RESEARCH DESIGN AND METHODS— We studied 13 diabetic Indians, 13 nondiabetic Indians, and 13 nondiabetic Northern European Americans who were matched for age, BMI, and sex. The primary comparisons were insulin sensitivity by hyperinsulinemic-euglycemic clamp and skeletal muscle mitochondrial capacity for oxidative phosphorylation (OXPHOS) by measuring mitochondrial DNA copy number (mtDNA), OXPHOS gene transcripts, citrate synthase activity, and maximal mitochondrial ATP production rate (MAPR). Other factors that may cause insulin resistance were also measured.

RESULTS— The glucose infusion rates required to maintain identical glucose levels during the similar insulin infusion rates were substantially lower in diabetic Indians than in the nondiabetic participants (P < 0.001), and they were lower in nondiabetic Indians than in nondiabetic Northern European Americans (P < 0.002). mtDNA (P < 0.02), OXPHOS gene transcripts (P < 0.01), citrate synthase, and MAPR (P < 0.03) were higher in Indians irrespective of their diabetic status. Intramuscular triglyceride, C-reactive protein, interleukin-6, and tumor necrosis factor- concentrations were higher, whereas adiponectin concentrations were lower in diabetic Indians.

CONCLUSIONS— Despite being more insulin resistant, diabetic Indians had similar muscle OXPHOS capacity as nondiabetic Indians, demonstrating that diabetes per se does not cause mitochondrial dysfunction. Indians irrespective of their diabetic status had higher OXPHOS capacity than Northern European Americans, although Indians were substantially more insulin resistant, indicating a dissociation between mitochondrial dysfunction and insulin resistance.

RESEARCH DESIGN AND METHODS— Male CD-1 mice were fed from conception to adulthood with either a high soy–containing diet or a soy-free diet. Serum levels of circulating isoflavones, ghrelin, leptin, free fatty acids, triglycerides, and cholesterol were quantified. Tissue samples were analyzed by quantitative RT-PCR and Western blotting to investigate changes of gene expression and phosphorylation state of key metabolic proteins. Glucose and insulin tolerance tests and euglycemic-hyperinsulinemic clamp were used to assess changes in insulin sensitivity and glucose uptake. In addition, insulin secretion was determined by in situ pancreas perfusion.

RESULTS— In peripheral tissues of soy-fed mice, especially in white adipose tissue, phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase was increased, and expression of genes implicated in peroxisomal fatty acid oxidation and mitochondrial biogenesis was upregulated. Soy-fed mice also showed reduced serum insulin levels and pancreatic insulin content and improved insulin sensitivity due to increased glucose uptake into skeletal muscle. Thus, mice fed with a soy-rich diet have improved adipose and glucose metabolism.

CONCLUSIONS— Dietary soy could prove useful to prevent obesity and associated disorders. Activation of the AMPK pathway by dietary soy is likely involved and may mediate the beneficial effects of dietary soy in peripheral tissues.

RESEARCH DESIGN AND METHODS— Twelve women (BMI 29.6 ± 6.6 kg/m²) received an identical, intravenous bolus dose of [1-¹⁴C]oleate followed by timed subcutaneous fat biopsies (abdominal and femoral) and then omental fat biopsy during tubal ligation surgery. Regional fat masses were assessed by combining dual-energy X-ray absorptiometry and computed tomography scanning. Separately, we assessed the fraction of FFA tracer entering VLDL-TG over the time representing the delay in collecting omental fat.

RESULTS— Site-specific fat specific activity (SA) (dpm/g lipid) decreased as a function of fat mass in both upper-body subcutaneous (UBSQ) and visceral fat depots. These patterns are consistent with dilution of a relatively fixed amount of FFA tracer within progressively greater amounts of fat. Interestingly, femoral SA did not vary as a function of lower-body subcutaneous (LBSQ) fat mass. [1-¹⁴C]oleate storage per million LBSQ adipocytes was positively associated with LBSQ fat mass, but no significant relationships were observed in UBSQ or visceral fat depot. The fraction of [1-¹⁴C]oleate stored in UBSQ, LBSQ, and visceral fat was 6.7 ± 3.2, 4.9 ± 3.4, and 1.0 ± 0.3%, respectively. Only ~4% of the tracer traversed VLDL-TG over 9.5 h.

CONCLUSIONS— The increase in FFA tracer storage per adipocyte as a function of LBSQ fat mass implies that LBSQ adipocytes, in contrast to UBSQ and omental adipocytes, store more FFA in women with greater adiposity. The direct FFA storage pathway might play a role in favoring lower-body fat accumulation in women.

RESEARCH DESIGN AND METHODS— AS160 mRNA expression was measured in mouse and human islets and fluorescence-activated cell sorted β-cells and compared in islets from control subjects versus individuals with type 2 diabetes. For knockdown experiments, transformed mouse insulin-secreting MIN6B1 cells were transfected with pSUPER-GFP plasmid encoding a small hairpin RNA against insulin receptor substrate (IRS)-2, AS160, or a negative control. Primary mouse islet cells were transfected with AS160 small interfering RNA.

RESULTS— AS160 was expressed in human and mouse pancreatic β-cells and phosphorylated after glucose stimulation. AS160 mRNA expression was downregulated in pancreatic islets from individuals with type 2 diabetes. In MIN6B1 cells, glucose induced phosphorylation of Akt and AS160, and this was mediated by insulin receptor/IRS-2/phosphatidylinositol 3-kinase independently of changes in cytosolic Ca²⁺. Knockdown of AS160 resulted in increased basal insulin secretion, whereas glucose-stimulated insulin release was abolished. Furthermore, β-cells with decreased AS160 showed increased apoptosis and loss of glucose-induced proliferation.

