Effect of an acute long-duration exercise bout on skeletal muscle lipid droplet morphology, GLUT 4 protein, and perilipin protein expression.

PURPOSE: Smaller lipid droplet morphology and GLUT 4 protein expression have been associated with greater muscle oxidative capacity and glucose uptake, respectively. The main purpose of this study was to determine the effect of an acute long-duration exercise bout on skeletal muscle lipid droplet morphology, GLUT4, perilipin 3, and perilipin 5 expressions. METHODS: Twenty healthy men (age 24.0 ± 1.0 years, BMI 23.6 ± 0.4 kg/m2) were recruited for the study. The participants were subjected to an acute bout of exercise on a cycle ergometer at 50% VO2max until they reached a total energy expenditure of 650 kcal. The study was conducted after an overnight fast. Vastus lateralis muscle biopsies were obtained before and immediately after exercise for immunohistochemical analysis to determine lipid, perilipin 3, perilipin 5, and GLUT4 protein contents while GLUT 4 mRNA was quantified using RT-qPCR. RESULTS: Lipid droplet size decreased whereas total intramyocellular lipid content tended to reduce (p = 0.07) after an acute bout of endurance exercise. The density of smaller lipid droplets in the peripheral sarcoplasmic region significantly increased (0.584 ± 0.04 to 0.638 ± 0.08 AU; p = 0.01) while larger lipid droplets significantly decreased (p < 0.05). GLUT4 mRNA tended to increase (p = 0.05). There were no significant changes in GLUT 4, perilipin 3, and perilipin 5 protein levels. CONCLUSION: The study demonstrates that exercise may impact metabolism by enhancing the quantity of smaller lipid droplets over larger lipid droplets.

Mass-Forming Gastric Heterotopia of the Rectum: A Series of 3 Cases from a Single Tertiary Health Center.

BACKGROUND Gastric heterotopia is a benign entity found throughout the gastrointestinal tract but is rarely identified in the rectum. Since 1939, only 94 cases have ever been identified, and it can present as a mass formation with symptomatology that mimics colorectal malignancy. In some instances, malignancy has been shown to arise within rectal gastric heterotopia. Here, we present 3 cases from the past 20-year period of rectal gastric heterotopia at a single tertiary institution. CASE REPORT A 25-year-old man (case 1), a 58-year-old woman (case 2), and a 33-year-old man (case 3) were found to have polypoid mass-like lesions greater than 1.0 cm within the rectum. Following biopsy, pathology showed gastric oxyntic mucosa flanked by colorectal mucosa, thus indicating gastric heterotopia. Presenting symptoms from all patients consisted of unspecified anal pain, hematochezia, or a combination of both. All patients were treated with endoscopic mucosal resection (EMR), which provided relief of symptoms and confirmed no evidence of invasive malignancy. CONCLUSIONS Rectal gastric heterotopia can mimic malignancy and in very rare instances can harbor high-grade dysplasia as well as invasive carcinoma. EMR seems to be a definitive treatment that offers relief to patient symptomatology and reassurance that any dysplasia is identified and removed.

Four weeks of electrical stimulation improves glucose tolerance in a sedentary overweight or obese Hispanic population.

Introduction/purpose: Most US adults (54%) do not meet the minimum exercise recommendations by the American College of Sports Medicine. Neuromuscular electrical stimulation (NMES) is a novel alternate strategy to induce muscle contraction. However, the effectiveness of NMES to improve insulin sensitivity and energy expenditure is unclear. The purpose of this study was to investigate the effects of 4 weeks of NMES on glucose tolerance in a sedentary overweight or obese population. Methods: Participants (n = 10; age: 36.8 ± 3.8 years; BMI = 32 ± 1.3 kg/m2) were randomized into either control or NMES group. All participants received bilateral quadriceps stimulation (12 sessions; 30 min/session; three times/week at 50 Hz and 300 µs pulse width) altering pulse amplitude to either provide low-intensity sensory level (control; tingling sensation) or at high-intensity neuromuscular level (NMES; maximum tolerable levels with visible muscle contraction). Glucose tolerance was assessed by a 3-h oral glucose tolerance test (OGTT), and substrate utilization was measured by indirect calorimetry and body composition via dual X-ray absorptiometry at baseline and after 4 weeks of NMES intervention. Results: Control and NMES groups had comparable fasting blood glucose, glucose tolerance, substrate utilization, and muscle mass at baseline. Four weeks of NMES resulted in a significant improvement in glucose tolerance measured by OGTT, whereas no change was observed in the control group. There was no change in substrate utilization and muscle mass in both control and NMES groups. Conclusion: NMES is a novel and effective strategy to improve glucose tolerance in an at-risk overweight or obese sedentary population.

