Blood Cholesterol and Triglycerides Associate with Right Ventricular Function in Pulmonary Hypertension.

Background: Blood lipids are dysregulated in pulmonary hypertension (PH). Lower high-density lipoproteins cholesterol (HDL-C) and low-density lipoproteins cholesterol (LDL-C) are associated with disease severity and death in PH. Right ventricle (RV) dysfunction and failure are the major determinants of morbidity and mortality in PH. This study aims to test the hypothesis that dyslipidemia is associated with RV dysfunction in PH. Methods: We enrolled healthy control subjects (n=12) and individuals with PH (n=30) (age: 18-65 years old). Clinical characteristics, echocardiogram, 2-[18F] fluoro-2-deoxy-D-glucose positron emission tomography (PET) scan, blood lipids, including total cholesterol (TC), triglycerides (TG), lipoproteins (LDL-C and HDL-C), and N-terminal pro-B type Natriuretic Peptide (NT-proBNP) were determined. Results: Individuals with PH had lower HDL-C [PH, 41±12; control, 56±16 mg/dL, p<0.01] and higher TG to HDL-C ratio [PH, 3.6±3.1; control, 2.2±2.2, p<0.01] as compared to controls. TC, TG, and LDL-C were similar between PH and controls. Lower TC and TG were associated with worse RV function measured by RV strain (R=-0.43, p=0.02 and R=-0.37, p=0.05 respectively), RV fractional area change (R=0.51, p<0.01 and R=0.48, p<0.01 respectively), RV end-systolic area (R=-0.63, p<0.001 and R=-0.48, p<0.01 respectively), RV end-diastolic area: R=-0.58, p<0.001 and R=-0.41, p=0.03 respectively), and RV glucose uptake by PET (R=-0.46, p=0.01 and R=-0.30, p=0.10 respectively). NT-proBNP was negatively correlated with TC (R=-0.61, p=0.01) and TG (R=-0.62, p<0.02) in PH. Conclusion: These findings confirm dyslipidemia is associated with worse right ventricular function in PH.

Method for depletion of mitochondria DNA in human bronchial epithelial cells.

Mitochondria are increasingly recognized to play a role in the airway inflammation of asthma. Model systems to study the role of mitochondrial gene expression in bronchial epithelium are lacking. Here, we create custom bronchial epithelial cell lines that are depleted of mitochondrial DNA. One week of ethidium bromide (EtBr) treatment led to ∼95 % reduction of mtDNA copy number (mtDNA-CN) in cells, which was further reduced by addition of 25 µM 2',3'-dideoxycytidin (ddC). Treatment for up to three weeks with EtBr and ddC led to near complete loss of mtDNA. The basal oxygen consumption rate (OCR) of mtDNA-depleted BET-1A and BEAS-2B cells dropped to near zero. Glycolysis measured by extracellular acidification rate (ECAR) increased ∼two-fold in cells when mtDNA was eliminated. BET-1A ρ0 and BEAS-2B ρ0 cells were cultured for two months, frozen and thawed, cultured for two more months, and maintained near zero mtDNA-CN. Mitochondrial DNA-depleted BET-1A ρ0 and BEAS-2B ρ0 cell lines are viable, lack the capacity for aerobic respiration, and increase glycolysis.•BET-1A and BEAS-2B cells were treated with ethidium bromide (EtBr) with or without 2',3'-dideoxycytidine (ddC) to create cells lacking mitochondrial DNA (mtDNA).•Cells' mtDNA copy number relative to nuclear DNA (nDNA) were verified by quantitative polymerase chain reaction (qPCR).•Cells were also assessed for oxidative phosphorylation by measures of oxygen consumption using the Seahorse analyzer.

Method for Depletion of Mitochondria DNA in Human Bronchial Epithelial Cells.

