Adipogenic progenitors from obese human skeletal muscle give rise to functional white adipocytes that contribute to insulin resistance.

BACKGROUND/OBJECTIVES: Recent reports indicate that inter/intramuscular adipose tissue (IMAT), composed by adipocytes underneath the deep fascia of the muscles, is positively correlated with aging, obesity and insulin resistance in humans. However, no molecular/cellular evidence is available to support these interactions. The current study aimed to better characterize human skeletal muscle-derived adipogenic progenitors obtained from obese volunteers and investigate the impact of derived adipocytes on insulin action in primary skeletal muscle cells. METHODS: Primary cultured stroma-vascular fraction (SVF) obtained from vastus lateralis muscle biopsies of middle-aged obese subjects was immunoseparated (magnetic beads or flow cytometry). The characteristics and/or metabolic phenotype of CD56(+), CD56(-) and CD56(-)CD15(+) cellular fractions were investigated by complementary approaches (flow cytometry, cytology, quantitative PCR and metabolic assays). The effects of conditioned media from CD56(-)CD15(+) cells differentiated into adipocytes on insulin action and signaling in human primary myotubes was also examined. RESULTS: Our data indicate that CD56(+) and CD56(-) cellular fractions isolated from cultured SVF of human muscle contain two distinct committed progenitors: CD56(+) cells (that is, satellite cells) as myogenic progenitors and CD15(+) cells as adipogenic progenitors, respectively. CD56(-)CD15(+)-derived adipocytes display the phenotype and metabolic properties of white adipocytes. Secretions of CD56(-)CD15(+) cells differentiated into functional white adipocytes reduced insulin-mediated non-oxidative glucose disposal (P=0.0002) and insulin signaling. CONCLUSIONS: Using in-vitro models, we show for the first time that secretions of skeletal muscle adipocytes are able to impair insulin action and signaling of muscle fibers. This paracrine effect could explain, at least in part, the negative association between high levels of IMAT and insulin sensitivity in obesity and aging.

Impaired atrial natriuretic peptide-mediated lipolysis in obesity.

BACKGROUND: Catecholamines and natriuretic peptides (NPs) are the only hormones with pronounced lipolytic effects in human white adipose tissue. Although catecholamine-induced lipolysis is well known to be impaired in obesity and insulin resistance, it is not known whether the effect of NPs is also altered. METHODS: Catecholamine- and atrial NP (ANP)-induced lipolysis was investigated in abdominal subcutaneous adipocytes in vitro and in situ by microdialysis. RESULTS: In a cohort of 122 women, both catecholamine- and ANP-induced lipolysis in vitro was markedly attenuated in obesity (n=87), but normalized after substantial body weight loss (n=52). The impairment of lipolysis differed between the two hormones when expressing lipolysis per lipid weight, the ratio of stimulated over basal (spontaneous) lipolysis rate or per number of adipocytes. Thus, while the response to catecholamines was lower when expressed as the former two measures, it was higher when expressed per cell number, a consequence of the significantly larger fat cell size in obesity. In contrast, although ANP-induced lipolysis was also attenuated when expressed per lipid weight or the ratio stimulated/basal, it was similar between non-obese and obese subjects when expressed per cell number suggesting that the lipolytic effect of ANP may be even more sensitive to the effects of obesity than catecholamines. Obesity was characterized by a decrease in the protein expression of the signaling NP A receptor (NPRA) and a trend toward increased levels of the clearance receptor NPRC. The impairment in ANP-induced lipolysis observed in vitro was corroborated by microdialysis experiments in situ in a smaller cohort of lean and overweight men. CONCLUSIONS: ANP- and catecholamine-induced lipolysis is reversibly attenuated in obesity. The pro-lipolytic effects of ANP are relatively more impaired compared with that of catecholamines, which may in part be due to specific changes in NP receptor expression.

Effect of endurance training on skeletal muscle myokine expression in obese men: identification of apelin as a novel myokine.

