independent transgenic mouse lines that display pancreas-specific expression of human amylin or a nonaggregating homolog, [^25,28,29Pro]human amylin, in an FVB/n background.RESULTS—Diabetes occurred in hemizygous human amylin transgenic mice from 6 weeks after birth. Glucose tolerance was impaired during the mid- and end-diabetic phases, in which progressive β-cell loss paralleled decreasing pancreatic and plasma insulin and amylin. Peripheral insulin resistance was absent because glucose uptake rates were equivalent in isolated soleus muscles from transgenic and control animals. Even in advanced diabetes, islets lacked amyloid deposits. In islets from nontransgenic mice, glucagon and somatostatin cells were present mainly at the periphery and insulin cells were mainly in the core; in contrast, all three cell types were distributed throughout the islet in transgenic animals. [^25,28,29Pro]human amylin transgenic mice developed neither β-cell degeneration nor glucose intolerance.CONCLUSIONS—Overexpression of fibrillogenic human amylin in these human amylin transgenic mice caused β-cell degeneration and diabetes through mechanisms independent from both peripheral insulin resistance and islet amyloid. These findings are consistent with β-cell death evoked by misfolded but soluble cytotoxic species, such as those formed by human amylin in vitro.Increasing evidence indicates that decreased β-cell mass contributes to the impaired insulin secretion characteristic of type 2 diabetes (1–3). Amylin, also referred to as islet amyloid polypeptide, is a 37-amino acid polypeptide (4,5) secreted from pancreatic islet β-cells whose aggregation results in islet amyloid formation in type 2 diabetes (6). Islet amyloid has been reported in 40–90% of pancreases from type 2 diabetic subjects studied post mortem (7–11) and has been linked to both decreased β-cell mass and β-cell dysfunction (12,13). In vitro, human amylin causes apoptosis of islet β-cells, and there is growing evidence that this pathogenic process may contribute to the β-cell deficit in type 2 diabetes (1,2,14,15). However, it remains unresolved whether islet amyloid contributes to the etiopathogenesis of type 2 diabetes or, by contrast, occurs only as a consequence of the disease.Several independent lines of human amylin transgenic mice have been developed to investigate the role of amylin and islet amyloid in the pathogenesis of type 2 diabetes (16–19). The findings and conclusions from phenotypic characterization studies are wide ranging and sometimes at variance. Transgenic animals developed by several research groups did not develop spontaneous diabetes or insulin resistance or exhibit evidence of islet amyloid formation, suggesting that overexpression of human amylin alone was not sufficient to contribute to diabetes development and islet amyloid formation in those models (16–18). In contrast, Janson et al. (19) showed development of spontaneous diabetes in the absence of islet amyloid in homozygous individuals from a further transgenic mouse model, consistent with the view that overexpression of human amylin is sufficient for diabetes development but not islet amyloid formation in that model. It was previously thought that overexpression of human amylin might be sufficient for islet amyloid formation, but some studies have suggested that insulin resistance might also be necessary (20–22).Evidence concerning the role of human amylin in the processes that lead to or cause diabetes remains conflicting, and a clear role for human amylin–mediated β-cell death has not been established at this time, at least in part due to conflicting evidence from the different lines of human amylin transgenic mice. Previous reports have described the noticeable lack of correlation between amyloid deposition and hyperglycemia in other transgenic models of amylin-induced diabetes (21,23). Islets from homozygous individuals from the FVB/n-based line reported by Janson et al. (19) demonstrated a pattern of β-cell loss that closely reflects that in islets from human type 2 diabetic patients (1,3,9), but hemizygous animals from that line reportedly do not develop diabetes.Here, we report a transgenic human amylin mouse model (L13) in which hemizygous individuals developed early-onset diabetes without peripheral insulin resistance and islet amyloid formation. We demonstrate that the disappearance of functional β-cells during the progression of diabetes in this model contributes to the pathogenesis of diabetes. The absence of islet amyloid in the pancreas of transgenic mice before diabetes onset and during its progression, despite the high secretion rates of human amylin, shows that islet amyloid is not required for islet β-cell degeneration and loss of physiological insulin secretion. These findings are consistent with the reports of Janson et al. (19) and provide strong support for continuing exploration of the mechanism by which human amylin evokes β-cell death and contributes to the failure of insulin secretion in type 2 diabetes.     

Selective small-molecule agonists of G protein-coupled receptor 40 promote glucose-dependent insulin secretion and reduce blood glucose in mice

