Association of 18 Confirmed Susceptibility Loci for Type 2 Diabetes With Indices of Insulin Release, Proinsulin Conversion, and Insulin Sensitivity in 5,327 Nondiabetic Finnish Men

OBJECTIVE

We investigated the effects of 18 confirmed type 2 diabetes risk single nucleotide polymorphisms (SNPs) on insulin sensitivity, insulin secretion, and conversion of proinsulin to insulin.

RESEARCH DESIGN AND METHODS

A total of 5,327 nondiabetic men (age 58 ± 7 years, BMI 27.0 ± 3.8 kg/m²) from a large population-based cohort were included. Oral glucose tolerance tests and genotyping of SNPs in or near PPARG, KCNJ11, TCF7L2, SLC30A8, HHEX, LOC387761, CDKN2B, IGF2BP2, CDKAL1, HNF1B, WFS1, JAZF1, CDC123, TSPAN8, THADA, ADAMTS9, NOTCH2, KCNQ1, and MTNR1B were performed. HNF1B rs757210 was excluded because of failure to achieve Hardy-Weinberg equilibrium.

RESULTS

Six SNPs (TCF7L2, SLC30A8, HHEX, CDKN2B, CDKAL1, and MTNR1B) were significantly (P < 6.9 × 10⁻⁴) and two SNPs (KCNJ11 and IGF2BP2) were nominally (P < 0.05) associated with early-phase insulin release (InsAUC_0–30/GluAUC_0–30), adjusted for age, BMI, and insulin sensitivity (Matsuda ISI). Combined effects of these eight SNPs reached −32% reduction in InsAUC_0–30/GluAUC_0–30 in carriers of ≥11 vs. ≤3 weighted risk alleles. Four SNPs (SLC30A8, HHEX, CDKAL1, and TCF7L2) were significantly or nominally associated with indexes of proinsulin conversion. Three SNPs (KCNJ11, HHEX, and TSPAN8) were nominally associated with Matsuda ISI (adjusted for age and BMI). The effect of HHEX on Matsuda ISI became significant after additional adjustment for InsAUC_0–30/GluAUC_0–30. Nine SNPs did not show any associations with examined traits.

CONCLUSIONS

Eight type 2 diabetes–related loci were significantly or nominally associated with impaired early-phase insulin release. Effects of SLC30A8, HHEX, CDKAL1, and TCF7L2 on insulin release could be partially explained by impaired proinsulin conversion. HHEX might influence both insulin release and insulin sensitivity.Impaired insulin secretion and insulin resistance, two main pathophysiological mechanisms leading to type 2 diabetes, have a significant genetic component (1). Recent studies have confirmed 20 genetic loci reproducibly associated with type 2 diabetes (2–13). Three were previously known (PPARG, KCNJ11, and TCF7L2), whereas 17 loci were recently discovered either by genome-wide association studies (SLC30A8, HHEX-IDE, LOC387761, CDKN2A/2B, IGF2BP2, CDKAL1, FTO, JAZF1, CDC123/CAMK1D, TSPAN8/LGR5, THADA, ADAMTS9, NOTCH2, KCNQ1, and MTNR1B), or candidate gene approach (WFS1 and HNF1B). The mechanisms by which these genes contribute to the development of type 2 diabetes are not fully understood.PPARG is the only gene from the 20 confirmed loci previously associated with insulin sensitivity (14,15). Association with impaired β-cell function has been reported for 14 loci (KCNJ11, SLC30A8, HHEX-IDE, CDKN2A/2B, IGF2BP2, CDKAL1, TCF7L2, WFS1, HNF1B, JAZF1, CDC123/CAMK1D, TSPAN8/LGR5, KCNQ1, and MTNR1B) (6,12,13,16–38). Although associations of variants in HHEX (16–22), CDKAL1 (6,21–26), TCF7L2 (22,27–30), and MTNR1B (13,31,32) with impaired insulin secretion seem to be consistent across different studies, information concerning other genes is limited (12,18–25,27,33–38). The mechanisms by which variants in these genes affect insulin secretion are unknown. However, a few recent studies suggested that variants in TCF7L2 (22,39–42), SLC30A8 (22), CDKAL1 (22), and MTNR1B (31) might influence insulin secretion by affecting the conversion of proinsulin to insulin. Variants of FTO have been shown to confer risk for type 2 diabetes through their association with obesity (7,16) and therefore were not included in this study.Large population-based studies can help to elucidate the underlying mechanisms by which single nucleotide polymorphisms (SNPs) of different risk genes predispose to type 2 diabetes. Therefore, we investigated confirmed type 2 diabetes–related loci for their associations with insulin sensitivity, insulin secretion, and conversion of proinsulin to insulin in a population-based sample of 5,327 nondiabetic Finnish men.     

SH2B1 Enhances Insulin Sensitivity by Both Stimulating the Insulin Receptor and Inhibiting Tyrosine Dephosphorylation of Insulin Receptor Substrate Proteins

OBJECTIVE

SH2B1 is a SH2 domain-containing adaptor protein expressed in both the central nervous system and peripheral tissues. Neuronal SH2B1 controls body weight; however, the functions of peripheral SH2B1 remain unknown. Here, we studied peripheral SH2B1 regulation of insulin sensitivity and glucose metabolism.

RESEARCH DESIGN AND METHODS

We generated TgKO mice expressing SH2B1 in the brain but not peripheral tissues. Various metabolic parameters and insulin signaling were examined in TgKO mice fed a high-fat diet (HFD). The effect of SH2B1 on the insulin receptor catalytic activity and insulin receptor substrate (IRS)-1/IRS-2 dephosphorylation was examined using in vitro kinase assays and in vitro dephosphorylation assays, respectively. SH2B1 was coexpressed with PTP1B, and insulin receptor–mediated phosphorylation of IRS-1 was examined.

RESULTS

Deletion of peripheral SH2B1 markedly exacerbated HFD-induced hyperglycemia, hyperinsulinemia, and glucose intolerance in TgKO mice. Insulin signaling was dramatically impaired in muscle, liver, and adipose tissue in TgKO mice. Deletion of SH2B1 impaired insulin signaling in primary hepatocytes, whereas SH2B1 overexpression stimulated insulin receptor autophosphorylation and tyrosine phosphorylation of IRSs. Purified SH2B1 stimulated insulin receptor catalytic activity in vitro. The SH2 domain of SH2B1 was both required and sufficient to promote insulin receptor activation. Insulin stimulated the binding of SH2B1 to IRS-1 or IRS-2. This physical interaction inhibited tyrosine dephosphorylation of IRS-1 or IRS-2 and increased the ability of IRS proteins to activate the phosphatidylinositol 3-kinase pathway.