Exercise-Induced Improvements in Insulin Sensitivity Are Not Attenuated by a Family History of Type 2 Diabetes.

Introduction: A family history of type 2 diabetes (FH+) is a major risk factor for the development of insulin resistance and type 2 diabetes. However, it remains unknown whether exercise-induced improvements in insulin sensitivity and metabolic flexibility are impacted by a FH+. Therefore, we investigated whether improvements in insulin sensitivity, metabolic flexibility, body composition, aerobic fitness and muscle strength are limited by a FH+ following eight weeks of combined exercise training compared to individuals without a family history of type 2 diabetes (FH-). Methods: Twenty (n = 10 FH-, n = 10 FH+) young, healthy, sedentary, normoglycemic, Mexican-American males (age: FH- 22.50 ± 0.81, FH+ 23.41 ± 0.86 years; BMI: FH- 27.91 ± 1.55, FH+ 26.64 ± 1.02 kg/m2) underwent eight weeks of combined aerobic and resistance exercise training three times/week (35 min aerobic followed by six full-body resistance exercises). Insulin sensitivity was assessed via hyperinsulinemic euglycemic clamps. Metabolic flexibility was assessed by the change in respiratory quotient from fasted to insulin-stimulated states. Body composition was determined using dual-energy x-ray absorptiometry. Aerobic fitness was determined by a graded exercise test, and upper- and lower-body strength were assessed via one-repetition maximum bench press and leg strength dynamometer, respectively. Results: Insulin sensitivity, metabolic flexibility, aerobic fitness and strength were not different between groups (p > 0.05). Eight weeks of combined aerobic and resistance exercise training improved insulin sensitivity (FH- p = 0.02, FH+ p = 0.002), increased fat free mass (FH- p = 0.006, FH+ p = 0.001), aerobic fitness (FH- p = 0.03, FH+ p = 0.002), and upper- (FH- p = 0.0001, FH+ p = 0.0001) and lower-body strength (FH- p = 0.0009, FH+ p = 0.0003), but did not change metabolic flexibility (p > 0.05) in both groups. Exercise-induced improvements in metabolic outcomes were similar between groups. Conclusions: Insulin sensitivity, metabolic flexibility, aerobic fitness and strength were not compromised by a FH+. Additionally, a FH+ is not a limiting factor for exercise-induced improvements in insulin sensitivity, aerobic fitness, body composition, and strength in normoglycemic young Mexican-American men.

Novel Cytogenetic Characterization of Pleomorphic Hyalinizing Angiectatic Tumor (PHAT).

OBJECTIVES: Pleomorphic hyalinizing angiectatic tumor (PHAT) is a rare soft tissue tumor that, despite its characteristic marked pleomorphism, is slow growing and of intermediate grade malignancy. PHAT is not known to metastasize, but is locally aggressive with a post-excision recurrence rate of up to 50%. Two other soft tissue tumors, hemosiderotic fibrolipomatous tumor (HFLT) and myxoinflammatory fibroblastic sarcoma (MIFS), share some morphological features with PHAT, and all three have been found to possess a t(1;10) translocation. Thus, it has been suggested PHAT, HFLT, and MIFS exist within a spectrum of a single entity; however, there is only one case of PHAT with a full cytogenetic profile and this showed the t(1;10). We report a case of PHAT with a complete cytogenetic profile differing from the previously reported case. Our case demonstrates 47,XY,+7,der(7)(qter::?::q31::?::pter::?::cen::?::pter::?::q31::?::qter)x2[20]/46,XY[10] karyotype with the typical morphologic features and immunohistochemical staining pattern seen in PHAT. This suggests that PHAT may be a distinctly separate entity and not within the spectrum of HFLT and MIFS.

