Roles for Peroxisome Proliferator-activated Receptor γ (PPARγ) and PPARγ Coactivators 1α and 1β in Regulating Response of White and Brown Adipocytes to Hypoxia

Sirt3 (silent mating type information regulation 2, homolog 3), a member of the sirtuin family of protein deacetylases with multiple actions on metabolism and gene expression is expressed in association with brown adipocyte differentiation. Using Sirt3-null brown adipocytes, we determined that Sirt3 is required for an appropriate responsiveness of cells to noradrenergic, cAMP-mediated activation of the expression of brown adipose tissue thermogenic genes. The transcriptional coactivator Pgc-1α (peroxisome proliferator-activated receptor-γ coactivator-1α) induced Sirt3 gene expression in white adipocytes and embryonic fibroblasts as part of its overall induction of a brown adipose tissue-specific pattern of gene expression. In cells lacking Sirt3, Pgc-1α failed to fully induce the expression of brown fat-specific thermogenic genes. Pgc-1α activates Sirt3 gene transcription through coactivation of the orphan nuclear receptor Err (estrogen-related receptor)-α, which bound the proximal Sirt3 gene promoter region. Errα knockdown assays indicated that Errα is required for full induction of Sirt3 gene expression in response to Pgc-1α. The present results indicate that Pgc-1α controls Sirt3 gene expression and this action is an essential component of the overall mechanisms by which Pgc-1α induces the full acquisition of a brown adipocyte differentiated phenotype.

Distribution and Development of Brown Adipocytes in the Murine and Human Adipose Organ

beta3-Adrenergic receptors (beta3-ARs) are expressed predominantly on white and brown adipocytes, and acute treatment of mice with CL 316,243, a potent and highly selective beta3-AR agonist, produces a 2-fold increase in energy expenditure, a 50-100-fold increase in insulin levels, and a 40-50% reduction in food intake. Recently, we generated gene knockout mice lacking functional beta3-ARs and demonstrated that each of these responses were mediated exclusively by beta3-ARs. However, the tissue site responsible for producing these actions is unknown. In the present study, genetically engineered mice were created in which beta3-ARs are expressed exclusively in white and brown adipocytes (WAT+BAT-mice), or in brown adipocytes only (BAT-mice). This was accomplished by injecting tissue-specific beta3-AR transgenic constructs into mouse zygotes homozygous for the beta3-AR knockout allele. Control, knockout, WAT+BAT, and BAT-mice were then treated acutely with CL, and the effects on various parameters were assessed. As previously observed, all effects of CL were completely absent in gene knockout mice lacking beta3-ARs. The effects on O2 consumption, insulin secretion, and food intake were completely rescued with transgenic re-expression of beta3-ARs in white and brown adipocytes (WAT+BAT-mice), demonstrating that each of these responses is mediated exclusively by beta3-ARs in white and/or brown adipocytes, and that beta3-ARs in other tissue sites were not required. Importantly, transgenic re-expression of beta3-ARs in brown adipocytes only (BAT-mice) failed to rescue, in any way, CL-mediated effects on insulin levels and food intake and only minimally restored effects on oxygen consumption, indicating that any effect on insulin secretion and food intake, and a full stimulation of oxygen consumption required the presence of beta3-ARs in white adipocytes. The mechanisms by which beta3-AR agonist stimulation of white adipocytes produces these responses are unknown but may involve novel mediators not previously known to effect these processes.

