Mutations in the high-affinity sulfonylurea receptor (SUR)-1 cause one of the severe recessively inherited diffuse forms of congenital hyperinsulinism or, when associated with loss of heterozygosity, focal adenomatosis. We hypothesized that SUR1 mutations would render the beta-cell insensitive to sulfonylureas and to glucose. Stimulated insulin responses were compared among eight patients with diffuse hyperinsulinism (two mutations), six carrier parents, and ten normal adults. In the patients with diffuse hyperinsulinism, the acute insulin response to intravenous tolbutamide was absent and did not overlap with the responses seen in either adult group. There was positive, albeit significantly blunted, acute insulin response to intravenous dextrose in the patients with diffuse hyperinsulinism. Graded infusions of glucose, to raise and then lower plasma glucose concentrations over 4 h, caused similar rises in blood glucose but lower peak insulin levels in the hyperinsulinemic patients. Loss of acute insulin response to tolbutamide can identify children with diffuse SUR1 defects. The greater response to glucose than to tolbutamide indicates that ATP-sensitive potassium (KATP) channel-independent pathways are involved in glucose-mediated insulin release in patients with diffuse SUR1 defects. The diminished glucose responsiveness suggests that SUR1 mutations and lack of KATP channel activity may contribute to the late development of diabetes in patients with hyperinsulinism independently of subtotal pancreatectomy.

Prolonged exposure of isolated islets to supraphysiologic concentrations of palmitate decreases insulin gene expression in the presence of elevated glucose levels. This study was designed to determine whether or not this phenomenon is associated with a glucose-dependent increase in esterification of fatty acids into neutral lipids. Gene expression of sn-glycerol-3-phosphate acyltransferase (GPAT), diacylglycerol acyltransferase (DGAT), and hormone-sensitive lipase (HSL), three key enzymes of lipid metabolism, was detected in isolated rat islets. Their levels of expression were not affected after a 72-h exposure to elevated glucose and palmitate. To determine the effects of glucose on palmitate-induced neutral lipid synthesis, isolated rat islets were cultured for 72 h with trace amounts of [14C]palmitate with or without 0.5 mmol/l unlabeled palmitate, at 2.8 or 16.7 mmol/l glucose. Glucose increased incorporation of [14C]palmitate into complex lipids. Addition of exogenous palmitate directed lipid metabolism toward neutral lipid synthesis. As a result, neutral lipid mass was increased upon prolonged incubation with elevated palmitate only in the presence of high glucose. The ability of palmitate to increase neutral lipid synthesis in the presence of high glucose was concentration-dependent in HIT cells and was inversely correlated to insulin mRNA levels. 2-Bromopalmitate, an inhibitor of fatty acid mitochondrial beta-oxidation, reproduced the inhibitory effect of palmitate on insulin mRNA levels. In contrast, palmitate methyl ester, which is not metabolized, and the medium-chain fatty acid octanoate, which is readily oxidized, did not affect insulin gene expression, suggesting that fatty-acid inhibition of insulin gene expression requires activation of the esterification pathway. These results demonstrate that inhibition of insulin gene expression upon prolonged exposure of islets to palmitate is associated with a glucose-dependent increase in esterification of fatty acids into neutral lipids.

Successful intraportal islet transplantation normalizes glucose metabolism in diabetic humans. To date, full function is not routinely achieved after islet transplantation in humans, with most grafts being characterized by only partial function. Moreover, the duration of full function is variable and cannot be sufficiently predicted with available methods. In contrast, most grafts retain partial function for a long time. We hypothesized that partial function can restore normal protein and lipid metabolism in diabetic individuals. We studied 45 diabetic patients after islet transplantation. Labeled glucose and leucine were infused to assess whole-body glucose and protein turnover in 1) 6 type 1 diabetic patients with full function after intraportal islet transplantation (FF group; C-peptide > 0.6 nmol/l; daily insulin dosage 0.03 +/- 0.02 U x kg(-1) body wt x day(-1); fasting plasma glucose < 7.7 mmol/l; HbA1c < or = 6.5%), 2) 17 patients with partial function (PF group; C-peptide > 0.16 nmol/l; insulin dosage < 0.4 U x kg(-1) body wt x day(-1)), 3) 9 patients with no function (NF group; C-peptide < 0.16 nmol/l; insulin dosage > 0.4 U x kg(-1) body wt x day(-1)), and 4) 6 patients with chronic uveitis as control subjects (CU group). Hepatic albumin synthesis was assessed in an additional five PF and five healthy volunteers by means of a primed-continuous infusion of [3,3,3-2H3]leucine. The insulin requirement was 97% lower than pretransplant levels for the FF group and 57% lower than pretransplant levels for the PF group. In the basal state, the PF group had a plasma glucose concentration slightly higher than that of the FF (P = 0.249) and CU groups (P = 0.08), but was improved with respect to the NF group (P < 0.01). Plasma leucine (101.1 +/- 5.9 micromol/l) and branched-chain amino acids (337.6 +/- 16.6 micromol/l) were similar in the PF, FF, and CU groups, and significantly lower than in the NF group (P < 0.01). During insulin infusion, the metabolic clearance rate of glucose was defective in the NF group versus in the other groups (P < 0.01). Both the basal and insulin-stimulated proteolytic and proteosynthetic rates were comparable in the PF, FF, and CU groups, but significantly higher in the NF group (P = 0.05). In addition, the PF group had a normal hepatic albumin synthesis. Plasma free fatty acid concentrations in the PF and FF groups were similar to those of the CU group, but the NF group showed a reduced insulin-dependent suppression during the clamp. We concluded that the restoration of approximately 60% of endogenous insulin secretion is capable of normalizing the alterations of protein and lipid metabolism in type 1 diabetic kidney recipients, notwithstanding chronic immunosuppressive therapy. The results of the present study indicate that "success" of islet transplantation may be best defined by a number of metabolic criteria, not just glucose concentration/metabolism alone.