BACKGROUND: It has been suggested that the metabolic benefits of physical exercise could be mediated by myokines. We examined here the effect of exercise training on skeletal muscle expression of a panel of myokines in humans. Pathways regulating myokine expression were investigated in human myotubes. METHODS: Eleven obese non-diabetic male subjects were enrolled in an 8-week endurance training program. Insulin sensitivity was assessed by an oral glucose tolerance test. Subcutaneous adipose tissue and Vastus lateralis muscle biopsy samples were collected before and after training. RNAs were prepared from adipose tissue and skeletal muscle. Primary culture of myoblasts was established. RESULTS: As expected, exercise training improved aerobic capacity and decreased fat mass. No significant change in interleukin 6, fibroblast growth factor 21, myostatin (MSTN) or irisin mRNA level was found in muscle after training. A twofold increase in apelin mRNA level was found in muscle but not in adipose tissue. No change in circulating myokine and adipokine plasma levels was observed in the resting state in response to training. Interestingly, apelin was significantly expressed and secreted in primary human myotubes. Apelin gene expression was upregulated by cyclic AMP and calcium, unlike the other myokines investigated. Importantly, changes in muscle apelin mRNA levels were positively related to whole-body insulin sensitivity improvement. CONCLUSION: Collectively, our data show that exercise training upregulates muscle apelin expression in obese subjects. Apelin expression is induced by exercise signaling pathways and secreted in vitro in human primary myotubes, and may behave as a novel exercise-regulated myokine with autocrine/paracrine action.

Semaphorin 3C is a novel adipokine linked to extracellular matrix composition.

AIMS/HYPOTHESIS: Alterations in white adipose tissue (WAT) function, including changes in protein (adipokine) secretion and extracellular matrix (ECM) composition, promote an insulin-resistant state. We set out to identify novel adipokines regulated by body fat mass in human subcutaneous WAT with potential roles in adipose function. METHODS: Adipose transcriptome data and secretome profiles from conditions with increased/decreased WAT mass were combined. WAT donors were predominantly women. In vitro effects were assessed using recombinant protein. Results were confirmed by quantitative PCR/ELISA, metabolic assays and immunochemistry in human WAT and adipocytes. RESULTS: We identified a hitherto uncharacterised adipokine, semaphorin 3C (SEMA3C), the expression of which correlated significantly with body weight, insulin resistance (HOMA of insulin resistance [HOMAIR], and the rate constant for the insulin tolerance test [KITT]) and adipose tissue morphology (hypertrophy vs hyperplasia). SEMA3C was primarily found in mature adipocytes and had no direct effect on human adipocyte differentiation, lipolysis, glucose transport or the expression of ß-oxidation genes. This could in part be explained by the significant downregulation of its cognate receptors during adipogenesis. In contrast, in pre-adipocytes, SEMA3C increased the production/secretion of several ECM components (fibronectin, elastin and collagen I) and matricellular factors (connective tissue growth factor, IL6 and transforming growth factor-ß1). Furthermore, the expression of SEMA3C in human WAT correlated positively with the degree of fibrosis in WAT. CONCLUSIONS/INTERPRETATION: SEMA3C is a novel adipokine regulated by weight changes. The correlation with WAT hypertrophy and fibrosis in vivo, as well as its effects on ECM production in human pre-adipocytes in vitro, together suggest that SEMA3C constitutes an adipocyte-derived paracrine signal that influences ECM composition and may play a pathophysiological role in human WAT.

Adipocyte lipases and lipid droplet-associated proteins: insight from transgenic mouse models.

Adipose tissue lipolysis is the catabolic process whereby stored triacylglycerol (TAG) is broken down by lipases into fatty acids and glycerol. Here, we review recent insights from transgenic mouse models. Genetic manipulations affecting lipases are considered first, followed by transgenic models of lipase co-factors and lastly non-lipase lipid droplet (LD)-associated proteins. The central role of hormone-sensitive lipase (HSL), long considered to be the sole rate-limiting enzyme of TAG hydrolysis, has been revised since the discovery of adipose triglyceride lipase (ATGL). It is now accepted that ATGL initiates TAG breakdown producing diacylglycerol, which is subsequently hydrolyzed by HSL. Furthermore, lipase activities are modulated by co-factors whose deletion causes severe metabolic disturbances. Another major advance has come from the description of the involvement of non-lipase proteins in the regulation of lipolysis. The role of perilipins has been extensively investigated. Other newly discovered LD-associated proteins have also been shown to regulate lipolysis.