OBJECTIVE— Acute activation of G protein–coupled receptor 40 (GPR40) by free fatty acids (FFAs) or synthetic GPR40 agonists enhances insulin secretion. However, it is still a matter of debate whether activation of GPR40 would be beneficial for the treatment of type 2 diabetes, since chronic exposure to FFAs impairs islet function. We sought to evaluate the specific role of GPR40 in islets and its potential as a therapeutic target using compounds that specifically activate GPR40.RESEARCH DESIGN AND METHODS— We developed a series of GPR40-selective small-molecule agonists and studied their acute and chronic effects on glucose-dependent insulin secretion (GDIS) in isolated islets, as well as effects on blood glucose levels during intraperitoneal glucose tolerance tests in wild-type and GPR40 knockout mice (GPR40^−/−).RESULTS— Small-molecule GPR40 agonists significantly enhanced GDIS in isolated islets and improved glucose tolerance in wild-type mice but not in GPR40^−/− mice. While a 72-h exposure to FFAs in tissue culture significantly impaired GDIS in islets from both wild-type and GPR40^−/− mice, similar exposure to the GPR40 agonist did not impair GDIS in islets from wild-type mice. Furthermore, the GPR40 agonist enhanced insulin secretion in perfused pancreata from neonatal streptozotocin-induced diabetic rats and improved glucose levels in mice with high-fat diet–induced obesity acutely and chronically.CONCLUSIONS— GPR40 does not mediate the chronic toxic effects of FFAs on islet function. Pharmacological activation of GPR40 may potentiate GDIS in humans and be beneficial for overall glucose control in patients with type 2 diabetes.Loss of glucose-dependent insulin secretion (GDIS) from the pancreatic β-cell is responsible for the onset and progression of type 2 diabetes (1,2). Oral agents that stimulate insulin secretion, such as sulfonylureas and related ATP-sensitive K⁺ channel blockers, reduce blood glucose and have been used as a first-line type 2 diabetes therapy for nearly 30 years (3,4). However, these agents act to force the β-cell to secrete insulin continuously regardless of prevailing glucose levels, thereby promoting hypoglycemia and accelerating the loss of islet function and, eventually, diminished efficacy (5,6). Despite the availability of a range of agents for type 2 diabetes, many diabetic patients fail to achieve or to maintain glycemic targets (7–9). In addition, stricter glycemic guidelines have been proposed to help define a path toward diabetes prevention through identifying and treating the pre-diabetes state (10). Agents that induce GDIS have great potential to replace sulfonylureas as a first-line therapy for the treatment of type 2 diabetes. In particular, agents that have positive effects on arresting or even reversing β-cell demise would represent a major therapeutic advance toward addressing the lack of durability seen with current therapies and perhaps obviate the need for eventual insulin intervention (11–13). The recent emergence of glucagon-like peptide 1–based GDIS agents (14–16), including inhibitors of dipeptidyl peptidase-4 (17) and peptidase-stable analogs such as exendin-4 (18), is undoubtedly a major advance in such a direction. Nevertheless, it remains to be observed whether glucagon-like peptide 1–related agents truly exert durable beneficial effects on β-cell mass and function.The molecular pharmacology of lipid and lipid-like mediators that signal through G protein–coupled receptors (GPCRs) has expanded significantly over the past few years. To date, several orphan GPCRs have been paired with lysophospholipids, bile acids, arachidonic acid metabolites, dioleoyl phosphatidic acid, and short-, medium-, and long-chain free fatty acids (FFAs) (19–21). From these discoveries, GPCR 40 (GPR40), GPR119, and GPR120 have been reported to play a role in regulating GDIS and therefore have potential as novel targets for the treatment of type 2 diabetes (22–26). GPR40 is a G_q-coupled, family A GPCR that is highly expressed in β-cells of human and rodent islets. Several naturally occurring medium- to long-chain FFAs and some thiazolidinedione peroxisome proliferator–activated receptor-γ agonists specifically activate GPR40 (27,28). Activation of GPR40 by FFAs (29–32) or synthetic compounds (23,33) enhances insulin secretion through the amplification of intracellular calcium signaling.The pleiotropic effects of FFAs on the pancreatic β-cell are well known. The fact that FFAs are in vitro ligands for GPR40 is suggestive of the link to the wealth of existing literature data on the acute, stimulatory effects of FFAs on insulin release (34,35). However, FFAs also exert suppressive or detrimental effects on β-cells. Lipotoxicity of β-cells, a condition observed with chronic exposure to high FFA levels, results in impairment in their function and a resulting diminution in their insulin secretory capacity (36,37). Currently, there is an ongoing debate on whether GPR40 mediates the deleterious effects of FFAs on islet function (lipotoxicity) and whether an antagonist of GPR40 is preferable to an agonist for the treatment of type 2 diabetes (38,39). Since FFAs can both be metabolized within cells to act as intracellular signaling molecules (35) and activate more than one receptor (20), they cannot be used as specific and selective tools to unravel the role that GPR40 plays in the β-cell. It is therefore necessary to identify small molecules that specifically activate GPR40.In the following discussion, we will detail the identification and in vitro pharmacology of a novel series of synthetic GPR40 agonists. Using isolated islets from wild-type and homozygous GPR40 knockout (GPR40^−/−) mice (to confirm the on-target activity of small-molecule activators), we not only extended previous findings that acute activation of GPR40 enhances GDIS in pancreatic β-cells but also showed that long-term exposure to the GPR40 agonist, in contrast to FFAs, did not impair β-cell function, thus dissociating the activation of GPR40 from β-cell lipotoxicity. Finally, acute and subchronic dosing of the GPR40 agonist robustly reduced the blood glucose excursion during an intraperitoneal glucose tolerance test (IPGTT) in wild-type, but not GPR40^−/−, mice.     