Tissue Specificity in Trisomy 22 Mosaicism: A Tale of Caution for Interpretation of Chromosomal Microarray Results.

OBJECTIVES: While the complete form of trisomy 22 is seemingly incompatible with life, the mosaic form is a rare syndrome associated with developmental delays, intellectual disability, and dysmorphic features. Due in part to the difficulty of analyzing chromosomal mosaicism, many instances either go undiagnosed or have their diagnosis delayed. We report a case of mosaic trisomy 22 in a diamnionic-dichorionic twin with marked growth discordance and intra-uterine growth restriction, diagnosed in a 2-year-old with developmental delays, sensorineural hearing loss, cardiac and gastrointestinal abnormalities, and osteopenia of prematurity. Evaluation with a chromosomal oligonucleotide microarray with SNP analysis did not detect any copy number variants. Fibroblast metaphase karyotype analysis from a skin biopsy, however, showed trisomy 22 which was confirmed by FISH. Follow-up peripheral blood karyotype analysis and FISH studies revealed a normal male karyotype. This case highlights an instance where classical cytogenetics from two separate tissue types can provide a diagnosis that is more cost-effective than microarray analysis in assessing pediatric developmental delay. Trisomy 22 is the second most common aneuploidy in spontaneous miscarriages and has a nondescript and variable phenotype, especially in cases of mosaicism. As such, this condition may be underdiagnosed using the current recommended testing algorithm. Chromosomal microarray is considered first tier testing in an unrecognized phenotype with whole exome or whole genome sequencing, often performed on peripheral blood, as second tier testing. Diagnoses such as mosaic trisomy 22 suggest the second tier of testing in undiagnosed cases should also include a recommendation to look at alternative tissue types.

Impact of prolonged overfeeding on skeletal muscle mitochondria in healthy individuals.

AIMS/HYPOTHESES: Reduced mitochondrial capacity in skeletal muscle has been observed in obesity and type 2 diabetes. In humans, the aetiology of this abnormality is not well understood but the possibility that it is secondary to the stress of nutrient overload has been suggested. To test this hypothesis, we examined whether sustained overfeeding decreases skeletal muscle mitochondrial content or impairs function. METHODS: Twenty-six healthy volunteers (21 men, 5 women, age 25.3 ± 4.5 years, BMI 25.5 ± 2.4 kg/m2) underwent a supervised protocol consisting of 8 weeks of high-fat overfeeding (40% over baseline energy requirements). Before and after overfeeding, we measured systemic fuel oxidation by indirect calorimetry and performed skeletal muscle biopsies to measure mitochondrial gene expression, content and function in vitro. Mitochondrial function in vivo was measured by 31P NMR spectroscopy. RESULTS: With overfeeding, volunteers gained 7.7 ± 1.8 kg (% change 9.8 ± 2.3). Overfeeding increased fasting NEFA, LDL-cholesterol and insulin concentrations. Indirect calorimetry showed a shift towards greater reliance on lipid oxidation. In skeletal muscle tissue, overfeeding increased ceramide content, lipid droplet content and perilipin-2 mRNA expression. Phosphorylation of AMP-activated protein kinase was decreased. Overfeeding increased mRNA expression of certain genes coding for mitochondrial proteins (CS, OGDH, CPT1B, UCP3, ANT1). Despite the stress of nutrient overload, mitochondrial content and mitochondrial respiration in muscle did not change after overfeeding. Similarly, overfeeding had no effect on either the emission of reactive oxygen species or on mitochondrial function in vivo. CONCLUSIONS/INTERPRETATION: Skeletal muscle mitochondria are significantly resilient to nutrient overload. The lower skeletal muscle mitochondrial oxidative capacity in human obesity is likely to be caused by reasons other than nutrient overload per se. TRIAL REGISTRATION: ClinicalTrials.gov NCT01672632.