CONCLUSIONS

SH2B1 is an endogenous insulin sensitizer. It directly binds to insulin receptors, IRS-1 and IRS-2, and enhances insulin sensitivity by promoting insulin receptor catalytic activity and by inhibiting tyrosine dephosphorylation of IRS proteins.Insulin decreases blood glucose both by promoting glucose uptake into skeletal muscle and adipose tissue and by suppressing hepatic glucose production. In type 2 diabetes, the ability of insulin to reduce blood glucose is impaired (insulin resistance) because of a combination of genetic and environmental factors, resulting in hyperglycemia. Insulin resistance is not only the hallmark but also a determinant of type 2 diabetes.Insulin binds to and activates the insulin receptor. Insulin receptor tyrosyl phosphorylates insulin receptor substrates (IRS-1, -2, -3, and -4). IRS proteins, particularly IRS-1 and IRS-2, initiate and coordinate multiple downstream pathways, including the phosphatidylinositol 3-kinase/Akt pathway (1). Genetic deletion of IRS-1, IRS-2, or Akt2 causes insulin resistance in mice, indicating that the IRS protein/phosphatidylinositol 3-kinase/Akt2 pathway is required for regulation of glucose homeostasis by insulin (2–5). Insulin receptor and IRS proteins are negatively regulated by various intracellular molecules, including PTP1B, Grb10, Grb14, SOCS1, SOCS3, JNK, PKCθ, S6K, and IKKβ (6–23). The relative contribution of these negative regulators to the progression of insulin resistance has been extensively studied (6–24). However, insulin signaling is likely to also be modulated by positive regulators. In this study, we demonstrate that SH2B1 is a novel endogenous insulin sensitizer.SH2B1 is a member of the SH2B family of adapter proteins that also includes SH2B2 (APS) and SH2B3 (Lnk). SH2B1 and SH2B2 are expressed in multiple tissues, including insulin target tissues (e.g., skeletal muscle, adipose tissue, liver, and the brain); by contrast, SH2B3 expression is restricted to hematopoietic tissue (25,26). Structurally, SH2B family members have an NH₂-terminal dimerization domain, a central pleckstrin homology domain, and a COOH-terminal Src homology 2 (SH2) domain. The dimerization domain mediates homodimerization or heterodimerization between different SH2B proteins (27). SH2B1 and SH2B2 bind via their SH2 domains to a variety of tyrosine phosphorylated proteins, including JAK2 and insulin receptor, in cultured cells (28). Genetic deletion of SH2B1 results in marked leptin resistance, obesity, insulin resistance, and type 2 diabetes in mice, demonstrating that SH2B1 is required for the maintenance of normal body weight, insulin sensitivity, and glucose metabolism (29–32). Surprisingly, SH2B2-null mice have normal body weight and slightly improved insulin sensitivity (32,33), suggesting that SH2B1 and SH2B2 have distinct functions in vivo. However, it remains unclear whether SH2B1 cell autonomously regulates insulin sensitivity in peripheral insulin target tissues because systemic deletion of SH2B1 causes obesity, which may cause insulin resistance in SH2B1-null mice.We generated a mouse model in which recombinant SH2B1 is specifically expressed in the brain of SH2B1-null mice (TgKO) using transgenic approaches (31). Neuron-specific restoration of SH2B1 corrects both leptin resistance and obesity, suggesting that neuronal SH2B1 regulates energy balance and body weight by enhancing leptin sensitivity (31). Consistent with these conclusions, polymorphisms in the SH2B1 loci are linked to leptin resistance and obesity in humans (34–36). In this work, we demonstrate that deletion of SH2B1 in peripheral tissues impairs insulin sensitivity independent of obesity in TgKO mice. Moreover, we demonstrate that SH2B1 directly promotes insulin responses by stimulating insulin receptor catalytic activity and by protecting IRS proteins from tyrosine dephosphorylation.     

Additive Effects of Genetic Variation in GCK and G6PC2 on Insulin Secretion and Fasting Glucose

OBJECTIVE

Glucokinase (GCK) and glucose-6-phosphatase catalytic subunit 2 (G6PC2) regulate the glucose-cycling step in pancreatic β-cells and may regulate insulin secretion. GCK rs1799884 and G6PC2 rs560887 have been independently associated with fasting glucose, but their interaction on glucose-insulin relationships is not well characterized.

RESEARCH DESIGN AND METHODS

We tested whether these variants are associated with diabetes-related quantitative traits in Mexican Americans from the BetaGene Study and attempted to replicate our findings in Finnish men from the METabolic Syndrome in Men (METSIM) Study.

RESULTS

rs1799884 was not associated with any quantitative trait (corrected P > 0.1), whereas rs560887 was significantly associated with the oral glucose tolerance test 30-min incremental insulin response (30′ Δinsulin, corrected P = 0.021). We found no association between quantitative traits and the multiplicative interaction between rs1799884 and rs560887 (P > 0.26). However, the additive effect of these single nucleotide polymorphisms was associated with fasting glucose (corrected P = 0.03) and 30′ Δinsulin (corrected P = 0.027). This additive association was replicated in METSIM (fasting glucose, P = 3.5 × 10⁻¹⁰ 30′ Δinsulin, P = 0.028). When we examined the relationship between fasting glucose and 30′ Δinsulin stratified by GCK and G6PC2, we noted divergent changes in these quantitative traits for GCK but parallel changes for G6PC2. We observed a similar pattern in METSIM.

CONCLUSIONS

Our data suggest that variation in GCK and G6PC2 have additive effects on both fasting glucose and insulin secretion.Genome-wide association (GWA) studies have identified several loci for type 2 diabetes (1–5) and type 2 diabetes–related quantitative traits (6–20). Two of these loci, glucokinase (GCK) (21–23) and glucose-6-phosphatase catalytic subunit 2 (G6PC2) (9,10), regulate the critical glucose-sensing mechanism within pancreatic β-cells. Mutations in GCK confer susceptibility to maturity-onset diabetes of the young (MODY)-2 (24–26), and a −30 GCK promoter variant (rs1799884) has been shown to be associated with β-cell function (21), fasting glucose, and birth weight (23). Chen et al. (9) demonstrated an association between the G6PC2 region and fasting glucose, an observation replicated by Bouatia-Naji et al. (10). Although fasting glucose levels are associated with both GCK and G6PC2, there has been no evidence that genetic variation at these two loci contribute a risk for type 2 diabetes, suggesting contribution to mild elevations in glycemia.GCK phosphorylates glucose to glucose-6-phosphate, whereas G6PC2 dephosphorylates glucose-6-phosphate back to glucose, forming a glucose cycle previously demonstrated to exist within pancreatic β-cells (27,28). The important role of GCK in glucose sensing by pancreatic islets has been demonstrated by numerous studies, and other studies suggest a role for glucose cycling in insulin secretion and diabetes (28–30), implying that the balance between GCK and G6PC2 activity is important for determining glycolytic flux, ATP production, and subsequent insulin secretion. This was validated by a demonstration that direct manipulation of glucose cycling alters insulin secretion (31,32).BetaGene is a study in which we are performing detailed phenotyping of Mexican American probands with recent gestational diabetes mellitus (GDM) and their family members to obtain quantitative estimates of body composition, insulin sensitivity (S_I), acute insulin response (AIR), and β-cell compensation (disposition index) with the goal of identifying genes influencing variations in type 2 diabetes–related quantitative traits (33–35). Based on the evidence that variation in GCK (rs1799884) and G6PC2 (rs560887) are independently associated with fasting glucose concentrations and both are crucial to glucose cycling in β-cells, we hypothesized that interaction between these loci may be associated not just with fasting glucose but also with measures of insulin secretion or β-cell function. We tested this hypothesis in the BetaGene Study and, for replication, in a separate sample of Finnish men participating in the METabolic Syndrome in Men (METSIM) Study (36).     

Insulin Reciprocally Regulates Glucagon Secretion in Humans

OBJECTIVE

We tested the hypothesis that an increase in insulin per se, i.e., in the absence of zinc, suppresses glucagon secretion during euglycemia and that a decrease in insulin per se stimulates glucagon secretion during hypoglycemia in humans.

RESEARCH DESIGN AND METHODS

We measured plasma glucagon concentrations in patients with type 1 diabetes infused with the zinc-free insulin glulisine on three occasions. Glulisine was infused with clamped euglycemia (∼95 mg/dl [5.3 mmol/l]) from 0 to 60 min on all three occasions. Then, glulisine was discontinued with clamped euglycemia or with clamped hypoglycemia (∼55 mg/dl [3.0 mmol/l]) or continued with clamped hypoglycemia from 60 to 180 min.

RESULTS

Plasma glucagon concentrations were suppressed by −13 ± 3, −9 ± 3, and −12 ± 2 pg/ml (−3.7 ± 0.9, −2.6 ± 0.9, and −3.4 ± 0.6 pmol/l), respectively, (all P < 0.01) during zinc-free hyperinsulinemic euglycemia over the first 60 min. Glucagon levels remained suppressed following a decrease in zinc-free insulin with euglycemia (−14 ± 3 pg/ml [−4.0 ± 0.9 pmol/l]) and during sustained hyperinsulinemia with hypoglycemia (−14 ± 2 pg/ml [−4.0 ± 0.6 pmol/l]) but increased to −3 ± 3 pg/ml (−0.9 ± 0.9 pmol/l) (P < 0.01) following a decrease in zinc-free insulin with hypoglycemia over the next 120 min.