Roles of IP3R and RyR Ca2+ Channels in Endoplasmic Reticulum Stress and ��-Cell Death

OBJECTIVE—Endoplasmic reticulum (ER) stress has been implicated in the pathogenesis of diabetes, but the roles of specific ER Ca²⁺ release channels in the ER stress–associated apoptosis pathway remain unknown. Here, we examined the effects of stimulating or inhibiting the ER-resident inositol trisphosphate receptors (IP₃Rs) and the ryanodine receptors (RyRs) on the induction of β-cell ER stress and apoptosis.RESEARCH DESIGN AND METHODS—Kinetics of β-cell death were tracked by imaging propidium iodide incorporation and caspase-3 activity in real time. ER stress and apoptosis were assessed by Western blot. Mitochondrial membrane potential was monitored by flow cytometry. Cytosolic Ca²⁺ was imaged using fura-2, and genetically encoded fluorescence resonance energy transfer (FRET)–based probes were used to measure Ca²⁺ in ER and mitochondria.RESULTS—Neither RyR nor IP₃R inhibition, alone or in combination, caused robust death within 24 h. In contrast, blocking sarco/endoplasmic reticulum ATPase (SERCA) pumps depleted ER Ca²⁺ and induced marked phosphorylation of PKR-like ER kinase (PERK) and eukaryotic initiation factor-2α (eIF2α), C/EBP homologous protein (CHOP)–associated ER stress, caspase-3 activation, and death. Notably, ER stress following SERCA inhibition was attenuated by blocking IP₃Rs and RyRs. Conversely, stimulation of ER Ca²⁺ release channels accelerated thapsigargin-induced ER depletion and apoptosis. SERCA block also activated caspase-9 and induced perturbations of the mitochondrial membrane potential, resulting eventually in the loss of mitochondrial polarization.CONCLUSIONS—This study demonstrates that the activity of ER Ca²⁺ channels regulates the susceptibility of β-cells to ER stress resulting from impaired SERCA function. Our results also suggest the involvement of mitochondria in β-cell apoptosis associated with dysfunctional β-cell ER Ca²⁺ homeostasis and ER stress.Inappropriate activation of cell death pathways in the pancreatic β-cell is involved in the pathogenesis of type 1 diabetes, type 2 diabetes, and rare diabetic disorders such as maturity-onset diabetes of the young, Wolcott-Rallison syndrome, and Wolfram syndrome (1–5). β-Cell apoptosis also hampers clinical islet transplantation (6). The endoplasmic reticulum (ER) plays a key role in multiple programmed cell death pathways (7–9). Apoptosis caused by ER stress has been associated with diabetes (1,2,5,10) and can be induced by the accumulation of unfolded proteins resulting from disrupted Ca²⁺-dependent chaperone function in the ER (1,11). Both thapsigargin, a potent and specific inhibitor of sarco/endoplasmic reticulum ATPase (SERCA), and endogenous factors that downregulate SERCA, evoke ER stress and apoptosis in β-cells (12,13). However, the detailed mechanisms underlying Ca²⁺-dependent apoptosis and the roles played by specific β-cell ER Ca²⁺ channels and pumps in ER stress remain unclear.In addition to multiple SERCA isoforms (14), the β-cell ER expresses several classes of intracellular Ca²⁺-releasing channels, including the inositol trisphosphate receptors (IP₃Rs) and the ryanodine receptors (RyRs) (15–19). In the diabetic state, the expression of these receptors is known to be modulated in several cell types, including β-cells (15,20–22). We have previously shown that long-term inhibition of RyR2 in low glucose leads to programmed β-cell death involving calpain-10, but not caspase-3; conversely, RyR inhibition protected islets under conditions of chronic hyperglycemia (17). We have also shown that RyR inhibition significantly reduces the ratio of ATP to ADP in MIN6 β-cells (23), an event that could conceivably activate ER stress (24,25). Furthermore, studies of other cells types have suggested that ER stress–associated damage can be affected by inhibitors of RyRs (26) or IP₃Rs (27). Despite these important questions and links, studies on the roles of RyRs and IP₃Rs in β-cell ER stress have not been published to date.In the present study, we investigated whether disrupting β-cell ER Ca²⁺ homeostasis by blocking Ca²⁺ release through IP₃Rs and RyRs is sufficient to induce ER stress. We also tested the hypothesis that stimulating or inhibiting these channels would alter ER stress or apoptosis triggered by ER Ca²⁺ depletion following SERCA inhibition. Our results demonstrate that while blocking ER Ca²⁺ release channels does not induce a major ER stress response, Ca²⁺ flux from both RyRs and IP₃Rs can modulate β-cell apoptosis and ER stress resulting from impaired SERCA function.     

m.3243A>G Mutation in Mitochondrial DNA Leads to Decreased Insulin Sensitivity in Skeletal Muscle and to Progressive ��-Cell Dysfunction