CONCLUSIONS— This study shows for the first time that AS160, previously recognized as a key player in insulin signaling in skeletal muscle and adipose tissue, is also a major effector of protein kinase B/Akt signaling in the β-cell.

RESEARCH DESIGN AND METHODS— Isolated human, rat, and mouse islets and the rat insulin-secreting INS-1E cells were incubated with ex-4 in the presence or absence of IL-1β. JNK activity was assessed by solid-phase JNK kinase assay and quantification of c-Jun expression. Cell apoptosis was determined by scoring cells displaying pycnotic nuclei.

RESULTS— Ex-4 inhibited induction of the JNK pathway elicited by IL-1β. This effect was mimicked with the use of cAMP-raising agents isobutylmethylxanthine and forskolin and required activation of the protein kinase A. Inhibition of the JNK pathway by ex-4 or IBMX and forskolin was concomitant with a rise in the levels of islet-brain 1 (IB1), a potent blocker of the stress-induced JNK pathway. In fact, ex-4 as well as IBMX and forskolin induced expression of IB1 at the promoter level through cAMP response element binding transcription factor 1. Suppression of IB1 levels with the use of RNA interference strategy impaired the protective effects of ex-4 against apoptosis induced by IL-1β.

CONCLUSIONS— The data establish the requirement of IB1 in the protective action of ex-4 against apoptosis elicited by IL-1β and highlight the GLP-1 mimetics as new potent inhibitors of the JNK signaling induced by cytokines.

RESEARCH DESIGN AND METHODS— 3T3-L1 adipocytes were exposed to HNE and/or infected with control adenovirus or adenovirus expressing FALDH.

RESULTS— Treatment of 3T3-L1 adipocytes with HNE at nontoxic concentrations leads to a pronounced decrease in insulin receptor substrate (IRS)-1/-2 proteins and in insulin-induced IRS and insulin receptor β (IRβ) tyrosine phosphorylation. Remarkably, we detect increased binding of HNE to IRS-1/-2–generating HNE-IRS adducts, which likely impair IRS function and favor their degradation. Phosphatidylinositol 3-kinase and protein kinase B activities are also downregulated upon HNE treatment, resulting in blunted metabolic responses. Moreover, FALDH, by reducing adduct formation, partially restores HNE-generated decrease in insulin-induced IRS-1 tyrosine phosphorylation and metabolic responses. Moreover, rosiglitazone could have an antioxidant effect because it blocks the noxious HNE action on IRS-1 by increasing FALDH gene expression. Collectively, our data show that FALDH improves insulin action in HNE-treated 3T3-L1 adipocytes.

CONCLUSION— Oxidative stress induced by reactive aldehydes, such as HNE, is implicated in the development of insulin resistance in 3T3-L1 adipocytes, which is alleviated by FALDH. Hence, detoxifying enzymes could play a crucial role in blocking progression of insulin resistance to diabetes.

RESEARCH DESIGN AND METHODS— We generated a strain of mice carrying a targeted disruption of Stat3 gene in the liver (L-Stat3^–/– mice). Hepatocytes of the L-Stat3^–/– mice were isolated to establish cell lines for mechanistic studies. Nuclear translocation and DNA-protein interaction of STAT3 was analyzed with immunofluorescent and chromatin immunoprecipitation methods, respectively. Levels of glucose, insulin, leptin, and glucagon were profiled, and putative downstream molecules of STAT3 were examined in the presence of various stimuli in L-Stat3^–/– and control mice.

RESULTS— STAT3 was found to sensitize the insulin signaling through suppression of GSK-3β, a negative regulator of insulin signaling pathway. During feeding, both mRNA and protein levels of GSK-3β decreased in Stat3^f/+ mice, which reflected the need of hepatocytes for insulin to induce glycogen synthesis. In contrast, the L-Stat3^–/– mice lost this control and showed a monophasic increase in the GSK-3β level in response to insulin. Administration of GSK-3β inhibitors lithium chloride and L803-mts restored glucose homeostasis and rescued the glucose intolerance and impaired insulin response in L-Stat3^–/– mice.

CONCLUSIONS— These data indicate that STAT3 sensitizes insulin signaling by negatively regulating GSK-3β. Inactivation of STAT3 in the liver contributes significantly to the pathogenesis of insulin resistance.

RESEARCH DESIGN AND METHODS— Primary rat β-cells and islet cells from wild-type, toll-like receptor (TLR) 3, type I interferon receptor (IFNAR1), or interferon regulatory factor (IRF)-3 knockout mice were exposed to external dsRNA (external polyinosinic-polycytidylic acid [PICex]) or were transfected with dsRNA ([PICin]).

RESULTS— TLR3 signaling mediated PICex-induced nuclear factor-B (NF-B) and IRF-3 activation and β-cell apoptosis. PICin activated NF-B and IRF-3 in a TLR3-independent manner, induced eukaryotic initiation factor 2 phosphorylation, and triggered a massive production of interferon (IFN)-β. This contributed to β-cell death, as islet cells from IFNAR1^–/– or IRF-3^–/– mice were protected against PICin-induced apoptosis.

CONCLUSIONS— PICex and PICin trigger β-cell apoptosis via the TLR3 pathway or IRF-3 signaling, respectively. Execution of PICin-mediated apoptosis depends on autocrine effects of type I IFNs.