Intramyocellular Lipid Droplet Size Rather Than Total Lipid Content is Related to Insulin Sensitivity After 8 Weeks of Overfeeding.

OBJECTIVE: Intramyocellular lipid (IMCL) is inversely related to insulin sensitivity in sedentary populations, yet no prospective studies in humans have examined IMCL accumulation with overfeeding. METHODS: Twenty-nine males were overfed a high-fat diet (140% caloric intake, 44% from fat) for 8 weeks. Measures of IMCL, whole-body fat oxidation from a 24-hour metabolic chamber, muscle protein extracts, and muscle ceramide measures were obtained before and after the intervention. RESULTS: Eight weeks of overfeeding did not increase overall IMCL. The content of smaller lipid droplets peripherally located in the myofiber decreased, while increases in larger droplets correlated inversely with glucose disposal rate. Overfeeding resulted in inhibition of Akt activity, which correlated with the reductions in smaller, peripherally located lipid droplets and drastic increases in ceramide content. Additionally, peripherally located lipid droplets were associated with more efficient lipid oxidation. Finally, participants who maintained a greater number of smaller, peripherally located lipid droplets displayed a better resistance to weight gain with overfeeding. CONCLUSIONS: These results show that lipid droplet size and location rather than mere IMCL content are important to understanding insulin sensitivity.

Skeletal Muscle PGC1α -1 Nucleosome Position and -260 nt DNA Methylation Determine Exercise Response and Prevent Ectopic Lipid Accumulation in Men.

Endurance exercise has been shown to improve lipid oxidation and increase mitochondrial content in skeletal muscle, two features that have shown dependence on increased expression of the peroxisome proliferator-activated receptor-γ coactivator 1α (PGC1α). It is also hypothesized that exercise-related alterations in PGC1α expression occur through epigenetic regulation of nucleosome positioning in association with differential DNA methylation status within the PGC1α promoter. In this study, we show that when primary human myotubes from obese patients with type 2 diabetes are exposed to lipolytic stimulus (palmitate, forskolin, inomycin) in vitro, nucleosome occupancy surrounding the -260 nucleotide (nt) region, a known regulatory DNA methylation site, is reduced. This finding is reproduced in vivo in the vastus lateralis from 11 healthy males after a single, long endurance exercise bout in which participants expended 650 kcal. Additionally, we show a significant positive correlation between fold change of PGC1α messenger RNA expression and -1 nucleosome repositioning away from the -260 nt methylation site in skeletal muscle tissue following exercise. Finally, we found that when exercise participants are divided into high and low responders based on the -260 nt methylation status, the -1 nucleosome is repositioned away from the regulatory -260 nt methylation site in high responders, those exhibiting a significant decrease in -260 nt methylation, but not in low responders. Additionally, high but not low responders showed a significant decrease in intramyocellular lipid content after exercise. These findings suggest a potential target for epigenetic modification of the PGC1α promoter to stimulate the therapeutic effects of endurance exercise in skeletal muscle.

Myokine Expression in Muscle and Myotubes in Response to Exercise Stimulation.