OBJECTIVE—To study insulin sensitivity and perfusion in skeletal muscle together with the β-cell function in subjects with the m.3243A>G mutation in mitochondrial DNA, the most common cause of mitochondrial diabetes.RESEARCH DESIGN AND METHODS—We measured skeletal muscle glucose uptake and perfusion using positron emission tomography and 2-[¹⁸F]fluoro-2-deoxyglucose and [¹⁵O]H₂O during euglycemic hyperinsulinemia in 15 patients with m.3243A>G. These patients included five subjects with no diabetes as defined by the oral glucose tolerance test (OGTT) (group 1), three with GHb <6.1% and newly found diabetes by OGTT (group 2), and seven with a previously diagnosed diabetes (group 3). Control subjects consisted of 13 healthy individuals who were similar to the carriers of m.3243A>G with respect to age and physical activity. β-Cell function was assessed using the OGTT and subsequent mathematical modeling.RESULTS—Skeletal muscle glucose uptake was significantly lower in groups 1, 2, and 3 than in the control subjects. The glucose sensitivity of β-cells in group 1 patients was similar to that of the control subjects, whereas in group 2 and 3 patients, the glucose sensitivity was significantly lower. The insulin secretion parameters correlated strongly with the proportion of m.3243A>G mutation in muscle.CONCLUSIONS—Our findings show that subjects with m.3243A>G are insulin resistant in skeletal muscle even when β-cell function is not markedly impaired or glucose control compromised. We suggest that both the skeletal muscle insulin sensitivity and the β-cell function are affected before the onset of the mitochondrial diabetes caused by the m.3243A>G mutation.Impaired insulin sensitivity characterizes adult-onset diabetes and has been attributed to decreased insulin-stimulated glucose uptake in major metabolic tissues such as skeletal muscle, liver, and adipose tissue (1,2). It predicts diabetes strongly in subjects with high hereditary risk (3). Decreased glucose uptake in skeletal muscle is the major determinant of impaired insulin sensitivity, because skeletal muscle is the tissue that accounts for the majority of insulin-stimulated glucose uptake in diabetes and in nondiabetic subjects (4). Impaired insulin sensitivity has been correlated with decreased mitochondrial function and with decreased expression of genes involved in mitochondrial oxidative phosphorylation in skeletal muscle (5,6). Interestingly, similar findings in oxidative phosphorylation have recently been made in healthy insulin-resistant subjects with high hereditary predisposition for diabetes (7).In addition to the impaired insulin sensitivity, a gradual β-cell failure is pivotal to the onset of diabetes in adulthood (8,9). It is noteworthy that most gene variants associated with adult-onset diabetes influence the β-cell insulin secretion (10). Genes that contribute to mitochondrial oxidative phosphorylation are located both in the nuclear DNA and in the maternally inherited mitochondrial DNA (mtDNA) (11). Intrinsic or acquired causes that could impair oxidative phosphorylation in the mitochondrion have been proposed to impair both skeletal muscle insulin sensitivity and β-cell function (12,13).The involvement of mtDNA mutations in the hereditary forms of diabetes is evident (14,15). The mtDNA m.3243A>G mutation accounts for 1–2% of adult-onset diabetes, and it has been estimated that most carriers of this mutation develop diabetes during their adulthood (16). This renders the m.3243A>G mutation an interesting pathogenic model for decreased mitochondrial function in adult-onset diabetes. The m.3243A>G mutation is heteroplasmic, i.e., the mutant allele and the wild-type allele co-occur in mitochondria, and the proportion of mutated mtDNA varies across patients and tissues (11). The hetero-plasmy is known to modify the phenotype in patients with m.3243A>G (17), but previous studies on glucose metabolism have not included mutation heteroplasmy as a variable. Furthermore, such studies have been small, so that more than four subjects with m.3243A>G have been examined in only a few of them (18–24). Most of these studies have revealed defects in insulin secretion (18–21,24). Hyperinsulinemic clamp technique has been applied in only two previous studies on m.3243A>G subjects, but these studies could not identify peripheral insulin resistance as the primary pathogenic factor (18,19).The aims of this study were 1) to characterize insulin secretion and sensitivity in patients with a substantial m.3243A>G mutation load and in age-matched healthy subjects, and 2) by using this disease model, to further enlighten the role of mitochondria in the pathogenesis of diabetes. We assessed whole-body glucose uptake by using the hyperinsulinemic clamp technique together with regional measurements of muscle perfusion and glucose uptake by positron emission tomography (PET). Model-based analysis of β-cell function was carried out, and measurements were correlated with mutation heteroplasmy.     

Somatostatin secreted by islet delta-cells fulfills multiple roles as a paracrine regulator of islet function