RESEARCH DESIGN AND METHODS— A newly developed immunoassay was used to measure serum FGF21 levels in 232 Chinese subjects recruited from our previous cross-sectional studies. The mRNA expression levels of FGF21 in the liver and adipose tissues were quantified by real-time PCR.

RESULTS— Serum FGF21 levels in overweight/obese subjects were significantly higher than in lean individuals. Serum FGF21 correlated positively with adiposity, fasting insulin, and triglycerides but negatively with HDL cholesterol, after adjusting for age and BMI. Logistic regression analysis demonstrated an independent association between serum FGF21 and the metabolic syndrome. Furthermore, the increased risk of the metabolic syndrome associated with high serum FGF21 was over and above the effects of individual components of the metabolic syndrome. Our in vitro study detected a differentiation-dependent expression of FGF21 in 3T3-L1 adipocytes and human adipocytes. In db/db obese mice, FGF21 mRNA expression was markedly increased in both the liver and adipose tissue compared with that in their lean littermates. Furthermore, FGF21 expression in subcutaneous fat correlated well with its circulating concentrations in humans.

CONCLUSIONS— FGF21 is a novel adipokine associated with obesity-related metabolic complications in humans. The paradoxical increase of serum FGF21 in obese individuals, which may be explained by a compensatory response or resistance to FGF21, warrants further investigation.

RESEARCH DESIGN AND METHODS— We investigated these two hypotheses by quantifying glucose metabolism and the accumulation of macrophages in adipose tissue of control and MCP-1–deficient (Mcp1^–/–) mice after feeding the animals a high-fat diet for 10 or 16 weeks.

RESULTS— We first established that the two strains were in the same genetic background and that macrophage recruitment into inflamed peritoneum was markedly reduced in the MCP-1–deficient animals. In striking contrast, independent studies at two different facilities at either an early or late time point failed to detect any impairment in macrophage accumulation in adipose tissue of fat-fed Mcp1^–/– mice. Immunoblot analysis revealed higher levels of Mac2, a macrophage-specific protein, in multiple fat depots of Mcp1^–/– mice fed a high-fat diet. These mice also had significantly more adipose tissue than control mice, but their glucose metabolism was similar.

CONCLUSIONS— Our observations suggest that MCP-1 does not play a prominent a role in promoting macrophage recruitment into adipose tissue or in systemic insulin resistance.

METHODS— We used 3T3-L1 adipocytes and diet-induced obese (DIO) mice to examine the role of obesity and insulin resistance in the regulation and/or dysregulation of intracellular ECs.

RESULTS— For the first time, we provide evidence that insulin is a major regulator of EC metabolism. Insulin treatment reduced intracellular ECs (2-arachidonylglycerol [2-AG] and anandamide [AEA]) in 3T3-L1 adipocytes. This corresponded with insulin-sensitive expression changes in enzymes of EC metabolism. In insulin-resistant adipocytes, patterns of insulin-induced enzyme expression were disturbed in a manner consistent with elevated EC synthesis and reduced EC degradation. Expression profiling of adipocytes from DIO mice largely recapitulated in vitro changes, suggesting that insulin resistance affects the EC system in vivo. In mice, expression changes of EC synthesis and degradation enzymes were accompanied by increased plasma EC concentrations (2-AG and AEA) and elevated adipose tissue 2-AG.

CONCLUSIONS— Our findings suggest that insulin-resistant adipocytes fail to regulate EC metabolism and decrease intracellular EC levels in response to insulin stimulation. These novel observations offer a mechanism whereby obese insulin-resistant individuals exhibit increased concentrations of ECs.

RESEARCH DESIGN AND METHODS— We followed 306 nondiabetic Japanese Americans over 10–11 years. Baseline variables included BMI; waist circumference; and abdominal, thoracic, and thigh fat areas measured by CT. Total fat area was estimated by the sum of all of these fat areas. Visceral adiposity was measured as intra-abdominal fat area at the umbilicus level. Total subcutaneous fat area was defined as total fat area minus intra-abdominal fat area. Insulin resistance was evaluated by homeostasis model assessment for insulin resistance (HOMA-IR), fasting plasma insulin level, Matsuda index, and area under the oral glucose tolerance test curve (AUC) of insulin.

RESULTS— Both baseline intra-abdominal fat area (P = 0.002) and HOMA-IR (P < 0.001) were independently associated with increased HOMA-IR at 10–11 years in a multiple linear regression model after adjustment for abdominal subcutaneous fat area, age, sex, 2-h plasma glucose level, and incremental insulin response. Intra-abdominal fat area remained a significant predictor of increased HOMA-IR at 10–11 years even after adjustment for total subcutaneous fat area, total fat area, BMI, or waist circumference, but no other measure of CT-measured regional or total adiposity was significantly related with HOMA-IR at 10–11 years in models that contained intra-abdominal fat area. Similar results were obtained for predicting future fasting plasma insulin level, Matsuda index, and AUC of insulin.

CONCLUSIONS— Greater visceral adiposity is associated with an increase in future insulin resistance.

RESEARCH DESIGN AND METHODS—We analyzed 177 LADA and 978 type 2 diabetic patients with different disease duration, collected in a nationwide Italian survey, the Non–Insulin Requiring Autoimmune Diabetes (NIRAD) study aimed at assessing prevalence and characteristics of autoimmune diabetes in type 2 diabetic patients and 106 newly diagnosed type 1 diabetic patients (53 children, 53 adults). By radioimmunoassay, we analyzed humoral immunoreactivity to seven IA-2 constructs: IA-2_{PTP (687–979)}, IA-2_(761–964), IA-2_(256–760), IA-2_{JM (601–630)}, IA-2_{IC (605–979)}, IA-2_{BDC (256–556:630–979)}, and IA-2_{FL (1–979)}.