Apolipoprotein A-II (apoA-II) is the second most abundant protein in HDLs. Genetic studies in humans have provided evidence of linkage of the apoA-II gene locus to plasma free fatty acid (FFA) levels and to type 2 diabetes, and transgenic mice overexpressing mouse apoA-II have elevated levels of both FFA and triglycerides. We now show that apoA-II promotes insulin resistance and has diverse effects on fat homeostasis. ApoA-II transgenic mice have increased adipose mass and higher plasma leptin levels than C57BL/6J control mice. Fasting glucose levels were similar between apoA-II transgenic and control mice, but plasma insulin levels were elevated approximately twofold in the apoA-II transgenic mice. Compared with control mice, apoA-II transgenic mice exhibited a delay in plasma clearance of a glucose bolus. Adipose tissue isolated from fasted apoA-II transgenic mice exhibited a 50% decrease in triglyceride hydrolysis compared with adipose tissue from control mice. This is consistent with a normal response of adipose tissue to the increased insulin levels in the apoA-II transgenic mice and may partially explain the increased fat deposition. Skeletal muscle isolated from fasted apoA-II transgenic mice exhibited reduced uptake of 2-deoxyglucose compared with muscles isolated from control mice. Our observations indicate that a primary disturbance in lipoprotein metabolism can result in several traits associated with insulin resistance, consistent with the hypothesis that insulin resistance and type 2 diabetes can, under certain circumstances, be related primarily to altered lipid metabolism rather than glucose metabolism.

Evidence is accumulating that low levels of IGF-I play a role in the pathogenesis of type 2 diabetes and cardiovascular diseases. We examined the role of a genetic polymorphism in the promoter region of the IGF-I gene in relation to circulating IGF-I levels and growth measured as body height, and we studied the relationship of this polymorphism with type 2 diabetes and myocardial infarction. The relation between the IGF-I polymorphism and body height was assessed in a population-based sample of 900 subjects from the Rotterdam Study. Within each genotype stratum, 50 subjects were randomly selected for a study of the relation of this polymorphism with serum IGF-I levels. To assess the risk for type 2 diabetes, we studied 220 patients and 596 normoglycemic control subjects. For myocardial infarction, 477 patients with evidence of myocardial infarction on electrocardiogram and 808 control subjects were studied. A 192-bp allele was present in 88% of the population, suggesting that this is the wild-type allele from which all other alleles originated. Body height was, on average, 2.7 cm lower (95% CI for difference -4.6 to -0.8 cm, P = 0.004), and serum IGF-I concentrations were 18% lower (95% CI for difference -6.0 to -1.3 mmol/l, P = 0.003) in subjects who did not carry the 192-bp allele. In noncarriers of the 192-bp allele, an increased relative risk for type 2 diabetes (1.7 [95% CI 1.1-2.7]) and for myocardial infarction (1.7 [95% CI 1.1-2.5]) was found. In patients with type 2 diabetes, the relative risk for myocardial infarction in subjects without the 192-bp allele was 3.4 (95% CI 1.1-11.3). Our study suggests that a genetically determined exposure to relatively low IGF-I levels is associated with an increased risk for type 2 diabetes and myocardial infarction.

