Differential time responses in inflammatory and oxidative stress markers after a marathon: An observational study.

Acute and adaptive changes in systemic markers of oxidatively generated nucleic acid modifications (i.e., 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) and 8-oxo-7,8-dihydroguanosine (8-oxoGuo)) as well as inflammatory cytokines (i.e., C-reactive protein, interleukin-6, interleukin-10, and tumour necrosis factor alpha), a liver hormone (i.e., fibroblast growth factor 21 (FGF21)), and bone metabolism markers (sclerostin, osteocalcin, C-terminal telopeptide, and N-terminal propeptide of type 1 procollagen) were investigated following a marathon in 20 study participants. Immediate changes were observed in inflammatory cytokines, FGF21, and bone metabolism markers following the marathon. In contrast, no immediate changes in urinary excretion of 8-oxodG and 8-oxoGuo were evident. Four days after the marathon, decreased urinary excretion of 8-oxodG (-2.9 (95% CI -4.8;-1.1) nmol/24 h, P < 0.01) and 8-oxoGuo (-5.8 (95% CI -10.3;-1.3) nmol/24 h, P = 0.02) was observed. The excretion rate of 8-oxodG remained decreased 7 days after the marathon compared to baseline (-2.3 (95%CI -4.3;-0.4) nmol/24 h, P = 0.02), whereas the excretion rate of 8-oxoGuo was normalized. In conclusion marathon participation immediately induced a considerable inflammatory response, but did not increase excretion rates of oxidatively generated nucleic acid modifications. In fact, a delayed decrease in oxidatively generated nucleic acid modifications was observed suggesting adaptive antioxidative effects following exercise. ABBREVIATIONS: 8-oxodG: 8-oxo-7,8-dihydro-2'-deoxyguanosine; 8-oxoGuo: 8-oxo-7,8-dihydroguanosine; CI: confidence interval; CTX: C-terminal telopeptide of type 1 collagen; DXA: dual-energy X-ray absorptiometry; ELISA: enzyme-linked immunosorbent assay; FGF21: Fibroblast growth factor 21; h: hour; hsCRP: high sensitivity C-reactive protein; IL: interleukin; IQR: interquartile range; MS: mass spectrometry: P1NP: N-terminal propeptide of type 1 procollagen; TNFα: tumour necrosis factor alpha; UPLC: ultra-performance liquid chromatography.

PPARG Pro12Ala Ala carriers exhibit greater improvements in peripheral insulin sensitivity in response to 12 weeks of aerobic exercise training.

The Ala allele of PPARG Pro12Ala ( rs1801282 ) is associated with greater improvements to the glucose metabolism in exercise studies, but whether this extends to peripheral insulin sensitivity is unknown. Our objective was to investigate the effect of PPARG Pro12Ala on exercise-induced changes in peripheral insulin sensitivity. A total of 124 (91 Pro homozygotes and 33 Ala carriers) previously physically inactive healthy young men and women with overweight or class 1 obesity who completed a 12 wk aerobic exercise intervention were included in the analysis. All participants underwent a hyperinsulinemic euglycemic clamp before and after the 12 wk intervention. The prescribed exercise frequency was 5-7 days/wk, and the exercise energy expenditure was 2,100 4,200 kcal/wk for men and 1,600 kcal/wk for women. Insulin sensitivity improved significantly in both genotype groups. However, Ala carriers had a 1.13-fold (95% confidence interval 1.01; 1.26, P = 0.032) greater improvement in insulin sensitivity from baseline compared with Pro homozygotes. Our data support that PPARG Pro12Ala modifies the effect of aerobic exercise on peripheral insulin sensitivity.

Rac1 governs exercise-stimulated glucose uptake in skeletal muscle through regulation of GLUT4 translocation in mice.