Complementary action of the PGC-1 coactivators in mitochondrial biogenesis and brown fat differentiation

The Role of C/EBP Genes in Adipocyte Differentiation

β-Adrenergic Receptors, Diet-induced Thermogenesis, and Obesity

In response to cold, norepinephrine (NE)-induced triacylglycerol hydrolysis (lipolysis) in adipocytes of brown adipose tissue (BAT) provides fatty acid substrates to mitochondria for heat generation (adaptive thermogenesis). NE-induced lipolysis is mediated by protein kinase A (PKA)-dependent phosphorylation of perilipin, a lipid droplet-associated protein that is the major regulator of lipolysis. We investigated the role of perilipin PKA phosphorylation in BAT NE-stimulated thermogenesis using a novel mouse model in which a mutant form of perilipin, lacking all six PKA phosphorylation sites, is expressed in adipocytes of perilipin knockout (Peri KO) mice. Here, we show that despite a normal mitochondrial respiratory capacity, NE-induced lipolysis is abrogated in the interscapular brown adipose tissue (IBAT) of these mice. This lipolytic constraint is accompanied by a dramatic blunting ( approximately 70%) of the in vivo thermal response to NE. Thus, in the presence of perilipin, PKA-mediated perilipin phosphorylation is essential for NE-dependent lipolysis and full adaptive thermogenesis in BAT. In IBAT of Peri KO mice, increased basal lipolysis attributable to the absence of perilipin is sufficient to support a rapid NE-stimulated temperature increase ( approximately 3.0 degrees C) comparable to that in wild-type mice. This observation suggests that one or more NE-dependent mechanism downstream of perilipin phosphorylation is required to initiate and/or sustain the IBAT thermal response.

Brown adipose tissue expends energy in the form of heat via the mitochondrial uncoupling protein UCP1. Recent studies showed that brown adipose tissue is present in adult humans and may be exploited for its anti-obesity and anti-diabetes actions. Apelin is an adipocyte-derived hormone that plays important roles in energy metabolism. Here, we report that apelin-APJ signaling promotes brown adipocyte differentiation by increasing the expressions of brown adipogenic and thermogenic transcriptional factors via the PI3K/Akt and AMPK signaling pathways. It is also found that apelin relieves the TNFα inhibition on brown adipogenesis. In addition, apelin increases the basal activity of brown adipocytes, as evidenced by the increased PGC1α and UCP1 expressions, mitochondrial biogenesis, and oxygen consumption. Finally, we provide both in vitro and in vivo evidence that apelin is able to increase the brown-like characteristics in white adipocytes. This study, for the first time, reveals the brown adipogenic and browning effects of apelin and suggests a potential therapeutic route to combat obesity and related metabolic disorders.

Glycerol-3-phosphate acyltransferases (GPATs; EC2.3.1.15) catalyze the first step in the de novo synthesis of neutral lipids (triglycerides) and glycerophospholipids. The existence of multiple enzyme isoforms with GPAT activity was predicted many years ago when GPAT activities with distinct kinetic profiles and sensitivity to inhibitors were characterized in two subcellular compartments, mitochondria and microsomes. We now know that mammals have at least four GPAT isoforms with distinct tissue distribution and function. GPAT1 is the major mitochondrial GPAT isoform and is characterized by its resistance to sulfhydryl-modifying reagents, such as N-ethylmaleimide (NEM). GPAT2 is a minor NEM-sensitive mitochondrial isoform. The activity referred to as microsomal GPAT is encoded by two closely related genes, GPAT3 and GPAT4. GPAT isoforms are important regulators of cellular triglyceride and phospholipid content, and may channel fatty acids toward particular metabolic fates. Overexpression and knock-out studies suggest that GPAT isoforms can play important roles in the development of hepatic steatosis, insulin resistance, and obesity; GPAT isoforms are also important for lactation. This review summarizes the current state of knowledge on mammalian GPAT isoforms.

Irisin in humans: recent advances and questions for future research

Brown adipose tissue involution associated with progressive restriction in progenitor competence.