OBJECTIVE— Somatostatin (SST) is secreted by islet δ-cells and by extraislet neuroendocrine cells. SST receptors have been identified on α- and β-cells, and exogenous SST inhibits insulin and glucagon secretion, consistent with a role for SST in regulating α- and β-cell function. However, the specific intraislet function of δ-cell SST remains uncertain. We have used Sst^−/− mice to investigate the role of δ-cell SST in the regulation of insulin and glucagon secretion in vitro and in vivo.RESEARCH DESIGN AND METHODS— Islet morphology was assessed by histological analysis. Hormone levels were measured by radioimmunoassay in control and Sst^−/− mice in vivo and from isolated islets in vitro.RESULTS— Islet size and organization did not differ between Sst^−/− and control islets, nor did islet glucagon or insulin content. Sst^−/− mice showed enhanced insulin and glucagon secretory responses in vivo. In vitro stimulus-induced insulin and glucagon secretion was enhanced from perifused Sst^−/− islets compared with control islets and was inhibited by exogenous SST in Sst^−/− but not control islets. No difference in the switch-off rate of glucose-stimulated insulin secretion was observed between genotypes, but the cholinergic agonist carbamylcholine enhanced glucose-induced insulin secretion to a lesser extent in Sst^−/− islets compared with controls. Glucose suppressed glucagon secretion from control but not Sst^−/− islets.CONCLUSIONS— We suggest that δ-cell SST exerts a tonic inhibitory influence on insulin and glucagon secretion, which may facilitate the islet response to cholinergic activation. In addition, δ-cell SST is implicated in the nutrient-induced suppression of glucagon secretion.Islets of Langerhans are heterogeneous cell aggregates containing β-, α-, δ-, and PP cells, which secrete insulin, glucagon, somatostatin (SST), and pancreatic polypeptide, respectively. The different cell types within the islet are affected by changes in the extracellular glucose concentration. Thus, elevations in circulating glucose stimulate insulin secretion from islet β-cells and inhibit glucagon secretion from α-cells as part of the reciprocal regulation of blood glucose by insulin and glucagon. SST secretion from islet δ-cells is also stimulated by increased extracellular glucose, although the threshold concentration for δ-cells to respond to glucose is lower than that for β-cells (1), and the ionic events in stimulus-response coupling differ between β- and δ-cells (2).Rodent islets have a defined architecture with a β-cell core surrounded by a mantle of non–β-cells (3), whereas human islets do not show such pronounced anatomical subdivisions (4). The anatomical organization of islets is important for their correct functioning, and there is much evidence to suggest that cell-cell interactions within islets are crucial for normal function (5–9). Interactions between islet β-cells have been investigated extensively, and we have previously demonstrated the importance of homotypic β-cell interactions in the regulation of insulin secretion (10,11). Similarly, there have been numerous studies of possible interactions between α- and β-cells within islets (9,12–15). Less attention has been paid to the possible intraislet roles of δ-cell–derived SST. δ-Cells comprise 5–10% of the islet endocrine cells (3), but the peptide hormone SST is also synthesized and secreted by neuroendocrine cells in the central nervous system and the gastrointestinal system, and the latter is the major contributor to circulating SST (16,17). SST often acts as an inhibitory regulator of endocrine systems, for example, as a hypothalamic factor to suppress growth hormone secretion from the anterior pituitary (18), or as a local inhibitor of the release of gastrointestinal peptide hormones (19). SST receptors (SSTR1–5) have been identified on both α- and β-cells (20), and exogenously administered SST or SST analogs inhibit glucose-induced insulin secretion and arginine-induced glucagon secretion both in vitro and in vivo (21–24). In addition, studies using SSTR-deficient mice (SSTR1, -2, or -5) revealed changes in both basal and stimulated insulin secretion (21,25,26). However, studies using SSTR knockout models are difficult to interpret because mouse islets express all five SSTRs (20) and reduced expression of one receptor subtype may be compensated for by the overexpression of another. Therefore, although current data are consistent with a negative regulatory role for SST in islet secretory function, it is unclear whether this effect can be ascribed to circulating SST or to locally released δ-cell SST.The study of δ-cell function within islets is complicated by the possibility of multiple heterotypic interactions between islet cell types in experiments using intact primary islets. Studies of islet SST have also been hampered by the lack of selective and potent SST receptor antagonists. To investigate the role of locally released δ-cell SST, we have therefore used a mouse model in which disruption of the SST gene produced a SST-deficient phenotype (27). Our results suggest that locally released δ-cell SST exerts a tonic inhibitory influence on insulin and glucagon secretion and that this inhibitory effect of δ-cell SST may be involved in facilitating the insulin secretory response to cholinergic activation and the nutrient-induced suppression of glucagon secretion.     

Regulation of calcium-permeable TRPV2 channel by insulin in pancreatic beta-cells