KEY POINT: Exercise increases skeletal muscle energy turnover and one of the important substrates for the working muscle is glucose taken up from the blood. The GTPase Rac1 can be activated by muscle contraction and has been found to be necessary for insulin-stimulated glucose uptake, although its role in exercise-stimulated glucose uptake is unknown. We show that Rac1 regulates the translocation of the glucose transporter GLUT4 to the plasma membrane in skeletal muscle during exercise. We find that Rac1 knockout mice display significantly reduced glucose uptake in skeletal muscle during exercise. ABSTRACT: Exercise increases skeletal muscle energy turnover and one of the important substrates for the working muscle is glucose taken up from the blood. Despite extensive efforts, the signalling mechanisms vital for glucose uptake during exercise are not yet fully understood, although the GTPase Rac1 is a candidate molecule. The present study investigated the role of Rac1 in muscle glucose uptake and substrate utilization during treadmill exercise in mice in vivo. Exercise-induced uptake of radiolabelled 2-deoxyglucose at 65% of maximum running capacity was blocked in soleus muscle and decreased by 80% and 60% in gastrocnemius and tibialis anterior muscles, respectively, in muscle-specific inducible Rac1 knockout (mKO) mice compared to wild-type littermates. By developing an assay to quantify endogenous GLUT4 translocation, we observed that GLUT4 content at the sarcolemma in response to exercise was reduced in Rac1 mKO muscle. Our findings implicate Rac1 as a regulatory element critical for controlling glucose uptake during exercise via regulation of GLUT4 translocation.

Three months of strictly controlled daily endurance exercise reduces thrombin generation and fibrinolytic risk markers in younger moderately overweight men.

PURPOSE: Physical activity is associated with a decreased risk of cardiovascular disease, but dose dependency of long-term physical exercise on biomarkers within coagulation and fibrinolysis is unknown. We aimed to investigate effects of two doses of daily endurance exercise on biomarkers of the haemostatic balance in overweight men. METHODS: Haemostatic variables were investigated in 53 healthy, younger (20-40 years), moderately overweight (BMI 25-30 kg/m(2)) men randomly assigned to 3 months of strictly controlled endurance exercise at two different doses corresponding to an energy expenditure of 600 kcal/day (HIGH), 300 kcal/day (MOD), or to maintain their habitual lifestyle (CON). Fasting blood samples were collected before and after the intervention and analysed for thrombin generation (endogenous thrombin potential, ETP) and concentrations of tissue-type plasminogen activator (t-PA), plasminogen activator inhibitor type 1 (PAI-1), and von Willebrand factor (vWF). RESULTS: We observed significant within-group decreases in ETP (MOD 7 %; HIGH 6 %) and in t-PA (MOD 22 %; HIGH 21 %) and PAI-1 (MOD 16 %; HIGH 32 %) in both training groups, and no changes in the CON group. At 3 months, between-group differences were observed for ETP (p = 0.016) and t-PA (p = 0.012) due to significantly lower values in MOD and HIGH compared with CON. Borderline significant between-group differences were observed for PAI-1 (p = 0.082). A significant increase was observed in vWF in HIGH, but with no between-group differences. CONCLUSIONS: Our results demonstrate an effect of 3 months of daily endurance exercise on biomarkers of the haemostatic balance in the direction of reduced cardiovascular risk, independent of exercise dose.

Skeletal muscle apolipoprotein B expression reduces muscular triglyceride accumulation.

BACKGROUND: Lipid accumulation in skeletal muscle is associated with impaired insulin sensitivity in type 2 diabetes. In cardiac myocytes, lipoprotein secretion controlled by apolipoproteinB (apoB) and microsomal triglyceride transfer protein (MTP) affects lipid homeostasis. DESIGN: In this study, we investigated whether expression of a human apoB transgene affects triglyceride accumulation and insulin sensitivity in skeletal muscle in fat fed obese mice. RESULTS: Expression of apoB and MTP mRNA and the human apoB transgene was seen in skeletal muscle of the transgene mice. Human apoB transgenic mice accumulated 28% less triglycerides in skeletal myocytes after one year of fat-feeding as compared with WT mice (32 ± 5, n = 10 vs. 44 ± 4 nmol/mg ww, n = 13, p = 0.04). Moreover, expression of human apoB in fat-fed mice was associated with 32% (p = 0.02) and 37% (p = 0.01) lower plasma insulin levels after 9 and 12 months, respectively, improved intra peritoneal glucose tolerance after 6 months, and a trend towards increased insulin-stimulated glucose uptake in isolated skeletal muscle. CONCLUSIONS: The data suggests that overexpression of apoB decreases skeletal muscle lipid accumulation and attenuates peripheral insulin resistance in obese mice.

