Brown adipocyte progenitor population is modified in obese and diabetic skeletal muscle.

Brown adipose tissue mitochondria express the unique thermogenic uncoupling protein-1. Recently, brown adipocyte progenitors have been identified in the CD34+ cell population of human skeletal muscle. The aims of this study were firstly to determine if obesity and diabetes have altered amounts of muscle brown adipocyte progenitors and, secondly, to establish if the latter are correlated with clinical parameters of obesity and diabetes. Body mass index (BMI), plasma glucose, insulin, cholesterol and triglycerides as well as homeostasis model assessment were measured in lean (n=10), obese (n=18) and obese-diabetic (n=15) subjects and muscle biopsies were taken from the rectus abdominus. CD34 being also expressed on endothelial cells, we measured CD31, another endothelial marker, and expressed the brown adipocyte progenitors, as the CD34/CD31 mRNA ratio. The latter was significantly reduced in the obese vs lean subjects suggesting a smaller pool of brown adipocyte progenitors. More strikingly, for lean and obese subjects negative correlations were observed between the CD34/CD31 mRNA ratios and BMI, fasting insulin levels and homeostasis model assessment. These correlations highlight the potential physiological relevance of the muscle CD34/CD31 mRNA ratio.

Total beta-adrenoceptor deficiency results in cardiac hypotrophy and negative inotropy.

The present study investigated cardiac function in hearts of mice with total deficiency of the beta1-, beta2- and beta3-adrenoceptors (TKO) in comparison to wildtype mice (WT). We investigated cardiac morphology and echocardiographic function, measured protein expression of Ca2+-regulatory proteins, SERCA 2a activity, myofibrillar function, and performed running wheel tests. Heart weight and heart-to-body weight ratio were significantly smaller in TKO as compared to WT. This was accompanied by a decrease in the size of the cardiomyocytes in TKO. Heart rate and ejection fraction were significantly diminished in TKO as compared to WT. Protein expressions of SERCA 2a, ryanodine receptor and Na+/Ca2)-exchanger were similar in TKO and WT mice, but phospholamban protein expression was increased. PKA-dependent phosphorylation of phospholamban at serine 16 was absent and CaMKII-dependent phosphorylation at threonine 17 was decreased in TKO. All alterations were paralleled by a decrease in SERCA 2a-activity. A similar maximal calcium-dependent tension but an increased myofibrillar calcium-sensitivity was measured in TKO as compared to WT. We did not observe relevant functional impairments of TKO in running wheel tests. In the absence of beta-agonistic stimulation, SERCA 2a activity is mainly regulated by alterations of phospholamban expression and phosphorylation. The decreased SERCA 2a activity following beta-adrenoceptor deficiency may be partly compensated by an increased myofibrillar calcium-sensitivity.

The emergence of cold-induced brown adipocytes in mouse white fat depots is determined predominantly by white to brown adipocyte transdifferentiation.

The origin of brown adipocytes arising in white adipose tissue (WAT) after cold acclimatization is unclear. Here, we demonstrate that several UCP1-immunoreactive brown adipocytes occurring in WAT after cold acclimatization have a mixed morphology (paucilocular adipocytes). These cells also had a mixed mitochondrioma with classic "brown" and "white" mitochondria, suggesting intermediate steps in the process of direct transformation of white into brown adipocytes (transdifferentiation). Quantitative electron microscopy disclosed that cold exposure (6 degrees C for 10 days) did not induce an increase in WAT preadipocytes. beta(3)-adrenoceptor-knockout mice had a blunted brown adipocyte occurrence upon cold acclimatization. Administration of the beta(3)-adrenoceptor agonist CL316,243 induced the occurrence of brown adipocytes, with the typical morphological features found after cold acclimatization. In contrast, administration of the beta(1)-adrenoceptor agonist xamoterol increased only the number of preadipocytes. These findings indicate that transdifferentiation depends on beta(3)-adrenoceptor activation, whereas preadipocyte recruitment is mediated by beta(1)-adrenoceptor. RT-qPCR experiments disclosed that cold exposure induced enhanced expression of the thermogenic genes and of genes expressed selectively in brown adipose tissue (iBAT) and in both interscapular BAT and WAT. beta(3)-adrenoceptor suppression blunted their expression only in WAT. Furthermore, cold acclimatization induced an increased WAT expression of the gene coding for C/EBPalpha (an antimitotic protein), whereas Ccna1 expression (related to cell proliferation) was unchanged. Overall, our data strongly suggest that the cold-induced emergence of brown adipocytes in WAT predominantly reflects beta(3)-adrenoceptor-mediated transdifferentiation.