CONCLUSIONS

These data indicate that an increase in insulin per se suppresses glucagon secretion and a decrease in insulin per se, in concert with a low glucose concentration, stimulates glucagon secretion. Thus, they document that insulin is a β-cell secretory product that, in concert with glucose and among other signals, reciprocally regulates α-cell glucagon secretion in humans.The regulation of pancreatic islet α-cell glucagon secretion by nutrients, hormones, neurotransmitters, and drugs is complex and incompletely understood (1–8). It involves direct signaling of α-cells (1) and indirect signaling of α-cells by β-cell (2–4) and δ-cell (5) secretory products, the autonomic nervous system (6,7), and gut incretins (8). Among the intraislet mechanisms, there is evidence that indirect reciprocal β-cell–mediated signaling of α-cells normally predominates over direct α-cell signaling in the regulation of glucagon secretion in humans (9–13). The physiological concept is as follows: 1) A β-cell secretory product, or products, tonically restrains α-cell glucagon secretion during postabsorptive euglycemia. 2) A decrease in β-cell secretion, in concert with a low α-cell glucose concentration, signals an increase in α-cell glucagon secretion during hypoglycemia (9–12). 3) An increase in β-cell secretion negates direct α-cell stimulation and thus results in no change or even suppression of α-cell glucagon secretion following a mixed meal (13).Among the various candidate signaling molecules—insulin, zinc, γ-aminobutyric acid, and amylin among others (4)—there is evidence that insulin is a β-cell secretory product that normally restrains basal α-cell glucagon secretion (14). First, administered insulin suppresses glucagon secretion in several species (4,14,15) including humans (16). However, most available insulin preparations contain zinc and zinc is cosecreted with insulin from β-cells and it has been reported that a decrease in pancreatic arterial zinc, but not in insulin depleted of zinc, increases glucagon secretion during hypoglycemia in streptozotocin diabetic rats, leading the authors to conclude that the inhibitory β-cell secretory product is zinc—not insulin (17). Second, perfusion of the rat (3) and the human (18) pancreas (and incubation of rat islets [15]) with an antibody to insulin increases glucagon release. Because insulin circulates as a zinc-free monomer (19), that finding seemingly implicates insulin directly as an α-cell inhibitory factor and suggests that zinc is not the only β-cell product that reciprocally regulates α-cell glucagon secretion. However, it could be reasoned that the antibody binds insulin-zinc complexes before they dissociate at physiological pH and with dilution. Third, findings—that siRNA-mediated knockdown of insulin receptors prevented the effect of low glucose concentrations to increase glucagon release from isolated mouse islets (20) and that blockade of insulin signaling with the phosphatidylinositol 3-kinase inhibitor wortmannin prevented the effect of high glucose concentrations to decrease glucagon release from isolated rat and human islets (21)—also appear to implicate insulin as the relevant β-cell secretory product.We tested the hypothesis that an increase in insulin per se, i.e., in the absence of an increase in zinc, suppresses glucagon secretion during euglycemia and that a decrease in insulin per se, in concert with low glucose concentrations, stimulates glucagon secretion in humans. To do so, we studied patients with type 1 diabetes, individuals with essentially no endogenous insulin secretion and no α-cell glucagon secretory response to hyperinsulinemic hypoglycemia (22–29) but viable α-cells as evidenced by a glucagon secretory response to administered amino acids (22,30–32), on three separate occasions. Plasma glucagon concentrations were measured during infusion of the zinc-free insulin glulisine with clamped euglycemia over 60 min on all three occasions. Then, glucagon concentrations were measured following discontinuation of glulisine, to produce a sharp decrease in systemic and therefore α-cell zinc-free insulin levels, over 120 min with clamped euglycemia on one occasion or clamped hypoglycemia on another occasion. On a third occasion, glucagon levels were measured during sustained glulisine infusion with clamped hypoglycemia. The data document 1) a decrease in plasma glucagon during zinc-free hyperinsulinemic euglycemia and 2) an increase in plasma glucagon following a decrease in zinc-free insulin during hypoglycemia but not following a decrease in insulin without hypoglycemia or during hypoglycemia without a decrease in insulin.     

Changes in Insulin Sensitivity and Insulin Release in Relation to Glycemia and Glucose Tolerance in 6,414 Finnish Men

OBJECTIVE

We evaluated insulin sensitivity and insulin secretion across the entire range of fasting (FPG) and 2-h plasma glucose (PG), and we investigated the differences in insulin sensitivity and insulin release in different glucose tolerance categories.

RESEARCH DESIGN AND METHODS

A total of 6,414 Finnish men (aged 57 ± 7 years, BMI 27.0 ± 3.9 kg/m²) from our ongoing population-based METSIM (Metabolic Syndrome in Men) study were included. Of these subjects, 2,168 had normal glucose tolerance, 2,859 isolated impaired fasting glucose (IFG), 217 isolated impaired glucose tolerance (IGT), 701 a combination of IFG and IGT, and 469 newly diagnosed type 2 diabetes.

RESULTS

The Matsuda index of insulin sensitivity decreased substantially within the normal range of FPG (−17%) and 2-h PG (−37%) and was approximately −65 and −53% in the diabetic range of FPG and 2-h PG, respectively, compared with the reference range (FPG and 2-h PG <5.0 mmol/l). Early-phase insulin release declined by only approximately −5% within the normal range of FPG and 2-h PG but decreased significantly in the diabetic range of FPG (by 32–70%) and 2-h PG (by 33–51%). Changes in insulin sensitivity and insulin secretion in relation to hyperglycemia were independent of obesity. The predominant feature of isolated IGT was impaired peripheral insulin sensitivity. Isolated IFG was characterized by impaired early and total insulin release.

CONCLUSIONS

Peripheral insulin sensitivity was already decreased substantially at low PG levels within the normoglycemic range, whereas impairment in insulin secretion was observed mainly in the diabetic range of FPG and 2-h PG. Obesity did not affect changes in insulin sensitivity or insulin secretion in relation to hyperglycemia.Type 2 diabetes is preceded by a long pre-diabetic state, characterized by mild elevation of fasting and/or postprandial glucose levels. This asymptomatic phase may last for years, and about one-third of these individuals finally develop type 2 diabetes (1). The pre-diabetic state, defined by an oral glucose tolerance test (OGTT), includes impaired fasting glucose (IFG), impaired glucose tolerance (IGT), or their combination (2). Epidemiological studies have shown that IFG and IGT represent two distinct subgroups of abnormal glucose tolerance (1,3–5) that differ in their age and sex distribution (6,7) and associated cardiovascular risk (8). Therefore, IFG and IGT are likely to have different pathophysiologies.Impaired insulin secretion and impaired insulin action are the two main pathophysiological disturbances leading to abnormal glucose tolerance. Previous studies on the role of impaired insulin secretion and insulin resistance in the development of IFG and IGT have yielded contradictory results (4–23). Inconsistencies across the studies are explained by differences in study populations, study designs and methods to assess insulin resistance and insulin secretion, and most importantly by a small sample size. Categorization of glucose tolerance is based on arbitrary cutoff points of glucose levels, and therefore different subgroups cannot fully account for changes in β-cell function and insulin action with increasing glycemia. Only a few studies have examined insulin secretion and/or insulin sensitivity as a function of glucose concentrations (13,24–28). These studies have been, however, relatively small, and most of them were conducted in non-Caucasian populations.The aim of this study was to evaluate insulin sensitivity and insulin secretion across the entire range of fasting and 2-h plasma glucose (PG) from normal glucose tolerance (NGT) to type 2 diabetes to understand better the pathophysiology of the pre-diabetic state. Furthermore, we investigated the differences in insulin sensitivity and insulin release in different glucose tolerance subgroups. To address these questions, we collected a large sample of carefully phenotyped middle-aged Finnish men.     