ATP is co-localized with norepinephrine at the sympathetic nerve terminals and may be released simultaneously upon neuronal stimulation, which results in activation of purinergic receptors. To examine whether leptin synthesis and lipolysis are influenced by P2 purinergic receptor activation, the effects of ATP and other nucleotides on leptin secretion and glycerol release have been investigated in differentiated rat white adipocytes. Firstly, insulin-induced leptin secretion was inhibited by nucleotide treatment with the following efficacy order: 3'-O-(4-benzoyl)benzoyl ATP (BzATP) > ATP >> UTP. Secondly, treatment of adipocytes with ATP increased both intracellular Ca(2+) concentration and cAMP content. Intracellular calcium concentration was increased by ATP and UTP, but not BzATP, an effect attributed to phospholipase C-coupled P2Y(2). On the other hand, cAMP was generated by treatment with BzATP and ATPgammaS, but not UTP, indicating functional expression of adenylyl cyclase-coupled P2Y(11) receptors in white adipocytes. Thirdly, lipolysis was significantly activated by BzATP and ATP, which correlated with the characteristics of the P2Y(11) subtype. Taken together, the data presented here suggest that white adipocytes express at least two different types of P2Y receptors and that activation of P2Y(11) receptor might be involved in inhibition of leptin production and stimulation of lipolysis, suggesting that purinergic transmission can play an important role in white adipocyte physiology.

Knockdown of NPY Expression in the Dorsomedial Hypothalamus Promotes Development of Brown Adipocytes and Prevents Diet-Induced Obesity

SIRT3 is one of the seven mammalian sirtuin homologs of the yeast Sir2 gene, which mediates the effect of caloric restriction on life span extension in yeast and Caenorhabditis elegans. Because adipose tissue is essential in energy homeostasis and also plays a role in life span determination, we decided to investigate the function of sirtuin members in fat. We report here that murine SIRT3 is expressed in brown adipose tissue and is localized on the mitochondria inner membrane. Caloric restriction activates SIRT3 expression in both white and brown adipose. Additionally, cold exposure up-regulates SIRT3 expression in brown fat, whereas elevated climate temperature reduces the expression. Enforced expression of SIRT3 in the HIB1B brown adipocytes enhances the expression of the uncoupling protein PGC-1alpha, UCP1, and a series of mitochondria-related genes. Both ADP-ribosyltransferase and deacetylase activities of SIRT3 are required for this action. Furthermore, the SIRT3 deacetylase mutant exhibits a dominant negative effect by inhibiting UCP1 expression. This inhibitive effect can be abolished by the coexpression of PGC-1alpha, indicating a major role of PGC-1alpha in the SIRT3 action. In addition, SIRT3 stimulates CREB phosphorylation, which reportedly activates PGC-1alpha promoter directly. Functionally, sustained expression of SIRT3 decreases membrane potential and reactive oxygen species production while increasing cellular respiration. Finally, SIRT3, along with genes related to mitochondrial function, is down-regulated in the brown adipose tissue of several genetically obese mice. In summary, our results demonstrate that SIRT3 activates mitochondria functions and plays an important role in adaptive thermogenesis in brown adipose.

ACSL1 (acyl-CoA synthetase 1), the major acyl-CoA synthetase of adipocytes, has been proposed to function in adipocytes as mediating free fatty acid influx, esterification, and storage as triglyceride. To test this hypothesis, ACSL1 was stably silenced (knockdown (kd)) in 3T3-L1 cells, differentiated into adipocytes, and evaluated for changes in lipid metabolism. Surprisingly, ACSL1-silenced adipocytes exhibited no significant changes in basal or insulin-stimulated long-chain fatty acid uptake, lipid droplet size, or tri-, di-, or monoacylglycerol levels when compared with a control adipocyte line. However, ACSL1 kd adipocytes displayed a 7-fold increase in basal and a approximately 15% increase in forskolin-stimulated fatty acid efflux without any change in glycerol release, indicating a role for the protein in fatty acid reesterification following lipolysis. Consistent with this proposition, ACSL1 kd cells exhibited a decrease in activation and phosphorylation of AMP-activated protein kinase and its primary substrate acetyl-CoA carboxylase. Moreover, ACSL1 kd adipocytes displayed an increase in phosphorylated protein kinase C and phosphorylated JNK, attenuated insulin signaling, and a decrease in insulin-stimulated glucose uptake. These findings identify a primary role of ACSL1 in adipocytes not in control of lipid influx, as previously considered, but in lipid efflux and fatty acid-induced insulin resistance.