Differentiation and characterization in primary culture of white adipose tissue brown adipocyte-like cells.

BACKGROUND: Rodent brown adipose tissue (BAT) is considered the main effector of adaptative thermogenesis as it contains a unique mitochondrial uncoupling protein, termed as uncoupling protein-1 (UCP1). The emergence of ectopic brown adipocytes in the white adipose tissue (WAT), called recruitment, might play an important role in the prevention of obesity. The recruitment phenomenon has until now been investigated mostly in vivo. OBJECTIVES: This study is an attempt to mimic in vitro the recruitment phenomenon. It consisted in culturing the stroma vascular fractions of mouse BAT and WAT in a brown adipocyte differentiation medium. The multilocular cells obtained, referred to as BAT(B) and WAT(B) adipocytes, respectively, were compared. RESULTS: The BAT(B) and WAT(B) adipocytes were morphologically different. The expressions of UCP1, peroxisome proliferator-activated receptor-gamma coactivator-1alpha (PGC-1alpha), leptin and resistin mRNAs were low in WAT(B) adipocytes as compared with those in BAT(B) adipocytes. The expressions of UCP1 and PGC-1alpha proteins were, however, much higher in WAT(B) adipocytes, amounting 51% and 36% of those in BAT(B) adipocytes. The patterns of expression of UCP1, PGC-1alpha and leptin in the BAT(B) and in WAT(B) adipocytes were different with a higher relative expression of PGC-1alpha in the latter. Rosiglitazone increased UCP1 mRNA expression 4.5-fold in the BAT(B) and significantly more, 7.9-fold, in the WAT(B) adipocytes. Retinoic acid and triiodothyronine increased UCP1 mRNA expression in the BAT(B) adipocytes 1.6- and 2-fold, respectively but, surprisingly, slightly decreased UCP1 mRNA expression in the WAT(B) adipocytes. CONCLUSIONS: The study suggests that the nature and possibly the origin of WAT brown adipocytes is different from that of BAT brown adipocytes. It proposes an in vitro approach that could prove very useful to better characterize the WAT brown adipocyte-like cells.

[Metabolic syndrome, a mitochondrial disease?]. / Syndrome métabotique, une maladie mitochondriale?

The metabolic syndrome is a cluster of metabolic risk factors including: atherogenic dyslipidemia, elevated blood pressure, high plasma glucose and a prothrombotic and proinflammatory state, frequently associated to overweight. Impaired cell metabolism has been suggested as a relevant pathophysiological process. Indeed, the accumulation of intracellular fatty acylCoA and diacylglycerol, which then activate critical signal transduction pathways that ultimatly lead to suppression of insulin signalisation. Therefore a defect in mitochondrial function may be responsible for insulin resistance. Moreover, mitochondrial dysfunction has been found to take place in organs such as skeletal muscle, liver, pancreas and smoth vascular cells suggesting that mitochondrial defect could play a critical role in the occurence of cardiovascular diseases.

Effects of leptin on energy metabolism in beta-less mice.

OBJECTIVE: To investigate the impact of beta-adrenoceptor deficiency on the metabolic effects of leptin. MEASUREMENTS: Leptin was infused subcutaneously through an osmotic minipump in wild-type (WT) and beta(1)/beta(2)/beta(3)-adrenoceptor knockout (beta-less) mice and its effects on food intake, energy expenditure, carbohydrate and lipid utilization as well as on the levels of expression of the brown adipose tissue (BAT), thermogenic marker uncoupling protein-1 (UCP1) and type II deiodinase (D2) mRNAs were compared. RESULTS: Leptin treatment decreased food intake by 23% in both the WT and the beta-less mice. In pair-fed animals being used as controls, leptin treatment was found to increase energy expenditure in WT, but not in beta-less mice. No difference was observed in carbohydrate or fat utilization between leptin-treated WT and beta-less mice. Leptin increased UCP1 and D2 mRNA levels in WT mouse BAT 1.7- and 3-fold, respectively, but had no effect on the expression of these genes in beta-less mouse BAT. CONCLUSION: The stimulatory effects of leptin on oxygen consumption, BAT UCP1 and D2 expression require functional beta-adrenoceptors, but its inhibitory effect on food intake and its stimulatory effect on fat utilization is independent of the beta-adrenoceptor signalling.