OBJECTIVE—Calcium-permeable cation channel TRPV2 is expressed in pancreatic β-cells. We investigated regulation and function of TRPV2 in β-cells.RESEARCH DESIGN AND METHODS—Translocation of TRPV2 was assessed in MIN6 cells and cultured mouse β-cells by transfecting TRPV2 fused to green fluorescent protein or TRPV2 containing c-Myc tag in the extracellular domain. Calcium entry was assessed by monitoring fura-2 fluorescence.RESULTS—In MIN6 cells, TRPV2 was observed mainly in cytoplasm in an unstimulated condition. Addition of exogenous insulin induced translocation and insertion of TRPV2 to the plasma membrane. Consistent with these observations, insulin increased calcium entry, which was inhibited by tranilast, an inhibitor of TRPV2, or by knockdown of TRPV2 using shRNA. A high concentration of glucose also induced translocation of TRPV2, which was blocked by nefedipine, diazoxide, and somatostatin, agents blocking glucose-induced insulin secretion. Knockdown of the insulin receptor attenuated insulin-induced translocation of TRPV2. Similarly, the effect of insulin on TRPV2 translocation was not observed in a β-cell line derived from islets obtained from a β-cell–specific insulin receptor knockout mouse. Knockdown of TRPV2 or addition of tranilast significantly inhibited insulin secretion induced by a high concentration of glucose. Likewise, cell growth induced by serum and glucose was inhibited by tranilast or by knockdown of TRPV2. Finally, insulin-induced translocation of TRPV2 was observed in cultured mouse β-cells, and knockdown of TRPV2 reduced insulin secretion induced by glucose.CONCLUSIONS—TRPV2 is regulated by insulin and is involved in the autocrine action of this hormone on β-cells.Insulin elicits pleiotropic actions in a variety of target cells and plays a pivotal role in regulating nutrient metabolism. Recent studies have revealed that the insulin signal is necessary to maintain the normal function of pancreatic β-cells. Thus, deletion of the insulin receptor (IR) in β-cells impairs insulin secretion and results in glucose intolerance (1). In β-cells of βIRKO mice, glucose-induced insulin secretion is reduced, which is accompanied by reduction of the expression of GLUT2 and glucokinase (1). However, insulin secretion induced by glyceraldehyde and KCl is also reduced in islets obtained from a βIRKO mouse (2), which cannot be explained simply by reduction of GLUT2 and/or glucokinase expression. Because addition of anti-insulin antibody immediately reduces insulin secretion from islets (3), it is likely that insulin modifies a molecule(s) involved in insulin secretion by a nongenomic mechanism. In accordance with these observations, knockdown of IR attenuates glucose-induced insulin secretion in MIN6 cells (4). In addition, postnatal β-cell growth is impaired in βIRKO mice. Consequently, the mechanism by which insulin maintains β-cell function is not totally known at present. It is thought that there must be a target molecule(s) of insulin that regulates secretion and possibly growth of β-cells.Transient receptor potential (TRP) (5) is a calcium-permeable channel expressed in Drosophila. A large number of mammalian homologues have been identified, and they regulate various cellular functions (6,7). Among them, calcium-permeable cation channels resembling the vanilloid receptor channel (TRPV1) (8) have common features and now are classified as members of the TRPV subfamily (9). The TRPV subfamily consists of six members, which function as cellular sensors responding to changes in the temperature, osmolarity, and mechanical stresses, and they are also regulated by various ligands (9).TRPV2 is regulated by heat, growth factors, and other ligands (10–13). An intriguing feature of TRPV2 is that its intracellular localization is changed by various stimulations. For example, IGF-I induces translocation of TRPV2 from an intracellular compartment to the plasma membrane (11). Regarding its expression, TRPV2 is abundantly expressed in neurons, neuroendocrine cells in the gastrointestinal tract, and blood cells such as macrophages (14). In the pancreas, TRPV2 is expressed in β-cells and ductal cells. In this regard, we previously reported that TRPV2 is expressed in an insulinoma cell line MIN6 (11). When MIN6 cells are cultured in a serum-free condition, immunoreactivity of TRPV2 is localized in an intracellular compartment. Addition of serum induces translocation of TRPV2 to the plasma membrane (11).In the present study, we further investigated regulation of TRPV2 in β-cells. Because these cells secrete insulin, and the mode of action of insulin resembles that of IGF-I, special attention was paid to the effect of insulin and insulin secretagogues on the localization of TRPV2. The results indicate that TRPV2 is regulated by insulin in an autocrine manner in β-cells. TRPV2 functions as an insulin-mediated regulator of calcium entry.     

Plasma fetuin-A levels and the risk of type 2 diabetes

OBJECTIVE—The liver-secreted protein fetuin-A induces insulin resistance in animals, and circulating fetuin-A is elevated in insulin resistance and fatty liver in humans. We investigated whether plasma fetuin-A levels predict the incidence of type 2 diabetes in a large prospective, population-based study.RESEARCH DESIGN AND METHODS—A case-cohort study within the European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam study comprising 27,548 subjects was designed. We randomly selected a subcohort of 2,500 individuals of whom 2,164 were diabetes free at baseline and had anamnestic, anthropometrical, and metabolic data for analysis. Of the 849 incident diabetic case subjects identified in the full cohort during 7 years of follow-up, 703 remained for analyses after similar exclusions.RESULTS—Plasma fetuin-A levels were positively associated with diabetes risk after adjustment for age (relative risk [RR] for extreme quintiles 1.75 [95% CI 1.32–2.31]; RR for 10 μg/ml 1.04 [1.03–1.06]). The association remained significant after adjustment for sex, BMI, waist circumference, and lifestyle risk factors (RR for 10 μg/ml 1.03 [1.01–1.06]). Adjustment for glucose, triglycerides, HDL cholesterol, A1C, γ-glutamyltransferase, or high-sensitivity C-reactive protein or mutual adjustment for these biomarkers did not appreciably change this result (RR for 10 μg/ml full adjusted model 1.05 [1.02–1.07]). Furthermore, fetuin-A was associated with increased diabetes risk particularly in individuals with elevated plasma glucose.CONCLUSIONS—Our data suggest that fetuin-A is an independent risk factor of type 2 diabetes.Type 2 diabetes represents a major global public health threat and, together with obesity, constitutes an important contributor to the predicted decline in life expectancy (1). The pathophysiology of type 2 diabetes is complex: In addition to impaired insulin secretion from β-cells, reduced insulin sensitivity was found to play a predominant role in the pathogenesis of the disease (2). Several circulating proteins have been shown to be involved in the regulation of insulin sensitivity such as adiponectin (3,4), retinol binding protein 4 (5,6), and fetuin-A (former name for the human protein α₂-Heremans-Schmid glycoprotein, AHSG). Fetuin-A is an endogenous inhibitor of the insulin-stimulated insulin receptor tyrosine kinase (7–9). Administration of fetuin-A to rodents inhibited insulin-stimulated tyrosine phosphorylation of the insulin receptor and insulin receptor substrate-1 in rat liver and skeletal muscle (7). In addition, fetuin-A knockout mice exhibited increased insulin sensitivity and were resistant to the adipogenic effect of a high-fat diet (10), supporting the hypothesis that fetuin-A is involved in the pathophysiology of insulin resistance in rodents.In agreement with these data, we and others have recently shown that high levels of circulating fetuin-A are associated with insulin resistance in humans (11,12), suggesting that fetuin-A may represent a novel mechanism involved in the pathophysiology of type 2 diabetes. In the present study, we investigated whether circulating fetuin-A predicted the incidence of type 2 diabetes, independently of established risk factors, in the large European Prospective Investigation into Cancer and Nutrition (EPIC)-Potsdam Study.     