The impact of obesity on secretion of adiponectin multimeric isoforms differs in visceral and subcutaneous adipose tissue.

OBJECTIVE: Hypoadiponectinemia observed in obesity is associated with insulin resistance, diabetes and atherosclerosis. The aim of the present study was to investigate secretion of adiponectin and its multimeric isoforms by explants derived from subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) in obese and non-obese subjects. DESIGN: Paired samples of SAT and VAT and blood samples were obtained from 23 subjects (10 non-obese and 13 obese) undergoing elective abdominal surgery. Total adiponectin quantities and adiponectin isoforms were measured in conditioned media of explants derived from SAT and VAT using enzyme-linked immunosorbent assay and non-denaturing western blot, respectively. RESULTS: Total adiponectin plasma levels were lower in obese than in non-obese subjects (P<0.05). Secretion of total adiponectin in adipose tissue (AT) explants was lower in obese than in non-obese subjects in SAT (P<0.05) but not in VAT. In both, SAT and VAT, the most abundant isoform released into conditioned media was the high-molecular weight (HMW) form. Its relative proportion in relation to total adiponectin was higher in conditioned media of explants from both fat depots when compared with plasma (P<0.001). The proportion of secreted HMW vs total adiponectin was higher in VAT than in SAT explants in the group of non-obese individuals (49.3±3.1% in VAT vs 40.6±2.8% in SAT; P<0.01), whereas no difference between the two depots was found in obese subjects (46.2±3.0 % in VAT vs 46.0±2.4 % in SAT). CONCLUSION: Obesity is associated with the decrease of total adiponectin secretion in SAT. The profile of adiponectin isoforms secreted by SAT and VAT explants differs from that in plasma. Secretion of total adiponectin and HMW isoform of adiponectin are different in obese and non-obese subjects in relation to AT depot.

Circulating ACE is a predictor of weight loss maintenance not only in overweight and obese women, but also in men.

BACKGROUND: Circulating angiotensin-converting enzyme (ACE) was identified as a predictor of weight loss maintenance in overweight/obese women of the Diogenes project. OBJECTIVE: To investigate whether ACE acted also as a predictor in men of the Diogenes study and to compare it with that in women. DESIGN: Subjects, who lost ≥ 8% of body weight induced by low-caloric diet in an 8-week weight loss period, were assigned to weight loss maintenance with dietary intervention for 6 months. SUBJECTS: 125 overweight/obese healthy men from eight European countries who completed whole intervention. MEASUREMENTS: Concentrations and activity of serum ACE at baseline and after the 8-week weight loss, in addition to anthropometric and physiological parameters. RESULTS: Serum ACE concentration decreased by 11.3 ± 10.6% during the weight loss period in men. A greater reduction is associated with less body weight regain during the maintenance period (r=0.227, P=0.012). ACE change was able to predict a weight regain ≤ 20% after 6 months, with an odds ratio of 1.59 (95% confidence interval (CI): 1.09-2.33, P=0.016) for every 10% reduction, which was independent of body mass index and weight loss. The prediction power was weaker in men than in women, but without a significant sex difference (P=0.137). In pooled subjects (N=218), the odds ratio was 1.96 (95% CI: 1.46-2.64, P<0.001). CONCLUSIONS: A greater reduction of ACE during weight loss is favorable for weight maintenance in both men and women. This can offer useful information for personalized advice to improve weight loss maintenance. It also confirms the role of ACE in the metabolic pathways of weight regulation.

Macrophage gene expression is related to obesity and the metabolic syndrome in human subcutaneous fat as well as in visceral fat.

AIMS/HYPOTHESIS: Our goal was to identify a set of human adipose tissue macrophage (ATM)-specific markers and investigate whether their gene expression in subcutaneous adipose tissue (SAT) as well as in visceral adipose tissue (VAT) is related to obesity and to the occurrence of the metabolic syndrome. METHODS: ATM-specific markers were identified by DNA microarray analysis of adipose tissue cell types isolated from SAT of lean and obese individuals. We then analysed gene expression of these markers by reverse transcription quantitative PCR in paired samples of SAT and VAT from 53 women stratified into four groups (lean, overweight, obese and obese with the metabolic syndrome). Anthropometric measurements, euglycaemic-hyperinsulinaemic clamp, blood analysis and computed tomography scans were performed. RESULTS: A panel of 24 genes was selected as ATM-specific markers based on overexpression in ATM compared with other adipose tissue cell types. In SAT and VAT, gene expression of ATM markers was lowest in lean and highest in the metabolic syndrome group. mRNA levels in the two fat depots were negatively correlated with glucose disposal rate and positively associated with indices of adiposity and the metabolic syndrome. CONCLUSIONS/INTERPRETATION: In humans, expression of ATM-specific genes increases with the degree of adiposity and correlates with markers of insulin resistance and the metabolic syndrome to a similar degree in SAT and in VAT.