Compliance with physical exercise: using a multidisciplinary approach within a dose-dependent exercise study of moderately overweight men.

AIMS: Sixty-one healthy, sedentary, moderately overweight young men participated in a randomised controlled trial to examine the effects of two different doses of endurance exercise on health behaviour and exercise compliance. METHODS: Participants were randomised to a sedentary control group, a moderate (MOD; 300 kcal/day) or a high-dose (HIGH; 600 kcal/day) endurance exercise group for 12 weeks. A sub-set of the subjects were interviewed using pre-determined, qualitative questions to elucidate physical activity and health behaviour. In combination with the Theory of Planned Behaviour (TPB), a post hoc thematic analysis was conducted to connect qualitative and quantitative data in a joint analysis. RESULTS: Of the subjects interviewed, exercise compliance expressed as 95% CI was [96.8; 103%] in the MOD group and [82.9; 99.6%] in the HIGH group. The different doses of daily exercise equally improved various metabolic health parameters. The MOD group was untroubled by the exercise load and had a positive attitude towards exercise. The HIGH group expressed increased fatigue, less positivity and perceived exercise as time-consuming. The MOD group described themselves as more energetic, and thereby may have increased physical activity levels in areas of their everyday lives that were not related to the intervention. CONCLUSIONS: A multidisciplinary approach provided explanations for similar effects of two different doses of exercise. This could not have been determined via either qualitative or quantitative methodology alone. The preconditions of the TBP were fulfilled, and it represents a methodological model to explain the high degree of compliance and motivation to exercise.

Contraction-induced lipolysis is not impaired by inhibition of hormone-sensitive lipase in skeletal muscle.

In skeletal muscle hormone-sensitive lipase (HSL) has long been accepted to be the principal enzyme responsible for lipolysis of intramyocellular triacylglycerol (IMTG) during contractions. However, this notion is based on in vitro lipase activity data, which may not reflect the in vivo lipolytic activity. We investigated lipolysis of IMTG in soleus muscles electrically stimulated to contract ex vivo during acute pharmacological inhibition of HSL in rat muscles and in muscles from HSL knockout (HSL-KO) mice. Measurements of IMTG are complicated by the presence of adipocytes located between the muscle fibres. To circumvent the problem with this contamination we analysed intramyocellular lipid droplet content histochemically. At maximal inhibition of HSL in rat muscles, contraction-induced breakdown of IMTG was identical to that seen in control muscles (P < 0.001). In response to contractions IMTG staining decreased significantly in both HSL-KO and WT muscles (P < 0.05). In vitro TG hydrolase activity data revealed that adipose triglyceride lipase (ATGL) and HSL collectively account for ∼98% of the TG hydrolase activity in mouse skeletal muscle, other TG lipases accordingly being of negligible importance for lipolysis of IMTG. The present study is the first to demonstrate that contraction-induced lipolysis of IMTG occurs in the absence of HSL activity in rat and mouse skeletal muscle. Furthermore, the results suggest that ATGL is activated and plays a major role in lipolysis of IMTG during muscle contractions.

Differential effects of endurance training and weight loss on plasma adiponectin multimers and adipose tissue macrophages in younger, moderately overweight men.

Obese individuals are characterized by low circulating adiponectin concentrations and an increased number of macrophages in adipose tissue, which is believed to be causally associated with chronic low-grade inflammation and insulin resistance. Regular physical exercise decreases overall morbidity in obese subjects, which may be due to modulations of inflammatory pathways. In this randomized clinical trial we investigated the separate effects of endurance training-induced weight loss, diet-induced weight loss, and endurance training per se (without weight loss) on plasma adiponectin multimer composition (Western blotting) and adipose tissue macrophage content (immunohistochemistry) in young, moderately overweight men. Weight loss and endurance training per se decreased whole body fat percentage in an additive manner. No intervention-induced changes were observed for plasma total adiponectin. Surprisingly, endurance training, irrespectively of any associated weight loss, shifted the adiponectin multimer distribution toward a lower molecular weight (21% decrease in HMW/LMW, P = 0.015), whereas diet-induced weight loss shifted the distribution toward a higher molecular weight (42% increase in HMW/MMW, P < 0.001). Furthermore, endurance training per se increased the number of anti-inflammatory CD163⁺ macrophages [from 12.7 ± 2.1 (means ± SE) to 16.1 ± 3.1 CD163⁺ cells/100 adipocytes, P = 0.013], whereas diet-induced weight loss tended to decrease CD68⁺ macrophages in subcutaneous abdominal adipose tissue. Thus regular physical exercise influences systemic and adipose tissue inflammatory pathways differently than diet-induced weight loss in younger, moderately overweight men. Our data suggest that some of the health benefits of a physically active lifestyle may occur through modulations of anti- rather than pro-inflammatory pathways in young, overweight men.