PURPOSE: Myokines have been shown to affect muscle physiology and exert systemic effects. We endeavored to investigate a panel of myokine mRNA expression after a single exercise bout (studies 1 and 2) to measure myokine mRNA in primary human myotubes in an in vitro exercise model (study 2). METHODS: Vastus lateralis muscle biopsies were obtained from 20 healthy males (age, 24.0 ± 4.5 yr; BMI, 23.6 ± 1.8 kg·m)(-2) before and after a single exercise bout (650 kcal at 50% V˙O2max). Primary myotubes from active and sedentary male donors were treated with a pharmacological cocktail (palmitate, forskolin, and ionomycin (PFI)) to mimic exercise-stimulated contractions in vitro. RESULTS: Interleukin 6 and 8 (IL-6 and IL-8), leukocyte-inducing factor, and connective tissue growth factor (CTGF) mRNA levels increased approximately 10-fold after a single exercise bout (all P < 0.001), whereas myostatin levels decreased (P < 0.05). Key correlations between myokine expression and parameters of muscle and whole-body physiology were found: myostatin versus skeletal muscle citrate synthase activity (r = -0.69, P < 0.001), V˙O2max (r = -0.64, P = 0.002) and the percentage of Type I fibers (r = -0.55, P = 0.01); IL-6 versus the RER (r = 0.45, P = 0.04), homeostatic model assessment of insulin resistance (r = 0.44, P = 0.05), and serum lactate (r = 0.50, P = 0.02). Myokine expressions in myotubes from sedentary donors for CTGF and myostatin decreased, whereas IL-6 and IL-8 increased after PFI treatment. In myotubes from active donors, myokine expression increased for IL-6, CTGF, and myostatin but decreased for IL-8 after PFI treatment. CONCLUSION: These data offer insight into the differences in regulation of myokine expression and their possible physiologic relationships.

Higher circulating leukocytes in women with PCOS is reversed by aerobic exercise.

Polycystic ovary syndrome (PCOS) is characterized by insulin resistance, elevated circulating leukocytes, and hypothesized to have higher adipose tissue inflammation. Aerobic exercise reduces circulating leukocytes and improves insulin sensitivity in obese individuals, but the effect of exercise on inflammation in PCOS is not known. We investigated circulating leukocytes, insulin sensitivity by euglycemic-hyperinsulinemic clamp, serum pro- and anti-inflammatory markers (hsCRP, TNF-α, total and high molecular weight adiponectin), and abdominal subcutaneous adipose tissue (SAT) gene expression of proinflammatory markers in 8 PCOS women and 8 obese control females matched for BMI. Additionally, in a prospective study, the 8 women with PCOS underwent a 16-week aerobic exercise regimen with the same measures performed post-intervention. Compared to controls, white blood cell counts (WBC) were 30% higher (p = 0.04) and circulating total adiponectin levels were 150% lower (p = 0.03) in women with PCOS at baseline/pre-exercise conditions. SAT gene expression of macrophage migration inhibitory factor (MIF, p < 0.01) and interleukin-6 (IL-6, p < 0.05) were also lower in women with PCOS. In response to 16 weeks of aerobic exercise, insulin sensitivity improved (p < 0.01) and WBC counts decreased (p = 0.02). The exercise-induced change in WBC and circulating neutrophils correlated inversely with changes in glucose disposal rate (r = -0.73, p = 0.03; and r = -0.82, p = 0.01, respectively). Aerobic exercise reduced serum leptin (p < 0.05) after 4 weeks, trended to reduce the ratio of leptin-to-high molecular weight adiponectin (p < 0.1) by the 8th week, and significantly increased serum dehydroepiandrosterone sulfate (DHEA-S, p < 0.001) after 16 weeks. In conclusion, women with PCOS have higher circulating leukocytes compared to controls, which can be reversed by aerobic exercise and is associated with improvements in insulin sensitivity.

The sirtuins: Markers of metabolic health.

The sirtuins represent a class of proteins first discovered orthologus to the yeast silent information regulator 2 protein that have been retained in mammalian species. Currently, seven sirtuins have been identified in humans, and their functions currently surpass their originally identified role as histone deacetylase and chromatin silencers to encompass nutrient sensing and metabolic function. All seven sirtuins require NAD(+) in order to carry out their enzymatic activity, and thus become activated in conditions of nutrient depletion, starvation, and cellular stress. Caloric restriction and increased physical activity have been postulated, though perhaps controversially, to mediate sirtuin function. Here, we review the current literature surrounding the functions of the seven human sirtuins, mediators of their function, and the roles they play in metabolic health related to dietary and physical activity interventions. Despite the controversy surrounding sirtuin function with regard to longevity, we have aimed to show that regardless of its effects on aging, sirtuin function is pivotal to pathways involving metabolic health, and should therefore be investigated with regard to improving metabolic diseases such as obesity and type 2 diabetes.

