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Exercise is a first-line treatment for type 2 diabetes and preserves β-cell function by hitherto unknown mechanisms. We postulated that proteins from contracting skeletal muscle may act as cellular signals to regulate pancreatic β-cell function. We employed electric pulse stimulation (EPS) to induce contraction in C2C12 myotubes and found that treatment of β-cells with EPS-conditioned media (EPS-CM) enhanced glucose-stimulated insulin secretion (GSIS). Transcriptomics and subsequent targeted validation revealed Growth Differentiation Factor 15 (GDF15) as a central component of the skeletal muscle secretome. Exposure to recombinant GDF15 enhanced GSIS in cells, islets, and mice. GDF15 enhanced GSIS by upregulating the insulin secretion pathway in β-cells, which was abrogated in the presence of a GDF15 neutralizing antibody. The effect of GDF15 on GSIS was also observed in islets from GFRAL-deficient mice. Circulating GDF15 was incrementally elevated in patients with pre- and type 2 diabetes and positively associated with C-peptide in humans with overweight/obesity. Six weeks of high intensity exercise training increased circulating GDF15 concentrations, which positively correlated with improvements in β-cell function in patients with type 2 diabetes. Taken together, GDF15 can function as a contraction-induced protein that enhances GSIS through activating the canonical signaling pathway in a GFRAL-independent manner.

Reductions in β-cell number and function contribute to the onset type 2 diabetes (T2D). Roux-en-Y gastric bypass (RYGB) surgery can resolve T2D within days of operation, indicating a weight-independent mechanism of glycemic control. We hypothesized that RYGB normalizes glucose homeostasis by restoring β-cell structure and function. Male Zucker Diabetic Fatty (fa/fa; ZDF) rats were randomized to sham surgery (n = 16), RYGB surgery (n = 16), or pair feeding (n = 16). Age-matched lean (fa/+) rats (n = 8) were included as a secondary control. Postprandial metabolism was assessed by oral glucose tolerance testing before and 27 days after surgery. Fasting and postprandial plasma GLP-1 was determined by mixed meal tolerance testing. Fasting plasma glucagon was also measured. β-cell function was determined in isolated islets by a glucose-stimulated insulin secretion assay. Insulin and glucagon positive areas were evaluated in pancreatic sections by immunohistochemistry. RYGB reduced body weight (P < 0.05) and improved glucose tolerance (P < 0.05) compared with sham surgery. RYGB reduced fasting glucose compared with both sham (P < 0.01) and pair-fed controls (P < 0.01). Postprandial GLP-1 (P < 0.05) was elevated after RYGB compared with sham surgery. RYGB islets stimulated with 20 mM glucose had higher insulin secretion than both sham and pair-fed controls (P < 0.01) and did not differ from lean controls. Insulin content was greater after RYGB compared with the sham (P < 0.05) and pair-fed (P < 0.05) controls. RYGB improves insulin secretion and pancreatic islet function, which may contribute to the remission of type 2 diabetes following bariatric surgery. NEW & NOTEWORTHY The onset and progression of type 2 diabetes (T2D) results from failure to secrete sufficient amounts of insulin to overcome peripheral insulin resistance. Here, we demonstrate that Roux-en-Y gastric bypass (RYGB) restores islet function and morphology compared to sham and pair-fed controls in ZDF rats. The improvements in islet function were largely attributable to enhanced insulin content and secretory function in response to glucose stimulation.

BACKGROUND: Metabolic surgery has beneficial metabolic effects, including remission of type 2 diabetes. We hypothesized that duodenojejunal bypass (DJB) surgery can protect against development of type 1 diabetes (T1D) by enhancing regulation of cellular and molecular pathways that control glucose homeostasis. METHODS: BBDP/Wor rats, which are prone to develop spontaneous autoimmune T1D, underwent loop DJB (n=15) or sham (n=15) surgery at a median age of 41 days, before development of diabetes. At T1D diagnosis, a subcutaneous insulin pellet was implanted, oral glucose tolerance test was performed 21 days later, and tissues were collected 25 days after onset of T1D. Pancreas and liver tissues were assessed by histology and RT-qPCR. Fecal microbiota composition was analyzed by 16S V4 sequencing. RESULTS: Postoperatively, DJB rats weighed less than sham rats (287.8 vs 329.9 g, P=0.04). In both groups, 14 of 15 rats developed T1D, at similar age of onset (87 days in DJB vs 81 days in sham, P=0.17). There was no difference in oral glucose tolerance, fasting and stimulated plasma insulin and c-peptide levels, and immunohistochemical analysis of insulin positive cells in the pancreas. DJB rats needed 1.3±0.4 insulin implants vs 1.9±0.5 in sham rats (P=0.002). Fasting and glucose stimulated glucagon-like peptide 1 (GLP-1) secretion was elevated after DJB surgery. DJB rats had reduced markers of metabolic stress in liver. After DJB, the fecal microbiome changed significantly, including increases in Akkermansia and Ruminococcus, while the changes were minimal in sham rats. CONCLUSION: DJB does not protect against autoimmune T1D in BBDP/Wor rats, but reduces the need for exogenous insulin and facilitates other metabolic benefits including weight loss, increased GLP-1 secretion, reduced hepatic stress, and altered gut microbiome.