Dietary intervention-induced weight loss decreases macrophage content in adipose tissue of obese women.

OBJECTIVE: Accumulation of adipose tissue macrophages (ATMs) is observed in obesity and may participate in the development of insulin resistance and obesity-related complications. The aim of our study was to investigate the effect of long-term dietary intervention on ATM content in human adipose tissue. DESIGN: We performed a multi-phase longitudinal study. SUBJECTS AND MEASUREMENTS: A total of 27 obese pre-menopausal women (age 39 ± 2 years, body mass index 33.7 ± 0.5 kg m(-2)) underwent a 6-month dietary intervention consisting of two periods: 4 weeks of very low-calorie diet (VLCD) followed by weight stabilization composed of 2 months of low-calorie diet and 3 to 4 months of weight maintenance diet. At baseline and at the end of each dietary period, samples of subcutaneous adipose tissue (SAT) were obtained by needle biopsy and blood samples were drawn. ATMs were determined by flow cytometry using combinations of cell surface markers. Selected cytokine and chemokine plasma levels were measured using enzyme-linked immunosorbent assay. In addition, in a subgroup of 16 subjects, gene expression profiling of macrophage markers in SAT was performed using real-time PCR. RESULTS: Dietary intervention led to a significant decrease in body weight, plasma insulin and C-reactive protein levels. After VLCD, ATM content defined by CD45+/14+/206+ did not change, whereas it decreased at the end of the intervention. This decrease was associated with a downregulation of macrophage marker mRNA levels (CD14, CD163, CD68 and LYVE-1 (lymphatic vessel endothelial hyaluronan receptor-1)) and plasma levels of monocyte-chemoattractant protein-1 (MCP-1) and CXCL5 (chemokine (C-X-C motif) ligand 5). During the whole dietary intervention, the proportion of two ATM subpopulations distinguished by the CD16 marker was not changed. CONCLUSION: A 6-month weight-reducing dietary intervention, but not VLCD, promotes a decrease in the number of the whole ATM population with no change in the relative distribution of ATM subsets.

Revisiting the Hallmarks of Aging to Identify Markers of Biological Age.

The Geroscience aims at a better understanding of the biological processes of aging, to prevent and/or delay the onset of chronic diseases and disability as well as to reduce the severity of these adverse clinical outcomes. Geroscience thus open up new perspectives of care to live a healthy aging, that is to say without dependency. To date, life expectancy in healthy aging is not increasing as fast as lifespan. The identification of biomarkers of aging is critical to predict adverse outcomes during aging, to implement interventions to reduce them, and to monitor the response to these interventions. In this narrative review, we gathered information about biomarkers of aging under the perspective of Geroscience. Based on the current literature, for each hallmark of biological aging, we proposed a putative biomarker of healthy aging, chosen for their association with mortality, age-related chronic diseases, frailty and/or functional loss. We also discussed how they could be validated as useful predictive biomarkers.

Transcription of the human uncoupling protein 3 gene is governed by a complex interplay between the promoter and intronic sequences.