RESULTS—IA-2_(256–760) fragment was identified as the marker with the highest sensitivity for detection of humoral IA-2 immunoreactivity in LADA patients, identifying IA-2 autoantibodies in ~30% of GAD antibody (GADA)-positive LADA patients and in 3.4% of GADA-negative type 2 diabetic patients. LADA IA-2_(256–760)A positivity was associated with an increased frequency of autoimmune diabetes HLA-susceptible genotypes and with a higher risk for developing thyroid autoimmunity compared with autoantibody-negative type 2 diabetic patients. At disease diagnosis, adult-onset type 1 diabetic and LADA patients showed a lower IA-2 COOH-terminal immunoreactivity compared with childhood-onset type 1 diabetic patients.

CONCLUSIONS—IA-2 immunoreactivity in LADA patients has thus far been underestimated, and IA-2_(256–760) autoantibody detection may represent a novel diagnostic tool for the identification of islet autoimmunity in these patients.

RESEARCH DESIGN AND METHODS—We isolated islets from mice lacking Bid, Bax, or Bak and those overexpressing Bcl-2 and exposed them to Fas ligand, tumor necrosis factor (TNF)-, and proinflammatory cytokines or cytotoxic stimuli that activate the mitochondrial apoptotic pathway (staurosporine, etoposide, -radiation, tunicamycin, and thapsigargin). Nuclear fragmentation was measured by flow cytometry.

RESULTS—Development and function of islets were not affected by loss of Bid, and Bid-deficient islets were as susceptible as wild-type islets to cytotoxic stimuli that cause apoptosis via the mitochondrial pathway. In contrast, Bid-deficient islets and those overexpressing antiapoptotic Bcl-2 were protected from Fas ligand–induced apoptosis. Bid-deficient islets were also resistant to apoptosis induced by TNF- plus cycloheximide and were partially resistant to proinflammatory cytokine-induced death. Loss of the multi-BH domain proapoptotic Bax or Bak protected islets partially from death receptor–induced apoptosis.

CONCLUSIONS—These results demonstrate that Bid is essential for death receptor–induced apoptosis of islets, similar to its demonstrated role in hepatocytes. This indicates that blocking Bid activity may be useful for protection of islets from immune-mediated attack and possibly also in other pathological states in which β-cells are destroyed.

RESEARCH DESIGN AND METHODS—Data were assembled from analysis of reported effects of mutagenesis of GAD65 on its reactivity with a panel of 11 human monoclonal antibodies (mAbs), supplemented by use of recombinant Fab to cross-inhibit reactivity with GAD65 by radioimmunoprecipitation of the same mAbs.

RESULTS—The COOH-terminal region on GAD65 was the major autoantigenic site. B-cell epitopes were distributed within two separate clusters around different faces of the COOH-terminal domain. Inclusion of epitope sites in the pyridoxal phosphate–and NH₂-terminal domains was attributed to the juxtaposition of all three domains in the crystal structure. Epitope preferences of different mAbs to GAD65 aligned with different clinical expressions of type 1 diabetes. Epitopes for four of five known reactive T-cell sequences restricted by HLA DRB1*0401 were aligned to solvent-exposed regions of the GAD65 structure and colocalized within the two B-cell epitope clusters. The continuous COOH-terminal epitope region of GAD65 was structurally highly flexible and therefore differed markedly from the equivalent region of GAD67.

CONCLUSIONS—Structural features could explain the differing antigenicity, and perhaps immunogenicity, of GAD65 versus GAD67. The proximity of B- and T-cell epitopes within the GAD65 structure suggests that antigen-antibody complexes may influence antigen processing by accessory cells and thereby T-cell reactivity.

RESEARCH DESIGN AND METHODS—NOD mice expressing transforming growth factor-β (TGF-β) specifically in the β-cells were infected with CB4, and the functional role of Tregs in disease protection was measured. Systemic treatments with TGF-β were used to assess its therapeutic potential.

RESULTS—Here, we report that Tregs induced after CB4 infection in the presence of TGF-β prevented type 1 diabetes. The capacity to directly infect pancreatic β-cells correlated with increased numbers of pancreatic Tregs, suggesting that presentation of β-cell antigen is integral to induction of diabetogenic protective Tregs. Furthermore, the presence of these viral induced Tregs correlated with protection from type 1 diabetes without altering the antiviral response. Finally, when TGF-β was administered systemically to NOD mice after infection, the incidence of type 1 diabetes was reduced, thereby signifying a potential therapeutic role for TGF-β.

CONCLUSIONS—We demonstrate manipulations of the immune response that result in Treg-mediated protection from type 1 diabetes without concomitant loss of the capacity to control viral infection.

RESEARCH DESIGN AND METHODS—We took advantage of a recently validated islet-specific CD8⁺ T-cell -interferon enzyme-linked immunospot (ISL8Spot) assay to follow responses against preproinsulin (PPI), GAD, insulinoma-associated protein 2 (IA-2), and islet-specific glucose-6-phosphatase catalytic subunit-related protein (IGRP) epitopes in 15 HLA-A2⁺ adult type 1 diabetic patients close to diagnosis and at a second time point 7–16 months later.