Impaired glucose effectiveness (i.e., a diminished ability of glucose per se to facilitate its own metabolism), increased gluconeogenesis, and endogenous glucose release are, together with insulin resistance and beta-cell abnormalities, established features of type 2 diabetes. To explore aspects of the pathophysiology behind type 2 diabetes, we assessed in a group of healthy people prone to develop type 2 diabetes (n = 23), namely first-degree relatives of type 2 diabetic patients (FDR), 1) endogenous glucose release and fasting gluconeogenesis measured using the 2H2O technique and 2) glucose effectiveness. The FDR group was insulin resistant when compared with an age-, sex-, and BMI-matched control group without a family history of type 2 diabetes (n = 14) (M value, clamp: 6.07 +/- 0.48 vs. 8.06 +/- 0.69 mg x kg(-1) lean body weight (lbw) x min(-1); P = 0.02). Fasting rates of gluconeogenesis (1.28 +/- 0.06 vs. 1.41 +/- 0.07 mg x kg(-1) lbw x min(-1); FDR vs. control subjects, P = 0.18) did not differ in the two groups and accounted for 53 +/- 2 and 60 +/- 3% of total endogenous glucose release. Glucose effectiveness was examined using a combined somatostatin and insulin infusion (0.17 vs. 0.14 mU x kg(-1) x min(-1), FDR vs. control subjects), the latter replacing serum insulin at near baseline levels. In addition, a 360-min labeled glucose infusion was given to simulate a prandial glucose profile. After glucose infusion, the integrated plasma glucose response above baseline (1,817 +/- 94 vs. 1,789 +/- 141 mmol/l per 6 h), the ability of glucose to simulate its own uptake (1.50 +/- 0.13 vs. 1.32 +/- 0.16 ml x kg(-1) lbw x min(-1)), and the ability of glucose per se to suppress endogenous glucose release did not differ between the FDR and control group. In conclusion, in contrast to overt type 2 diabetic patients, healthy people at high risk of developing type 2 diabetes are characterized by normal glucose effectiveness at near-basal insulinemia and normal fasting rates of gluconeogenesis.

To examine the mechanism by which metformin lowers endogenous glucose production in type 2 diabetic patients, we studied seven type 2 diabetic subjects, with fasting hyperglycemia (15.5 +/- 1.3 mmol/l), before and after 3 months of metformin treatment. Seven healthy subjects, matched for sex, age, and BMI, served as control subjects. Rates of net hepatic glycogenolysis, estimated by 13C nuclear magnetic resonance spectroscopy, were combined with estimates of contributions to glucose production of gluconeogenesis and glycogenolysis, measured by labeling of blood glucose by 2H from ingested 2H2O. Glucose production was measured using [6,6-2H2]glucose. The rate of glucose production was twice as high in the diabetic subjects as in control subjects (0.70 +/- 0.05 vs. 0.36 +/- 0.03 mmol x m(-2) min(-1), P < 0.0001). Metformin reduced that rate by 24% (to 0.53 +/- 0.03 mmol x m(-2) x min(-1), P = 0.0009) and fasting plasma glucose concentration by 30% (to 10.8 +/- 0.9 mmol/l, P = 0.0002). The rate of gluconeogenesis was three times higher in the diabetic subjects than in the control subjects (0.59 +/- 0.03 vs. 0.18 +/- 0.03 mmol x m(-2) min(-1) and metformin reduced that rate by 36% (to 0.38 +/- 0.03 mmol x m(-2) x min(-1), P = 0.01). By the 2H2O method, there was a twofold increase in rates of gluconeogenesis in diabetic subjects (0.42 +/- 0.04 mmol m(-2) x min(-1), which decreased by 33% after metformin treatment (0.28 +/- 0.03 mmol x m(-2) x min(-1), P = 0.0002). There was no glycogen cycling in the control subjects, but in the diabetic subjects, glycogen cycling contributed to 25% of glucose production and explains the differences between the two methods used. In conclusion, patients with poorly controlled type 2 diabetes have increased rates of endogenous glucose production, which can be attributed to increased rates of gluconeogenesis. Metformin lowered the rate of glucose production in these patients through a reduction in gluconeogenesis.

In response to hypoglycemia, healthy individuals rapidly antagonize insulin action on glucose and lipid metabolism, but the effects on protein metabolism are unclear. Because amino acids are an important substrate for gluconeogenesis and a fuel alternative to glucose for oxidation, we evaluated whether hypoglycemia antagonizes the hypoaminoacidemic and the antiproteolytic effects of insulin and changes the de novo synthesis of glutamine, a gluconeogenic amino acid. To this purpose, in 7 healthy subjects, we performed 2 studies, 3.5 h each, at similar insulin but different glucose concentrations (i.e., 4.9 +/- 0.1 mmol/l [euglycemic clamp] or 2.9 +/- 0.2 mmol/l [hypoglycemic clamp]). As expected, hypoglycemia antagonized the insulin suppression of glucose production achieved in euglycemia (from 21 +/- 15 to 116 +/- 12% of basal, P < 0.001), the stimulation of glucose uptake (from 207 +/- 28 to 103 +/- 7% of basal, P < 0.01) and the suppression of circulating free fatty acids (from 30 +/- 5 to 80 +/- 17% of basal, P < 0.001). In contrast, hypoglycemia increased the insulin suppression of circulating leucine (from 63 +/- 1 to 46 +/- 2% of basal, P < 0.001) and phenylalanine (from 79 +/- 3 to 64 +/- 3% of basal, P < 0.001) concentrations. Hypoglycemia did not change the insulin suppression of proteolysis (from 79 +/- 2 to 82 +/- 4% of basal, P < 0.001). However, hypoglycemia doubled the insulin suppression of the glutamine concentrations (from 84 +/- 3 to 63 +/- 3% of basal, P < 0.01) in the absence of significant changes in the glutamine rate of appearance, but it also caused an imbalance between glutamine uptake and release. This study demonstrates that successful counterregulation does not affect proteolysis. Moreover, it does not increase the availability of circulating amino acids by de novo synthesis. In contrast, despite the lower concentration of circulating amino acids, hypoglycemia increases the uptake of glutamine that can be used for gluconeogenesis and as a fuel alternative to glucose.