Introduction: Mitochondria are increasingly recognized to play a role in the airway inflammation of asthma. Model systems to study the role of mitochondrial gene expression in bronchial epithelium are lacking. Here, we create custom bronchial epithelial cell lines derived from primary airway epithelium that are depleted of mitochondrial DNA. Methods: We treated BET-1A and BEAS-2B cells with ethidium bromide (EtBr) with or without 2',3'-dideoxycytidine (ddC) to create cells lacking mitochondrial DNA (mtDNA). Cells' mtDNA copy number were verified by quantitative polymerase chain reaction (qPCR) in comparison to nuclear DNA (nDNA). Cells were also assessed for oxidative phosphorylation by measures of oxygen consumption using the Seahorse analyzer. Results: One week of EtBr treatment led to ~95% reduction of mtDNA copy number (mtDNA-CN) in cells (mtDNA-CN, mean±SE, baseline vs. treatment: BEAS-2B, 820 ± 62 vs. 56 ± 9; BET-1A, 957 ± 52 vs. 73 ± 2), which was further reduced by addition of 25 µM ddC (mtDNA-CN: BEAS-2B, 2.8; BET-1A, 47.9). Treatment for up to three weeks with EtBr and ddC led to near complete loss of mtDNA (mtDNA-CN: BEAS-2B, 0.1; BET-1A, 0.3). The basal oxygen consumption rate (OCR) of mtDNA-depleted BET-1A and BEAS-2B cells dropped to near zero. Glycolysis measured by extracellular acidification rate (ECAR) increased ~two-fold in cells when mtDNA was eliminated [ECAR (mpH/min/103 cells), baseline vs. treatment: BEAS-2B, 0.50 ± 0.03 vs. 0.94 ± 0.10 P=0.005; BET-1A, 0.80 ± 0.04 vs. 1.14 ± 0.06 P=0.001]. Conclusion: Mitochondrial DNA-depleted BET-1A ρ0 and BEAS-2B ρ0 cell lines are viable, lack the capacity for aerobic respiration, and increase glycolysis. This cell model system can be used to further test mitochondrial mechanisms of inflammation in bronchial epithelial cells.

GDF15 Mediates the Effect of Skeletal Muscle Contraction on Glucose-Stimulated Insulin Secretion.

Exercise is a first-line treatment for type 2 diabetes and preserves ß-cell function by hitherto unknown mechanisms. We postulated that proteins from contracting skeletal muscle may act as cellular signals to regulate pancreatic ß-cell function. We used electric pulse stimulation (EPS) to induce contraction in C2C12 myotubes and found that treatment of ß-cells with EPS-conditioned medium 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 ß-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 or obesity. Six weeks of high-intensity exercise training increased circulating GDF15 concentrations, which positively correlated with improvements in ß-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. ARTICLE HIGHLIGHTS: Exercise improves glucose-stimulated insulin secretion through direct interorgan communication. Contracting skeletal muscle releases growth differentiation factor 15 (GDF15), which is required to synergistically enhance glucose-stimulated insulin secretion. GDF15 enhances glucose-stimulated insulin secretion by activating the canonical insulin release pathway. Increased levels of circulating GDF15 after exercise training are related to improvements in ß-cell function in patients with type 2 diabetes.

Lipids activate skeletal muscle mitochondrial fission and quality control networks to induce insulin resistance in humans.

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 ±â€¯1.7 years; BMI:22.7 ±â€¯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.

Resting Energy Expenditure Is Elevated in Asthma.

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 < 0.01) and neutrophil (2.8 ± 0.9 vs. 3.6 ± 1.4, p = 0.02) counts that correlated with REE (both p < 0.01). Interestingly, despite higher REE, asthmatics reported consuming fewer calories (25.1 ± 7.5 vs. 20.3 ± 6.0 kcals/kg/day, p < 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.

Roux-en-Y gastric bypass restores islet function and morphology independent of body weight in ZDF rats.

Reductions in ß-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 ß-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. ß-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.

Metabolic effects of duodenojejunal bypass surgery in a rat model of type 1 diabetes.

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.

BAM15-mediated mitochondrial uncoupling protects against obesity and improves glycemic control.

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.

Effects of gastric bypass surgery on expression of glucose transporters and fibrotic biomarkers in kidney of diabetic fatty rats.

BACKGROUND: Diabetic nephropathy is the leading cause of chronic kidney disease. Observational studies suggest Roux-en-Y gastric bypass (RYGB) reduces progression of diabetic nephropathy. OBJECTIVES: To unravel the mechanisms by which RYGB is beneficial and protective for diabetic nephropathy. SETTING: Academic laboratories. METHODS: 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. RESULTS: 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 (Tgfß) 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. CONCLUSIONS: 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/TGFß pathway.

Exercise training remodels human skeletal muscle mitochondrial fission and fusion machinery towards a pro-elongation phenotype.

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 < 0.01), as well as reductions in body weight, BMI, fat mass and fasting glucose (all P < 0.001). When normalized for changes in mitochondrial content, exercise reduced skeletal muscle FIS1 and Parkin (P < 0.05), while having no significant effect on MFN1, MFN2, OPA1, and OMA1 expression. Exercise also improved the ratio of fusion to fission proteins (P < 0.05), which positively correlated with improvements in glucose disposal (r2  = 0.59, P < 0.05). CONCLUSIONS: Exercise training alters the expression of mitochondrial fusion and fission proteins, promoting a more fused, tubular network. These changes may contribute to the improvements in insulin sensitivity and substrate utilization that are observed after exercise training.

Mitochondrial dynamics in skeletal muscle insulin resistance and type 2 diabetes.

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.

2-Octylcyanoacrylate for the prevention of anastomotic leak.