AIMS/HYPOTHESIS: Uncoupling protein (UCP) 3 is an inner mitochondrial membrane transporter mainly produced in skeletal muscle in humans. UCP3 plays a role in fatty acid metabolism and energy homeostasis and modulates insulin sensitivity. In humans, UCP3 content is higher in fast-twitch glycolytic muscle than in slow-twitch oxidative muscle and is dysregulated in type 2 diabetes. Here, we studied the molecular mechanisms determining human UCP3 levels in skeletal muscle and their regulation by fasting in transgenic mice. METHODS: We produced a series of transgenic lines with constructs bearing different putative regulatory regions of the human UCP3 gene, including promoter and intron sequences. UCP3 mRNA and reporter gene expression and activity were measured in different skeletal muscles and tissues. RESULTS: The profile of expression and the response to fasting and thyroid hormone of human UCP3 mRNA in transgenic mice with 16 kb of the human UCP3 gene were similar to that of the endogenous human gene. Various parts of the UCP3 promoter did not confer expression in transgenic lines. Inclusion of intron 1 resulted in an expression profile in skeletal muscle that was identical to that of human UCP3 mRNA. Further dissection of intron 1 revealed that distinct regions were involved in skeletal muscle expression, distribution among fibre types and response to fasting. CONCLUSIONS/INTERPRETATION: The control of human UCP3 transcription in skeletal muscle is not solely conferred by the promoter, but depends on several cis-acting elements in intron 1, suggesting a complex interplay between the promoter and intronic sequences.

Adrenaline but not noradrenaline is a determinant of exercise-induced lipid mobilization in human subcutaneous adipose tissue.

The relative contribution of noradrenaline (norepinephrine) and adrenaline (epinephrine) in the control of lipid mobilization in subcutaneous adipose tissue (SCAT) during exercise was evaluated in men treated with a somatostatin analogue, octreotide. Eight lean and eight obese young men matched for age and physical fitness performed 60 min exercise bouts at 50% of their maximal oxygen consumption on two occasions: (1) during i.v. infusion of octreotide, and (2) during placebo infusion. Lipolysis and local blood flow changes in SCAT were evaluated using in situ microdialysis. Infusion of octreotide suppressed plasma insulin and growth hormone levels at rest and during exercise. It blocked the exercise-induced increase in plasma adrenaline while that of noradrenaline was unchanged. Plasma natriuretic peptides (NPs) level was higher at rest and during exercise under octreotide infusion in lean men. Under placebo, no difference was found in the exercise-induced increase in glycerol between the probe perfused with Ringer solution alone and that with phentolamine (an alpha-adrenergic receptor antagonist) in lean subjects while a greater increase in glycerol was observed in the obese subjects. Under placebo, propranolol infusion in the probe containing phentolamine reduced by about 45% exercise-induced glycerol release; this effect was fully suppressed under octreotide infusion while noradrenaline was still elevated and exercise-induced lipid mobilization maintained in both lean and obese individuals. In conclusion, blockade of beta-adrenergic receptors during exercise performed during infusion of octreotide (blocking the exercise-induced rise in adrenaline but not that of noradrenaline) does not alter the exercise-induced lipolysis. This suggests that adrenaline is the main adrenergic agent contributing to exercise-induced lipolysis in SCAT. Moreover, it is the combined action of insulin suppression and NPs release which explains the lipolytic response which remains under octreotide after full local blockade of fat cell adrenergic receptors. For the moment, it is unknown if results apply specifically to SCAT and exercise only or if conclusions could be extended to all forms of lipolysis in humans.

Macronutrient-specific effect of FTO rs9939609 in response to a 10-week randomized hypo-energetic diet among obese Europeans.

BACKGROUND: The A risk allele of rs9939609 of the fat mass- and obesity-associated gene (FTO) increases body fat mass. OBJECTIVE: To examine whether FTO rs9939609 affects obese individuals' response to a high-fat, low-carbohydrate (CHO) (HF) or low-fat, high-CHO (LF), hypo-energetic diet and whether the effect of the FTO variant depends on dietary fat and CHO content. DESIGN: In a 10-week, European, multi-centre dietary intervention study 771 obese women and men were randomized to either LF (20-25% of energy (%E) from fat, 60-65%E from CHO) or HF (40-45%E from fat, 40-45%E from CHO), hypo-energetic diet (measured resting metabolic rate multiplied by 1.3-600 kcal day(-1)). Body weight, fat mass (FM), fat-free mass (FFM), waist circumference (WC), resting energy expenditure (REE), fasting fat oxidation as % of REE (FatOx), insulin release (HOMA-beta) and a surrogate measure of insulin resistance (HOMA-IR) were measured at baseline and after the intervention. In all, 764 individuals were genotyped for FTO rs9939609. RESULTS: For A-allele carriers the drop-out rate was higher on HF than LF diet (in AT, P=0.002; in AT/AA combined, P=0.003). Among those individuals completing the intervention, we found no effect of FTO rs9939609 genotype on Deltaweight, DeltaFM, DeltaFFM, DeltaWC or DeltaFatOx. However, participants with TT had a smaller reduction in REE on LF than on HF diet (75 kcal/24 h; interaction: P=0.0055). These individuals also showed the greatest reduction in HOMA-beta and HOMA-IR (interaction: P=0.0083 and P=0.047). CONCLUSION: The FTO rs9939609 may interact with the macronutrient composition in weight loss diets in various ways; carriers of the A allele on LF diet appear to have a lower risk for drop out, and TT individuals have a smaller decrease in REE and greater decrease in HOMA-beta and HOMA-IR on LF than on HF diet.