We determined the beta-cell replicative rate, beta-cell apoptosis, cross-sectional beta-cell area, and pancreatic beta-cell mass throughout the entire postweaning lifespan (months 1, 3, 7, 10, 15, and 20) of Lewis rats. Beta-cell replication was progressively reduced in the initial months of life but remained stable after month 7 (month 1, 0.99 +/- 0.10%; month 3, 0.24 +/- 0.04%; month 7, 0.12 +/- 0.02%; month 10, 0.14 +/- 0.02%; month 15, 0.10 +/- 0.03%; month 20, 0.13 +/- 0.03%; analysis of variance [ANOVA], P < 0.001). Beta-cell apoptosis was low and did not change significantly from month 1 to 20 of life. Cross-sectional area of individual beta-cells increased progressively in the initial months, remained stable from month 7 to 15, and increased again on month 20. The estimated number of beta-cells per pancreas, calculated as the ratio of total beta-cell mass to individual beta-cell mass, tripled from month 1 to 7 but did not change significantly thereafter. Beta-cell mass increased approximately 8 times from month 1 to 20 (month 1, 2.04 +/- 0.28 mg; month 20, 15.5 +/- 2.32 mg; ANOVA, P < 0.001) and showed a strong and significant linear correlation with body weight (r = 0.98, P < 0.001). In summary, we have shown that beta-cell replication was maintained throughout the lifespan in normal rats, clearly establishing that the beta-cell birth rate does not fall to 0, even in very old rats. Beta-cell mass increased throughout the lifespan, closely matching the increment in total body weight at any time point. This increment was selective for beta-cells, since the growth of the endocrine non-beta-cell mass was limited to the initial months of life. Both beta-cell hypertrophy and hyperplasia contributed to increased beta-cell mass in young animals, but only beta-cell hypertrophy was responsible for the increased beta-cell mass found in old animals. This study provides a global perspective for understanding the dynamics of beta-cell mass in young, adult, and aged animals.

Glucagon-like peptide 1 (GLP-1), a hormonal activator of adenyl cyclase, stimulates insulin gene transcription, an effect mediated by the cAMP response element (CRE) of the rat insulin I gene promoter (RIP1). Here we demonstrate that the signaling mechanism underlying stimulatory effects of GLP-1 on insulin gene transcription results from protein kinase A (PKA)-independent activation of the RIP1 CRE. Although GLP-1 stimulates cAMP production in rat INS-1 insulinoma cells, we find accompanying activation of a -410-bp RIP1 luciferase construct (-410RIP1-LUC) to exist independently of this second messenger. GLP-1 produced a dose-dependent stimulation of -410RIP1-LUC (EC50 0.43 nmol/l), an effect reproduced by the GLP-1 receptor agonist exendin-4 and abolished by the antagonist exendin(9-39). Activation of RIP1 by GLP-1 was not affected by cotransfection with dominant-negative Gs alpha, was not blocked by cAMP antagonist Rp-cAMPS, and was insensitive to PKA antagonist H-89. Truncation of -410RIP1-LUC to generate -307-, -206-, and -166-bp constructs revealed 2 segments of RIP1 targeted by GLP-1. The first segment, not regulated by forskolin, was located between -410 and -307 bp of the promoter. The second segment, regulated by both GLP-1 and forskolin, included the CRE and was located between -206 and -166 bp. Consistent with these observations, stimulatory effects of GLP-1 at RIP1 were reduced after introduction of delta-182 and delta-183/180 inactivating deletions at the CRE. The action of GLP-1 at -410RIP1-LUC was also reduced by cotransfection with A-CREB, a genetically engineered isoform of the CRE binding protein CREB, which dimerizes with and prevents binding of basic-region-leucine-zipper (bZIP) transcription factors to the CRE. In contrast, the action of GLP-1 at the CRE was not blocked by cotransfection with M1-CREB, an isoform that lacks a consensus serine residue serving as substrate for PKA-mediated phosphorylation. On the basis of these studies, it is proposed that PKA-independent stimulatory actions of GLP-1 at RIP1 are mediated by bZIP transcription factors related in structure but not identical to CREB.