Background and aims A diminution in skeletal muscle mitochondrial function due to ectopic lipid accumulation and excess nutrient intake is thought to contribute to insulin resistance and the development of type 2 diabetes. However, the functional integrity of mitochondria in insulin-resistant skeletal muscle remains highly controversial. Methods 19 healthy adults (age:28.4 ± 1.7 years; BMI:22.7 ± 0.3 kg/m2) received an overnight intravenous infusion of lipid (20% Intralipid) or saline followed by a hyperinsulinemic-euglycemic clamp to assess insulin sensitivity using a randomized crossover design. Skeletal muscle biopsies were obtained after the overnight lipid infusion to evaluate activation of mitochondrial dynamics proteins, ex-vivo mitochondrial membrane potential, ex-vivo oxidative phosphorylation and electron transfer capacity, and mitochondrial ultrastructure. Results Overnight lipid infusion increased dynamin related protein 1 (DRP1) phosphorylation at serine 616 and PTEN-induced kinase 1 (PINK1) expression (P = 0.003 and P = 0.008, respectively) in skeletal muscle while reducing mitochondrial membrane potential (P = 0.042). The lipid infusion also increased mitochondrial-associated lipid droplet formation (P = 0.011), the number of dilated cristae, and the presence of autophagic vesicles without altering mitochondrial number or respiratory capacity. Additionally, lipid infusion suppressed peripheral glucose disposal (P = 0.004) and hepatic insulin sensitivity (P = 0.014). Conclusions These findings indicate that activation of mitochondrial fission and quality control occur early in the onset of insulin resistance in human skeletal muscle. Targeting mitochondrial dynamics and quality control represents a promising new pharmacological approach for treating insulin resistance and type 2 diabetes. Clinical trial registration NCT02697201 , ClinicalTrials.gov

Obesity is a leading cause of preventable death worldwide. Despite this, current strategies for the treatment of obesity remain ineffective at achieving long‐term weight control. This is due, in part, to difficulties in identifying tolerable and efficacious small molecules or biologics capable of regulating systemic nutrient homeostasis. Here, we demonstrate that BAM15, a mitochondrially targeted small molecule protonophore, stimulates energy expenditure and glucose and lipid metabolism to protect against diet‐induced obesity. Exposure to BAM15 in vitro enhanced mitochondrial respiratory kinetics, improved insulin action, and stimulated nutrient uptake by sustained activation of AMPK. C57BL/6J mice treated with BAM15 were resistant to weight gain. Furthermore, BAM15‐treated mice exhibited improved body composition and glycemic control independent of weight loss, effects attributable to drug targeting of lipid‐rich tissues. We provide the first phenotypic characterization and demonstration of pre‐clinical efficacy for BAM15 as a pharmacological approach for the treatment of obesity and related diseases., This study presents a novel therapy for treatment of obesity‐related diseases. Oral delivery of the mitochondrial protonophore BAM15 markedly reduced weight gain and fat accrual while improving glycemic control.

We are interested in understanding mechanisms that govern the protective role of exercise against lipid-induced insulin resistance, a key driver of type 2 diabetes. In this context, cell culture models provide a level of abstraction that aid in our understanding of cellular physiology. Here we describe the development of an in vitro myotube contraction system that provides this protective effect, and which we have harnessed to investigate lipid-induced insulin resistance. C2C12 myocytes were differentiated into contractile myotubes. A custom manufactured platinum electrode system and pulse stimulator, with polarity switching, provided an electrical pulse stimulus (EPS) (1 Hz, 6-ms pulse width, 1.5 V/mm, 16 h). Contractility was assessed by optical flow flied spot noise mapping and inhibited by application of ammonium acetate. Following EPS, myotubes were challenged with 0.5 mM palmitate for 4 h. Cells were then treated with or without insulin for glucose uptake (30 min), secondary insulin signaling activation (10 min), and phosphoinositide 3-kinase-α (PI3Kα) activity (5 min). Prolonged EPS increased non-insulin-stimulated glucose uptake (83%, P = 0.002), Akt (Thr308) phosphorylation ( P = 0.005), and insulin receptor substrate-1 (IRS-1)-associated PI3Kα activity ( P = 0.048). Palmitate reduced insulin-specific action on glucose uptake (−49%, P < 0.001) and inhibited insulin-stimulated Akt phosphorylation ( P = 0.049) and whole cell PI3Kα activity ( P = 0.009). The inhibitory effects of palmitate were completely absent with EPS pretreatment at the levels of glucose uptake, insulin responsiveness, Akt phosphorylation, and whole cell PI3Kα activity. This model suggests that muscle contraction alone is a sufficient stimulus to protect against lipid-induced insulin resistance as evidenced by changes in the proximal canonical insulin-signaling pathway.

Objective This study hypothesized that a low-glycemic diet combined with exercise would increase expression of nuclear regulators of fat transport and oxidation in insulin-resistant skeletal muscle. Method Nineteen subjects (64 ± 1 y; 34 ± 1 kg/m2) were randomized to receive isocaloric high-glycemic-index (HiGIX; 80 ± 0.6 units, n = 10) or low-glycemic-index (LoGIX; 40 ± 0.3 units, n = 9) diets combined with supervised exercise (1 h/d, 5 d/wk at ∼85% HRmax) for 12 weeks. Insulin sensitivity was determined by hyperinsulinemic-euglycemic clamp. Skeletal muscle biopsies were obtained before and after the intervention to assess fasting gene and protein expression. Results Weight loss was similar for both groups (9.5 ± 1.3 kg). Likewise, improvements in insulin sensitivity (P