Fructose-1,6-bisphosphatase overexpression in pancreatic beta-cells results in reduced insulin secretion: a new mechanism for fat-induced impairment of beta-cell function

OBJECTIVE—Fructose-1,6-bisphosphatase (FBPase) is a gluconeogenic enzyme that is upregulated in islets or pancreatic β-cell lines exposed to high fat. However, whether specific β-cell upregulation of FBPase can impair insulin secretory function is not known. The objective of this study therefore is to determine whether a specific increase in islet β-cell FBPase can result in reduced glucose-mediated insulin secretion.RESEARCH DESIGN AND METHODS—To test this hypothesis, we have generated three transgenic mouse lines overexpressing the human FBPase (huFBPase) gene specifically in pancreatic islet β-cells. In addition, to investigate the biochemical mechanism by which elevated FBPase affects insulin secretion, we made two pancreatic β-cell lines (MIN6) stably overexpressing huFBPase.RESULTS—FBPase transgenic mice showed reduced insulin secretion in response to an intravenous glucose bolus. Compared with the untransfected parental MIN6, FBPase-overexpressing cells showed a decreased cell proliferation rate and significantly depressed glucose-induced insulin secretion. These defects were associated with a decrease in the rate of glucose utilization, resulting in reduced cellular ATP levels.CONCLUSIONS—Taken together, these results suggest that upregulation of FBPase in pancreatic islet β-cells, as occurs in states of lipid oversupply and type 2 diabetes, contributes to insulin secretory dysfunction.Type 2 diabetes is characterized by a chronic elevation of plasma glucose concentration, causing complications such as retinopathy, neuropathy, and nephropathy and increasing the risk of cardiovascular disease and stroke. Although insulin resistance may be the initiating defect, hyperglycemia in type 2 diabetes results from a relative deficiency of circulating insulin (1). Progressive deterioration in β-cell function is likely to result from exposure to the diabetic milieu (i.e., hyperglycemia and hyperlipidemia), thus setting up a positive feedback loop in which hyperglycemia and/or hyperlipidemia impairs β-cell function, leading to further hyperglycemia (2–7).Chronic high fatty acid exposure results in increased basal and blunted glucose-mediated insulin secretion and reduced β-cell mass (2,5,8,9). This is associated with the pancreatic β-cells undergoing adaptive changes such that genes that are highly expressed under normal conditions, for example, insulin, PDX-1, and GLUT2, are underexpressed and genes that are poorly expressed in the pancreatic β-cells, such as hexokinase I, glucose-6-phosphatase, c-Myc, and acetate dehydrogenase, are shown to be upregulated (10,11).One of the genes that is upregulated in β-cell lines under conditions of high fatty acid exposure is fructose-1,6-bisphosphatase (FBPase) (10–12), a regulated enzyme in the gluconeogenic pathway that catalyzes the dephosphorylation of fructose-1,6-bisphosphate to fructose-6-phosphate. FBPase is abundant in the liver and the kidneys but is poorly expressed in the pancreatic β-cells under normal conditions. In addition, FBPase was upregulated fivefold in islets from the diabetes-susceptible obese BTBR mouse compared with the diabetes-resistant C57BL/6 mouse (13). We have previously shown that FBPase is upregulated in the liver of mice or rats fed a high-fat diet (14,15) and in the New Zealand Obese (NZO) mouse, an obese model of type 2 diabetes (14,16). We have recently demonstrated that transgenic mice with specific overexpression of FBPase in the liver displayed increased glycerol gluconeogenesis (17).From the abovementioned studies, it is evident that upregulation of FBPase is induced by fatty acids. However, whether an increase in FBPase alone can be detrimental to cellular function and in particular to β-cell insulin secretory rates has not been investigated.To determine whether an increase in FBPase impairs insulin secretion, we generated both transgenic mice and stably transfected pancreatic β-cell lines (MIN6) overexpressing the human FBPase (huFBPase) gene. We demonstrated that overexpression of FBPase in β-cells results in impaired glucose-stimulated insulin secretion, which is associated with decreased glucose metabolism, resulting in reduced cellular ATP levels and cell proliferation.     