Membrane-bound vascular cell adhesion molecule 1 (VCAM-1) allows the tethering and rolling of monocytes and lymphocytes as well as firm attachment and transendothelial migration of leukocytes. Soluble forms of VCAM (sVCAM-1) may serve as monitors of increased expression of membrane-bound VCAM-1 and thus may reflect progressive formation of atherosclerotic lesions. Levels of sVCAM-1 have been found to be increased among type 2 diabetic as compared with nondiabetic subjects. To study the association of plasma sVCAM-1 concentration and risk of cardiovascular and all-cause mortality among nondiabetic and diabetic subjects, we investigated an age-, sex-, and glucose-tolerance-stratified sample (n = 631) of a population-based cohort aged 50-75 years that was followed prospectively. Plasma levels of sVCAM-1 were determined in frozen -70 degrees C baseline samples. After 7.4 years (mean) of follow-up, 107 (17%) subjects had died (42 of cardiovascular causes). In the entire group, increased sVCAM-1 levels were significantly associated with increased risk of cardiovascular mortality (relative risks [RRs] per 100 ng/ml sVCAM-1 increase, 1.10 [1.05-1.15] after adjustment for age, sex, and glucose tolerance status). This RR was somewhat diminished by further adjustment for the presence of hypertension and cardiovascular disease; levels of total, HDL, and LDL cholesterol and homocysteine; the presence of microalbuminuria (a putative marker of endothelial dysfunction); levels of von Willebrand factor (a marker of endothelial dysfunction) and C-reactive protein (a marker of low-grade inflammation); and estimates of glomerular filtration rate. However, the RR remained statistically significant. The RR among type 2 diabetic subjects was 1.13 (1.07-1.20) per 100 ng/ml sVCAM-1 increase after adjustment for age and sex, which was somewhat higher but not significantly different from the RR in nondiabetic subjects (P value for interaction term, 0.12). Further adjustment for other risk factors gave similar results. In conclusion, levels of sVCAM-1 are independently associated with the risk of cardiovascular mortality in type 2 diabetic subjects and therefore might be useful for identifying subjects at increased cardiovascular risk. Increased plasma sVCAM-1 levels may reflect progressive formation of atherosclerotic lesions, or sVCAM-1 itself may have bioactive properties related to cardiovascular risk. Our data, however, argue against the hypotheses of sVCAM-1 levels simply being a marker of endothelial dysfunction, of low-grade inflammation, or of an impaired renal function.

Hypothalamic melanocortins are among several neuropeptides strongly implicated in the control of food intake. Agonists for melanocortin 4 (MC-4) receptors such as alpha-melanocyte-stimulating hormone (alpha-MSH), a product of proopiomelanocortin (POMC), reduce food intake, whereas hypothalamic agouti-related protein (AgRP) is a MC-4 receptor antagonist that increases food intake. To investigate whether reduced melanocortin signaling contributes to hyperphagia induced by uncontrolled diabetes, male Sprague-Dawley rats were studied 7 days after administration of streptozotocin (STZ) or vehicle. In addition, we wished to determine the effect of diabetes on muscle uncoupling protein 3 (UCP-3), a potential regulator of muscle energy metabolism. STZ diabetic rats were markedly hyperglycemic (31.3 +/- 1.0 mmol/l; P < 0.005) compared with nondiabetic controls (9.3 +/- 0.2 mmol/l). Insulin treatment partially corrected the hyperglycemia (18.8 +/- 2.5 mol/l; P < 0.005). Plasma leptin was markedly reduced in STZ diabetic rats (0.4 +/- 0.1 ng/ml; P < 0.005) compared with controls (3.0 +/- 0.4 ng/ml), an effect that was also partially reversed by insulin treatment (1.8 +/- 0.3 ng/ml). Untreated diabetic rats were hyperphagic, consuming 40% more food (48 +/- 1 g/day; P < 0.005) than controls (34 +/- 1 g/day). Hyperphagia was prevented by insulin treatment (32 +/- 2 g/day). In untreated diabetic rats, hypothalamic POMC mRNA expression (measured by in situ hybridization) was reduced by 80% (P < 0.005), whereas AgRP mRNA levels were increased by 60% (P < 0.01), suggesting a marked decrease of hypothalamic melanocortin signaling. The change in POMC, but not in AgRP, mRNA levels was partially reversed by insulin treatment. By comparison, the effects of diabetes to increase hypothalamic neuropeptide Y (NPY) expression and to decrease corticotropin-releasing hormone (CRH) expression were normalized by insulin treatment, whereas the expression of mRNA encoding the long form of the leptin receptor in the arcuate nucleus was unaltered by diabetes or insulin treatment. UCP-3 mRNA expression in gastrocnemius muscle from diabetic rats was increased fourfold (P < 0.005), and the increase was prevented by insulin treatment. The effect of uncontrolled diabetes to decrease POMC, while increasing AgRP gene expression, suggests that reduced hypothalamic melanocortin signaling, along with increased NPY and decreased CRH signaling, could contribute to diabetic hyperphagia. These responses, in concert with increased muscle UCP-3 expression, may also contribute to the catabolic effects of uncontrolled diabetes on fuel metabolism in peripheral tissues.