Diabetic nephropathy is the leading cause of chronic kidney disease. Observational studies suggest Roux-en-Y gastric bypass (RYGB) reduces progression of diabetic nephropathy.To unravel the mechanisms by which RYGB is beneficial and protective for diabetic nephropathy.Academic laboratories.Forty-eight Zucker diabetic fatty rats were randomized to RYGB, sham surgery (SHAM), or pair-fed (PF) groups. An oral glucose tolerance test was performed at 25 days post intervention and kidneys were harvested at 30 days. Primary outcome measures included expression of key genes and proteins in the glucose transport, oxidative stress, inflammation, and fibrosis pathways.Thirty days post intervention, RYGB rats weighed 349 ± 8 g, which was lower than SHAM (436 ± 14 g, P.001), but not PF (374 ± 18 g) rats. RYGB rats had lower fasting glucose than PF animals and improved homeostatic model assessment of insulin resistance compared with PF and SHAM groups. These enhanced metabolic outcomes were accompanied by reduced sodium-glucose co-transporter 1 (Sglt1) gene expression (-23% versus PF, P = .01) in the kidney of RYGB rats. Expression of Sglt2, Glut1, or Glut2 mRNA, or oxidative stress and inflammation markers did not differ significantly. However, RYGB surgery induced a 19% lower expression of transforming growth factor (Tgfβ) mRNA (P = .004) compared with SHAM treated animals. Notably, adenosine monophosphate-activated protein kinase phosphorylation was increased (P = .04) in kidneys of the RYGB surgery animals.Improvement of hyperglycemia after RYGB may reduce the glucose load on the kidney leading to a downregulation of specific glucose transporters. RYGB surgery may also attenuate kidney fibrosis through the adenosine monophosphate-activated protein kinase/TGFβ pathway.

Background: Asthma physiology affects respiratory function and inflammation, factors that may contribute to elevated resting energy expenditure (REE) and altered body composition. Objective: We hypothesized that asthma would present with elevated REE compared to weight-matched healthy controls. Methods: Adults with asthma (n = 41) and healthy controls (n = 20) underwent indirect calorimetry to measure REE, dual-energy X-ray absorptiometry (DEXA) to measure body composition, and 3-day diet records. Clinical assessments included spirometry, fractional exhaled nitric oxide (FENO), and a complete blood count. Results: Asthmatics had greater REE than controls amounting to an increase of ~100 kcals/day, even though body mass index (BMI) and body composition were similar between groups. Inclusion of asthma status and FENO in validated REE prediction equations led to improved estimates. Further, asthmatics had higher white blood cell (control vs. asthma (mean ± SD): 4.7 ± 1.1 vs. 5.9 ± 1.6, p &lt, 0.01) and neutrophil (2.8 ± 0.9 vs. 3.6 ± 1.4, p = 0.02) counts that correlated with REE (both p &lt, 0.01). Interestingly, despite higher REE, asthmatics reported consuming fewer calories (25.1 ± 7.5 vs. 20.3 ± 6.0 kcals/kg/day, p &lt, 0.01) and carbohydrates than controls. Conclusion: REE is elevated in adults with mild asthma, suggesting there is an association between REE and the pathophysiology of asthma.

Diabetes and obesity are associated with inflammasome-mediated low-grade, chronic inflammation that may induce pancreatic beta-cell dysfunction and apoptosis. We examined the effects of Roux-en-Y gastric bypass (RYGB) surgery on NOD-like receptor family, pyrin domain containing-3 (NLRP3) inflammasome-related genes from pancreatic islets of Zucker diabetic fatty rats. Islets were collected from Zucker diabetic fatty sham control and RYGB, 30 days after surgery. We assessed expression of genes that regulate glucose metabolism and the NLRP3 inflammasome (NLRP3, caspase-1, IL-1β, IL-18, apoptosis-associated speck-like protein), IL-6, and monocyte chemoattractant protein-1. Gene expression for NLRP3 (p

Overweight and obesity are global health problems placing an ever-increasing demand on health care systems. Brown adipose tissue (BAT) is present in significant amounts in adults. BAT has potential as a fuel for oxidation and dissipation as heat production, which makes it an attractive target for obesity therapy. BAT activation results in increased energy expenditure via thermogenesis. The role of BAT/beige adipocyte activation on whole body energy homeostasis, body weight management/regulation, and whole body glucose and lipid homeostasis remains unproven. This paper reviews knowledge on brown/beige adipocytes in energy expenditure and how it may impact obesity therapy and its comorbidities.

AIMS Mitochondria exist as a morphologically plastic network driven by cellular bioenergetic demand. Induction of fusion and fission machinery allows the organelle to regulate quality control and substrate flux. Physiological stressors promote fragmentation of the mitochondrial network, a process implicated in the onset of metabolic disease, including type 2 diabetes and obesity. It is well-known that exercise training improves skeletal muscle mitochondrial volume, number, and density. However, the effect of exercise training on muscle mitochondrial dynamics remains unclear. METHODS Ten sedentary adults (65.8 ± 4.6 years; 34.3 ± 2.4 kg/m2 ) underwent 12 weeks of supervised aerobic exercise training (5 day/wk, 85% of HRMAX ). Body composition, cardio-metabolic testing, hyperinsulinaemic-euglycaemic clamps, and skeletal muscle biopsies were performed before and after training. MFN1, MFN2, OPA1, OMA1, FIS1, Parkin, PGC-1α, and HSC70 protein expression was assessed via Western blot. RESULTS Exercise training led to improvements in insulin sensitivity, aerobic capacity, and fat oxidation (all P

β-cell dysfunction plays a major role in the onset of type 2 diabetes (T2D). Exercise improves or restores β-cell function in both humans and rodents with T2D. The specific mechanism is unclear. We hypothesized that exercise-induced myokines engage in cross-talk with the pancreas to help regulate β-cell function and mass. To test our hypothesis, differentiated C2C12 myotubes were subjected to electrical pulse stimulation (EPS) to simulate in vivo exercise. EPS-conditioned media from the contracting myotubes significantly elevated glucose-stimulated insulin secretion (GSIS) in β-cells vs. non-EPS control conditions (P In summary, these findings expand our understanding of skeletal muscle as an endocrine organ and identify GDF15 as a potential myokine for T2D therapy. Disclosure H. Zhang: None. A. Mulya: None. S. Nieuwoudt: None. R. McDowell: None. J.P. Kirwan: None.