Visfatin expression in subcutaneous adipose tissue of pre-menopausal women: relation to hormones and weight reduction.

BACKGROUND: A novel adipokine, visfatin, was found to be related to adiposity in humans and regulated by a number of hormonal signals. The aim of this study was to investigate the relationships of visfatin expression in adipose tissue with potential regulatory factors such as insulin, testosterone and tumor necrosis factor-alpha (TNF-alpha) and to elucidate the effect of a diet induced weight reduction on adipose tissue mRNA expression and plasma levels of visfatin. MATERIALS AND METHODS: Biopsies of subcutaneous abdominal adipose tissue (SCAAT) and plasma samples were obtained at the beginning of the study from 47 pre-menopausal women (age 38.7 +/- 1.7 years, body mass index (BMI) 27.9 +/- 1.4 kg m(-2)), consisting of 15 lean, 16 overweight and 16 obese subjects. The subgroup of 32 overweight/obese women (age 42.1 +/- 1.9 years, BMI 31.2 +/- 0.9 kg m(-2)) underwent a 12 week hypocaloric weight reducing diet and samples were obtained at the end of the diet. Biopsy samples were analysed for visfatin and TNF-alpha mRNA levels and plasma was analysed for relevant metabolites and hormones. RESULTS: In the group of 47 subjects visfatin mRNA expression in SCAAT was negatively correlated with plasma free testosterone (r = -0. 363, P < 0.05) and BMI (r = -0.558, P < 0.01) and positively associated with adipose tissue TNF-alpha mRNA expression (r = 0.688, P < 0.01). The diet resulted in the reduction of body weight and in the decrease of plasma insulin, free testosterone and TNF-alpha levels. In the group of overweight/obese subjects visfatin mRNA in SCAAT increased after the diet and the diet induced increase was positively correlated with the magnitude of body weight loss. CONCLUSION: Visfatin mRNA expression in SCAAT is associated with TNF-alpha expression, plasma free testosterone and BMI in pre-menopausal women. A weight reducing hypocaloric diet results in the increase of visfatin mRNA in SCAAT.

Retinol-binding protein 4 expression in visceral and subcutaneous fat in human obesity.

Retinol binding protein 4 (RBP4) is a novel adipokine which might be involved in the development of insulin resistance. The aim of the study was to investigate the expression of RBP4 mRNA in subcutaneous and visceral fat depots and the relationship between RBP4 plasma and mRNA levels relative to indices of adiposity and insulin resistance. In 59 Caucasian women (BMI 20 to 49 kg/m(2)) paired samples of subcutaneous and visceral fat were obtained for RBP4, leptin and GLUT 4 mRNA analysis using reverse transcription-quantitative PCR. Euglycemic hyperinsulinemic clamp and computed tomography scans were performed. RBP4 mRNA levels as well as GLUT 4 mRNA and leptin mRNA levels were lower (P<0.001, P<0.01 and P<0.001, respectively) in visceral compared to subcutaneous fat. No differences were found in RBP4 mRNA expression in the two fat depots or in RBP4 plasma levels between subgroups of non-obese subjects (n=26), obese subjects without metabolic syndrome (n=17) and with metabolic syndrome (n=16). No correlations between RBP4 mRNA or plasma levels relative to adiposity, glucose disposal rate and GLUT 4 mRNA expression in adipose tissue were found. There was a weak positive correlation between plasma RBP4 and plasma triglycerides (r = 0.30, p<0.05) and between plasma RBP4 and blood glucose (r = 0.26, p<0.05). Regardless of the state of adiposity or insulin resistance, RBP4 expression in humans was lower in visceral than in subcutaneous fat. We found no direct relationship between either RBP4 mRNA or its plasma levels and the adiposity or insulin resistance.