Interaction between the different types of cells within the islet of Langerhans is vital for adequate control of insulin release. Once insulin secretion becomes defective, as in type 2 diabetes, the most useful drugs to increase insulin release are sulfonylureas. It is well-known that sulfonylureas block K(ATP) channels, which results in depolarization of the membrane that provokes calcium influx and increases intracellular calcium concentration ([Ca2+]i), which thereby triggers insulin secretion. The sulfonamide diazoxide produces the opposite effect: it activates K(ATP) channels, resulting in a decreased insulin secretion. Despite such evidence, little is known about the effect of sulfonylureas and sulfonamides in non-beta-cells of the islet of Langerhans. In this article, we describe the effects of tolbutamide and diazoxide on [Ca2+]i in alpha-, beta-, and delta-cells within intact islets of Langerhans. Tolbutamide elicits an increase in [Ca2+li in beta- and delta-cells, regardless of glucose concentrations. Remarkably, tolbutamide is without effect in alpha-cells. When diazoxide is applied, glucose-induced [Ca2+]i oscillations in beta- and delta-cells are abolished, whereas [Ca2+]i oscillations in alpha-cells remain unaltered. Furthermore, the existence of sulfonylurea receptors is demonstrated in beta-cells but not in alpha-cells by using binding of glybenclamide-4,4-difluoro-4-bora-3a,4a-diaza-s-indacene (BODIPY) combined with immunostaining for insulin and glucagon.

Increased beta-cell sensitivity to glucose precedes the loss of glucose-induced insulin secretion in diabetic animals. Changes at the level of beta-cell glucose sensor have been described in these situations, but it is not clear whether they fully account for the increased insulin secretion. Using a euglycemic-normolipidemic 60% pancreatectomized (60%-Px) mouse model, we have studied the ionic mechanisms responsible for increased beta-cell glucose sensitivity. Two weeks after Px (Px14 group), Px mice maintained normoglycemia with a reduced beta-cell mass (0.88 +/- 0.18 mg) compared with control mice (1.41 +/- 0.21 mg). At this stage, the dose-response curve for glucose-induced insulin release showed a significant displacement to the left (P < 0.001). Islets from the Px14 group showed oscillatory electrical activity and cytosolic Ca2+ ([Ca2+]i) oscillations in response to glucose concentrations of 5.6 mmol/l compared with islets from the control group at 11.1 mmol/l. All the above changes were fully reversible both in vitro (after 48-h culture of islets from the Px14 group) and in vivo (after regeneration of beta-cell mass in islets studied 60 days after Px). No significant differences in the input resistance and ATP inhibition of ATP-sensitive K+ (K(ATP)) channels were found between beta-cells from the Px14 and control groups. The dose-response curve for glucose-induced MTT (C,N-diphenyl-N''-4,5-dimethyl thiazol 2 yl tetrazolium bromide) reduction showed a significant displacement to the left in islets from the Px14 group (P < 0.001). These results indicate that increased glucose sensitivity in terms of insulin secretion and Ca2+ signaling was not due to intrinsic modifications of K(ATP) channel properties, and suggest that the changes are most likely to be found in the glucose metabolism.

By virtue of its potential effects on rates of energy expenditure, uncoupling protein 3 (UCP3) is an obesity candidate gene. We identified nine sequence variants in UCP3, including Val9Met, Val102Ile, Arg282Cys, and a splice site mutation in the intron between exons 6 and 7. The splice mutation results in an inability to synthesize mRNA for the long isoform (UCP3L) of UCP3. Linkage (sib pair), association, and transmission disequilibrium testing studies on 942 African-Americans did not suggest a significant effect of UCP3 on body composition in this group. In vastus lateralis skeletal muscle of individuals homozygous for the splice mutation, no UCP3L mRNA was detectable; the short isoform (UCP3S) was present in an increased amount. In this muscle, we detected no alterations of in vitro mitochondrial coupling activity, mitochondrial respiratory enzyme activity, or systemic oxygen consumption or respiratory quotient at rest or during exercise. These genetic and physiologic data suggest the following possibilities: UCP3S has uncoupling capabilities equivalent to UCP3L; other UCPs may compensate for a deficiency of bioactive UCP3L; UCP3L does not function primarily as a mitochondrial uncoupling protein.