Sleep Restriction for 1 Week Reduces Insulin Sensitivity in Healthy Men

OBJECTIVE

Short sleep duration is associated with impaired glucose tolerance and an increased risk of diabetes. The effects of sleep restriction on insulin sensitivity have not been established. This study tests the hypothesis that decreasing nighttime sleep duration reduces insulin sensitivity and assesses the effects of a drug, modafinil, that increases alertness during wakefulness.

RESEARCH DESIGN AND METHODS

This 12-day inpatient General Clinical Research Center study included 20 healthy men (age 20–35 years and BMI 20–30 kg/m²). Subjects spent 10 h/night in bed for ≥8 nights including three inpatient nights (sleep-replete condition), followed by 5 h/night in bed for 7 nights (sleep-restricted condition). Subjects received 300 mg/day modafinil or placebo during sleep restriction. Diet and activity were controlled. On the last 2 days of each condition, we assessed glucose metabolism by intravenous glucose tolerance test (IVGTT) and euglycemic-hyperinsulinemic clamp. Salivary cortisol, 24-h urinary catecholamines, and neurobehavioral performance were measured.

RESULTS

IVGTT-derived insulin sensitivity was reduced by (means ± SD) 20 ± 24% after sleep restriction (P = 0.001), without significant alterations in the insulin secretory response. Similarly, insulin sensitivity assessed by clamp was reduced by 11 ± 5.5% (P < 0.04) after sleep restriction. Glucose tolerance and the disposition index were reduced by sleep restriction. These outcomes were not affected by modafinil treatment. Changes in insulin sensitivity did not correlate with changes in salivary cortisol (increase of 51 ± 8% with sleep restriction, P < 0.02), urinary catecholamines, or slow wave sleep.

CONCLUSIONS

Sleep restriction (5 h/night) for 1 week significantly reduces insulin sensitivity, raising concerns about effects of chronic insufficient sleep on disease processes associated with insulin resistance.The average sleep duration in the U.S. has fallen below 7 h per night, a drop of ∼2 h per night over the last century and >1 h per night over the last 40 years (1,2). Cross-sectional and longitudinal studies have demonstrated a link between short sleep duration or poor sleep quality and increased risk of obesity (3–7), diabetes (7–11), hypertension (12), cardiovascular disease (13,14), the metabolic syndrome (15), and early mortality (14,16–21). Short-term sleep restriction (4 h/night for 1 week in a laboratory setting) impaired glucose tolerance during a frequently sampled intravenous glucose tolerance test (IVGTT) in healthy subjects (22).In healthy subjects, the mechanisms leading to impaired glucose tolerance with short-term reductions in nightly sleep duration are unclear. Decreases in insulin secretion have been implicated, and sleep restriction increases cortisol levels, which could influence glucose tolerance (22). Further, insulin resistance has been reported in two very different models of disrupted sleep: sleep apnea (23) and experimental disruption of slow-wave sleep (24). In the latter model, the extent of slow-wave sleep disruption predicted reductions in insulin sensitivity (24).Our primary goal was to test the hypothesis that sleep restriction in healthy subjects reduces insulin sensitivity as assessed by the hyperinsulinemic-euglycemic clamp. Insulin secretion was assessed using IVGTTs. To identify possible mechanisms by which sleep restriction may affect insulin sensitivity, we assessed the relationships between changes in insulin sensitivity and changes in cortisol, catecholamines, and slow-wave sleep. Further, we tested the ability of modafinil to ameliorate the adverse effects of sleep restriction on insulin sensitivity. Modafanil activates central, wake-promoting dopaminergic and noradrenergic mechanisms (25,26) and ameliorates the adverse effects of sleep deprivation on alertness and performance (27–29)—impairments that have been attributed to reduced brain glucose utilization (30). Thus, we performed hyperinsulinemic-euglycemic clamps and intravenous glucose tolerance twice: at baseline in sleep-replete individuals and after 7 nights of sleep restriction (5 h in bed) in healthy individuals randomized to daily treatment with placebo or modafinil.     

Insulin resistance, defective insulin-mediated fatty acid suppression, and coronary artery calcification in subjects with and without type 1 diabetes: The CACTI study

OBJECTIVE

To assess insulin action on peripheral glucose utilization and nonesterified fatty acid (NEFA) suppression as a predictor of coronary artery calcification (CAC) in patients with type 1 diabetes and nondiabetic controls.

RESEARCH DESIGN AND METHODS

Insulin action was measured by a three-stage hyperinsulinemic-euglycemic clamp (4, 8, and 40 mU/m²/min) in 87 subjects from the Coronary Artery Calcification in Type 1 Diabetes cohort (40 diabetic, 47 nondiabetic; mean age 45 ± 8 years; 55% female).

RESULTS

Peripheral glucose utilization was lower in subjects with type 1 diabetes compared with nondiabetic controls: glucose infusion rate (mg/kg FFM/min) = 6.19 ± 0.72 vs. 12.71 ± 0.66, mean ± SE, P < 0.0001, after adjustment for age, sex, BMI, fasting glucose, and final clamp glucose and insulin. Insulin-induced NEFA suppression was also lower in type 1 diabetic compared with nondiabetic subjects: NEFA levels (μM) during 8 mU/m²/min insulin infusion = 370 ± 27 vs. 185 ± 25, P < 0.0001, after adjustment for age, sex, BMI, fasting glucose, and time point insulin. Lower glucose utilization and higher NEFA levels, correlated with CAC volume (r = −0.42, P < 0.0001 and r = 0.41, P < 0.0001, respectively) and predicted the presence of CAC (odds ratio [OR] = 0.45, 95% CI = 0.22–0.93, P = 0.03; OR = 2.4, 95% CI = 1.08–5.32, P = 0.032, respectively). Insulin resistance did not correlate with GHb or continuous glucose monitoring parameters.

CONCLUSIONS

Type 1 diabetic patients are insulin resistant compared with nondiabetic subjects, and the degree of resistance is not related to current glycemic control. Insulin resistance predicts the extent of coronary artery calcification and may contribute to the increased risk of cardiovascular disease in patients with type 1 diabetes as well as subjects without diabetes.Cardiovascular disease (CVD) remains the leading cause of death in individuals with type 1 diabetes (1–4). Although hyperglycemia appears to be the primary mediator of microvascular disease (5,6), its role in macrovascular disease is less clear (4). Tight glycemic control improves, but does not normalize CVD risk, and correlation of GHb to CVD risk remains controversial (7–15). In addition, standard prediction rules for CVD risk do not accurately predict CVD in type 1 diabetic populations (16). Thus, the mechanism of accelerated atherosclerosis in type 1 diabetes is unclear and identification of those patients at highest risk and most in need of aggressive risk factor modification is inaccurate.In the general population, insulin resistance has been implicated as an important contributor to accelerated atherosclerosis (17–25). Although type 1 diabetes is primarily a disease of insulin deficiency, previous studies have demonstrated insulin resistance and suggested that CVD may also be linked to insulin resistance in type 1 diabetes (10,26–32). As early as 1968, Martin et al. (30) demonstrated an “impaired glucose assimilation index” and an inverse association between this index and prevalent macrovascular disease in type 1 diabetic subjects. More recently, the Pittsburgh Epidemiology of Diabetes Complications Study (10) found no correlation between GHb and coronary artery disease outcomes. However, in addition to other known CVD risk factors, estimated glucose disposal rate correlated inversely with these outcomes. Similar correlations of estimated insulin resistance or a surrogate of insulin resistance (waist-to-hip ratio) to coronary artery disease were also found in the Diabetes Control and Complications Trial (DCCT) and the EURODIAB study (33). These data suggest that an estimate of insulin resistance may add to CVD risk prediction in type 1 diabetes. In addition, elevated nonesterified fatty acid (NEFA) levels have been proposed to mediate the increased atherosclerotic risk associated with insulin resistance in the general population (18,34–37). It is not known whether the defects in insulin action in type 1 diabetes extend beyond glucose utilization to NEFA suppression.The Coronary Artery Calcification in Type 1 Diabetes (CACTI) study has followed a cohort of type 1 diabetic subjects and similar nondiabetic controls with electron beam computed tomography for measurement of coronary artery calcification (CAC) and CVD outcomes for 6 years (15,38). We hypothesized that type 1 diabetic subjects would be more insulin resistant than nondiabetic controls in terms of both glucose utilization and NEFA suppression, and that both measures of insulin resistance would correlate with CAC, a marker of the extent of coronary atherosclerosis.     