Functional beta3-adrenoceptor in the human heart.

Beta3-adrenoceptors are involved in metabolism, gut relaxation, and vascular vasodilation. However, their existence and role in the human heart have not been documented. We investigated the effects of several beta-adrenoceptor agonists and antagonists on the mechanical properties of ventricular endomyocardial biopsies. In the presence of nadolol, a beta1- and beta2-adrenoceptor antagonist, isoprenaline produced consistent negative inotropic effects. Similar negative inotropic effects also resulted from the action of beta3-adrenoceptor agonists with an order of potency: BRL 37344 > SR 58611 approximately CL 316243 > CGP 12177. The dose-response curve to BRL 37344-decreasing myocardial contractility was not modified by pretreatment with nadolol, but was shifted to the right by bupranolol, a nonselective beta-adrenoceptor antagonist. Beta3-adrenoceptor agonists also induced a reduction in the amplitude and an acceleration in the repolarization phase of the human action potential. Beta3-adrenoceptor transcripts were detected in human ventricle by a polymerase chain reaction assay. These results indicate that: (a) beta3-adrenoceptors are present and functional in the human heart; and (b) these receptors are responsible for the unexpected negative inotropic effects of catecholamines and may be involved in pathophysiological mechanisms leading to heart failure.

Increased uncoupling protein-2 and -3 mRNA expression during fasting in obese and lean humans.

Uncoupling protein-2 and -3 (UCP2 and UCP3) are mitochondrial proteins that show high sequence homology with the brown adipocyte-specific UCP1. UCP1 induces heat production by uncoupling respiration from ATP synthesis. UCP2 is widely expressed in human tissues, whereas UCP3 expression seems restricted to skeletal muscle, an important site of thermogenesis in humans. We have investigated the regulation of UCP2 and UCP3 gene expression in skeletal muscle and adipose tissue from lean and obese humans. UCP2 and -3 mRNA levels were not correlated with body mass index (BMI) in skeletal muscle, but a positive correlation (r = 0.55, P < 0.01, n = 22) was found between UCP2 mRNA level in adipose tissue and BMI. The effect of fasting was investigated in eight lean and six obese subjects maintained on a hypocaloric diet (1,045 kJ/d) for 5 d. Calorie restriction induced a similar 2-2.5-fold increase in UCP2 and -3 mRNA levels in lean and obese subjects. To study the effect of insulin on UCP gene expression, six lean and five obese subjects underwent a 3-h euglycemic hyperinsulinemic clamp. Insulin infusion did not modify UCP2 and -3 mRNA levels. In conclusion, the similar induction of gene expression observed during fasting in lean and obese subjects shows that there is no major alteration of UCP2 and -3 gene regulation in adipose tissue and skeletal muscle of obese subjects. The increase in UCP2 and -3 mRNA levels suggests a role for these proteins in the metabolic adaptation to fasting.

Dynamic strength training improves insulin sensitivity without altering plasma levels and gene expression of adipokines in subcutaneous adipose tissue in obese men.

CONTEXT: Obesity is characterized by a low-grade inflammatory state, which could play a role in insulin resistance. Dynamic strength training improves insulin sensitivity. OBJECTIVE: The objective of this study was to investigate, in obese subjects, whether the insulin sensitizing effect of dynamic strength training is associated with changes in plasma levels and gene expression of adipokines potentially involved in the development of insulin resistance. DESIGN: Twelve obese male subjects were investigated before and at the end of 3 months of dynamic strength training. Insulin sensitivity was evaluated using euglycemic-hyperinsulinemic clamp. Blood samples and needle biopsy samples of sc abdominal adipose tissue were obtained. The plasma levels and adipose tissue mRNA levels of adiponectin, leptin, IL-1beta, IL-6, and TNF-alpha were determined. RESULTS: The training induced an increase in the whole-body glucose disposal rate by 24% (P = 0.04). The body weight was not altered during the training. Plasma levels of leptin decreased during the training (16.6 +/- 6.3 vs. 13.1 +/- 5.7 ng/ml) by 21% (P < 0.02), whereas no change in plasma levels of other adipokines and C-reactive protein was observed. Gene expression of the investigated adipokines was not changed in sc adipose tissue during the training. CONCLUSIONS: In obese subjects, the dynamic strength training resulted in an improvement of whole-body insulin sensitivity. The increase in insulin sensitivity was not associated with training-induced modifications of plasma levels or adipose tissue gene expression of adipokines supposedly involved in the development of insulin resistance.