Myocardial impulse propagation is impaired in right ventricular tissue of Zucker diabetic fatty (ZDF) rats.

BACKGROUND: Diabetes increases the risk of cardiovascular complications including arrhythmias, but the underlying mechanisms remain to be established. Decreased conduction velocity (CV), which is an independent risk factor for re-entry arrhythmias, is present in models with streptozotocin (STZ) induced type 1 diabetes. Whether CV is also disturbed in models of type 2 diabetes is currently unknown. METHODS: We used Zucker Diabetic Fatty (ZDF) rats, as a model of type 2 diabetes, and their lean controls Zucker Diabetic Lean (ZDL) rats to investigate CV and its response to the anti-arrhythmic peptide analogue AAP10. Gap junction remodeling was examined by immunofluorescence and western blotting. Cardiac histomorphometry was examined by Masson`s Trichrome staining and intracellular lipid accumulation was analyzed by Bodipy staining. RESULTS: CV was significantly slower in ZDF rats (56±1.9 cm/s) compared to non-diabetic controls (ZDL, 66±1.6 cm/s), but AAP10 did not affect CV in either group. The total amount of Connexin43 (C×43) was identical between ZDF and ZDL rats, but the amount of lateralized C×43 was significantly increased in ZDF rats (42±12 %) compared to ZDL rats (30±8%), p<0.04. Judged by electrophoretic mobility, C×43 phosphorylation was unchanged between ZDF and ZDL rats. Also, no differences in cardiomyocyte size or histomorphometry including fibrosis were observed between groups, but the volume of intracellular lipid droplets was 4.2 times higher in ZDF compared to ZDL rats (p<0.01). CONCLUSION: CV is reduced in type 2 diabetic ZDF rats. The CV disturbance may be partly explained by increased lateralization of C×43, but other factors are likely also involved. Our data indicates that lipotoxicity potentially may play a role in development of conduction disturbances and arrhythmias in type 2 diabetes.

Body fat loss and compensatory mechanisms in response to different doses of aerobic exercise--a randomized controlled trial in overweight sedentary males.

The amount of weight loss induced by exercise is often disappointing. A diet-induced negative energy balance triggers compensatory mechanisms, e.g., lower metabolic rate and increased appetite. However, knowledge about potential compensatory mechanisms triggered by increased aerobic exercise is limited. A randomized controlled trial was performed in healthy, sedentary, moderately overweight young men to examine the effects of increasing doses of aerobic exercise on body composition, accumulated energy balance, and the degree of compensation. Eighteen participants were randomized to a continuous sedentary control group, 21 to a moderate-exercise (MOD; 300 kcal/day), and 22 to a high-exercise (HIGH; 600 kcal/day) group for 13 wk, corresponding to ∼30 and 60 min of daily aerobic exercise, respectively. Body weight (MOD: -3.6 kg, P < 0.001; HIGH: -2.7 kg, P = 0.01) and fat mass (MOD: -4.0 kg, P < 0.001 and HIGH: -3.8 kg, P < 0.001) decreased similarly in both exercise groups. Although the exercise-induced energy expenditure in HIGH was twice that of MOD, the resulting accumulated energy balance, calculated from changes in body composition, was not different (MOD: -39.6 Mcal, HIGH: -34.3 Mcal, not significant). Energy balance was 83% more negative than expected in MOD, while it was 20% less negative than expected in HIGH. No statistically significant changes were found in energy intake or nonexercise physical activity that could explain the different compensatory responses associated with 30 vs. 60 min of daily aerobic exercise. In conclusion, a similar body fat loss was obtained regardless of exercise dose. A moderate dose of exercise induced a markedly greater than expected negative energy balance, while a higher dose induced a small but quantifiable degree of compensation.