Upregulation of peroxisome proliferator-activated receptor gamma coactivator gene (PGC1A) during weight loss is related to insulin sensitivity but not to energy expenditure.

AIMS/HYPOTHESIS: We investigated whether skeletal muscle peroxisome proliferator-activated receptor gamma coactivator-1 (PGC1A; also known as PPARGC1A) and its target mitofusin-2 (MFN2), as well as carnitine palmitoyltransferase-1 (CPT1; also known as carnitine palmitoyltransferase 1A [liver] [CPT1A]) and uncoupling protein (UCP)3, are involved in the improvement of insulin resistance and/or in the modification of energy expenditure during surgically induced massive weight loss. MATERIALS AND METHODS: Seventeen morbidly obese women (mean BMI: 45.9 +/- 4 kg/m(2)) were investigated before, and 3 and 12 months after, Roux-en-Y gastric bypass (RYGB). We evaluated insulin sensitivity by the euglycaemic-hyperinsulinaemic clamp, energy expenditure and substrate oxidation by indirect calorimetry, and muscle mRNA expression by PCR. RESULTS: Post-operatively, PGC1A was enhanced at 3 (p = 0.02) and 12 months (p = 0.03) as was MFN2 (p = 0.008 and p = 0.03 at 3 and 12 months respectively), whereas UCP3 was reduced (p = 0.03) at 12 months. CPT1 did not change. The expression of PGC1A and MFN2 were strongly (p < 0.0001) related. Insulin sensitivity, which increased after surgery (p = 0.002 at 3, p = 0.003 at 12 months), was significantly related to PGC1A and MFN2, but only MFN2 showed an independent influence in a multiple regression analysis. Energy expenditure was reduced at 3 months post-operatively (p = 0.001 vs before RYGB), remaining unchanged thereafter until 12 months. CPT1 and UCP3 were not significantly related to the modifications of energy expenditure or of lipid oxidation rate. CONCLUSIONS/INTERPRETATION: Weight loss upregulates PGC1A, which in turn stimulates MFN2 expression. MFN2 expression significantly and independently contributes to the improvement of insulin sensitivity. UCP3 and CPT1 do not seem to influence energy expenditure after RYGB.

beta-Adrenergic control of stearoyl-CoA desaturase 1 repression in relation to sympathoadrenal regulation of thermogenesis.

Mice lacking beta-adrenoceptors, which mediate the thermogenic effects of norepinephrine and epinephrine, show diminished thermogenesis and high susceptibility to obesity, whereas mice lacking stearoyl-CoA desaturase 1 (SCD1), which catalyzes the synthesis of monounsaturated fatty acids, show enhanced thermogenesis and high resistance to obesity. In testing whether beta-adrenergic control of thermogenesis might be mediated via repression of the SCD1 gene, we found that in mice lacking beta-adrenoceptors, the gene expression of SCD1 is elevated in liver, skeletal muscle and white adipose tissue. In none of these tissues/organs, however, could a link be found between increased sympathetic nervous system activity and diminished SCD1 gene expression when thermogenesis is increased in response to diet or cold, nor is the SCD1 transcript repressed by the administration of epinephrine. Taken together, these studies suggest that the elevated SCD1 transcript in tissues of mice lacking beta-adrenoceptors is not a direct effect of blunted beta-adrenergic signalling, and that beta-adrenergic control of SCD1 repression is unlikely to be a primary effector mechanism in sympathoadrenal regulation of thermogenesis. Whether approaches that target both SCD1 and molecular effectors of thermogenesis under beta-adrenergic control might be more effective than targeting SCD1 alone are potential avenues for future research in obesity management.

UCP3 protein expression is lower in type I, IIa and IIx muscle fiber types of endurance-trained compared to untrained subjects.