The C3a Anaphylatoxin Receptor Is a Key Mediator of Insulin Resistance and Functions by Modulating Adipose Tissue Macrophage Infiltration and Activation

OBJECTIVE

Significant new data suggest that metabolic disorders such as diabetes, obesity, and atherosclerosis all posses an important inflammatory component. Infiltrating macrophages contribute to both tissue-specific and systemic inflammation, which promotes insulin resistance. The complement cascade is involved in the inflammatory cascade initiated by the innate and adaptive immune response. A mouse genomic F2 cross biology was performed and identified several causal genes linked to type 2 diabetes, including the complement pathway.

RESEARCH DESIGN AND METHODS

We therefore sought to investigate the effect of a C3a receptor (C3aR) deletion on insulin resistance, obesity, and macrophage function utilizing both the normal-diet (ND) and a diet-induced obesity mouse model.

RESULTS

We demonstrate that high C3aR expression is found in white adipose tissue and increases upon high-fat diet (HFD) feeding. Both adipocytes and macrophages within the white adipose tissue express significant amounts of C3aR. C3aR^−/− mice on HFD are transiently resistant to diet-induced obesity during an 8-week period. Metabolic profiling suggests that they are also protected from HFD-induced insulin resistance and liver steatosis. C3aR^−/− mice had improved insulin sensitivity on both ND and HFD as seen by an insulin tolerance test and an oral glucose tolerance test. Adipose tissue analysis revealed a striking decrease in macrophage infiltration with a concomitant reduction in both tissue and plasma proinflammatory cytokine production. Furthermore, C3aR^−/− macrophages polarized to the M1 phenotype showed a considerable decrease in proinflammatory mediators.

CONCLUSIONS

Overall, our results suggest that the C3aR in macrophages, and potentially adipocytes, plays an important role in adipose tissue homeostasis and insulin resistance.The complement system is an integral part of both the innate and adaptive immune response involved in the defense against invading pathogens (1). Complement activation culminates in a massive amplification of the immune response leading to increased cell lysis, phagocytosis, and inflammation (1). C3 is the most abundant component of the complement cascade and the convergent point of all three major complement activation pathways. C3 is cleaved into C3a and C3b by the classical and lectin pathways, and iC3b is generated by the alternative pathway (2,3). C3a has potent anaphylatoxin activity, directly triggering degranulation of mast cells, inflammation, chemotaxis, activation of leukocytes, as well as increasing vascular permeability and smooth muscle contraction (3). C3a mediates its downstream signaling effects by binding to the C3a receptor (C3aR), a Gi-coupled G protein–coupled receptor. Several studies have demonstrated a role for C3a and C3aR in asthma, sepsis, liver regeneration, and autoimmune encephalomyelitis (1,3). Therefore, targeting C3aR may be an attractive therapeutic option for the treatment of several inflammatory diseases.Increasing literature suggests that metabolic disorders such as diabetes, obesity, and atherosclerosis also possess an important inflammatory component (4–7). Several seminal reports have demonstrated that resident macrophages can constitute as much as 40% of the cell population of adipose tissue (7–9) and can significantly affect insulin resistance (10–18). Several proinflammatory cytokines, growth factors, acute-phase proteins, and hormones are produced by the adipose tissue and implicated in insulin resistance and vascular homeostasis (4–7,19). An integrated genomics approach was performed with several mouse strains to infer causal relationships between gene expression and complex genetic diseases such as obesity/diabetes. This approach identified the C3aR gene as being causal for omental fat pad mass (20). The C3aR^−/− mice were shown to have decreased adiposity as compared with wild-type mice on a regular diet (20). Monocytes and macrophages express the C3aR (21–28). Increased C3a levels also correlate with obesity, diabetes, cholesterol, and lipid levels (29–34). We therefore sought to investigate the specific role of the C3aR in insulin resistance, obesity, and macrophage function utilizing both normal diet and the diet-induced obesity model.     

Virtual Optimization of Nasal Insulin Therapy Predicts Immunization Frequency to Be Crucial for Diabetes Protection

OBJECTIVE

Development of antigen-specific strategies to treat or prevent type 1 diabetes has been slow and difficult because of the lack of experimental tools and defined biomarkers that account for the underlying therapeutic mechanisms.

RESEARCH DESIGN AND METHODS

The type 1 diabetes PhysioLab platform, a large-scale mathematical model of disease pathogenesis in the nonobese diabetic (NOD) mouse, was used to investigate the possible mechanisms underlying the efficacy of nasal insulin B:9-23 peptide therapy. The experimental aim was to evaluate the impact of dose, frequency of administration, and age at treatment on Treg induction and optimal therapeutic outcome.

RESULTS

In virtual NOD mice, treatment efficacy was predicted to depend primarily on the immunization frequency and stage of the disease and to a lesser extent on the dose. Whereas low-frequency immunization protected from diabetes atrributed to Treg and interleukin (IL)-10 induction in the pancreas 1–2 weeks after treatment, high-frequency immunization failed. These predictions were confirmed with wet-lab approaches, where only low-frequency immunization started at an early disease stage in the NOD mouse resulted in significant protection from diabetes by inducing IL-10 and Treg.

CONCLUSIONS

Here, the advantage of applying computer modeling in optimizing the therapeutic efficacy of nasal insulin immunotherapy was confirmed. In silico modeling was able to streamline the experimental design and to identify the particular time frame at which biomarkers associated with protection in live NODs were induced. These results support the development and application of humanized platforms for the design of clinical trials (i.e., for the ongoing nasal insulin prevention studies).Type 1 diabetes is a complex and multifactorial autoimmune disease, in which, (pro-) insulin-specific T-cell responses have been described in lymphocytes obtained from nonobese diabetic (NOD) mice and (pre-)diabetic patients (1–4). In NOD mice, the insulin B:9-23 peptide sequence is a dominant epitope, and a single amino acid substitution in position 16 (B16:A) confers protection from the disease (5–7). Antigen-specific immunotherapies with whole insulin and B:9-23 peptide have been successful in preventing diabetes in NOD mice when administered via the subcutaneous, oral, or nasal route or via intramuscular DNA vaccination (8–18). The success in halting disease progression in prediabetic mice prompted physicians to establish similar protocols to test safety and efficacy in human prediabetic or diabetic subjects. To this end, clinical trials with nasal or oral whole insulin were conducted, which proved to be safe. However, diabetes progression was only slightly affected in a subset of insulin antibody–positive patients treated with oral insulin in the Diabetes Prevention Trial–Type 1 (19–24). In contrast, nasal insulin phase I–II trials in Finland (21,24) and in Australia (20), during which insulin was administered daily, failed to provide therapeutic efficacy. Highlighting the difficulties we are facing to rationally translate antigen-specific therapies to humans, there are conflicting reports on the efficacy of nasal B:9-23 peptide immunization in the NOD mouse. Taken together, these studies suggest that the manner by which insulin therapy is administered is important (25,26). Many variables may influence efficacy, including dose, frequency of administration, and the stage of the disease. Systematic investigation of each of these variables and combinations thereof is experimentally impractical because of the time constraints of in vivo studies, therefore necessitating biosimulation approaches.The type 1 diabetes PhysioLab platform is a top-down, outcome-focused, large-scale mathematical model composed of ordinary differential and algebraic equations (27,28). This model reproduces type 1 diabetes pathogenesis in female NOD mice from birth until disease onset, with extensive representation of critical biological processes that were described in the literature and take place in the pancreas, the pancreatic-draining lymph nodes (PDLN), the gut, the nasal-associated lymphoid tissue (NALT), and the peripheral blood.In the present study, we used a cohort of virtual NOD mice (VM) with diversity in underlying pathophysiology to investigate how variations in dose, frequency, and age at treatment initiation may impact the efficacy of nasal B:9-23 peptide therapy. The VM program was designed based on the following assumptions: 1) induction of Treg is beneficial for disease prevention, 2) induction of Th1 responses (interferon [IFN] γ) is detrimental for diabetes progression, whereas 3) induction of Th2 responses (interleukin [IL]-10 and IL-4) favor disease protection. In silico investigation of the underlying mechanisms predicted that too frequent nasal B:9-23 immunization would inhibit IL-10 induction and the generation of adaptive Treg (aTreg), which should be critical in mediating protection. The model also aided in defining the optimal time frame in which Treg and IL-10 would be induced after immunization, thereby mapping the timing for both as crucial biomarkers.Laboratory experiments confirmed many of these predictions, establishing immunization frequency and induction of IL-10⁺ Treg as important considerations for the design of future clinical trials. Interestingly, in the initial and follow-up Peakman studies (2,29), it became evident that naturally occurring circulating islet-specific IL-10–producing cells regulate proinflammatory Th1 responses in healthy individuals by linked suppression. This suggests than in certain circumstances, given the heterogeneity of human type 1 diabetes, IL-10 production could be sufficient in sustaining prolonged antigen-specific tolerance. Future and ongoing trials might benefit from less frequent immunization, for example the ongoing nasal insulin studies in Australia (20). Moving forward, better defining the immunological parameters that could serve as biomarkers is of particular importance for improving the previously failed clinical trials (19–23,30) and for increasing the chance of success (31,32).     

