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.     

Muscle Contraction, but Not Insulin, Increases Microvascular Blood Volume in the Presence of Free Fatty Acid�CInduced Insulin Resistance

OBJECTIVE

Insulin and contraction each increase muscle microvascular blood volume (MBV) and glucose uptake. Inhibiting nitric oxide synthase blocks insulin''s but not contraction''s effects. We examined whether contraction could augment the MBV increase seen with physiologic hyperinsulinemia and whether free fatty acid (FFA)-induced insulin resistance differentially affects contraction- versus insulin-mediated increases in MBV.

RESEARCH DESIGN AND METHODS

Rats were fasted overnight. Plasma FFAs were increased by intralipid/heparin infusion (3 h), insulin was increased with a euglycemic clamp (3 mU · min⁻¹ · kg⁻¹), and hindlimb muscle contraction was electrically stimulated. Muscle MBV was measured using contrast-enhanced ultrasound. Insulin transport into muscle was measured using ¹²⁵I-insulin. BQ-123 (0.4 mg/h) was used to block the endothelin-1 (ET-1) receptor A.

RESULTS

Superimposing contraction on physiologic hyperinsulinemia increased MBV within 10 min by 37 and 67% for 0.1 or 1 Hz, respectively (P < 0.01). FFA elevation alone did not affect MBV, whereas 0.1 Hz stimulation doubled MBV (P < 0.05) and increased muscle insulin uptake (P < 0.05) despite high FFA. Physiologic hyperinsulinemia during FFA elevation paradoxically decreased MBV (P < 0.05). This MBV decrease was reversed by either 0.1 Hz contraction or ET-1 receptor A antagonism, and the combination raised MBV above basal.

CONCLUSIONS

Contraction recruits microvasculature beyond that seen with physiologic hyperinsulinemia by a distinct mechanism that is not blocked by FFA-induced vascular insulin resistance. The paradoxical MBV decline seen with insulin plus FFA may result from differential inhibition of insulin-stimulated nitric oxide–dependent vasodilation relative to ET-1 vasoconstriction. Our results implicate ET-1 as a potential mediator of FFA-induced vascular insulin resistance.Insulin delivery to muscle interstitium is reported to be rate limiting for overall muscle insulin action (1,2). Insulin promotes its own access to muscle interstitium by increasing blood flow (3), by recruiting microvasculature (4,5) to expand the endothelial transporting surface available, and perhaps by also stimulating its own endothelial transport (6). Insulin''s entry to muscle interstitium is delayed in insulin-resistant states (7). This implicates insulin''s vascular actions as a significant regulator of overall insulin action in muscle.Elevated plasma concentrations of free fatty acids (FFAs), as occur with obesity and type 2 diabetes, increase cellular lipid concentrations and are associated with insulin resistance in skeletal muscle, liver, and fat (8,9). Experimentally, increased dietary fat (10–12) or acute infusion of a lipid emulsion induces insulin resistance (13–16). Increased intramyocellular lipid content in the context of obesity and type 2 diabetes could be one factor that contributes to muscle insulin resistance. Postprandially or in response to a euglycemic-insulin clamp, plasma (FFA) falls in insulin-sensitive individuals (17–19). This response is impaired in states of insulin resistance (8,17,19,20).Insulin also increases muscle blood flow and recruits microvasculature in both humans (21–24) and animals (4,25–27); both processes are inhibited by nitric oxide synthases (NOS) blockade (27). Raising plasma FFAs initiates hemodynamic effects that include decreased compliance, increased blood pressure and heart rate, and increased vascular resistance (28–31). Raising plasma (FFA) blunts insulin''s NOS-dependent effects to mediate increases in both muscle microvascular blood volume (MBV) and glucose uptake (14,32,33). Thus, FFAs exert acute vascular as well as metabolic actions.Insulin (34) and muscle contraction can each increase MBV and total flow in skeletal muscle (35–37). In addition, Wheatley et al. (38) observed that in the Zucker rat, insulin-mediated increases in MBV are blunted, but contraction-induced increases in MBV persisted. This suggests that exercise might recruit microvasculature via a mechanism that is distinct from that of insulin. Supporting this, we have recently shown that like insulin, brief low-frequency isometric contraction of the rat hindlimb (0.1 Hz, 10 min) robustly increases MBV without any observed increase in total femoral blood flow (FBF) and, unlike insulin''s effect, this process is nitric oxide (NO)-independent (39).In this study, we addressed whether 1) low-frequency contraction enhances muscle MBV and ³H-2-deoxyglucose (³H-2-DG) uptake beyond the effect of physiologic hyperinsulinemia; 2) lipid infusion differentially affects contraction- versus insulin-mediated increases in MBV; and 3) lipid infusion blunts combined insulin- and contraction-mediated effects on MBV.     