Metformin (Met) is the most commonly prescribed antidiabetic agent worldwide. Although it has been marketed in the U.S. for over two decades, the exact molecular mechanisms of Met remain controversial. In addition to suppression of hepatic gluconeogenesis, increasing evidence suggests Met induces extrahepatic tissue-specific effects, such as in skeletal muscle (SKM), where the mitochondrion is believed to be the primary cellular target. We investigated the effects of Met on mitochondrial pathways in human SKM cells isolated by percutaneous needle biopsy from lean control, obese nondiabetic, and type 2 diabetic (T2D) volunteers. Mitochondrial function was assessed by Seahorse XF24 analyzer in SKM cells treated with or without Met for 24 hours. Glycolytic rate increased in Met-treated SKM cells from lean (P Disclosure J. Sacks: None. A. Mulya: None. C.E. Fealy: None. K.M. Fitzgerald: None. E. Huang: None. J.P. Kirwan: None.

The role of mitochondria in insulin resistance and type 2 diabetes (T2D) is the subject of much debate. Recent evidence has suggested the presence of altered skeletal muscle mitochondrial dynamics, both increased fission and reduced fusion, in the etiology of insulin resistance and T2D, however, it is unclear what role, if any, changes in mitochondrial dynamics play in the induction of insulin resistance. We have proposed that increased mitochondrial fission may represent an early mechanistic link between the mitochondria and the onset of skeletal muscle insulin resistance. To test this hypothesis we recruited healthy humans to receive, in a randomized crossover design, an overnight saline (control) or lipid infusion (20% Intralipid) at 0.55 ml/kg/h, followed by a 2h, 90 mg/dL, 40 mU/m2 hyperinsulinemic-euglycemic clamp to assess insulin sensitivity. Changes to mitochondrial dynamics protein expression were assessed by western blot, ex vivo mitochondrial membrane potential (TMRM staining) by confocal microscopy, oxidative capacity by respirometry (Oroboros O2k), and mitochondrial morphology by transmission electron microscopy, in skeletal muscle biopsy samples following the overnight infusions. Preliminary data (n=8; 5M, 3F; Age: 26±1 years; BMI: 22.6±0.7; VO2max: 44.0±2.5) indicate that lipid infusion results in a tendency for reduced insulin sensitivity (P=0.08) and mitochondrial membrane potential (P=0.09), and an increase in Complex I state 3 respiration (P Disclosure C.E. Fealy: None. C.L. Axelrod: None. M.L. Erickson: None. E. Huang: None. A. Hari: None. A. Mulya: None. H. Fujioka: None. C.L. Hoppel: None. J.P. Kirwan: None.

Introduction: The integrity, morphology, structure and functionality of skeletal muscle mitochondria in patients with type 2 diabetes (T2D) is heavily disputed. Thus, the purpose of this investigation was to characterize mitochondrial phenotypes across the insulin resistance spectrum. Methods: 30 sedentary adults aged 18-55 were grouped as lean (BMI 21±0.6, N=13), obese (BMI 32±0.9, N=12), or T2D (BMI 33±2.5, N=5) based on oral glucose tolerance tests and body mass index (BMI). All subjects completed a 3-day, inpatient stay consisting of cardio-metabolic testing, body composition analysis and a hyperinsulinemic-euglycemic clamp study. On the day of the clamp, muscle specimens were obtained basally and under insulin-stimulated conditions. Mitochondrial membrane potential (δψm) was measured by confocal microscopy using the cationic fluorophore TMRM. Oxidative phosphorylation (OXPHOS) was assessed in permeabilized fibers via high resolution respirometry (Oroboros Oxygraph-2k). Muscle mitochondrial ultrastructure and content were determined by transmission electron microscopy (TEM) of fixed muscle sections. Results: There was a significant, step-wise decrease in insulin-stimulated glucose uptake across the metabolic spectrum (P Conclusion: Collectively, these findings provide direct evidence of mitochondrial adaptation in insulin resistant and diabetic skeletal muscle. Disclosure C.L. Axelrod: None. C.E. Fealy: None. A. Mulya: None. E. Huang: None. H. Fujioka: None. B. Burguera: None. C.L. Hoppel: None. J.P. Kirwan: None.

The traditional view of mitochondria as isolated, spherical, energy producing organelles, is undergoing a revolutionary change. Emerging data show that mitochondria form a dynamic reticulum that is regulated by cycles of fission and fusion. The discovery of proteins that modulate these activities has led to important advances in understanding human disease. Here, we review the latest evidence that connects the emerging field of mitochondrial dynamics to skeletal muscle insulin resistance and propose some potential mechanisms that may explain the long debated link between mitochondria and the development of type 2 diabetes.