Preserved postprandial suppression of bone turnover markers, despite increased fasting levels, in postmenopausal women.

CONTEXT: Menopause leads to an increased bone turnover associated with a high risk of fractures. Bone turnover is inhibited by meal intake, to some extent mediated by gut hormones, and interventions based on these endocrine changes may have potential in future prevention of osteoporosis. OBJECTIVE: To investigate whether postmenopausal women exhibit postprandial suppression of bone turnover markers to the same extent as premenopausal women, despite higher fasting levels. Furthermore, to assess whether menopausal differences in bone turnover markers are related to postmenopausal changes in plasma gut hormone levels. METHODS: A cross-sectional study of 21 premenopausal, 9 perimenopausal, and 24 postmenopausal women between 45 and 60 years of age. Serum/plasma levels of bone turnover markers and gut hormones were investigated during a 120 min oral glucose tolerance test. Bone turnover markers included N-terminal propeptide of type-I procollagen (PINP, bone formation marker) and carboxyterminal collagen I crosslinks (CTX-I, bone resorption marker). Gut hormone secretion was evaluated from responses of glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2) and glucose-dependent insulinotropic polypeptide (GIP). RESULTS: Fasting levels of s-CTX-I were increased in peri- and postmenopausal women compared to premenopausal women (p = 0.001). Despite higher fasting levels, the relative postprandial s-CTX-I suppression was comparable across menopausal status (p = 0.14). Fasting levels of s-PINP were also increased in postmenopausal women compared to premenopausal women (p < 0.001) with comparable and modest s-PINP suppression over menopause (p = 0.13). Postprandial plasma GLP-1 (p = 0.006) and GLP-2 (p = 0.01) were significantly increased in postmenopausal women compared to premenopausal women while GIP responses were slightly increased in the perimenopausal group (p = 0.02) but comparable between pre- and postmenopausal women. None of the postprandial gut hormone increases predicted postprandial bone turnover suppression in these women. CONCLUSIONS: Glucose-induced suppression of bone turnover markers is preserved in postmenopausal women, despite significantly higher fasting values, indicating that CTX-I lowering treatments based on these postprandial mechanisms might be a feasible strategy to prevent postmenopausal osteoporosis.

Changes in abdominal subcutaneous adipose tissue phenotype following menopause is associated with increased visceral fat mass.

Menopause is associated with a redistribution of adipose tissue towards central adiposity, known to cause insulin resistance. In this cross-sectional study of 33 women between 45 and 60 years, we assessed adipose tissue inflammation and morphology in subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) across menopause and related this to menopausal differences in adipose tissue distribution and insulin resistance. We collected paired SAT and VAT biopsies from all women and combined this with anthropometric measurements and estimated whole-body insulin sensitivity. We found that menopause was associated with changes in adipose tissue phenotype related to metabolic dysfunction. In SAT, postmenopausal women showed adipocyte hypertrophy, increased inflammation, hypoxia and fibrosis. The postmenopausal changes in SAT was associated with increased visceral fat accumulation. In VAT, menopause was associated with adipocyte hypertrophy, immune cell infiltration and fibrosis. The postmenopausal changes in VAT phenotype was associated with decreased insulin sensitivity. Based on these findings we suggest, that menopause is associated with changes in adipose tissue phenotype related to metabolic dysfunction in both SAT and VAT. Whereas increased SAT inflammation in the context of menopause is associated with VAT accumulation, VAT morphology is related to insulin resistance.

Cardiac and metabolic changes in long-term high fructose-fat fed rats with severe obesity and extensive intramyocardial lipid accumulation.