Insulin Acutely Inhibits Intestinal Lipoprotein Secretion in Humans in Part by Suppressing Plasma Free Fatty Acids

OBJECTIVE

Intestinal lipoprotein production has recently been shown to be increased in insulin resistance, but it is not known whether it is regulated by insulin in humans. Here, we investigated the effect of acute hyperinsulinemia on intestinal (and hepatic) lipoprotein production in six healthy men in the presence and absence of concomitant suppression of plasma free fatty acids (FFAs).

RESEARCH DESIGN AND METHODS

Each subject underwent the following three lipoprotein turnover studies, in random order, 4–6 weeks apart: 1) insulin and glucose infusion (euglycemic-hyperinsulinemic clamp) to induce hyperinsulinemia, 2) insulin and glucose infusion plus Intralipid and heparin infusion to prevent the insulin-induced suppression of plasma FFAs, and 3) saline control.

RESULTS

VLDL1 and VLDL2-apoB48 and -apoB100 production rates were suppressed by 47–62% by insulin, with no change in clearance. When the decline in FFAs was prevented by concomitant infusion of Intralipid and heparin, the production rates of VLDL1 and VLDL2-apoB48 and -apoB100 were intermediate between insulin and glucose infusion and saline control.

CONCLUSIONS

This is the first demonstration in humans that intestinal apoB48-containing lipoprotein production is acutely suppressed by insulin, which may involve insulin''s direct effects and insulin-mediated suppression of circulating FFAs.Dyslipidemia is a well-recognized feature of insulin resistance and type 2 diabetes and is a common risk factor for atherosclerotic cardiovascular disease. Hypertriglyceridemia, low plasma concentrations of HDL, and qualitative changes in LDL comprise the typical dyslipidemia, which is felt to play an important but not exclusive role in accelerated atherosclerosis of affected individuals (1,2). Overproduction of large, triglyceride-rich, apolipoprotein (apo)B100-containing, hepatic VLDL1 particles has been well documented in animal models and in humans with insulin resistance and type 2 diabetes and contributes to dyslipidemia (3,4). In addition, postprandial hyperlipidemia and elevated plasma concentrations of intestinal apoB48-containing particles have been demonstrated in insulin-resistant states (5–9). We and others have recently shown that insulin-resistant animal models and humans have overproduction of intestinal, apoB48-containing lipoproteins (10–12). Whereas numerous factors are known to regulate hepatic lipoprotein particle overproduction, less is known about factors that regulate intestinal lipoprotein production in insulin-resistant conditions.We have recently shown that acute elevation of plasma free fatty acids (FFAs) stimulates not only hepatic (13) but also intestinally derived apoB48-containing lipoprotein particles in fed humans (14), demonstrating that at least one of the factors involved in the regulation of hepatic lipoprotein production also regulates intestinal lipoprotein particle production. Insulin has been shown both in vitro (9,15,16) and in vivo (13,17–22) in animals and humans to acutely suppress hepatic apoB100-containing lipoprotein particle production. This acute suppressive effect on VLDL in fasting individuals has been shown to be in part dependent on the FFA suppression induced by acute hyperinsulinemia in vivo (13,21) and is due predominantly to suppression of the VLDL1 fraction with no (22) or an opposite (17) effect on VLDL2. Insulin-resistant hyperinsulinemic, obese humans and those with type 2 diabetes are resistant to the acute inhibitory effect of insulin on VLDL production (18,20), as are primary cultured hepatocytes derived from insulin-resistant rats (23). At least part of the effect of insulin is direct, occurring by co- and posttranslational mechanisms through increasing posttranslational protein degradation (24). Incubation of human fetal small intestinal cells with insulin has also been shown to reduce chylomicron secretion (25). Recent studies have shown that intestinal lipoprotein production in chow-fed hamsters is responsive to the acute inhibitory effect of insulin, whereas enterocytes derived from insulin-resistant, fructose-fed hamsters are resistant to this acute suppressive effect of insulin through an aberrant intestinal insulin-signaling cascade (26). The responsiveness of intestinal lipoprotein secretion to acute hyperinsulinemia has not previously been examined in humans.In the present study, we used the euglycemic-hyperinsulinemic clamp technique to examine the effects of acute hyperinsulinemia on VLDL1 and VLDL2 intestinal (apoB48) and hepatic (apoB100) lipoprotein production in six healthy men in a constant fed state. We found that insulin infusion suppresses both VLDL1 and 2 apoB48 and apoB100 concentrations as a result of suppression of VLDL1 lipoprotein secretion, with consequently less VLDL2 formed from VLDL1. When Intralipid and heparin were coinfused to prevent insulin-induced suppression of plasma FFAs, production rates of these lipoproteins were still suppressed, although to a lesser extent. These results indicate that insulin directly suppresses both intestinal and hepatic lipoprotein production in humans.     

Euglycemic Infusion of Insulin Detemir Compared With Human Insulin Appears to Increase Direct Current Brain Potential Response and Reduces Food Intake While Inducing Similar Systemic Effects

OBJECTIVE

In the treatment of diabetic patients, the long-acting insulin analog insulin detemir is less prone to induce weight gain than other insulin formulations. Assuming that because of its pharmacologic properties, detemir displays stronger central nervous anorexigenic efficacy than human insulin, we compared acute effects of human insulin and detemir on electroencephalography (EEG) measures and food intake.

RESEARCH DESIGN AND METHODS

Frontocortical EEG direct current (DC) potentials were recorded in 15 healthy men during two hyperinsulinemic-euglycemic clamps that included an insulin bolus injection (human insulin, 17.75 mU/kg body wt; detemir, 90 mU/kg body wt) followed by a steady 90-min infusion (1.0 vs. 2.0 mU · kg⁻¹ · min⁻¹). A higher dosage was chosen for detemir to compensate for its delay in impact relative to human insulin and to elicit similar systemic effects. At 20 min after infusion, subjects were allowed to eat ad libitum from a test buffet.