AIM High-fat diets are known to contribute to the development of obesity and related co-morbidities including non-alcoholic fatty liver disease (NAFLD). The accumulation of hepatic lipid may increase endoplasmic reticulum (ER) stress and contribute to non-alcoholic steatohepatitis and metabolic disease. We hypothesized that bariatric surgery would counter the effects of a high-fat diet (HFD) on obesity-associated NAFLD. METHODS Sixteen of 24 male Sprague Dawley rats were randomized to Sham (N = 8) or Roux-en-Y gastric bypass (RYGB) surgery (N = 8) and compared to Lean controls (N = 8). Obese rats were maintained on a HFD throughout the study. Insulin resistance (HOMA-IR), and hepatic steatosis, triglyceride accumulation, ER stress and apoptosis were assessed at 90 days post-surgery. RESULTS Despite eating a HFD for 90 days post-surgery, the RYGB group lost weight (-20.7 ± 6%, P

BACKGROUND Anastomotic leak after colorectal surgery is a significant cause of morbidity and mortality. The aim of this study was to evaluate the impact of a reinforced colo-colonic anastomosis with tissue adhesive, 2-octylcyanoacrylate (2-OCA), on the integrity of anastomotic healing as measured by anastomotic bursting pressure. METHODS Sixty-eight female Sprague-Dawley rats underwent a rectosigmoid colon transection and a sutured end-to-end anastomosis followed by randomization to receive no further intervention or reinforcement with the tissue adhesive, 2-OCA. After seven postoperative days, a macroscopic assessment of the anastomosis, mechanical assessment to determine anastomotic bursting pressure, and a detailed semi-quantitative histopathologic healing assessment were performed. RESULTS Thirty-four animals were randomized to each group. Study characteristics did not differ between the groups. There was also no difference in the degree of adhesions present postoperatively. Although there was no difference between the net proximal and distal luminal areas in the two groups (0.37 cm2versus 0.55 cm2, P = 0.26), the 2-OCA group exhibited evidence of stricture in 15% of anastomoses as compared with 3% in the suture-only group (P

Background & Aims Activation of Fas death receptor results in apoptosis in multiple organs, particularly liver, in a process dependent on Bid cleavage. Mice injected with an anti-Fas antibody die within hours of acute liver failure associated with massive apoptosis and hemorrhage. Our aim was to investigate the crosstalk of apoptotic and inflammatory pathways and the contribution of selective hepatocellular apoptosis during in vivo Fas activation. Methods We generated hepatocyte-specific Bid deficient mice ( hBid −/− ). Acute liver injury was induced by Fas-activating antibody (Jo2) in a time-course study. Results In contrast to controls, nearly all Jo2 injected hBid −/− survived. Their livers showed complete protection against hepatocellular apoptosis with minimal focal hemorrhagic changes and mainly non-parenchymal cell apoptosis. In agreement, the hepatocytes had no mitochondrial cytochrome c release in cytosol, or caspase 3 activation. hBid −/− livers showed marked increase in acute inflammatory foci composed of neutrophils and monocytes associated with the increased expression of proinflammatory chemokines and cytokines, in the manner dependent on non-canonical interleukin-1β activation and amplified in the absence of caspase-3 activation. In addition, hBid −/− mice were completely protected from hepatotoxicity and the infiltrated cells were cleared 2weeks post single Jo2 injection. Conclusions Hepatocyte Bid suppression is critical for the resistance to the lethal effects of Fas activation in vivo . Fas signaling induces differential activation of non-canonical interleukin-1β maturation, amplified in the absence of apoptotic Bid-mitochondrial loop, in hepatocytes. These findings may have important pathophysiological and therapeutic implications in a variety of liver disorders associated with Fas activation.

Fetuin-A is synthesized in the liver and may be associated with nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes. Lifestyle-induced weight loss reduces fetuin-A, but the effect of exercise alone is unknown. We determined the effect of short-term exercise training on plasma fetuin-A in 13 (50.5 ± 3.4 yr) obese adults (body mass index, 33.3 ± 0.9 kg/m2) with clinically diagnosed NAFLD. Subjects participated in 7 days of supervised exercise training (60 min/day at ∼85% maximum heart rate) and were instructed to maintain their normal caloric and macronutrient intake. Insulin resistance was assessed by an oral glucose tolerance test. Hepatic triglyceride content (HTGC) was determined by proton MRI. We used C2C12 skeletal muscle cells to examine the direct effect of fetuin-A on 2-deoxyglucose uptake, insulin signaling [phosphorylation of Akt and AS160 (pAkt and pAS160, respectively)], and glucose transporter-4 (GLUT-4) translocation. Insulin resistance was reduced by 29% ( P < 0.05), and glucose area under the curve (AUC) was decreased by 13% ( P < 0.01) after the 7 days of exercise. Furthermore, circulating fetuin-A was decreased by 11% (4.2 ± 03 vs. 3.6 ± 0.2 nM; P < 0.02), and this change correlated with reduced insulin resistance ( r = 0.62; P < 0.04) and glucose AUC ( r = 0.58; P < 0.04). Importantly, the exercise program did not change body weight ( P = 0.12), HTGC ( P = 0.73), or aerobic capacity ( P = 0.14). In vitro experiments revealed that fetuin-A decreased skeletal muscle glucose uptake by downregulating pAkt and pAS160 and subsequent GLUT-4 translocation to the plasma membrane. Together, our findings highlight a role for fetuin-A in skeletal muscle insulin resistance and suggest that part of the exercise-induced improvement in glucose tolerance in patients with NAFLD may be due to lowering fetuin-A.