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.     

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.     

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.     

Pigment epithelium-derived factor regulates lipid metabolism via adipose triglyceride lipase

OBJECTIVE

Pigment epithelium–derived factor (PEDF) is an adipocyte-secreted factor involved in the development of insulin resistance in obesity. Previous studies have identified PEDF as a regulator of triacylglycerol metabolism in the liver that may act through adipose triglyceride lipase (ATGL). We used ATGL^−/− mice to determine the role of PEDF in regulating lipid and glucose metabolism.

RESEARCH DESIGN AND METHODS

Recombinant PEDF was administered to ATGL^−/− and wild-type mice, and whole-body energy metabolism was studied by indirect calorimetry. Adipose tissue lipolysis and skeletal muscle fatty acid metabolism was determined in isolated tissue preparations. Muscle lipids were assessed by electrospray ionization–tandem mass spectrometry. Whole-body insulin sensitivity and skeletal muscle glucose uptake were assessed.

RESULTS

PEDF impaired the capacity to adjust substrate selection, resulting in a delayed diurnal decline in the respiratory exchange ratio, and suppressed daily fatty acid oxidation. PEDF enhanced adipocyte lipolysis and triacylglycerol lipase activity in skeletal muscle. Muscle fatty acid uptake and storage were unaffected, whereas fatty acid oxidation was impaired. These changes in lipid metabolism were abrogated in ATGL^−/− mice and were not attributable to hypothalamic actions. ATGL^−/− mice were also refractory to PEDF-mediated insulin resistance, but this was not related to changes in lipid species in skeletal muscle.

CONCLUSIONS

The results are the first direct demonstration that 1) PEDF influences systemic fatty acid metabolism by promoting lipolysis in an ATGL-dependent manner and reducing fatty acid oxidation and 2) ATGL is required for the negative effects of PEDF on insulin action.Adipose tissue biology is markedly affected by obesity, and its endocrine role has been extensively investigated. Studies using proteomic approaches estimate that ∼90–260 individual proteins are released by adipocytes (1–4). Several adipose-secreted factors that are elevated in obesity are implicated in the pathogenesis of metabolic dysfunction and insulin resistance, including tumor necrosis factor-α (5), interleukin-6 (6), resistin (7), and retinol-binding protein 4 (8). These data support a major role for adipose tissue in regulating whole-body fatty acid metabolism and insulin action.Pigment epithelium–derived factor (PEDF, SerpinF1) is upregulated in individuals with the metabolic syndrome (9,10) and patients with type 2 diabetes (11,12). Although PEDF is best known for its antiangiogenic and neuroprotective functions (13), recent work has implicated PEDF in the development of obesity-related insulin resistance (3,14). PEDF induced proinflammatory signaling, increased adipocyte lipolysis, and promoted lipid accumulation in muscle and liver that was associated with insulin resistance (3).PEDF is thought to exert its biologic actions by binding to a cell surface receptor (15,16). A recently identified cell surface receptor that possesses phospholipase activity was reported in retinal pigment epithelial cells (17). Surprisingly, this putative PEDF receptor was reported to be adipose triglyceride lipase (ATGL), a highly conserved triacylglycerol lipase that is critical for the maintenance of lipid and glucose homeostasis (18–23). Others have shown that recombinant PEDF is transported into cells and colocalizes with ATGL at lipid droplets, and coimmunoprecipitation studies indicate that ATGL interacts with PEDF (24). PEDF-deficient mice have hepatic steatosis, and some evidence supports the premise that the PEDF–ATGL nexus may be important in conveying PEDF’s modulation of lipid metabolism (24).In the current study, we used pharmacologic and genetic models to examine the role of PEDF in systemic fatty acid metabolism. We tested the hypothesis that ATGL is required for the metabolic actions of PEDF.     