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 < 0.0001). Histologically, the presence of only fibroblasts density was statistically more evident in the 2-OCA group compared with the sutured-only anastomosis (P = 0.0183). There was not a significant increase in mechanical strength in the 2-OCA group (238.9 mm Hg) versus in the suture-only group (231.8 mm Hg). There was no difference in the rate of anastomotic leak in the 2-OCA as compared with the suture-only group (9.1 versus 8.8%). CONCLUSIONS: Application of 2-OCA to reinforce a colo-colonic anastomosis clinically provides no benefit to its mechanical strength and detrimentally increases the rate of obstruction and/or stricture in this in vivo model.

Effect of Roux-en-Y gastric bypass on liver mitochondrial dynamics in a rat model of obesity.

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 < 0.05), mitofusin 2 (Mfn2; P < 0.01), and optic atrophy 1 (OPA1; P < 0.05) increased following RYGB surgery. Biogenesis regulators, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α; P < 0.01) and nuclear respiratory factor 1 (Nrf1; P < 0.05), also increased in the RYGB group, as well as mitophagy marker, BCL-2 interacting protein 3 (Bnip3; P < 0.01). Protein expression for Mfn1 (P < 0.001), PGC1α (P < 0.05), BNIP3 (P < 0.0001), and mitochondrial complexes I-V (P < 0.01) was also increased by RYGB, and Mfn1 expression negatively correlated with body weight, insulin resistance, and fasting plasma insulin. In the RYGB group, citrate synthase activity was increased (P < 0.02) and reactive oxygen species (ROS) was decreased compared to the Sham control group (P < 0.05), although total antioxidant capacity was unchanged between groups. These data are the first to show an association between RYGB surgery and improved markers of liver mitochondrial dynamics. These observed improvements may be related to weight loss and reduced energetic demand on the liver, which could facilitate normalization of glucose homeostasis and protect against hepatic steatosis.

In vitro contraction protects against palmitate-induced insulin resistance in C2C12 myotubes.

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.

Exercise training-induced improvement in skeletal muscle PGC-1α-mediated fat metabolism is independent of dietary glycemic index.

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 < 0.002) and PPARγ (P < 0.002), PGC-1α (P = 0.003), CD36 (P = 0.003), FABP3 (mRNA, P = 0.01 and protein, P = 0.02), and CPT1B (mRNA, P = 0.03 and protein, P = 0.008) expression were similar for both interventions. Increased insulin sensitivity correlated with increased PGC-1α expression (P = 0.04), and increased fasting fat oxidation correlated with increased FABP3 (P = 0.04) and CPT1B (P = 0.05) expression. CONCLUSIONS: An exercise/diet program resulting in 8% to 10% weight loss improved insulin sensitivity and key molecular mechanisms in skeletal muscle that are controlled by PGC-1α. These effects were independent of the glycemic index of the diets.

Effect of Roux-en-Y Gastric Bypass on the NLRP3 Inflammasome in Pancreatic Islets from Zucker Diabetic Fatty Rats.

PURPOSE: 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. MATERIALS AND METHODS: 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-1ß, IL-18, apoptosis-associated speck-like protein), IL-6, and monocyte chemoattractant protein-1. RESULTS: Gene expression for NLRP3 (p < 0.02), IL-1ß (p < 0.04), and IL-6 (p < 0.01) was reduced by RYGB and positively correlated with change in body weight. IL-1ß positively correlated with glucose AUC response. CONCLUSION: Suppression of the NLRP3 inflammasome in pancreatic islets may contribute to improved glycemic control after RYGB.

Brown and Beige Adipose Tissue: Therapy for Obesity and Its Comorbidities?

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.

Effect of Roux-en-Y Gastric Bypass on the NLRP3 Inflammasome in Adipose Tissue from Obese Rats.

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-1ß, 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-1ß, 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-1ß, 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-1ß 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.

Microparticles release by adipocytes act as "find-me" signals to promote macrophage migration.

Macrophage infiltration of adipose tissue during weight gain is a central event leading to the metabolic complications of obesity. However, what are the mechanisms attracting professional phagocytes to obese adipose tissue remains poorly understood. Here, we demonstrate that adipocyte-derived microparticles (MPs) are critical "find-me" signals for recruitment of monocytes and macrophages. Supernatants from stressed adipocytes stimulated the attraction of monocyte cells and primary macrophages. The activation of caspase 3 was required for release of these signals. Adipocytes exposed to saturated fatty acids showed marked release of MPs into the supernatant while common genetic mouse models of obesity demonstrate high levels of circulating adipocyte-derived MPs. The release of MPs was highly regulated and dependent on caspase 3 and Rho-associated kinase. Further analysis identified these MPs as a central chemoattractant in vitro and in vivo. In addition, intravenously transplanting circulating MPs from the ob/ob mice lead to activation of monocytes in circulation and adipose tissue of the wild type mice. These data identify adipocyte-derived MPs as novel "find me" signals that contributes to macrophage infiltration associated with obesity.