Apolipoprotein M (apoM) is a novel apolipoprotein that is reportedly necessary for pre beta HDL formation; however, its detailed function remains unknown. We investigated the biogenesis and properties of apoM and its effects on the initial steps of nascent pre beta HDL assembly by ABCA1 in HEK293 cells. Transiently transfected apoM was localized primarily in the endomembrane compartment. Pulse-chase analyses demonstrated that apoM is inefficiently secreted, relative to human serum albumin, and that approximately 50% remains membrane-associated after extraction with sodium carbonate, pH 11.5. To investigate the role of apoM in nascent pre beta HDL formation, ABCA1-expressing or control cells, transfected with empty vector, apoM, or C-terminal epitope-tagged apoM (apoM-C-FLAG), were incubated with (125)I-apoA-I for 24 h. Conditioned media were harvested and fractionated by fast-protein liquid chromatography (FPLC) to monitor HDL particle size. Pre beta HDL particles were formed effectively in the absence of apoM expression; however, increased apoM expression stimulated the formation of larger-sized nascent pre beta HDLs. Immunoprecipitation with anti-apoA-I antibody followed by apoM Western blot analysis revealed that little secreted apoM was physically associated with pre beta HDL. Our results suggest that apoM is an atypical secretory protein that is not necessary for ABCA1-dependent pre beta HDL formation but does stimulate the formation of larger-sized pre beta HDL. We propose that apoM may function catalytically at an intracellular site to transfer lipid onto pre beta HDL during or after their formation by ABCA1.

Bariatric surgery provides significant and durable improvements in glycemic control and hepatic steatosis, but the underlying mechanisms that drive improvements in these metabolic parameters remain to be fully elucidated. Recently, alterations in mitochondrial morphology have shown a direct link to nutrient adaptations in obesity. Here, we evaluate the effects of Roux‐en‐Y gastric bypass (RYGB) surgery on markers of liver mitochondrial dynamics in a diet‐induced obesity Sprague‐Dawley (SD) rat model. Livers were harvested from adult male SD rats 90‐days after either Sham or RYGB surgery and continuous high‐fat feeding. We assessed expression of mitochondrial proteins involved in fusion, fission, mitochondrial autophagy (mitophagy) and biogenesis, as well as differences in citrate synthase activity and markers of oxidative stress. Gene expression for mitochondrial fusion genes, mitofusin 1 (Mfn1; P

We investigated the in vivo metabolic fate of pre-beta HDL particles in human apolipoprotein A-I transgenic (hA-I (Tg)) mice. Pre-beta HDL tracers were assembled by incubation of [(125)I]tyramine cellobiose-labeled apolipoprotein A-I (apoA-I) with HEK293 cells expressing ABCA1. Radiolabeled pre-beta HDLs of increasing size (pre-beta1, -2, -3, and -4 HDLs) were isolated by fast-protein liquid chromatography and injected into hA-I (Tg)-recipient mice, after which plasma decay, in vivo remodeling, and tissue uptake were monitored. Pre-beta2, -3, and -4 had similar plasma die-away rates, whereas pre-beta1 HDL was removed 7-fold more rapidly. Radiolabel recovered in liver and kidney 24 h after tracer injection suggested increased (P < 0.001) liver and decreased kidney catabolism as pre-beta HDL size increased. In plasma, pre-beta1 and -2 were rapidly (

Macrophage-specific Abca1 knock-out (Abca1(-)(M)(/-)(M)) mice were generated to determine the role of macrophage ABCA1 expression in plasma lipoprotein concentrations and the innate immune response of macrophages. Plasma lipid and lipoprotein concentrations in chow-fed Abca1(-)(M)(/-)(M) and wild-type (WT) mice were indistinguishable. Compared with WT macrophages, Abca1(-)(M)(/-)(M) macrophages had a95% reduction in ABCA1 protein, failed to efflux lipid to apoA-I, and had a significant increase in free cholesterol (FC) and membrane lipid rafts without induction of endoplasmic reticulum stress. Lipopolysaccharide (LPS)-treated Abca1(-)(M)(/-)(M) macrophages exhibited enhanced expression of pro-inflammatory cytokines and increased activation of the NF-kappaB and MAPK pathways, which could be diminished by silencing MyD88 or by chemical inhibition of NF-kappaB or MAPK. In vivo LPS injection also resulted in a higher pro-inflammatory response in Abca1(-)(M)(/-)(M) mice compared with WT mice. Furthermore, cholesterol depletion of macrophages with methyl-beta-cyclodextrin normalized FC content between the two genotypes and their response to LPS; cholesterol repletion of macrophages resulted in increased cellular FC accumulation and enhanced cellular response to LPS. Our results suggest that macrophage ABCA1 expression may protect against atherosclerosis by facilitating the net removal of excess lipid from macrophages and dampening pro-inflammatory MyD88-dependent signaling pathways by reduction of cell membrane FC and lipid raft content.

Objectives— The aim of this study was to determine the role of ATP binding cassette transporter A1 (ABCA1) on generation of different-sized nascent HDLs. Methods and Results— HEK293 cells stably-transfected with ABCA1 (HEK293-ABCA1) or non-transfected (control) cells were incubated with lipid free 125 I-apoA-I for 24 hours. Incubation of apoA-I with HEK293-ABCA1 cells, but not control cells, led to the formation of heterogeneous-sized, pre-β migrating nascent HDL subpopulations (pre-β1 to -4) that varied in size (7.1 to 15.7 nm), lipid, and apoA-I content. Kinetic studies suggested that all subpopulations were formed simultaneously, with no evidence for a precursor-product relationship between smaller and larger-sized particles. When isolated nascent pre-β HDLs (pre-β1 to -4) were added back to HEK293-ABCA1 cells, their ability to bind to ABCA1 and efflux lipid was severely compromised. Heat-denaturation of pre-β1 HDL resulted in partial recovery of ABCA1 binding, suggesting that initial interaction of apoA-I with ABCA1 results in a constrained conformation of apoA-I that decreases subsequent binding. Conclusions— Interaction of apoA-I with ABCA1 results in the simultaneous generation of pre-β HDLs of discrete size and chemical composition. These nascent particles are poor substrates for subsequent lipidation by ABCA1 and presumably require additional non-ABCA1-mediated lipidation for further maturation.