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.     

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.     

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.     

Lipoprotein Subfraction Cholesterol Distribution Is Proatherogenic in Women With Type 1 Diabetes and Insulin Resistance

OBJECTIVE

Individuals with type 1 diabetes have a less atherogenic fasting lipid profile than those without diabetes but paradoxically have increased rates of cardiovascular disease (CVD). We investigated differences in lipoprotein subfraction cholesterol distribution and insulin resistance between subjects with and without type 1 diabetes to better understand the etiology of increased CVD risk.

RESEARCH DESIGN AND METHODS

Fast protein liquid chromatography was used to fractionate lipoprotein cholesterol distribution in a substudy of the Coronary Artery Calcification in Type 1 Diabetes (CACTI) study (n = 82, age 46 ± 8 years, 52% female, 49% with type 1 diabetes for 23 ± 8 years). Insulin resistance was assessed by a hyperinsulinemic-euglycemic clamp.

RESULTS

Among men, those with type 1 diabetes had less VLDL and more HDL cholesterol than control subjects (P < 0.05), but among women, those with diabetes had a shift in cholesterol to denser LDL, despite more statin use. Among control subjects, men had more cholesterol distributed as VLDL and LDL but less as HDL than women; however, among those with type 1 diabetes, there was no sex difference. Within sex and diabetes strata, a more atherogenic cholesterol distribution by insulin resistance was seen in men with and without diabetes, but only in women with type 1 diabetes.

CONCLUSIONS

The expected sex-based less atherogenic lipoprotein cholesterol distribution was not seen in women with type 1 diabetes. Moreover, insulin resistance was associated with a more atherogenic lipoprotein cholesterol distribution in all men and in women with type 1 diabetes. This lipoprotein cholesterol distribution may contribute to sex-based differences in CVD in type 1 diabetes.Cardiovascular disease (CVD) is the major cause of mortality in type 1 diabetes, and, in addition to glycemic control and blood pressure, dyslipidemia is an important and modifiable CVD risk factor (1–8). Curiously, despite higher rates of CVD in type 1 diabetes, including a relative loss of sex protection in women with type 1 diabetes (9–11), and the role of dyslipidemia as a determinant of CVD (8), individuals with type 1 diabetes have similar or less atherogenic lipid profiles than age-, sex-, and BMI-matched nondiabetic subjects (5,12). This paradox is well known (13), but few data exist to explain this phenomenon. Lipoprotein differences in type 1 diabetes have been investigated in the Diabetes Control and Complications Trial (DCCT)/Epidemiology of Diabetes Interventions and Complications (EDIC) study but lack a nondiabetic comparison group (14–17).Furthermore, it has been known for nearly 40 years that insulin resistance is a prominent CVD risk factor in type 1 diabetes (18,19), and recent studies have demonstrated increased insulin resistance in people with type 1 diabetes compared with age-, sex-, and BMI-matched nondiabetic control subjects (20,21). The hyperinsulinemic-euglycemic clamp is considered the “gold standard” method for measuring insulin resistance in a wide variety of circumstances (22), especially in individuals with type 1 diabetes, in whom prediction models of insulin resistance that rely on glucose and insulin levels cannot be used. The effect of insulin resistance on lipoproteins has been investigated in individuals with type 2 diabetes and in those without diabetes (23–25), but to our knowledge, the effect of insulin resistance on lipoproteins has not been investigated in individuals with type 1 diabetes.Therefore, to examine beyond the ability of the standard fasting lipid profile to assess CVD risk, we investigated differences in lipoprotein subfraction cholesterol distribution between subjects with and without type 1 diabetes and how insulin resistance affects this distribution. We hypothesized that differences would exist in lipoprotein subfraction cholesterol distribution by type 1 diabetes status, and, furthermore, we hypothesized that subjects with more insulin resistance as measured by a hyperinsulinemic-euglycemic clamp would have a more atherogenic lipoprotein subfraction cholesterol distribution.     