RESULTS—CD8⁺ T-cell reactivities were less frequent at follow-up, as 28.6% of responses tested positive at type 1 diabetes diagnosis vs. 13.2% after a median of 11 months (P = 0.003). While GAD and IA-2 autoantibody (aAb) titers were unchanged in 75% of cases, the fraction of patients responding to PPI and/or GAD epitopes by ISL8Spot decreased from 60–67 to 20% (P < 0.02). The previously subdominant IA-2_206–214 and IGRP_265–273 peptides were newly targeted, thus becoming the immunodominant epitopes.

CONCLUSIONS—Shifts both in frequency and in immunodominance of CD8⁺ T-cell responses occur more rapidly than do changes in aAb titers. These different kinetics may suggest complementary clinical applications for T-cell and aAb measurements.

RESEARCH DESIGN AND METHODS—We generated mice possessing a monoclonal population of T-cells expressing 1 of 17 T-cell receptors (TCR) specific for either known autoantigens (GAD65, insulinoma-associated protein 2 (IA2), IA2β/phogrin, and insulin), unknown islet antigens, or control antigens on a NOD.scid background using retroviral-mediated stem cell gene transfer and 2A-linked multicistronic retroviral vectors (referred to herein as retrogenic [Rg] mice). The TCR Rg approach provides a mechanism by which T-cells with broad phenotypic differences can be directly compared.

RESULTS—Neither GAD- nor IA2-specific TCRs mediated T-cell islet infiltration or diabetes even though T-cells developed in these Rg mice and responded to their cognate epitope. IA2β/phogrin and insulin-specific Rg T-cells produced variable levels of insulitis, with one TCR producing delayed diabetes. Three TCRs specific for unknown islet antigens produced a hierarchy of insulitogenic and diabetogenic potential (BDC-2.5 > NY4.1 > BDC-6.9), while a fourth (BDC-10.1) mediated dramatically accelerated disease, with all mice diabetic by day 33, well before full T-cell reconstitution (days 42–56). Remarkably, as few as 1,000 BDC-10.1 Rg T-cells caused rapid diabetes following adoptive transfer into NOD.scid mice.

CONCLUSIONS—Our data show that relatively few autoantigen-specific TCRs can mediate islet infiltration and β-cell destruction on their own and that autoreactivity does not necessarily imply pathogenicity.

RESEARCH DESIGN & METHODS—Effects of the DPP-IV inhibitor MK0431 (sitagliptin) on glycemic control and functional islet mass in a streptozotocin (STZ)-induced type 1 diabetes mouse model were determined with metabolic studies and microPET imaging.

RESULTS—The type 1 diabetes mouse model exhibited elevated plasma DPP-IV levels that were substantially inhibited in mice on an MK0431 diet. Residual β-cell mass was extremely low in STZ-induced diabetic mice, and although active GLP-1 levels were increased by the MK0431 diet, there were no significant effects on glycemic control. After islet transplantation, mice fed normal diet rapidly lost their ability to regulate blood glucose, reflecting the suboptimal islet transplant. By contrast, the MK0431 group fully regulated blood glucose throughout the study, and PET imaging demonstrated a profound protective effect of MK0431 on islet graft size.

CONCLUSIONS—Treatment with a DPP-IV inhibitor can prolong islet graft retention in an animal model of type 1 diabetes.

RESEARCH DESIGN AND METHODS—Isoglycemic oral (75 g) and intravenous glucose administration was performed in 51 subjects (24 with normal glucose tolerance [NGT], 17 with impaired glucose tolerance [IGT], and 10 with type 2 diabetes) with a wide range of BMI (20–61 kg/m²). C-peptide deconvolution was used to reconstruct insulin secretion rates, and β-cell glucose sensitivity (slope of the insulin secretion/glucose concentration dose-response curve) was determined by mathematical modeling. The incretin effect was defined as the oral-to-intravenous ratio of responses. In 8 subjects with NGT and 10 with diabetes, oral glucose appearance was measured by the double-tracer technique.

RESULTS—The incretin effect on total insulin secretion and β-cell glucose sensitivity and the GLP-1 response to oral glucose were significantly reduced in diabetes compared with NGT or IGT (P ≤ 0.05). The results were similar when subjects were stratified by BMI tertile (P ≤ 0.05). In the whole dataset, each manifestation of the incretin effect was inversely related to both glucose tolerance (2-h plasma glucose levels) and BMI (partial r = 0.27–0.59, P ≤ 0.05) in an independent, additive manner. Oral glucose appearance did not differ between diabetes and NGT and was positively related to the GLP-1 response (r = 0.53, P < 0.01). Glucagon suppression during the oral glucose tolerance test was blunted in diabetic patients.

CONCLUSIONS—Potentiation of insulin secretion, glucose sensing, glucagon-like peptide-1 release, and glucagon suppression are physiological manifestations of the incretin effect. Glucose tolerance and obesity impair the incretin effect independently of one another.

RESEARCH DESIGN AND METHODS—A euinsulinemic hyperglycemic clamp at 5, 10, and 15 mmol/l glucose was given in increasing steps as a single "spike" or oscillating between basal and high levels over 24 h in normal subjects and type 2 diabetic patients. Flow-mediated dilatation, a marker of endothelial function, and plasma 3-nitrotyrosine and 24-h urinary excretion rates of free 8-iso PGF2, two markers of oxidative stress, were measured over 48 h postclamp.

RESULTS—Glucose at two different levels (10 and 15 mmol/l) resulted in a concentration-dependent fasting blood glucose–independent induction of both endothelial dysfunction and oxidative stress in both normal and type 2 diabetic patients. Oscillating glucose between 5 and 15 mmol/l every 6 h for 24 h resulted in further significant increases in endothelial dysfunction and oxidative stress compared with either continuous 10 or 15 mmol/l glucose.