Reduction of plasma LCAT activity has been observed in several conditions in which the size of HDL particles is increased; however, the mechanism of this reduction remains elusive. We investigated the plasma activity, mass, and in vivo catabolism of LCAT and its association with HDL particles in human apolipoprotein A-I transgenic, scavenger receptor class B type I knockout (hA-I Tg SR-BI−/−) mice. Compared with hA-I Tg mice, hA-I Tg SR-BI−/− mice had a 4-fold higher total plasma cholesterol concentration, which occurred predominantly in 13–18 nm diameter HDL particles, a significant reduction in plasma esterified cholesterol-total cholesterol (EC/TC) ratio, and significantly lower plasma LCAT activity, suggesting a decrease in LCAT protein. However, LCAT protein in plasma, hepatic mRNA for LCAT, and in vivo turnover of 35S-radiolabeled LCAT were similar in both genotypes of mice. HDL from hA-I Tg SR-BI−/− mice was enriched in sphingomyelin (SM), relative to phosphatidylcholine, and had less associated [35S]LCAT radiolabel and endogenous LCAT activity compared with HDL from hA-I Tg mice. We conclude that the decreased EC/TC ratio in the plasma of hA-I Tg SR-BI−/− mice is attributed to a reduction in LCAT reactivity with SM-enriched HDL particles.

Objective Obesity is associated with low-grade chronic inflammation. We hypothesized that Roux-en-Y gastric bypass (RYGB) surgery would reduce activation of the NLRP3 inflammasome in metabolically active adipose tissue (AT) of obese rats, and this change would be related to decreases in body weight and improved glycemic control. Methods Omental, mesenteric and subcutaneous fat depots were collected from Sprague-Dawley rats: Sham control and RYGB; 90-days after surgery. NLRP3, caspase–1, apoptosis-associated speck-like protein (ASC), IL–1β, IL–18, IL–6 and MCP–1 gene and protein expression were quantified. Glucose metabolism was assessed by oral glucose tolerance test (OGTT). Results Compared to Sham surgery controls, RYGB surgery decreased IL–6, MCP–1, NLRP3, IL–18, caspase–1 and ASC in omental fat, and decreased IL–6, MCP1, IL–1β, IL–18, caspase–1 and ASC gene expression in mesenteric fat. We observed differential gene expression between visceral and subcutaneous fat for IL–6 and IL–1β, both being downregulated by RYGB in visceral, and upregulated in subcutaneous depots. These changes in gene expression were accompanied by a decrease in NLRP3, ASC, IL–18, caspase–1 and IL–1β protein expression in omental tissue. We found a positive correlation between caspase–1, ASC, MCP–1, IL–18 and IL–6 gene expression following surgery and glucose AUC response in omental fat, while the change in glucose AUC response correlated with caspase–1 gene expression in subcutaneous fat. Conclusion This study demonstrates that bariatric surgery reverses inflammation in visceral adipose tissue by suppressing NLRP3 inflammasome activation. These are the first data to implicate the NLRP3 inflammasome in diabetes remission after RYGB surgery.

Atherosclerosis development is accelerated severalfold in patients with Type 2 diabetes. In the initial stages of disease, monocytes transmigrate into the subendothelial space and differentiate into foam cells. Scavenger receptors and ATP binding cassette (ABC) Transporters play an important role in foam cell formation as they regulate the influx and efflux of oxidized lipids. Here, we show that peritoneal macrophages isolated from Type 2 diabetic db/db mice have decreased expression of the ABC transporter ABCG1 and increased expression of the scavenger receptor CD36. We found a 2-fold increase in accumulation of esterified cholesterol in diabetic db/db macrophages compared with wild-type control macrophages. Diabetic db/db macrophages also had impaired cholesterol efflux to high density lipoprotein but not to lipid-free apo A-I, suggesting that the increased esterified cholesterol in diabetic db/db macrophages was due to a selective loss of ABCG1-mediated efflux to high density lipoprotein. Additionally, we were able to confirm down-regulation of ABCG1 using C57BL/6J peritoneal macrophages cultured in elevated glucose in vitro (25 mM glucose for 7 days), suggesting that ABCG1 expression in diabetic macrophages is regulated by chronic exposure to elevated glucose. Diabetic KK(ay) mice were also studied and were found to have decreased ABCG1 expression without an increase in CD36. These observations demonstrate that ABCG1 plays a major role in macrophage cholesterol efflux and that decreased ABCG1 function can facilitate foam cell formation in Type 2 diabetic mice.

Patients homozygous for Tangier disease have a near absence of plasma HDL as a result of mutations in ABCA1 and hypercatabolize normal HDL particles. To determine the relationship between ABCA1 expression and HDL catabolism, we investigated intravascular remodeling, plasma clearance, and organ-specific uptake of HDL in mice expressing the human apolipoprotein A-I (apoA-I) transgene in the Abca1 knockout background. Small HDL particles (7.5 nm), radiolabeled with (125)I-tyramine cellobiose, were injected into recipient mice to quantify plasma turnover and the organ uptake of tracer. Small HDL tracer was remodeled to 8.2 nm diameter particles within 5 min in human apolipoprotein A-I transgenic (hA-I(Tg)) mice (control) and knockout mice. Decay of tracer from plasma was 1.6-fold more rapid in knockout mice (P0.05) and kidney uptake was twice that of controls, with no difference in liver uptake. We also observed 2-fold greater hepatic expression of ABCA1 protein in hA-I(Tg) mice compared with nontransgenic mice, suggesting that overexpression of human apoA-I stabilized hepatic ABCA1 protein in vivo. We conclude that ABCA1 is not required for in vivo remodeling of small HDLs to larger HDL subfractions and that the hypercatabolism of normal HDL particles in knockout mice is attributable to a selective catabolism of HDL apoA-I by the kidney.