Uncoupling protein 3 (UCP3) is a muscle mitochondrial protein believed to uncouple the respiratory chain, producing heat and reducing aerobic ATP production. Our aim was to quantify and compare the UCP3 protein levels in type I, IIa and IIx skeletal muscle fibers of endurance-trained (Tr) and healthy untrained (UTr) individuals. UCP3 protein content was quantified using Western blot and immunofluorescence. Skeletal muscle fiber type was determined by both an enzymatic ATPase stain and immunofluorescence. UCP3 protein expression measured in skeletal muscle biopsies was 46% lower ( P=0.01) in the Tr compared to the UTr group. UCP3 protein expression in the different muscle fibers was expressed as follows; IIx>IIa>I in the fibers for both groups ( P<0.0167) but was lower in all fiber types of the Tr when compared to the UTr subjects ( P<0.001). Our results show that training status did not change the skeletal muscle fiber hierarchical UCP3 protein expression in the different fiber types. However, it affected UCP3 content more in type I and type IIa than in the type IIx muscle fibers. We suggest that this decrease may be in relation to the relative improvement in the antioxidant defense systems of the skeletal muscle fibers and that it might, as a consequence, participate in the training induced improvement in mechanical efficiency.

Slow component of [V]O(2) kinetics: the effect of training status, fibre type, UCP3 mRNA and citrate synthase activity.

OBJECTIVE: In healthy individuals performing constant-load exercise at intensities above the lactate threshold a secondary rise in pulmonary oxygen uptake ([V]O(2)) occurs. [V]O(2) reaches a maximum and exhaustion rapidly prevails for a range of work rates lower than the maximal work rate achieved during a conventional rapid-incremental test. This phenomenon is called the slow component (SC) of [V]O(2) kinetics and represents an increase in [V]O(2) without an increase in work rate. Although still under debate, the magnitude of the SC is believed to be associated with the percentage of type II muscle fibres and their recruitment. In this study we investigated the relationship between the magnitude of the relative SC, citrate synthase activity, UCP2 and UCP3 mRNA levels and muscle fibre composition in both endurance-trained and recreationally active subjects. METHOD: The magnitude of the relative SC was measured in 12 endurance-trained (Tr) and 15 recreationally active (RA) male subjects. The magnitude of the relative SC was determined as the difference between the end-exercise [V]O(2) and 3 min [V]O(2) divided by the difference between end-exercise [V]O(2) and baseline [V]O(2). UCP2 and UCP3 mRNA expression in the vastus lateralis was measured by RT-PCR with beta-actin mRNA used as an internal control. These values were also normalized against cytochrome-b mRNA to control for training induced changes in mitochondria when comparing the Tr and RA groups. Type I, IIa and IIx skeletal muscle fibre composition was determined using a routine myosin ATPase histochemical staining technique. Citrate synthase (CS) activity was measured using spectrophotometric detection. RESULTS: The magnitude of the relative SC of the Tr group had the highest correlation with citrate synthase activity (r=-0.90, P<0.001) and that of the RA group with [V]O(2) peak (r=-0.68, P<0.01). For the Tr group other correlations with the magnitude of the relative SC included UCP3 mRNA levels (r=0.69, P<0.05) and the percentage of type I fibres (r=-0.58, P<0.05), while for the RA group they included UCP3 mRNA (r=0.58, P<0.05) and the percentage of type IIa muscle fibres (r=0.59, P<0.05). The Tr subjects had a lower relative SC (P=0.04) and a lower expression of UCP2 (P=0.04), and UCP3 mRNA (P=0.01) than the RA subjects. When the groups were combined the magnitude of the relative SC correlated with UCP3 mRNA (r=0.70, P<0.01), percentage of type IIa muscle fibres (r=0.60, P<0.01) and [V]O(2) peak (r=-0.73, P<0.01). Additionally UCP3 mRNA correlated with the percentage of type IIa muscle fibres (r=0.63, P<0.001). CONCLUSION: Citrate synthase activity and [V]O(2) peak are indicators of aerobic fitness. The high negative correlations between the magnitude of the relative SC and citrate synthase activity and [V]O(2) peak, of the Tr and RA subjects, respectively, suggests that the magnitude of the relative SC is inversely correlated with aerobic fitness. Additionally the correlations between UCP3 mRNA and the magnitude of the relative SC for both groups individually and combined suggest that the uncoupling activity of the UCP3 protein may also influence the magnitude of the relative SC.