CONCLUSIONS—These data suggest that oscillating glucose can have more deleterious effects than constant high glucose on endothelial function and oxidative stress, two key players in favoring cardiovascular complications in diabetes. Concomitant vitamin C infusion can reverse this impairment.

RESEARCH DESIGN AND METHODS—FOXO1, PEPCK, and G6PC mRNA levels were evaluated in 84 subjects: 26 with steatohepatitis, 28 with steatosis alone, 14 with normal liver histology without metabolic alterations, and 16 with hepatitis C virus chronic hepatitis, of whom 8 were with and 8 were without steatosis. Protein expression and regulation of FOXO1 and upstream insulin signaling were analyzed in a subset.

RESULTS—Expression of PEPCK was higher in steatohepatitis compared with steatosis alone and normal liver, and it was correlated with the homeostasis model assessment of insulin resistance (HOMA-IR) index. FOXO1 mRNA levels were higher in steatohepatitis, correlated with PEPCK and G6PC mRNA and with HOMA-IR. FOXO1 upregulation was confirmed at protein levels in steatohepatitis and, in the presence of oxidative stress, was associated with decreased Ser²⁵⁶ phosphorylation, decreased Akt1, and increased Jun NH₂-terminal kinase-1 activity. Consistently, immunohistochemistry showed increased FOXO1 expression and nuclear localization in steatohepatitis. FOXO1 mRNA levels correlated with nonalcoholic steatohepatitis activity score and were modulated by drugs counteracting hepatic lipogenesis.

CONCLUSIONS—FOXO1 expression and activity are increased in patients with steatohepatitis, and mRNA levels are correlated with hepatic insulin resistance.

RESEARCH DESIGN AND METHODS—To test this hypothesis, we quantified expression of the GABA synthetic enzyme, glutamic acid decarboxylase (GAD), in the VMH of control and recurrently hypoglycemic rats. Subsequently, we used microdialysis and microinjection techniques to assess changes in VMH GABA levels and the effects of GABA_A receptor blockade on counterregulatory responses to a standardized hypoglycemic stimulus.

RESULTS—Quantitative RT-PCR and immunoblots in recurrently hypoglycemic animals revealed that GAD₆₅ mRNA and protein were increased 33 and 580%, respectively. Basal VMH GABA concentrations were more than threefold higher in recurrently hypoglycemic animals. Furthermore, whereas VMH GABA levels decreased in both control and recurrently hypoglycemic animals with the onset of hypoglycemia, the fall was not significant in recurrently hypoglycemic rats. During hypoglycemia, recurrently hypoglycemic rats exhibited a 49–63% reduction in glucagon and epinephrine release. These changes were reversed by delivery of a GABA_A receptor antagonist to the VMH.

CONCLUSIONS—Our data suggest that recurrent hypoglycemia increases GABAergic inhibitory tone in the VMH and that this, in turn, suppresses glucagon and sympathoadrenal responses to subsequent bouts of acute hypoglycemia. Thus, hypoglycemia-associated autonomic failure may be due in part to a relative excess of the inhibitory neurotransmitter, GABA, within the VMH.

RESEARCH DESIGN AND METHODS—The counterregulatory response to insulin-induced hypoglycemia was evaluated in Sprague-Dawley rats after bilateral VMH injections of 1) a GK activator drug (compound A) to increase VMH GK activity, 2) low-dose alloxan (4 µg) to acutely inhibit GK activity, 3) high-dose alloxan (24 µg), or 4) an adenovirus expressing GK short hairpin RNA (shRNA) to chronically reduce GK expression and activity.

RESULTS—Compound A increased VMH GK activity sixfold in vitro and reduced the epinephrine, norepinephrine, and glucagon responses to insulin-induced hypoglycemia by 40–62% when injected into the VMH in vivo. On the other hand, acute and chronic reductions of VMH GK mRNA or activity had a lesser and more selective effect on increasing primarily the epinephrine response to insulin-induced hypoglycemia by 23–50%.

CONCLUSIONS—These studies suggest that VMH GK activity is an important regulator of the counterregulatory response to insulin-induced hypoglycemia and that a drug that specifically inhibited the rise in hypothalamic GK activity after insulin-induced hypoglycemia might improve the dampened counterregulatory response seen in tightly controlled diabetic subjects.

RESEARCH DESIGN AND METHODS—Chronically cannulated rats underwent afferent ablation of the portal vein (PV) or portal and superior mesenteric veins (PMV) or sham operation (control). One week later, animals underwent hyperinsulinemic-hypoglycemic clamps in which the hypoglycemic nadir, 2.48 ± 0.06 mmol/l, was reached at a rate of decline in glucose of –0.09 or –0.21 mmol · l^–1 · min^–1 (PMV and control only). Additional PMV and control animals received an intravenous injection of the glucopenic agent 2-deoxyglucose.

RESULTS—Inducing hypoglycemia slowly, at a rate of –0.09 mmol · l^–1 · min^–1, resulted in a 26-fold increase in epinephrine (23.39 ± 0.62 nmol/l) and 12-fold increase in norepinephrine (11.42 ± 0.92 nmol/l) for controls (P < 0.001). The epinephrine response to hypoglycemia was suppressed by 91% in PMV (2.09 ± 0.07 nmol/l) vs. 61% in PV (9.05 ± 1.59 nmol/l) (P < 0.001). The norepinephrine response to hypoglycemia was suppressed by 94 and 80% in PMV and PV, respectively, compared with that in controls. In contrast, when arterial glucose was lowered to 2.49 ± 0.06 mmol/l within 20 min, no significant differences were observed in the catecholamine responses for PMV and controls over the first 45 min of hypoglycemia (20–65 min). Only at min 105 were catecholamines significantly lower for PMV vs. controls. Injection of 2-deoxyglucose induced a very rapid sympathoadrenal response with no significant differences between PMV and controls.