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).     

AMP-activated Protein Kinase ��2 Subunit Is Required for the Preservation of Hepatic Insulin Sensitivity by n-3 Polyunsaturated Fatty Acids

OBJECTIVE

The induction of obesity, dyslipidemia, and insulin resistance by high-fat diet in rodents can be prevented by n-3 long-chain polyunsaturated fatty acids (LC-PUFAs). We tested a hypothesis whether AMP-activated protein kinase (AMPK) has a role in the beneficial effects of n-3 LC-PUFAs.

RESEARCH DESIGN AND METHODS

Mice with a whole-body deletion of the α2 catalytic subunit of AMPK (AMPKα2^−/−) and their wild-type littermates were fed on either a low-fat chow, or a corn oil-based high-fat diet (cHF), or a cHF diet with 15% lipids replaced by n-3 LC-PUFA concentrate (cHF+F).

RESULTS

Feeding a cHF diet induced obesity, dyslipidemia, hepatic steatosis, and whole-body insulin resistance in mice of both genotypes. Although cHF+F feeding increased hepatic AMPKα2 activity, the body weight gain, dyslipidemia, and the accumulation of hepatic triglycerides were prevented by the cHF+F diet to a similar degree in both AMPKα2^−/− and wild-type mice in ad libitum-fed state. However, preservation of hepatic insulin sensitivity by n-3 LC-PUFAs required functional AMPKα2 and correlated with the induction of adiponectin and reduction in liver diacylglycerol content. Under hyperinsulinemic-euglycemic conditions, AMPKα2 was essential for preserving low levels of both hepatic and plasma triglycerides, as well as plasma free fatty acids, in response to the n-3 LC-PUFA treatment.