RESULTS

Mean glucose infusions to maintain euglycemia (P > 0.93) and blood glucose concentrations (P > 0.34) did not differ between conditions. Detemir infusion induced a negative DC-potential shift, averaging −372.2 μV from 21 to 90 min that was not observed during human insulin infusion (146.5 μV, P = 0.02). Detemir, in comparison with human insulin, reduced subsequent food intake by 303 kcal (1,257 vs. 1,560, P < 0.04).

CONCLUSIONS

While inducing comparable peripheral effects, detemir exerts stronger acute effects on brain functions than human insulin and triggers a relative decrease in food consumption, suggesting an enhanced anorexigenic impact of detemir compared with human insulin on central nervous networks that control nutrient uptake.Systemic insulin accessing the brain via an active, saturable transport mechanism (1) is assumed to contribute to the central nervous regulation of energy homeostasis (2). Experimental administration of insulin to the central nervous system inhibits food intake and reduces body fat content in animals (3,4) and humans (5,6), suggesting that circulating insulin provides negative, anorexigenic feedback on the amount of body fat to the brain. The long-acting insulin analog insulin detemir, because of the acylation of a 14-carbon fatty acid (myristic acid) to lysine at locus B29, displays increased self-association and reversible albumin binding (7,8), which delays absorption of the molecule and thereby reduces the risk of hypoglycemic episodes (9,10). Insulin therapy using detemir has been frequently found to induce weight-sparing effects in comparison with other insulins, curtailing body weight gain in patients with type 2 diabetes (11,12) and maintaining stable body weight in type 1 diabetic patients (9,13,14). The mechanisms behind this favorable effect of detemir are unclear. Because of its pharmacologic properties, detemir might cross the blood-brain barrier faster and in higher quantities than other insulins and induce stronger effects on brain functions (15,16). Supporting this assumption, detemir in comparison with human insulin has been found to amplify the central nervous impact of hypoglycemia (16,17) and to exert stronger magnetoencephalographic effects in overweight humans (15) who display relative central nervous insulin resistance (18–20). To investigate the relevance of enhanced central nervous detemir action in the regulation of food intake, we assessed the effects of euglycemic intravenous infusion of detemir in comparison with human insulin on electroencephalography (EEG) direct current (DC) potentials that are sensitive to changes in systemic insulin concentrations (21) as well as on free-choice food intake. As we aimed at comparing the brain impact of peripherally equipotent doses of detemir and human insulin, care was taken for both infusions to induce similar effects on systemic glucose homeostasis.     

Influence of Insulin in the Ventromedial Hypothalamus on Pancreatic Glucagon Secretion In Vivo

OBJECTIVE

Insulin released by the β-cell is thought to act locally to regulate glucagon secretion. The possibility that insulin might also act centrally to modulate islet glucagon secretion has received little attention.

RESEARCH DESIGN AND METHODS

Initially the counterregulatory response to identical hypoglycemia was compared during intravenous insulin and phloridzin infusion in awake chronically catheterized nondiabetic rats. To explore whether the disparate glucagon responses seen were in part due to changes in ventromedial hypothalamus (VMH) exposure to insulin, bilateral guide cannulas were inserted to the level of the VMH and 8 days later rats received a VMH microinjection of either 1) anti-insulin affibody, 2) control affibody, 3) artificial extracellular fluid, 4) insulin (50 μU), 5) insulin receptor antagonist (S961), or 6) anti-insulin affibody plus a γ-aminobutyric acid A (GABA_A) receptor agonist muscimol, prior to a hypoglycemic clamp or under baseline conditions.

RESULTS

As expected, insulin-induced hypoglycemia produced a threefold increase in plasma glucagon. However, the glucagon response was fourfold to fivefold greater when circulating insulin did not increase, despite equivalent hypoglycemia and C-peptide suppression. In contrast, epinephrine responses were not altered. The phloridzin-hypoglycemia induced glucagon increase was attenuated (40%) by VMH insulin microinjection. Conversely, local VMH blockade of insulin amplified glucagon twofold to threefold during insulin-induced hypoglycemia. Furthermore, local blockade of basal insulin levels or insulin receptors within the VMH caused an immediate twofold increase in fasting glucagon levels that was prevented by coinjection to the VMH of a GABA_A receptor agonist.

CONCLUSIONS

Together, these data suggest that insulin''s inhibitory effect on α-cell glucagon release is in part mediated at the level of the VMH under both normoglycemic and hypoglycemic conditions.The pancreatic β-cell is the primary regulator of glucose homeostasis and glucagon secretion (1,2). In the presence of changes in blood glucose concentration, local changes in insulin together with zinc and/or γ-aminobutyric acid (GABA) release from the β-cell are thought to act in concert to regulate α-cell glucagon secretion (3,4). However, during hypoglycemia the central nervous system (5,6), and the VMH in particular, also plays a critical role in stimulating the release of glucagon and other counterregulatory hormones, via changes in the activity of specialized glucose-sensing neurons (7–10). Moreover, mice with selective loss of glutamate release in steroidogenic factor 1 neurons, which are expressed exclusively in the VMH, show a markedly impaired glucagon secretory response to acute hypoglycemia (11). These and other studies suggest that central regulation of pancreatic α-cell glucagon secretion may play a greater role than previously anticipated, at least during insulin-induced hypoglycemia (2,6–8).The fact that the VMH may directly influence α-cell glucagon release, and that glucose-sensing neurons in the VMH appear to use similar mechanisms for detecting fluctuations in glucose to the pancreatic α- and β-cells (7,8), suggested that insulin might play a similar regulatory role through a central mechanism. The possibility that insulin might exert a central effect is consistent with evidence that brain insulin levels mirror those in the circulation (12), that glucose-sensing neurons express insulin receptors, and that insulin alters the firing rate of these neurons (8,13–15). Moreover, insulin has been reported to act on the hypothalamus to regulate hepatic glucose production (16). However, studies in humans evaluating whether the level of circulating insulin during exogenous insulin administration influences counterregulatory hormone responses have yielded conflicting results. It has been reported that higher levels of plasma insulin may amplify (17,18) or inhibit (19–21) epinephrine responses during hypoglycemia. One study noted a significant effect of circulating insulin on accompanying glucagon responses, specifically an inhibitory action (20). Whether insulin acts centrally to influence glucose counterregulation is also uncertain. In animal studies, insulin delivered directly into the brain in very large doses has been reported to amplify counterregulatory hormone secretion (22–24) or to have no direct effect (25).This study was undertaken to determine whether insulin acts within the ventromedial hypothalamus (VMH) to modulate the secretion of glucoregulatory hormones. For this purpose, we either blocked insulin action or administered insulin to the VMH, during hypoglycemia produced in the presence or absence of elevated circulating insulin levels, respectively. In addition, we examined whether insulin acts locally in the VMH under basal conditions to regulate glucoregulatory hormone secretion in the absence of elevated levels of insulin. Our findings provide in vivo evidence for the first time that insulin acts within the VMH to influence the release of glucagon from α-cells both during hypoglycemia and under basal conditions.     