CONCLUSIONS—The critical locus for hypoglycemic detection shifts away from the portal-mesenteric vein to some other loci (e.g., the brain) when hypoglycemia develops rapidly.

RESEARCH DESIGN AND METHODS—Wild-type mice, 5-lipoxygenase–deficient mice, and 12/15-lipoxygenase–deficient mice were assessed 1) after 9 months of diabetes for retinal histopathology and leukotriene receptor expression and 2) after 3 months of diabetes for leukostasis and retinal superoxide generation.

RESULTS—Diabetic wild-type mice developed the expected degeneration of retinal capillaries and pericytes and increases in both leukostasis and superoxide production (P < 0.006). We found no evidence of diabetes-induced degeneration of retinal ganglion cells in these animals. The vascular histopathology was significantly inhibited in 5-lipoxygenase–deficient mice, but not in 12/15-lipoxygenase–deficient mice. Retinas from diabetic 5-lipoxygenase–deficient mice also had significantly less leukostasis, superoxide production, and nuclear factor-B (NF-B) expression (all P < 0.006), whereas retinas from diabetic 12/15-lipoxygenase–deficient mice had significantly less leukostasis (P < 0.005) but not superoxide production or NF- B expression. Retinas from diabetic wild-type mice were enriched with receptors for the 5-lipoxygenase metabolite leukotriene B₄. Diabetes-induced histological and biochemical alterations were significantly reduced in 5-lipoxygenase–deficient mice, but not 12/15-lipoxygenase–deficient mice.

CONCLUSIONS—5-Lipoxygenase represents a novel pathway for therapeutic intervention of diabetic retinopathy.

RESEARCH DESIGN AND METHODS—We used selective synthetic PPAR and PPAR agonists and investigated their angiogenic potentials in vitro and in vivo.

RESULTS—Activation of PPAR and PPAR leads to endothelial tube formation in an endothelial/interstitial cell co-culture assay. This effect is associated with increased production of the angiogenic cytokine vascular endothelial growth factor (VEGF). Neovascularization also occurs in vivo, when PPAR and PPAR agonists are used in the murine corneal angiogenic model. No vascular growth is detectable when PPAR and PPAR agonists are respectively used in PPAR knockout mice and mice treated with a specific PPAR inhibitor, demonstrating that this angiogenic response is PPAR mediated. PPAR- and PPAR-induced angiogenesis is associated with local VEGF production and does not differ in extent and morphology from that induced by VEGF. In addition, PPAR- and PPAR-induced in vitro and in vivo angiogenesis may be significantly decreased by inhibiting VEGF activity. Finally, in corneas treated with PPAR and PPAR agonists, there is increased phosphorylation of endothelial nitric oxide synthase and Akt.

CONCLUSIONS—These findings demonstrate that PPAR and PPAR activation stimulates neoangiogenesis through a VEGF-dependent mechanism. Neoangiogenesis is a crucial pathological event in type 2 diabetes. The ability of PPAR and PPAR agonists to induce neoangiogenesis might have important implications for the clinical and therapeutic management of type 2 diabetes.

RESEARCH DESIGN AND METHODS—Regulation of PPAR target genes by telmisartan was studied by real-time PCR and Western immunoblotting in vitro and in vivo in liver/skeletal muscle of mice with diet-induced obesity. Activation of the PPAR ligand binding domain (LBD) was investigated using transactivation assays.

RESULTS—Telmisartan significantly induced the PPAR target genes carnitine palmitoyl transferase 1A (CPT1A) in human HepG2 cells and acyl-CoA synthetase long-chain family member 1 (ACSL1) in murine AML12 cells in the micromolar range. Telmisartan-induced CPT1A stimulation was markedly reduced after small interfering RNA–mediated knockdown of PPAR. Telmisartan consistently activated the PPAR-LBD as a partial PPAR agonist. Despite high in vitro concentrations required for PPAR activation, telmisartan (3 mg · kg^–1 · day^–1) potently increased ACSL1 and CPT1A expression in liver from diet-induced obese mice associated with a marked decrease of hepatic and serum triglycerides. Muscular CPT1B expression was not affected. Tissue specificity of telmisartan-induced PPAR target gene induction may be the result of previously reported high hepatic concentrations of telmisartan.

CONCLUSIONS—The present study identifies the ARB/PPAR modulator telmisartan as a partial PPAR agonist. As a result of its particular pharmacokinetic profile, PPAR activation by telmisartan seems to be restricted to the liver. Hepatic PPAR activation may provide an explanation for telmisartan's antidyslipidemic actions observed in recent clinical trials.

RESEARCH DESIGN AND METHODS—AMPK phosphorylation was examined in L6 myotubes and LKB1^–/– cells, with or without the Ca²⁺/calmodulin-dependent protein kinase kinase (CAMKK) inhibitor STO-609. Oxygen consumption was measured in L6 myotubes and isolated muscle mitochondria. The effect of a BBR derivative, dihydroberberine (dhBBR), on adiposity and glucose metabolism was examined in rodents fed a high-fat diet.