Metabolic syndrome and obesity-related diseases are affecting more and more people in the Western world. The basis for an effective treatment of these patients is a better understanding of the underlying pathophysiology. Here, we characterize fructose- and fat-fed rats (FFFRs) as a new animal model of metabolic syndrome. Sprague-Dawley rats were fed a 60 kcal/100 kcal fat diet with 10% fructose in the drinking water. After 6, 12, 18, 24, 36, and 48 wk of feeding, blood pressure, glucose tolerance, plasma insulin, glucose, and lipid levels were measured. Cardiac function was examined by in vivo pressure volume measurements, and intramyocardial lipid accumulation was analyzed by confocal microscopy. Cardiac AMP-activated kinase (AMPK) and hepatic phosphoenolpyruvate carboxykinase (PEPCK) levels were measured by Western blotting. Finally, an ischemia-reperfusion study was performed after 56 wk of feeding. FFFRs developed severe obesity, decreased glucose tolerance, increased serum insulin and triglyceride levels, and an initial increased fasting glucose, which returned to control levels after 24 wk of feeding. The diet had no effect on blood pressure but decreased hepatic PEPCK levels. FFFRs showed significant intramyocardial lipid accumulation, and cardiac hypertrophy became pronounced between 24 and 36 wk of feeding. FFFRs showed no signs of cardiac dysfunction during unstressed conditions, but their hearts were much more vulnerable to ischemia-reperfusion and had a decreased level of phosphorylated AMPK at 6 wk of feeding. This study characterizes a new animal model of the metabolic syndrome that could be beneficial in future studies of metabolic syndrome and cardiac complications.

Low-Grade Inflammation Is Not Present in Former Obese Males but Adipose Tissue Macrophage Infiltration Persists.

Macrophage infiltration in two subcutaneous adipose tissue depots and systemic low-grade inflammation were studied in post-obese (PO), obese (O), and control (C) subjects. Young males were recruited into PO: (n = 10, weight-loss avg. 26%, BMI: 26.6 ± 0.7, mean ±SEM kg/m2), O: (n = 10, BMI: 33.8 ± 1.0kg/m2) and C: (n = 10, BMI: 26.6 ± 0.6 kg/m2). PO and C were matched by BMI. Blood and abdominal and gluteal subcutaneous adipose tissue were obtained in the overnight fasted state. Plasma concentrations of IL-6 and CRP were higher (p < 0.05) in O than in PO and C, TNF-α was higher (p < 0.05) only in O compared to PO and IL-18 was similar between groups. The number of CD68+ macrophages was higher (p < 0.05) in the gluteal than the abdominal depot, and higher (p < 0.05) in O and PO compared to C in both depots. The content of CD163+ macrophages was similar between depots but was higher (p < 0.05) in PO compared to C and O in the gluteal depot. In post obese men with a long-term sustained weight loss, systemic low-grade inflammation was similar to non-obese controls despite a higher subcutaneous adipose tissue CD68+ macrophage content. Interestingly, the anti-inflammatory CD163+ macrophage adipose tissue content was consistently higher in post obese than obese and controls.

Ectopic Lipid Deposition Is Associated With Insulin Resistance in Postmenopausal Women.

Context: Menopause is associated with an increased incidence of insulin resistance and diabetes. Objective: The aim of this study was to explore the lipid deposition in liver and skeletal muscle and investigate the association with insulin sensitivity in postmenopausal and premenopausal women. Design and Setting: Single-center cross-sectional study of 55 healthy women between 45 and 60 years of age. We measured lipid deposition in the liver with magnetic resonance spectroscopy, intramuscular and intra-abdominal lipid deposition with MRI, body composition with a dual-energy X-ray absorptiometry scan, and insulin sensitivity with the composite Matsuda Index. Outcome Measures: We studied the association between fat distribution, ectopic lipid deposition, and insulin sensitivity in pre- and postmenopausal women. Results: Postmenopausal women had an increased lipid deposition in the liver [0.68% (0.44 to 0.99) vs 0.49% (0.38 to 0.64), P = 0.01] and skeletal muscle [3% (2 to 4) vs 2% (1 to 3), P = 0.001] and had a 28% lower Matsuda insulin sensitivity index during an oral glucose tolerance test (6.31 ± 3.48 vs 8.78 ± 4.67, P = 0.05) compared with premenopausal women. Total fat mass and leg fat mass were stronger predictors of ectopic lipid deposition, and visceral fat mass was a stronger predictor of both ectopic lipid deposition and insulin resistance in postmenopausal women compared with premenopausal women. Conclusions: For a given subcutaneous and visceral fat depot size, postmenopausal women show increased ectopic lipid deposition and insulin resistance compared with premenopausal women. It is suggested that lipid deposition in liver and skeletal muscle may represent important mechanistic links between the changes in fat depots and the increased incidence of insulin resistance seen after menopause.