CONCLUSIONS

Our results show that n-3 LC-PUFAs prevent hepatic insulin resistance in an AMPKα2-dependent manner and support the role of adiponectin and hepatic diacylglycerols in the regulation of insulin sensitivity. AMPKα2 is also essential for hypolipidemic and antisteatotic effects of n-3 LC-PUFA under insulin-stimulated conditions.Naturally occurring n-3 long-chain polyunsaturated fatty acids (LC-PUFAs)—namely, eicosapentaenoic acid (20:5n-3) and docosahexaenoic acid (22:6n-3)—which are abundant in sea fish, act as hypolipidemics, reduce cardiac events, and decrease the progression of atherosclerosis [reviewed in refs (1,2).]. Studies of obese humans have also demonstrated a reduction in adiposity after n-3 LC-PUFA supplementation (3,4). In rodents fed a high-fat diet, n-3 LC-PUFAs efficiently prevented the development of obesity, hepatic steatosis, and dyslipidemia (5–8), as well as impaired glucose tolerance (8–10). However, in diabetic patients, n-3 LC-PUFAs appear to have little effect on glycemic control (3,11,12).The hypolipidemic and antiobesity effects of n-3 LC-PUFAs depend on both the suppression of lipogenesis and the increase in fatty acid oxidation in several tissues, including the liver (13,14), adipose tissue (6), and intestine (15). This metabolic switch may reduce the accumulation of toxic fatty acid derivatives, while protecting insulin signaling in the liver and muscle (9,10,16). Our previous work has documented that the preservation of whole-body insulin sensitivity by n-3 LC-PUFAs in mice fed a high-fat diet mainly reflects improved hepatic insulin sensitivity (8). The effects of n-3 LC-PUFAs and their active metabolites (17,18) are mediated by peroxisome proliferator-activated receptors (PPAR), with PPAR-α and PPAR-δ (-β) being the main targets (14,16), although PPAR-γ, liver X receptor-α, hepatic nuclear factor-4, sterol regulatory element binding protein-1c (SREBP-1c) and carbohydrate-responsive element-binding protein are also involved (16,19–21).It has been demonstrated that n-3 LC-PUFAs enhanced AMP-activated protein kinase (AMPK) activity in the liver (22), intestine (23), and adipose tissue (18,24). AMPK is a heterotrimeric protein consisting of a catalytic α-subunit and regulatory β- and γ-subunits, with multiple isoforms identified for each subunit [α1, α2, β1, β2, γ1, γ2, and γ3; reviewed in ref (25)]. Experiments using whole-body AMPKα2 null [AMPKα2^−/−; ref (26)] mice showed the importance of the AMPKα2 subunit for whole-body insulin action, while liver-specific AMPKα2 knockout mice (27) as well as adenovirus-mediated activation of AMPKα2 in the liver (28) implicated the hepatic AMPKα2 isoform in the suppression of hepatic glucose production and maintenance of fasting blood glucose levels. Furthermore, AMPK controls metabolic fluxes in response to changing cellular energy levels, namely, the partitioning between lipid oxidation and lipogenesis (29,30).We hypothesized that the effects of n-3 LC-PUFA on insulin sensitivity and lipid metabolism in mice fed an obesogenic high-fat diet require a functional AMPKα2 isoform. To test this hypothesis in vivo, AMPKα2^−/− and wild-type mice were fed either a low-fat chow diet (Chow), a corn oil-based high-fat (cHF) diet, or cHF diet in which 15% of the lipids were replaced by n-3 LC-PUFA concentrate (cHF+F). Our results demonstrate an AMPKα2-dependent action of n-3 LC-PUFAs, in 1) the preservation of hepatic and muscle insulin sensitivity; 2) the changes in hepatic diacylglycerol content and composition; and 3) the antisteatotic effect in the liver and hypolipidemic effect under insulin-stimulated conditions, such as during hyperinsulinemic-euglycemic clamp, but not when the organism depends on lipids as substrates.     

Glucagon-Like Peptide 1/Glucagon Receptor Dual Agonism Reverses Obesity in Mice

OBJECTIVE

Oxyntomodulin (OXM) is a glucagon-like peptide 1 (GLP-1) receptor (GLP1R)/glucagon receptor (GCGR) dual agonist peptide that reduces body weight in obese subjects through increased energy expenditure and decreased energy intake. The metabolic effects of OXM have been attributed primarily to GLP1R agonism. We examined whether a long acting GLP1R/GCGR dual agonist peptide exerts metabolic effects in diet-induced obese mice that are distinct from those obtained with a GLP1R-selective agonist.

RESEARCH DESIGN AND METHODS

We developed a protease-resistant dual GLP1R/GCGR agonist, DualAG, and a corresponding GLP1R-selective agonist, GLPAG, matched for GLP1R agonist potency and pharmacokinetics. The metabolic effects of these two peptides with respect to weight loss, caloric reduction, glucose control, and lipid lowering, were compared upon chronic dosing in diet-induced obese (DIO) mice. Acute studies in DIO mice revealed metabolic pathways that were modulated independent of weight loss. Studies in Glp1r^−/− and Gcgr^−/− mice enabled delineation of the contribution of GLP1R versus GCGR activation to the pharmacology of DualAG.

RESULTS

Peptide DualAG exhibits superior weight loss, lipid-lowering activity, and antihyperglycemic efficacy comparable to GLPAG. Improvements in plasma metabolic parameters including insulin, leptin, and adiponectin were more pronounced upon chronic treatment with DualAG than with GLPAG. Dual receptor agonism also increased fatty acid oxidation and reduced hepatic steatosis in DIO mice. The antiobesity effects of DualAG require activation of both GLP1R and GCGR.