(P)atients with Tangier disease exhibit extremely low plasma HDL concentrations resulting from mutations in the ATP-binding cassette, sub-family A, member 1 (ABCA1) protein. ABCA1 controls the rate-limiting step in HDL particle assembly by mediating efflux of cholesterol and phospholipid from cells to lipid-free apoA-I, which forms nascent HDL particles. ABCA1 is widely expressed; however, the specific tissues involved in HDL biogenesis are unknown. To determine the role of the liver in HDL biogenesis, we generated mice with targeted deletion of the second nucleotide-binding domain of Abca1 in liver only (Abca1(-L/-L)). Abca1(-L/-L) mice had total plasma and HDL cholesterol concentrations that were 19% and 17% those of wild-type littermates, respectively. In vivo catabolism of HDL apoA-I from wild-type mice or human lipid-free apoA-I was 2-fold higher in Abca1(-L/-L) mice compared with controls due to a 2-fold increase in the catabolism of apoA-I by the kidney, with no change in liver catabolism. We conclude that in chow-fed mice, the liver is the single most important source of plasma HDL. Furthermore, hepatic, but not extrahepatic, Abca1 is critical in maintaining the circulation of mature HDL particles by direct lipidation of hepatic lipid-poor apoA-I, slowing its catabolism by the kidney and prolonging its plasma residence time

Objective: To determine the effect of exercise on chemerin in relation to changes in fat loss, insulin action, and dyslipidemia in older adults. Participants: Thirty older (65.9±0.9yr) obese adults (BMI:34.5±0.7kg/m2). Setting: Single-center, Cleveland Clinic. Design: Prospective clinical trial. Intervention: Twelve-weeks of exercise training (60minutes/day, 5day/week at ∼85% HRmax). Subjects were instructed to maintain habitual nutrient intake. Measurements: Plasma chemerin was analyzed using an enzyme-linked immunosorbent assay. Peripheral and hepatic insulin sensitivity was assessed using a euglycemic-hyperinsulinic clamp with glucose kinetics. First-phase and total glucose-stimulated insulin secretion (GSIS) was calculated from an oral glucose tolerance test. Fasting blood lipids (cholesterol, triglycerides), total/visceral fat (dual-x-ray absorptiometry and computerized tomography) and cardiorespiratory fitness (treadmill test) were also tested pre and post intervention. Results: Exercise increased fitness and reduced total/visceral fat, blood lipids, and firstphase GSIS (P

Defects in mitochondrial dynamics, the processes of fission, fusion, and mitochondrial autophagy, may contribute to metabolic disease including type 2 diabetes. Dynamin-related protein-1 (Drp1) is a GTPase protein that plays a central role in mitochondrial fission. We hypothesized that aerobic exercise training would decrease Drp1 Ser616phosphorylation and increase fat oxidation and insulin sensitivity in obese (body mass index: 34.6 ± 0.8 kg/m2) insulin-resistant adults. Seventeen subjects performed supervised exercise for 60 min/day, 5 days/wk at 80–85% of maximal heart rate for 12 wk. Insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp, and fat oxidation was determined by indirect calorimetry. Skeletal muscle biopsies were obtained from the vastus lateralis muscle before and after the 12-wk program. The exercise intervention increased insulin sensitivity 2.1 ± 0.2-fold ( P < 0.01) and fat oxidation 1.3 ± 0.3-fold ( P < 0.01). Phosphorylation of Drp1 at Ser616was decreased (pre vs. post: 0.81 ± 0.15 vs. 0.58 ± 0.14 arbitrary units; P < 0.05) following the intervention. Furthermore, reductions in Drp1 Ser616phosphorylation were negatively correlated with increases in fat oxidation ( r = −0.58; P < 0.05) and insulin sensitivity (rho = −0.52; P < 0.05). We also examined expression of genes related to mitochondrial dynamics. Dynamin1-like protein (DNM1L; P < 0.01), the gene that codes for Drp1, and Optic atrophy 1 (OPA1; P = 0.05) were significantly upregulated following the intervention, while there was a trend towards an increase in expression of both mitofusin protein MFN1 ( P = 0.08) and MFN2 ( P = 0.07). These are the first data to suggest that lifestyle-mediated improvements in substrate metabolism and insulin sensitivity in obese insulin-resistant adults may be regulated through decreased activation of the mitochondrial fission protein Drp1.

Dipeptidyl peptidase-4 (DPP-4) is a circulating glycoprotein that impairs insulin-stimulated glucose uptake and is linked to obesity and metabolic syndrome. However, the effect of exercise on plasma DPP-4 in adults with metabolic syndrome is unknown. Therefore, we determined the effect of exercise on DPP-4 and its role in explaining exercise-induced improvements in insulin sensitivity. Fourteen obese adults (67.9±1.2 years, BMI: 34.2±1.1kg/m(2)) with metabolic syndrome (ATP III criteria) underwent a 12-week supervised exercise intervention (60min/day for 5 days/week at ∼85% HRmax). Plasma DPP-4 was analyzed using an enzyme-linked immunosorbent assay. Insulin sensitivity was measured using the euglycemic-hyperinsulinemic clamp (40mU/m(2)/min) and estimated by HOMA-IR. Visceral fat (computerized tomography), 2-h glucose levels (75g oral glucose tolerance), and basal fat oxidation as well as aerobic fitness (indirect calorimetry) were also determined before and after exercise. The intervention reduced visceral fat, lowered blood pressure, glucose and lipids, and increased aerobic fitness (P