Effect of serial cell passaging in the retention of fiber type and mitochondrial content in primary human myotubes.

OBJECTIVE: The purpose of the study was to determine the effects of passaging on retention of donor phenotypic characteristics in primary human myotubes. METHODS: Primary muscle cultures and serial passaged myotubes from physically active, sedentary lean, and individuals with type 2 diabetes were established. Maximal ATP synthesis capacity (ATPmax) and resting ATP flux (ATPase) in vivo were measured by (31) P magnetic resonance spectroscopy, type-I fibers and intramyocelluar lipid (IMCL) in vastus lateralis tissue were determined using immunohistochemistry techniques, and oxidative phosphorylation complexes (OXPHOS) were measured by Western immunoblotting. Similar in vitro measures for lipid and type-I fibers were made in myotubes, along with mitochondrial content measured by MitoTracker. RESULTS: Passage 4 and 5 measures for myotubes correlated positively with in vivo measurements for percent type-I fibers (P4: R(2) = 0.39, p = 0.02; P5: R(2) = 0.48, p = 0.01), ATPmax (P4: R(2) = 0.30, p = 0.03; P5: R(2) = 0.22, p = 0.05), and OXPHOS (P4: R(2) = 0.44, p = 0.04; P5: R(2) = 0.59, p = 0.006). No correlations were observed for IMCL. However, passage 4 measures for myotubes correlated with passage 5 measures for percent type-I fibers (R(2) = 0.49, p = 0.01), IMCL (R(2) = 0.80, p < 0.001), and mitochondrial content (R(2) = 0.26, p = 0.03). CONCLUSIONS: Myotubes through the first two passages following immunopurification (referred to as passage 4 and 5) reflect the mitochondrial and type-I fiber content in vivo phenotype of the donor.

Perilipin 3 Differentially Regulates Skeletal Muscle Lipid Oxidation in Active, Sedentary, and Type 2 Diabetic Males.

CONTEXT: The role of perilipin 3 (PLIN3) on lipid oxidation is not fully understood. OBJECTIVE: We aimed to 1) determine whether skeletal muscle PLIN3 protein content is associated with lipid oxidation in humans, 2) understand the role of PLIN3 in lipid oxidation by knocking down PLIN3 protein content in primary human myotubes, and 3) compare PLIN3 content and its role in lipid oxidation in human primary skeletal muscle cultures established from sedentary, healthy lean (leans), type 2 diabetic (T2D), and physically active donors. DESIGN, PARTICIPANTS, AND INTERVENTION: This was a clinical investigation of 29 healthy, normoglycemic males and a cross-sectional study using primary human myotubes from five leans, four T2D, and four active donors. Energy expenditure, whole-body lipid oxidation, PLIN3 protein content in skeletal muscle tissue, and ex vivo muscle palmitate oxidation were measured. Myotubes underwent lipolytic stimulation (palmitate, forskolin, inomycin [PFI] cocktail), treatment with brefeldin A (BFA), and knockdown of PLIN3 using siRNA. SETTING: Experiments were performed in a Biomedical Research Institute. MAIN OUTCOME MEASURES: Protein content, 24-hour respiratory quotient (RQ), and ex vivo/in vitro lipid oxidations. RESULTS: PLIN3 protein content was associated with 24-h RQ (r = -0.44; P = .02) and skeletal muscle-specific ex vivo palmitate oxidation (r = 0.61; P = .02). PLIN3 knockdown showed drastic reductions in lipid oxidation in myotubes from leans. Lipolytic stimulation increased PLIN3 protein in cells from leans over T2Ds with little expression in active participants. Furthermore, treatment with BFA, known to inhibit coatomers that associate with PLIN3, reduced lipid oxidation in cells from lean and T2D, but not in active participants. CONCLUSIONS: Differential expression of PLIN3 and BFA sensitivity may explain differential lipid oxidation efficiency in skeletal muscle among these cohorts.