CONCLUSIONS

Sustained GLP1R/GCGR dual agonism reverses obesity in DIO mice and is a novel therapeutic approach to the treatment of obesity.Obesity is an important risk factor for type 2 diabetes, and ∼90% of patients with type 2 diabetes are overweight or obese (1). Among new therapies for type 2 diabetes, peptidyl mimetics of the gut-derived incretin hormone glucagon-like peptide 1 (GLP-1) stimulate insulin biosynthesis and secretion in a glucose-dependent manner (2,3) and cause modest weight loss in type 2 diabetic patients. The glucose-lowering and antiobesity effects of incretin-based therapies for type 2 diabetes have prompted evaluation of the therapeutic potential of other glucagon-family peptides, in particular oxyntomodulin (OXM). The OXM peptide is generated by post-translational processing of preproglucagon in the gut and is secreted postprandially from l-cells of the jejuno-ileum together with other preproglucagon-derived peptides including GLP-1 (4,5). In rodents, OXM reduces food intake and body weight, increases energy expenditure, and improves glucose metabolism (6–8). A 4-week clinical study in obese subjects demonstrated that repeated subcutaneous administration of OXM was well tolerated and caused significant weight loss with a concomitant reduction in food intake (9). An increase in activity-related energy expenditure was also noted in a separate study involving short-term treatment with the peptide (10).OXM activates both, the GLP-1 receptor (GLP1R) and glucagon receptor (GCGR) in vitro, albeit with 10- to 100-fold reduced potency compared with the cognate ligands GLP-1 and glucagon, respectively (11–13). It has been proposed that OXM modulates glucose and energy homeostasis solely by GLP1R agonism, because its acute metabolic effects in rodents are abolished by coadministration of the GLP1R antagonist exendin(9–39) and are not observed in Glp1r^−/− mice (7,8,14,15). Other aspects of OXM pharmacology, however, such as protective effects on murine islets and inhibition of gastric acid secretion appear to be independent of GLP1R signaling (14). In addition, pharmacological activation of GCGR by glucagon, a master regulator of fasting metabolism (16), decreases food intake in rodents and humans (17–19), suggesting a potential role for GCGR signaling in the pharmacology of OXM. Because both OXM and GLP-1 are labile in vivo (T_1/2 ∼12 min and 2–3 min, respectively) (20,21) and are substrates for the cell surface protease dipeptidyl peptidase 4 (DPP-4) (22), we developed two long-acting DPP-4–resistant OXM analogs as pharmacological agents to better investigate the differential pharmacology and therapeutic potential of dual GLP1R/GCGR agonism versus GLP1R-selective agonism. Peptide DualAG exhibits in vitro GLP1R and GCGR agonist potency comparable to that of native OXM and is conjugated to cholesterol via a Cys sidechain at the C-terminus for improved pharmacokinetics. Peptide GLPAG differs from DualAG by only one residue (Gln³→Glu) and is an equipotent GLP1R agonist, but has no significant GCGR agonist or antagonist activity in vitro. The objective of this study was to leverage the matched GLP1R agonist potencies and pharmacokinetics of peptides DualAG and GLPAG in comparing the metabolic effects and therapeutic potential of a dual GLP1R/GCGR agonist with a GLP1R-selective agonist in a mouse model of obesity.     

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).     

Perilipin 2 Improves Insulin Sensitivity in Skeletal Muscle Despite Elevated Intramuscular Lipid Levels