Central Resistin Overexposure Induces Insulin Resistance Through Toll-Like Receptor 4

Resistin promotes both inflammation and insulin resistance associated with energy homeostasis impairment. However, the resistin receptor and the molecular mechanisms mediating its effects in the hypothalamus, crucial for energy homeostasis control, and key insulin-sensitive tissues are still unknown. In the current study, we report that chronic resistin infusion in the lateral cerebral ventricle of normal rats markedly affects both hypothalamic and peripheral insulin responsiveness. Central resistin treatment inhibited insulin-dependent phosphorylation of insulin receptor (IR), AKT, and extracellular signal–related kinase 1/2 associated with reduced IR expression and with upregulation of suppressor of cytokine signaling-3 and phosphotyrosine phosphatase 1B, two negative regulators of insulin signaling. Additionally, central resistin promotes the activation of the serine kinases Jun NH₂-terminal kinase and p38 mitogen-activated protein kinase, enhances the serine phosphorylation of insulin receptor substrate-1, and increases the expression of the proinflammatory cytokine interleukin-6 in the hypothalamus and key peripheral insulin-sensitive tissues. Interestingly, we also report for the first time, to our knowledge, the direct binding of resistin to Toll-like receptor (TLR) 4 receptors in the hypothalamus, leading to the activation of the associated proinflammatory pathways. Taken together, our findings clearly identify TLR4 as the binding site for resistin in the hypothalamus and bring new insight into the molecular mechanisms involved in resistin-induced inflammation and insulin resistance in the whole animal.The hypothalamus integrates hormonal and metabolic signals to respond to energy body requirements through the regulation of energy homeostasis (1,2). The disruption of this regulatory loop promotes the onset of obesity, currently considered a worldwide epidemic. Obesity is linked to common metabolic diseases including insulin resistance, which constitutes a principal risk factor for type 2 diabetes (3–5). Accumulating evidence indicates that changes in adipose-secreted factors in obesity, including release of inflammatory cytokines, dramatically affect insulin sensitivity (3–7). Among these adipokines, resistin is described as a potential factor in obesity-mediated insulin resistance and type 2 diabetes. Resistin is a cysteine-rich 12.5-kDa polypeptide secreted by adipose tissue in rodents and by macrophages in humans (7,8), promoting inflammation and insulin resistance (9–12). Circulating resistin is increased in obese insulin-resistant rodents (6) and humans (7), and fasting decreases resistin mRNA expression (6,13). Peripheral administration or transgenic overexpression of resistin impairs insulin action in insulin-sensitive tissues (14–16). Conversely, deletion of the resistin gene or infusing of resistin antibodies or antisense oligonucleotides restores insulin responsiveness (6,17–19). In humans, recent studies have linked resistin to insulin resistance, atherosclerosis, and inflammation (12,20,21). More recently, it has been shown that resistin is expressed in the hypothalamus (22) and activates specific hypothalamic neurons (23). Central resistin also modulates glucose homeostasis, lipid metabolism, and food intake and impairs liver insulin sensitivity (24–27).Resistin also regulates the synthesis and secretion of key proinflammatory cytokines such as tumor necrosis factor-α, interleukin (IL)-6, and IL-12 in macrophages via a nuclear factor-κB–dependent pathway promoting insulin resistance (4,6,28,29). Moreover, recent studies have provided evidence for the contribution of Toll-like receptor-4 (TLR4) in the pathogenesis of obesity and insulin resistance. Saturated fatty acids activate both hypothalamic and peripheral TLR4 signaling, leading to proinflammatory cytokine production and endoplasmic reticulum stress (30–32). Conversely, TLR4 loss-of-function prevents saturated fatty acid-induced inflammation and insulin resistance (30,31,33). Resistin and TLR4 have been linked to a proinflammatory process in a human epithelial cell line in which resistin competes with lipopolysaccharide (LPS) for binding to TLR4 (34). Recently, an isoform of decorin was identified as a resistin receptor involved in white adipose tissue expansion (35). Another report has described that mouse resistin modulates glucose uptake and promotes adipogenesis in 3T3-L1 cells through the receptor tyrosine kinase-like orphan receptor-1 (36). In addition, in rheumatoid arthritis disease, resistin has been shown to use the IGF-1R signaling pathway (37). These data reveal a puzzling situation in which resistin could potentially interact with different receptors depending upon cellular model. However, in vivo at the neuronal level, the resistin receptor and its signaling have not yet been identified.Thus, we aimed to characterize hypothalamic resistin receptor and its signaling pathways involved in the impairment of insulin responsiveness. We show that resistin signals through TLR4 in the hypothalamus lead to the activation of Jun NH₂-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) signaling pathways by the recruitment of the adaptor proteins myeloid differentiating factor 88 (MyD88) and Toll/interleukin-1 receptor domain-containing adaptor protein (TIRAP), promoting overall inflammation. These findings reveal strong evidence for the direct role of hypothalamic TLR4 signal transduction in resistin-induced whole-body inflammation and insulin resistance.     

Roles of Insulin,Age, and Asymmetric Dimethylarginine on Nitric Oxide Synthesis In Vivo

We tested the effects of insulin on production of nitrous oxide (NO)-related substances (nitrites and nitrates [NOx]) after ¹⁵N-arginine intravenous infusion and on asymmetric dimethylarginine (ADMA) and symmetric dimethylarginine (SDMA) concentrations in conditions reportedly associated with altered NO availability, i.e., aging, hypertension, hypercholesterolemia, and type 2 diabetes mellitus (T2DM). A total of 26 male subjects (age 23–71 years, BMI 23–33 kg/m²), some of whom were affected by mixed pathologic features, were enrolled. NOx fractional synthesis rate (FSR) was lower in elderly (P < 0.015) and T2DM subjects (P < 0.03) than in matched control subjects. Hyperinsulinemia generally increased both NOx FSR and absolute synthesis rate (ASR) and reduced NOx, ADMA, and SDMA concentrations. Insulin sensitivity was impaired only in T2DM. With use of simple linear regression analysis across all subjects, age was inversely correlated with both NOx FSR (R² = 0.23, P < 0.015) and ASR (R² = 0.21, P < 0.02). NOx FSR inversely correlated with both ADMA and SDMA. With use of multiple regression analysis and various models, NOx FSR remained inversely associated with age and ADMA, whereas ASR was inversely associated with age and diabetes. No association with insulin sensitivity was found. We conclude that whole-body NOx production is decreased in aging and T2DM. Age, ADMA concentration, and T2DM, but not insulin resistance, appear as negative regulators of whole-body NOx production.Nitric oxide (NO) is a molecule with key functions in the cardiovascular, immune, and nervous systems (1). NO production is modified during physical exercise (2) and is altered in aging (3), diabetes (4,5), hypertension (6,7), and hypercholesterolemia (8,9). The understanding of the pathophysiological mechanism(s) underlying the altered NO metabolism in these diseases is important also for the development of therapeutic interventions aimed at improving vascular function.NO is synthesized from the guanidine group of arginine via the enzyme family NO synthases (NOS), which include three isoforms (10). One of these, the constitutive endothelial NOS (eNOS) enzyme, is stimulated by hormones (insulin and estrogens), physical exercise, and cofactors such as tetrahydrobiopterin (10). Conversely, it is inhibited by the endogenous methylarginines asymmetric dimethylarginine (ADMA), l-monomethylarginine (LMMA), and symmetric dimethylarginine (SDMA) (11,12). ADMA and LMMA inhibit both eNOS and arginine cellular transport, whereas SDMA inhibits arginine transport (11,12). Dimethylarginines are increasingly recognized as important markers or factors of endothelial dysfunction and cardiovascular disease (11). ADMA concentration is increased in diabetes, hypertension, hypercholesterolemia, and aging (11,13).Insulin is an important regulator of NO production, and insulin resistance is frequently associated with endothelial dysfunction (14). Insulin mediates both glucose entry into insulin-sensitive tissues and NO production via stimulation of protein kinase B/Akt (15), translocation of GLUT4 on cell membrane, and stimulation of eNOS (16). Since in insulin-resistant states insulin signaling is altered at the Akt level (17), any pathway downstream of Akt (including glucose metabolism and NOS activity) should be concomitantly affected. Furthermore, in many insulin-resistant states, ADMA levels are increased, too (18), and they may thus interfere with the insulin signaling on NOS activity and NO production.The relative roles of insulin sensitivity and of ADMA and SDMA concentrations, as well as of other potential interfering factors such as age, on NO production in vivo have never been comprehensively investigated. Therefore, this study was designed to measure whole-body insulin sensitivity (i.e., the insulin-stimulated glucose disposal), ADMA and SDMA concentrations, and basal and insulin-stimulated NO production (19) in human subjects over a wide range of insulin sensitivity and age either healthy or affected by hypertension, hypercholesterolemia, or type 2 diabetes mellitus (T2DM). NO production was determined by a precursor product, isotope dilution technique (5). A key target of this study was also to examine the possible correlates between production of nitrites and nitrates (NOx) and ADMA, SDMA, insulin sensitivity, and age.