Potential effects of aerobic exercise on the expression of perilipin 3 in the adipose tissue of women with polycystic ovary syndrome: a pilot study.

OBJECTIVE: Polycystic ovary syndrome (PCOS) is associated with reduced adipose tissue lipolysis that can be rescued by aerobic exercise. We aimed to identify differences in the gene expression of perilipins and associated targets in adipose tissue in women with PCOS before and after exercise. DESIGN AND METHODS: We conducted a cross-sectional study in eight women with PCOS and eight women matched for BMI and age with normal cycles. Women with PCOS also completed a 16-week prospective aerobic exercise-training study. Abdominal subcutaneous adipose tissue biopsies were collected, and primary adipose-derived stromal/stem cell cultures were established from women with PCOS before 16 weeks of aerobic exercise training (n=5) and controls (n=5). Gene expression was measured using real-time PCR, in vitro lipolysis was measured using radiolabeled oleate, and perilipin 3 (PLIN3) protein content was measured by western blotting analysis. RESULTS: The expression of PLIN1, PLIN3, and PLIN5, along with coatomers ARF1, ARFRP1, and ßCOP was ∼ 80% lower in women with PCOS (all P<0.05). Following exercise training, PLIN3 was the only perilipin to increase significantly (P<0.05), along with coatomers ARF1, ARFRP1, ßCOP, and SEC23A (all P<0.05). Furthermore, PLIN3 protein expression was undetectable in the cell cultures from women with PCOS vs controls. Following exercise training, in vitro adipose oleate oxidation, glycerol secretion, and PLIN3 protein expression were increased, along with reductions in triglyceride content and absence of large lipid droplet morphology. CONCLUSIONS: These findings suggest that PLIN3 and coatomer GTPases are important regulators of lipolysis and triglyceride storage in the adipose tissue of women with PCOS.

Effect of 8 weeks of overfeeding on ectopic fat deposition and insulin sensitivity: testing the "adipose tissue expandability" hypothesis.

OBJECTIVE: The presence of large subcutaneous adipocytes in obesity has been proposed to be linked with insulin resistance and type 2 diabetes through the "adipose tissue expandability" hypothesis, which holds that large adipocytes have a limited capacity for expansion, forcing lipids to be stored in nonadipose ectopic depots (skeletal muscle, liver), where they interfere with insulin signaling. This hypothesis has, however, been largely formulated by cross-sectional findings and to date has not been prospectively demonstrated in the development of insulin resistance in humans. RESEARCH DESIGN AND METHODS: Twenty-nine men (26.8 ± 5.4 years old; BMI 25.5 ± 2.3 kg/m(2)) were fed 40% more than their baseline requirement for 8 weeks. Before and after overfeeding, insulin sensitivity was determined using a two-step hyperinsulinemic-euglycemic clamp. Intrahepatic lipid (IHL) and intramyocellular lipid (IMCL) were measured by (1)H-MRS and abdominal fat by MRI. Subcutaneous abdominal adipose and skeletal muscle tissues were collected to measure adipocyte size and markers of tissue inflammation. RESULTS: Subjects gained 7.6 ± 2.1 kg (55% fat) and insulin sensitivity decreased 18% (P < 0.001) after overfeeding. IHL increased 46% from 1.5% to 2.2% (P = 0.002); however, IMCL did not change. There was no association between adipocyte size and ectopic lipid accumulation. Despite similar weight gain, subjects with smaller fat cells at baseline had a greater decrease in insulin sensitivity, which was linked with upregulated skeletal muscle tissue inflammation. CONCLUSIONS: In experimental substantial weight gain, the presence of larger adipocytes did not promote ectopic lipid accumulation. In contrast, smaller fat cells were associated with a worsened metabolic response to overfeeding.

Caveolin-1 expression and cavin stability regulate caveolae dynamics in adipocyte lipid store fluctuation.