White-to-brite conversion in human adipocytes promotes metabolic reprogramming towards fatty acid anabolic and catabolic pathways.

OBJECTIVE: Fat depots with thermogenic activity have been identified in humans. In mice, the appearance of thermogenic adipocytes within white adipose depots (so-called brown-in-white i.e., brite or beige adipocytes) protects from obesity and insulin resistance. Brite adipocytes may originate from direct conversion of white adipocytes. The purpose of this work was to characterize the metabolism of human brite adipocytes. METHODS: Human multipotent adipose-derived stem cells were differentiated into white adipocytes and then treated with peroxisome proliferator-activated receptor (PPAR)γ or PPARα agonists between day 14 and day 18. Gene expression profiling was determined using DNA microarrays and RT-qPCR. Variations of mRNA levels were confirmed in differentiated human preadipocytes from primary cultures. Fatty acid and glucose metabolism was investigated using radiolabelled tracers, Western blot analyses and assessment of oxygen consumption. Pyruvate dehydrogenase kinase 4 (PDK4) knockdown was achieved using siRNA. In vivo, wild type and PPARα-null mice were treated with a ß3-adrenergic receptor agonist (CL316,243) to induce appearance of brite adipocytes in white fat depot. Determination of mRNA and protein levels was performed on inguinal white adipose tissue. RESULTS: PPAR agonists promote a conversion of white adipocytes into cells displaying a brite molecular pattern. This conversion is associated with transcriptional changes leading to major metabolic adaptations. Fatty acid anabolism i.e., fatty acid esterification into triglycerides, and catabolism i.e., lipolysis and fatty acid oxidation, are increased. Glucose utilization is redirected from oxidation towards glycerol-3-phophate production for triglyceride synthesis. This metabolic shift is dependent on the activation of PDK4 through inactivation of the pyruvate dehydrogenase complex. In vivo, PDK4 expression is markedly induced in wild-type mice in response to CL316,243, while this increase is blunted in PPARα-null mice displaying an impaired britening response. CONCLUSIONS: Conversion of human white fat cells into brite adipocytes results in a major metabolic reprogramming inducing fatty acid anabolic and catabolic pathways. PDK4 redirects glucose from oxidation towards triglyceride synthesis and favors the use of fatty acids as energy source for uncoupling mitochondria.

A role for hormone-sensitive lipase in the selective mobilization of adipose tissue fatty acids.

The mobilization of fatty acids from rat and human fat cells is selective according to molecular structure, and notably carbon atom chain length. This study aimed at examining whether the release of individual fatty acids from triacylglycerols (TAG) by hormone-sensitive lipase (HSL) plays a role in the selectivity of fatty acid mobilization. Recombinant rat and human HSL were incubated with a lipid emulsion. The hydrolysis of 18 individual fatty acids, ranging in chain length from 12 to 24 carbon atoms and in unsaturation degree from 0 to 3 double bond(s), was measured by comparing the composition of non-esterified fatty acids (NEFA) to that of the original TAG. The relative hydrolysis (% in NEFA/% in TAG) differed between fatty acids, being about 5-fold and 3-fold higher for the most (18:1n-7) than for the least (24:0) readily released fatty acid by recombinant rat and human HSL, respectively. Relationships were found between the chain length of fatty acids and their relative hydrolysis. Among 12-24 carbon atom saturated fatty acids, the relative hydrolysis markedly decreased (by about 5- and 3-times for recombinant rat and human HSL, respectively) with increasing chain length. We conclude that fatty acids are selectively released from TAG by HSL according to carbon atom chain length. These data provide insight on the mechanism by which fatty acids are selectively mobilized from fat cells.