The adipocyte hormone leptin reduces food intake in normal animals. During uncontrolled type 1 diabetes, plasma leptin levels fall, whereas food intake increases. To test the hypothesis that low leptin levels contribute to diabetic hyperphagia, we investigated the effect on food intake of replacement of leptin at basal plasma concentrations for 7 days in Long-Evans rats with uncontrolled diabetes induced by streptozotocin (STZ). One group of STZ diabetic rats received saline (STZ + Sal) (n = 11), while the other group (STZ + Lep) (n = 15) received a subcutaneous infusion of recombinant rat leptin (100 microg x kg(-1) x day(-1)) via osmotic minipumps. A nondiabetic control group (Con) (n = 11) received saline only. In the STZ + Sal group, plasma leptin levels decreased by 75% (P < 0.05) from 2.4+/-0.5 on the day before STZ/citrate buffer vehicle (Veh) injection (day 0) to 0.6+/-0.2 ng/ml on day 7. In contrast, plasma leptin levels on days 3-7 were comparable to pretreatment values in both the STZ + Lep group (day 0: 2.6+/-0.4 vs. day 7: 2.5+/-0.3 ng/ml, NS) and the Con group (day 0: 3.8+/-0.4 vs. day 7: 2.9+/-1.0 ng/ml, NS). In the STZ + Sal group, daily food intake increased gradually to values 43% above basal by day 7 (day 0: 24+/-2 to day 7: 33+/-3 g, P < 0.05), whereas food intake did not increase in either the STZ + Lep group (day 0: 24+/-1 vs. day 7: 21+/-2 g, NS), or the Con group (day 0: 23+/-1 vs. day 7: 23+/-2 g). Plasma glucose levels exceeded nondiabetic control values (7.7+/-0.2 mmol/l) in both diabetic groups, but were lower in the STZ + Lep group (17.2+/-1.8 mmol/l) than in the STZ + Sal group (24.3+/-1.1 mmol/l, P < 0.05). To determine if sensitivity to leptin-induced anorexia was affected by STZ treatment, a second experiment was performed in which the effect of intracerebroventricular leptin injection (at doses of 0.35, 1.0, or 3.5 microg) on food intake was measured 10 days after STZ or Veh treatment. Leptin suppressed both 4- and 24-h food intake in the two groups to an equal extent at every dose (by 15, 22, and 35%, respectively). These findings support the hypothesis that the effect of uncontrolled diabetes to lower leptin levels contributes to diabetic hyperphagia and that this effect is not due to altered leptin sensitivity.

The Epidemiology of Diabetes Interventions and Complications (EDIC) is a multicenter longitudinal observational study of the Diabetes Control and Complications Trial (DCCT) cohort. One of the major objectives of EDIC is to study the development and progression of atherosclerotic cardiovascular disease in type 1 diabetes. In this study, we evaluated the role of cardiovascular risk factors and antecedent therapy in the DCCT on carotid intima-media wall thickness (IMT) in type 1 diabetes. At approximately 18 months after the end of the DCCT, high-resolution B-mode ultrasonography was used to assess the carotid arteries of 1,325 patients with type 1 diabetes, 19-51 years of age, with duration of diabetes ranging from 6.3 to 26.1 years. An age- and sex-matched nondiabetic population (n = 153) was studied with the same protocol. The ultrasound protocol was carried out in 28 EDIC clinics by centrally trained and certified sonographers using one of three scanning systems. Determination of IMT from videotaped images was performed by a single reader at the Central Ultrasound Reading Unit. Univariate associations with greater IMT were strongest for older age and longer diabetes duration, greater waist-to-hip ratio (men only), higher blood pressure, higher LDL cholesterol, and smoking. The DCCT therapy group (intensive versus conventional) and HbA1c, measured at the time of the ultrasound or the mean HbA1c during the DCCT, were not significantly related to IMT. Multivariate analyses suggest that age, height, smoking, and BMI were the major predictors of common carotid IMT, whereas age, smoking, and LDL cholesterol predicted internal carotid IMT. There were significant differences between the IMT values of the internal carotid artery in the EDIC male cohort and similarly aged male nondiabetic control subjects. There were no significant differences between the IMT values in the EDIC female cohort and similarly aged female nondiabetic control subjects. At this point in the planned 10-year follow-up of the DCCT cohort, neither intensive therapy nor HbA1c level appears to influence the early signs of atherosclerosis. Traditional risk factors, including age, smoking, and LDL cholesterol, were related to IMT. As the cohort is only now entering the age interval during which rapid progression and clinical expression of atherosclerosis are expected, further follow-up will help to determine the role of hyperglycemia, and its interaction with other risk factors, on the development of atherosclerosis.

Leptin induces weight loss in rodents via its effects on food intake and energy expenditure. High-fat diets induce weight gain, but the mechanism is not well understood. Previous studies have not found an effect of dietary fat content on fasting leptin. There is a nocturnal increase of leptin, however, which is related to insulin responses to meals. We have reported that adipocyte glucose utilization is involved in insulin-induced leptin secretion in vitro. Accordingly, high-fat, low-carbohydrate (HF/LC) meals, which induce smaller insulin and glucose responses, would produce lower leptin concentrations than low-fat, high-carbohydrate (LF/HC) meals. Blood samples were collected every 30-60 min for 24 h from 19 normal-weight (BMI, 24.2 +/- 0.7 kg/m2; percent body fat = 31 +/- 1%) women on 2 days (10 days apart) during which the subjects were randomized to consume three isocaloric 730-kcal meals containing either 60/20 or 20/60% of energy as fat/carbohydrate. Overall insulin and glycemic responses (24-h area under the curve [AUC]) were reduced by 55 and 61%, respectively, on the HF/LC day (P < 0.0001). During LF/HC feeding, there were larger increases of leptin 4-6 h after breakfast (38 +/- 7%, P < 0.001) and lunch (78 +/- 14%, P < 0.001) than after HF/LC meals (both P < 0.02). During LF/HC feeding, leptin increased from a morning baseline of 10.7 +/- 1.6 ng/ml to a nocturnal peak of 21.3 +/- 1.3 ng/ml (change, 10.6 +/- 1.3 ng/ml; percent change, 123 +/- 16%; P < 0.0001). The amplitudes of the nocturnal rise of leptin and the 24-h leptin AUC were 21 +/- 8% (P < 0.005) and 38 +/- 12% (P < 0.0025) larger, respectively, on the LF/HC day. In summary, consumption of HF/LC meals results in lowered 24-h circulating leptin concentrations. This result may be a consequence of decreased adipocyte glucose metabolism. Decreases of 24-h circulating leptin could contribute to the weight gain during consumption of high-fat diets.