Type 2 diabetes is characterized by excessive lipid storage in skeletal muscle. Excessive intramyocellular lipid (IMCL) storage exceeds intracellular needs and induces lipotoxic events, ultimately contributing to the development of insulin resistance. Lipid droplet (LD)–coating proteins may control proper lipid storage in skeletal muscle. Perilipin 2 (PLIN2/adipose differentiation–related protein [ADRP]) is one of the most abundantly expressed LD-coating proteins in skeletal muscle. Here we examined the role of PLIN2 in myocellular lipid handling and insulin sensitivity by investigating the effects of in vitro PLIN2 knockdown and in vitro and in vivo overexpression. PLIN2 knockdown decreased LD formation and triacylglycerol (TAG) storage, marginally increased fatty-acid (FA) oxidation, and increased incorporation of palmitate into diacylglycerols and phospholipids. PLIN2 overexpression in vitro increased intramyocellular TAG storage paralleled with improved insulin sensitivity. In vivo muscle-specific PLIN2 overexpression resulted in increased LD accumulation and blunted the high-fat diet–induced increase in protein content of the subunits of the oxidative phosphorylation (OXPHOS) chain. Diacylglycerol levels were unchanged, whereas ceramide levels were increased. Despite the increased IMCL accumulation, PLIN2 overexpression improved skeletal muscle insulin sensitivity. We conclude that PLIN2 is essential for lipid storage in skeletal muscle by enhancing the partitioning of excess FAs toward TAG storage in LDs, thereby blunting lipotoxicity-associated insulin resistance.Lipid droplets (LDs) serve an essential function in eukaryotic cells. Accordingly, intracellular lipid levels need to be tightly controlled. Indeed, inappropriate intracellular lipid storage leads to impaired cellular function. In obesity, lipids will overflow into the circulation as a result of lack of storage capacity in adipose tissue, and, as a consequence, lipids may accumulate ectopically in tissues, including skeletal muscle (intramyocellular lipids [IMCLs]). This ectopic fat storage exceeds intracellular demand and may result in lipotoxic events, including the development of insulin resistance (1,2). Paradoxically, IMCL is increased in both endurance-trained athletes and type 2 diabetic patients (3,4), indicating that ectopic lipid accumulation per se does not induce insulin resistance.Thus far, explanations for this athlete’s paradox have focused on lipid turnover, oxidative capacity, and levels of lipid intermediates (5–8). Interestingly, one exercise session was shown to prevent lipid-induced insulin resistance by partitioning more fatty acids (FAs) toward triacylglycerol (TAG) synthesis in skeletal muscle (9). Therefore, increasing the depot for TAG storage might improve insulin sensitivity. Intracellular TAG is stored in LDs, which are increasingly recognized as dynamic organelles. They are composed of a neutral lipid core containing TAG, diacylglycerol (DAG), cholesterolesters, retinol esters, and free cholesterol (10) surrounded by a phospholipid monolayer (11) and a protein coat, composed of a variety of LD-coating proteins (12). Accumulating evidence suggests that LD-coating proteins mediate LD dynamics, including LD synthesis, growth and fusion, intracellular transport, organelle interactions, and breakdown and lipolysis (13,14).The best-characterized family of LD-coating proteins is the perilipin (PLIN) protein family, including PLIN1, PLIN2 (adipophilipin and adipose differentiation–related protein [ADRP]), PLIN3 (tail-interacting protein, 47 kDa [TIP47]), PLIN4 (adipocyte protein S3-12), and PLIN5 (OXPAT, lipid droplet storage protein 5 [LSDP5]). Whereas PLIN1 expression is restricted to adipose tissue, where it plays a crucial role in the control of storage and degradation of LDs (15,16), PLIN2 is expressed in several tissues, including liver, small intestine, and skeletal muscle (17,18). PLIN2 in skeletal muscle was previously shown to colocalize with IMCL (19). Interestingly, skeletal muscle Plin2 gene expression was shown to be lower in patients with type 2 diabetes versus obese control subjects (20). Furthermore, weight loss as well as metformin treatment, both resulting in lower IMCL levels (21,22), were demonstrated to increase skeletal muscle PLIN2 levels in parallel with improved insulin sensitivity (23). PLIN2 may be involved in the protection against lipotoxicity by facilitating efficient IMCL storage in the form of TAG. However, loss- and gain-of-function studies to characterize PLIN2 function in skeletal muscle, required to obtain more functional insight into the role of PLIN2 in muscle, have not been performed to date. Here, we aimed to examine the role of PLIN2 in myocellular fat accumulation, lipotoxicity, and insulin sensitivity.