Imaging of insulin signaling in skeletal muscle of living mice shows major role of T-tubules.

Insulin stimulates glucose transport in skeletal muscle by glucose transporter GLUT4 translocation to sarcolemma and membrane invaginations, the t-tubules. Although muscle glucose uptake plays a key role in insulin resistance and type 2 diabetes, the dynamics of GLUT4 translocation and the signaling involved are not well described. We have now developed a confocal imaging technique to follow trafficking of green fluorescent protein-labeled proteins in living muscle fibers in situ in anesthetized mice. Using this technique, by imaging the dynamics of GLUT4 translocation and phosphatidylinositol 3,4,5 P(3) (PIP(3)) production in response to insulin, here, for the first time, we delineate the temporal and spatial distribution of these processes in a living animal. We find a 10-min delay of maximal GLUT4 recruitment and translocation to t-tubules compared with sarcolemma. Time-lapse imaging of a fluorescent dye after intravenous injection shows that this delay is similar to the time needed for insulin diffusion into the t-tubule system. Correspondingly, immunostaining of muscle fibers shows that insulin receptors are present throughout the t-tubule system. Finally, PIP(3) production, an early event in insulin signaling, progresses slowly along the t-tubules with a 10-min delay between maximal PIP(3) production at sarcolemma compared with deep t-tubules following the appearance of dye-labeled insulin. Our findings in living mice indicate a major role of the t-tubules in insulin signaling in skeletal muscle and show a diffusion-associated delay in insulin action between sarcolemma and inner t-tubules.

Macrophage Area Content and Phenotype in Hepatic and Adipose Tissue in Patients with Obesity Undergoing Roux-en-Y Gastric Bypass.

OBJECTIVE: To investigate hepatic and adipose tissue macrophage content in subjects with obesity and the role of adipose tissue macrophages in weight loss-induced improved insulin sensitivity (IS). METHODS: A cross-sectional and a longitudinal study were combined to investigate the role of macrophages in subcutaneous (SAT) and visceral (VAT) adipose tissue and the liver in obesity-induced impaired IS and improvements with weight loss. Macrophage markers (CD68, CD163, and CD206) in SAT, VAT, and the liver from patients with obesity were investigated. The same macrophage markers were investigated in SAT from 18 patients with obesity before and ∼18 months after a diet- and Roux-en-Y gastric bypass-induced weight loss. RESULTS: SAT macrophage markers did not decrease with weight loss, but macrophage concentration may have increased, concomitant with improved IS. Hepatic macrophage markers did not correlate to VAT mass or macrophage markers, but they were higher in patients with obesity compared with patients without obesity. Hepatic anti-inflammatory macrophage markers correlated positively with hepatic IS. VAT and SAT macrophage markers did not correlate. CONCLUSIONS: The results indicate that decreased SAT macrophage content is not a primary driver for weight loss-induced IS improvements, but a better hepatic CD163 and CD206 macrophage profile may contribute to improved glycemic control. SAT macrophage markers were not predictive for VAT macrophage markers.

Early and rapid development of insulin resistance, islet dysfunction and glucose intolerance after high-fat feeding in mice overexpressing phosphodiesterase 3B.

Inadequate islet adaptation to insulin resistance leads to glucose intolerance and type 2 diabetes. Here we investigate whether beta-cell cAMP is crucial for islet adaptation and prevention of glucose intolerance in mice. Mice with a beta-cell-specific, 2-fold overexpression of the cAMP-degrading enzyme phosphodiesterase 3B (RIP-PDE3B/2 mice) were metabolically challenged with a high-fat diet. We found that RIP-PDE3B/2 mice early and rapidly develop glucose intolerance and insulin resistance, as compared with wild-type littermates, after 2 months of high-fat feeding. This was evident from advanced fasting hyperinsulinemia and early development of hyper-glycemia, in spite of hyperinsulinemia, as well as impaired capacity of insulin to suppress plasma glucose in an insulin tolerance test. In vitro analyses of insulin-stimulated lipogenesis in adipocytes and glucose uptake in skeletal muscle did not reveal reduced insulin sensitivity in these tissues. Significant steatosis was noted in livers from high-fat-fed wild-type and RIP-PDE3B/2 mice and liver triacyl-glycerol content was 3-fold higher than in wild-type mice fed a control diet. Histochemical analysis revealed severe islet perturbations, such as centrally located alpha-cells and reduced immunostaining for insulin and GLUT2 in islets from RIP-PDE3B/2 mice. Additionally, in vitro experiments revealed that the insulin secretory response to glucagon-like peptide-1 stimulation was markedly reduced in islets from high-fat-fed RIP-PDE3B/2 mice. We conclude that accurate regulation of beta-cell cAMP is necessary for adequate islet adaptation to a perturbed metabolic environment and protective for the development of glucose intolerance and insulin resistance.