Overexpression of Carnitine Palmitoyltransferase-1 in Skeletal Muscle Is Sufficient to Enhance Fatty Acid Oxidation and Improve High-Fat Diet�CInduced Insulin Resistance

OBJECTIVE—Skeletal muscle insulin resistance is associated with lipid accumulation, but whether insulin resistance is due to reduced or enhanced flux of long-chain fatty acids into the mitochondria is both controversial and unclear. We hypothesized that skeletal muscle–specific overexpression of the muscle isoform of carnitine palmitoyltransferase 1 (CPT1), the enzyme that controls the entry of long-chain fatty acyl CoA into mitochondria, would enhance rates of fatty acid oxidation and improve insulin action in muscle in high-fat diet insulin-resistant rats.RESEARCH DESIGN AND METHODS—Rats were fed a standard (chow) or high-fat diet for 4 weeks. After 3 weeks, in vivo electrotransfer was used to overexpress the muscle isoform of CPT1 in the distal hindlimb muscles (tibialis anterior and extensor digitorum longus [EDL]). Skeletal muscle insulin action was examined in vivo during a hyperinsulinemic-euglycemic clamp.RESULTS—In vivo electrotransfer produced a physiologically relevant increase of ∼20% in enzyme activity; and although the high-fat diet produced insulin resistance in the sham-treated muscle, insulin action was improved in the CPT1-overexpressing muscle. This improvement was associated with a reduction in triacylglycerol content, the membrane-to-cytosolic ratio of diacylglycerol, and protein kinase C θ activity. Importantly, overexpression of CPT1 did not affect markers of mitochondrial capacity or function, nor did it alter skeletal muscle acylcarnitine profiles irrespective of diet.CONCLUSIONS—Our data provide clear evidence that a physiological increase in the capacity of long-chain fatty acyl CoA entry into mitochondria is sufficient to ameliorate lipid-induced insulin resistance in muscle.The pathogenesis of insulin resistance is a well-investigated area of research, but the precise molecular mechanisms that lead to this disorder are not fully understood. Emerging evidence suggests that insulin resistance, at least in skeletal muscle, is caused by dysregulated signaling processes secondary to lipid accumulation (1–4). Although the increase in lipid content is manifested as an increase in triacylglycerol (TAG), it is likely that elevated TAG may only serve as a marker of dysfunctional muscle fatty acid metabolism and that accumulation of bioactive lipids such as diacylglycerol (DAG) and/or ceramide is actually responsible for the insulin resistance (2,3,5). DAG can activate several isoforms of protein kinase C (PKC), which can impair insulin signal transduction via serine phosphorylation of insulin receptor substrate (IRS)-1 (6,7). Ceramides can cause insulin resistance by preventing insulin-stimulated Akt serine phosphorylation and activation and translocation of Akt to its substrate (8,9). In addition, ceramide initiates inflammatory signaling pathways, leading to the activation of both c-jun NH₂-terminal kinase (JNK) and nuclear factor κB/inducer of κ kinase (10), which have been implicated in the development of insulin resistance (11–13).Several factors may contribute to increased lipid deposition in muscle. An increase in fatty acid uptake without any change in oxidation could lead to cytosolic lipid accumulation (14). Conversely, an impaired ability to utilize fat as a fuel source because of reduced activity of enzymes of oxidative metabolism and fatty acid utilization could also result in increased cytosolic lipids (15–17). Recently, the concept of defective fatty acid oxidation causing insulin resistance has been challenged. Muoio and colleagues (18) have suggested that the increased flux of long-chain fatty acids into the mitochondria is not accompanied by complete β-oxidation because of the inability of the tricarboxylic acid (TCA) cycle to cope with the increase in the demand on fatty acid metabolism. This leads to intramitochondrial metabolite accumulation, mitochondrial stress, and cellular insulin resistance (18). Thus, the role of fatty acid oxidation in regulating insulin sensitivity is controversial and mechanisms remain unresolved. Carnitine palmitoyltransferase 1 (CPT1) is a mitochondrial transmembrane enzyme thought to be rate limiting for long-chain fatty acid entry into the mitochondria for β-oxidation (16,19). Inhibition of CPT1 with the chemical etomoxir increases lipid deposition and exacerbates insulin resistance when animals are placed on a high-fat diet (20), whereas overexpression of CPT1 protects myotubes against lipid-induced insulin resistance (21,22), arguing that alterations in fatty acid flux into the mitochondria are critical in regulating lipid effects on insulin sensitivity. Thus, to test whether increasing the capacity for fatty acid flux into the mitochondria is, in itself, sufficient to increase fat oxidation and alter insulin action, we used an approach in which we selectively overexpressed the muscle isoform of CPT1 in skeletal muscle in vivo. The results show that a physiological increase in CPT1 activity is sufficient to improve insulin resistance caused by a high-fat diet, suggesting that entry of long-chain fatty acids into the mitochondria is more critical in the regulation of fatty acid oxidation than the downstream capacity of the β-oxidation and TCA cycle.