RESULTS—We have made the following novel observations: 1) BBR dose-dependently inhibited respiration in L6 myotubes and muscle mitochondria, through a specific effect on respiratory complex I, similar to that observed with metformin and rosiglitazone; 2) activation of AMPK by BBR did not rely on the activity of either LKB1 or CAMKKβ, consistent with major regulation at the level of the AMPK phosphatase; and 3) a novel BBR derivative, dhBBR, was identified that displayed improved in vivo efficacy in terms of counteracting increased adiposity, tissue triglyceride accumulation, and insulin resistance in high-fat–fed rodents. This effect is likely due to enhanced oral bioavailability.

CONCLUSIONS—Complex I of the respiratory chain represents a major target for compounds that improve whole-body insulin sensitivity through increased AMPK activity. The identification of a novel derivative of BBR with improved in vivo efficacy highlights the potential importance of BBR as a novel therapy for the treatment of type 2 diabetes.

RESEARCH DESIGN AND METHODS—We tested the association between FTO genotype and 10 metabolic traits using data from 17,037 white European individuals. We compared the observed effect of FTO genotype on each trait to that expected given the FTO-BMI and BMI-trait associations.

RESULTS—Each copy of the FTO rs9939609 A allele was associated with higher fasting insulin (0.039 SD [95% CI 0.013–0.064]; P = 0.003), glucose (0.024 [0.001–0.048]; P = 0.044), and triglycerides (0.028 [0.003–0.052]; P = 0.025) and lower HDL cholesterol (0.032 [0.008–0.057]; P = 0.009). There was no evidence of these associations when adjusting for BMI. Associations with fasting alanine aminotransferase, -glutamyl-transferase, LDL cholesterol, A1C, and systolic and diastolic blood pressure were in the expected direction but did not reach P < 0.05. For all metabolic traits, effect sizes were consistent with those expected for the per allele change in BMI. FTO genotype was associated with a higher odds of metabolic syndrome (odds ratio 1.17 [95% CI 1.10–1.25]; P = 3 x 10^–6).

CONCLUSIONS—FTO genotype is associated with metabolic traits to an extent entirely consistent with its effect on BMI. Sample sizes of >12,000 individuals were needed to detect associations at P < 0.05. Our findings highlight the importance of using appropriately powered studies to assess the effects of a known diabetes or obesity variant on secondary traits correlated with these conditions.

RESEARCH DESIGN AND METHODS—The polymorphisms were genotyped in 7,683 white Danish subjects using Taqman allelic discrimination or chip-based matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, providing a statistical power of >99% to replicate previous findings. Data were analyzed in case-control and haplotype settings, and quantitative metabolic traits were examined for association. Moreover, epistatic effects between AHSG variants and insulin receptor substrate-1 (IRS1) and β-2-adrenergic receptor polymorphisms were investigated.

RESULTS—The –469T>G (rs2077119) and IVS6+98C>T (rs2518136) polymorphisms were associated with type 2 diabetes (P = 0.007 and P = 0.006, respectively, or P_corr = 0.04 and P_corr = 0.03, respectively, following correction for multiple hypothesis testing), and in a combined analysis of the present and a previous study –469T>G remained significant (odds ratio 0.90 [95% CI 0.84–0.97]; P = 0.007). Furthermore, two AHSG haplotypes were associated with dyslipidemia (P = 0.003 and P_corr = 0.009). Thr248Met (rs4917) tended to associate with lower fasting and post–oral glucose tolerance test serum insulin release (P = 0.02, P_corr = 0.1 for fasting and P = 0.04, P_corr = 0.2 for area under the insulin curve) and improved insulin sensitivity estimated by the homeostasis model assessment of insulin resistance (9.0 vs. 8.6 mmol · l^–1 · pmol^–1 · l^–1; P = 0.01, P_corr = 0.06). Indications of epistatic effects of AHSG variants with the IRS1 Gly971Arg polymorphism were observed for fasting serum triglyceride concentrations.

CONCLUSIONS—Based on present and previous findings, common variation in AHSG may contribute to the interindividual variation in metabolic traits.

RESEARCH DESIGN AND METHODS—To accomplish this, we studied whether LADA shares variation in the HLA locus or INS VNTR and PTPN22 genes with type 1 diabetes or the TCF7L2 gene with type 2 diabetes in 361 LADA, 718 type 1 diabetic, and 1,676 type 2 diabetic patients, as well as 1,704 healthy control subjects from Sweden and Finland.

RESULTS—LADA subjects showed, compared with type 2 diabetic patients, increased frequency of risk for the HLA-DQB1 *0201/*0302 genotype (27 vs. 6.9%; P < 1 x 10^–6), with similar frequency as with type 1 diabetes (36%). In addition, LADA subjects showed higher frequencies of protective HLA-DQB1 *0602(3)/X than type 1 diabetic patients (8.1 vs. 3.2%, P = 0.003). The AA genotype of rs689, referring to the class I allele in the INS VNTR, as well as the CT/TT genotypes of rs2476601 in the PTPN22 gene, were increased both in type 1 diabetic (P = 3 x 10^–14 and P = 1 x 10^–10, respectively) and LADA (P = 0.001 and P = 0.002) subjects compared with control subjects. Notably, the frequency of the type 2 diabetes–associated CT/TT genotypes of rs7903146 in the TCF7L2 were increased in LADA subjects (52.8%; P = 0.03), to the same extent as in type 2 diabetic subjects (54.1%, P = 3 x 10^–7), compared with control subjects (44.8%) and type 1 diabetic subjects (43.3%).

CONCLUSIONS—LADA shares genetic features with both type 1 (HLA, INS VNTR, and PTPN22) and type 2 (TCF7L2) diabetes, which justifies considering LADA as an admixture of the two major types of diabetes.