Myotonic dystrophy (MyD) is a multisystem autosomal dominant disorder associated with progressive muscle wasting and weakness. The striking metabolic abnormality in MyD is insulin resistance. The mechanism by which target tissues are insensitive to insulin action remains uncertain. In a recent study, plasma soluble tumor necrosis factor receptor (sTNFR)2 levels were found to be associated with muscle tissue mass and insulin resistance. Given these associations, we speculated that disorders of the muscle cell membrane could lead simultaneously to insulin insensitivity and sTNFR2 leakage in MyD. To test this hypothesis, we measured the levels of circulating sTNFR1 and sTNFR2 and insulin resistance in MyD patients. We studied 22 MyD patients and 24 age-, BMI-, and fat mass-matched control subjects. Both MyD men and women showed higher plasma insulin levels in the presence of comparable glucose concentrations than did control subjects. sTNFR2, but not sTNFR1, levels were approximately 1.5-fold higher in MyD patients. In parallel with these findings, the fasting insulin resistance index (FIRI) was also higher in MyD patients. In fact, in the whole population, fasting insulin and FIRI strongly correlated with sTNFR2 in both men (r = 0.77 and r = 0.81, P<0.0001, respectively) and women (r = 0.67 and r = 0.64, P = 0.001, respectively). sTNFR2 levels were also associated with the insulin sensitivity index (S(I)), calculated from an oral glucose tolerance test (OGTT) according to the method by Cederholm and Wibell (r = -0.43, P = 0.006). We constructed a multiple linear regression to predict FIRI, with BMI, waist-to-hip ratio, and sTNFR2 as independent variables. In this model, both BMI (P = 0.0014) and sTNFR2 (P = 0.0048) levels contributed independently to 46% of the variance of FIRI. In another model, in which FIRI was substituted for S(I) from the OGTT, both BMI (P = 0.0001) and sTNFR2 (P = 0.04) levels contributed independently to 48% of the variance of S(I) from the OGTT. Plasma cholesterol and triglyceride concentrations were significantly increased in MyD patients. sTNFR1 and sTNFR2 levels were found to be strongly associated with plasma cholesterol, LDL cholesterol, and triglycerides. sTNFR1 and sTNFR2 also correlated with serum creatine kinase activity in MyD patients (r = 0.57, P = 0.006; r = 0.75, P<0.0001, respectively). In conclusion, here we describe, for the first time to our knowledge, a relationship between insulin action and plasma sTNFR2 concentration in MyD patients. We have also found increased concentrations of plasma triglycerides and cholesterol levels in parallel with sTNFR1 and sTNFR2 concentrations in MyD patients. We speculate that the latter associations are dependent on, and secondary to, increased tumor necrosis factor (TNF)-alpha action. Whether TNF action is implicated in the pathogenesis of MyD or is a simple marker of disease activity awaits further studies.

Leptin administration has been shown to increase renal, adrenal, and lumbar sympathetic nerve activity. However, this generalized sympathoexcitatory activity is not always followed by an increase in arterial pressure. The present study tested the hypothesis that leptin induces a release of nitric oxide (NO) that opposes the pressor effect of sympathoexcitation. The effect of intravenous administration of leptin (10, 100, and 1,000 microg/kg body wt) or vehicle on blood pressure (BP), heart rate (HR), and serum nitrite/nitrate concentrations of anesthetized Wistar rats was examined. At 90 min after injection, the three leptin doses tested increased serum NO concentrations 20.5, 33.1, and 89.5%, respectively (P < 0.001 vs. baseline). The effect of leptin on NO concentrations was significantly dose-dependent on linear trend testing (P = 0.0001). In contrast, leptin did not change serum nitrite/nitrate concentrations of fa/fa rats. Leptin administration to Wistar rats under NO synthesis inhibition (N(omega)-nitro-L-arginine methyl ester [L-NAME]) produced a statistically significant increase (P < 0.05) in both systolic BP and mean arterial pressure as well as in HR (P < 0.01). Injection of leptin into rats with pharmacologically induced ganglionic blockade (chlorisondamine) was followed by a decrease in BP and HR to values significantly lower (P < 0.01) than those observed with chlorisondamine treatment alone. The leptin-induced hypotension observed in the setting of ganglionic blockade was blocked by L-NAME. These findings raise the possibility that the leptin-induced release of NO may contribute to the homeostasis of BP.