Assessment of six commercial plasma small RNA isolation kits using qRT-PCR and electrophoretic separation: higher recovery of microRNA following ultracentrifugation.

Growing interest in blood-borne microRNAs (miRNAs) as biomarkers has led to the introduction of a number of commercial kits for isolating small RNAs from plasma/serum. We sought to compare the efficacy of six such kits in isolating miRNAs from either whole plasma or a plasma-derived ultracentrifugation (UC) fraction from 2 healthy volunteers with some of the results being validated in 10 additional subjects. To assess the overall yield and concentration of isolated small RNAs, we measured the levels of one spiked-in and four endogenous miRNAs by quantitative reverse transcription and polymerase chain reaction (qRT-PCR). We also tested the performance of the Agilent Bioanalyzer small RNA assay with these RNA samples. Additionally, we tested the effects of hemolysis on measured miRNA levels in whole plasma and in the UC fraction. Both the efficiency of RNA isolation and the relative levels of specific miRNAs in different samples varied considerably between the tested extraction methods. Of all kits tested, the QIAGEN miRNeasy kits (Mini and Serum/Plasma kits) and the Macherey-Nagel NucleoSpin kit produced the highest RNA yields. The QIAGEN Exo kit produced lesser yields than what could be extracted from the UC fraction using the QIAGEN miRNeasy kits and the Macherey-Nagel NucleoSpin kit. Bioanalyzer results showed an average correlation of R 2 = 0.8 with endogenous miRNA qRT-PCR results, for sample concentrations >40 pg/µl. The levels of the endogenous miRNAs measured in the two volunteer samples were compared with those in a larger group of subjects (n = 10) and found to be typical. Our comparison favors the use of the QIAGEN Serum/Plasma kit and the Macherey-Nagel NucleoSpin kit for plasma miRNA applications. Furthermore, extraction of miRNAs from the UC fraction results in higher yield than extraction from whole plasma.

Expression profiling reveals differences in metabolic gene expression between exercise-induced cardiac effects and maladaptive cardiac hypertrophy.

While cardiac hypertrophy elicited by pathological stimuli eventually leads to cardiac dysfunction, exercise-induced hypertrophy does not. This suggests that a beneficial hypertrophic phenotype exists. In search of an underlying molecular substrate we used microarray technology to identify cardiac gene expression in response to exercise. Rats exercised for seven weeks on a treadmill were characterized by invasive blood pressure measurements and echocardiography. RNA was isolated from the left ventricle and analysed on DNA microarrays containing 8740 genes. Selected genes were analysed by quantitative PCR. The exercise program resulted in cardiac hypertrophy without impaired cardiac function. Principal component analysis identified an exercise-induced change in gene expression that was distinct from the program observed in maladaptive hypertrophy. Statistical analysis identified 267 upregulated genes and 62 downregulated genes in response to exercise. Expression changes in genes encoding extracellular matrix proteins, cytoskeletal elements, signalling factors and ribosomal proteins mimicked changes previously described in maladaptive hypertrophy. Our most striking observation was that expression changes of genes involved in beta-oxidation of fatty acids and glucose metabolism differentiate adaptive from maladaptive hypertrophy. Direct comparison to maladaptive hypertrophy was enabled by quantitative PCR of key metabolic enzymes including uncoupling protein 2 (UCP2) and fatty acid translocase (CD36). DNA microarray analysis of gene expression changes in exercise-induced cardiac hypertrophy suggests that a set of genes involved in fatty acid and glucose metabolism could be fundamental to the beneficial phenotype of exercise-induced hypertrophy, as these changes are absent or reversed in maladaptive hypertrophy.