Adipocytes specialized in the storage of energy as fat are among the most caveolae-enriched cell types. Loss of caveolae produces lipodystrophic diabetes in humans, which cannot be reversed by endothelial rescue of caveolin expression in mice, indicating major importance of adipocyte caveolae. However, how caveolae participate in fat cell functions is poorly understood. We investigated dynamic conditions of lipid store fluctuations and demonstrate reciprocal regulation of caveolae density and fat cell lipid droplet storage. We identified caveolin-1 expression as a crucial step in adipose cell lines and in mice to raise the density of caveolae, to increase adipocyte ability to accommodate larger lipid droplets, and to promote cell expansion by increased glucose utilization. In human subjects enrolled in a trial of 8 weeks of overfeeding to promote fattening, adipocyte expansion response correlated with initial caveolin-1 expression. Conversely, lipid mobilization in cultured adipocytes to induce lipid droplet shrinkage led to biphasic response of cavin-1 with ultimate loss of expression of cavin-1 and -3 and EHD2 by protein degradation, coincident with caveolae disassembly. We have identified the key steps in cavin/caveolin interplay regulating adipocyte caveolae dynamics. Our data establish that caveolae participate in a unique cell response connected to lipid store fluctuation, suggesting lipid-induced mechanotension in adipocytes.

Skeletal muscle perilipin 3 and coatomer proteins are increased following exercise and are associated with fat oxidation.

Lipid droplet-associated proteins such as perilipin 3 (PLIN3) and coatomer GTPase proteins (GBF1, ARF1, Sec23a, and ARFRP1) are expressed in skeletal muscle but little is known so far as to their regulation of lipolysis. We aimed here to explore the effects of lipolytic stimulation in vitro in primary human myotubes as well as in vivo following an acute exercise bout. In vitro lipolytic stimulation by epinephrine (100 µM) or by a lipolytic cocktail (30 µM palmitate, 4 µM forskolin, and 0.5 µM ionomycin, PFI) resulted in increases in PLIN3 protein content. Coatomer GTPases such as GBF1, ARF1, Sec23a, and ARFRP1 also increased in response to lipolytic stimuli. Furthermore, a long duration endurance exercise bout (20 males; age 24.0 ± 4.5 y; BMI 23.6 ± 1.8 kg/m(2)) increased PLIN3 protein in human skeletal muscle (p = 0.03) in proportion to ex vivo palmitate oxidation (r = 0.45, p = 0.04) and whole body in vivo fat oxidation (r = 0.52, p = 0.03). Protein content of ARF1 was increased (p = 0.04) while mRNA expression was increased for several other coatomers (GBF1, ARF1, and Sec23a, all p<0.05). These data provide novel observational insight into the possible relationships between lipolysis and PLIN3 along with these coatomoer GTPase proteins in human skeletal muscle.

Weight gain reveals dramatic increases in skeletal muscle extracellular matrix remodeling.

CONTEXT: In animal models of obesity, chronic inflammation and dysregulated extracellular matrix remodeling in adipose tissue leads to insulin resistance. Whether similar pathophysiology occurs in humans is not clear. OBJECTIVE: The aim of this study was to test whether 10% weight gain induced by overfeeding triggers inflammation and extracellular matrix remodeling (gene expression, protein, histology) in skeletal muscle and sc adipose tissue in humans. We also investigated whether such remodeling was associated with an impaired metabolic response (hyperinsulinemic-euglycemic clamp). DESIGN, SETTING, PARTICIPANTS, AND INTERVENTION: Twenty-nine free-living males were fed 40% over their baseline energy requirements for 8 weeks. RESULTS: Ten percent body weight gain prompted dramatic up-regulation of a repertoire of extracellular matrix remodeling genes in muscle and to a lesser degree in adipose tissue. The amount of extracellular matrix genes in the muscle were directly associated with the amount of lean tissue deposited during overfeeding. Despite weight gain and impaired insulin sensitivity, there was no change in local adipose tissue or systemic inflammation, but there was a slight increase in skeletal muscle inflammation. CONCLUSION: We propose that skeletal muscle extracellular matrix remodeling is another feature of the pathogenic milieu associated with energy excess and obesity, which, if disrupted, may contribute to the development of metabolic dysfunction.