Energy metabolism of adipose tissue--physiological aspects and target in obesity treatment

Body fat content is controlled, at least in part, by energy charge of adipocytes. In vitro studies indicated that lipogenesis as well as lipolysis depend on cellular ATP levels. Respiratory uncoupling may, through the depression of ATP synthesis, control lipid metabolism of adipose cells. Expression of some uncoupling proteins (UCP2 and UCP5) as well as other protonophoric transporters can be detected in the adipose tissue. Expression of other UCPs (UCP1 and UCP3) can be induced by pharmacological treatments that reduce adiposity. A negative correlation between the accumulation of fat and the expression of UCP2 in adipocytes was also found. Ectopic expression of UCP1 in the white fat of aP2-Ucp1 transgenic mice mitigated obesity induced by genetic or dietary factors. In these mice, changes in lipid metabolism of adipocytes were associated with the depression of intracellular energy charge. Recent data show that AMP-activated protein kinase may be involved in the complex changes elicited by respiratory uncoupling in adipocytes. Changes in energy metabolism of adipose tissue may mediate effects of treatments directed against adiposity, dyslipidemia, and insulin resistance.     

Transgenic UCP1 in white adipocytes modulates mitochondrial membrane potential

To test if mitochondrial uncoupling in white adipocytes is responsible for obesity resistance of the aP2-Ucp transgenic mice expressing ectopic uncoupling protein 1 (UCPI) in white fat, mitochondrial membrane potential (delta psi(m)) was estimated by flow cytometry in adipocytes isolated from gonadal fat. Ectopic UCP1 (approximately 0.8 mol UCP1/mol respiratory chain) decreased the delta psi(m) and rendered the potential sensitive to GDP and fatty acids. These ligands of UCP1 had no effect on delta psi(m) in white adipocytes from non-transgenic mice, suggesting that the function of endogenous UCP2 in adipocytes was not affected. The results support the hypothesis that mitochondrial uncoupling in white fat may prevent development of obesity.     

Effect of uncoupling protein-1 expression on 3T3-L1 adipocyte gene expression

The mitochondrial respiratory uncoupling protein 1 (UCP1) partially uncouples substrate oxidation and oxidative phosphorylation to promote the dissipation of cellular biochemical energy as heat in brown adipose tissue. We have recently shown that expression of UCP1 in 3T3-L1 white adipocytes reduces the accumulation of triglycerides. Here, we investigated the molecular basis underlying UCP1 expression in 3T3-L1 adipocytes. Gene expression data showed that forced UCP1 expression down-regulated several energy metabolism pathways; but ATP levels were constant. A metabolic flux analysis model was used to reflect the gene expression changes onto metabolic processes and concordance was observed in the down-regulation of energy consuming pathways. Our data suggest that adipocytes respond to long-term mitochondrial uncoupling by minimizing ATP utilization.     

Metabolic Flux Analysis of Mitochondrial Uncoupling in 3T3-L1 Adipocytes

Background

Increasing energy expenditure at the cellular level offers an attractive option to limit adiposity and improve whole body energy balance. In vivo and in vitro observations have correlated mitochondrial uncoupling protein-1 (UCP1) expression with reduced white adipose tissue triglyceride (TG) content. The metabolic basis for this correlation remains unclear.

Methodology/Principal Findings

This study tested the hypothesis that mitochondrial uncoupling requires the cell to compensate for the decreased oxidation phosphorylation efficiency by up-regulating lactate production, thus redirecting carbon flux away from TG synthesis. Metabolic flux analysis was used to characterize the effects of non-lethal, long-term mitochondrial uncoupling (up to 18 days) on the pathways of intermediary metabolism in differentiating 3T3-L1 adipocytes. Uncoupling was induced by forced expression of UCP1 and chemical (FCCP) treatment. Chemical uncoupling significantly decreased TG content by ca. 35%. A reduction in the ATP level suggested diminished oxidative phosphorylation efficiency in the uncoupled adipocytes. Flux analysis estimated significant up-regulation of glycolysis and down-regulation of fatty acid synthesis, with chemical uncoupling exerting quantitatively larger effects.

Conclusions/Significance

The results of this study support our hypothesis regarding uncoupling-induced redirection of carbon flux into glycolysis and lactate production, and suggest mitochondrial proton translocation as a potential target for controlling adipocyte lipid metabolism.     

Effects of forced uncoupling protein 1 expression in 3T3-L1 cells on mitochondrial function and lipid metabolism

Obesity-related increase in body fat mass is a risk factor for many diseases, including type 2 diabetes. Controlling adiposity by targeted modulation of adipocyte enzymes could offer an attractive alternative to current dietary approaches. Brown adipose tissue, which is present in rodents but not in adult humans, expresses the mitochondrial uncoupling protein 1 (UCP1) that promotes cellular energy dissipation as heat. Here, we report on the direct metabolic effects of forced UCP1 expression in white adipocytes derived from a murine (3T3-L1) preadipocyte cell line. After stable integration, the ucp1 gene product was continuously expressed during differentiation and reduced the total lipid accumulation by approximately 30% without affecting other adipocyte markers, such as cytosolic glycerol-3-phosphate dehydrogenase activity and leptin production. The expression of UCP1 also decreased glycerol output and increased glucose uptake, lactate output, and the sensitivity of cellular ATP content to nutrient removal. However, oxygen consumption and beta-oxidation were minimally affected. Together, our results suggest that the reduction in intracellular lipid by constitutive expression of UCP1 reflects a downregulation of fat synthesis rather than an upregulation of fatty acid oxidation.     

Stimulation of mitochondrial oxidative capacity in white fat independent of UCP1: A key to lean phenotype

We are facing a revival of the strategy to counteract obesity and associated metabolic disorders by inducing thermogenesis mediated by mitochondrial uncoupling protein-1 (UCP1). Thus, the main focus is on the adaptive non-shivering thermogenesis occurring both in the typical depots of brown adipose tissue (BAT) and in UCP1-containing cells that could be induced in white adipose tissue (WAT). Because contribution of WAT to resting metabolic rate is relatively small, the possibility to reduce adiposity by enhancing energy expenditure in classical white adipocytes is largely neglected. However, several pieces of evidence support a notion that induction of energy expenditure based on oxidation of fatty acids (FA) in WAT may be beneficial for health, namely: (i) studies in both humans and rodents document negative association between oxidative capacity of mitochondria in WAT and obesity; (ii) pharmacological activation of AMPK in rats as well as cold-acclimation of UCP1-ablated mice results in obesity resistance associated with increased oxidative capacity in WAT; and (iii) combined intervention using long-chain n-3 polyunsaturated FA (omega 3) and mild calorie restriction exerted synergism in the prevention of obesity in mice fed a high-fat diet; this was associated with strong hypolipidemic and insulin-sensitizing effects, as well as prevention of inflammation, and synergistic induction of mitochondrial oxidative phosphorylation (OXPHOS) and FA oxidation, specifically in epididymal WAT. Importantly, these changes occurred without induction of UCP1 and suggested the involvement of: (i) futile substrate cycle in white adipocytes, which is based on lipolysis of intracellular triacylglycerols and re-esterification of FA, in association with the induction of mitochondrial OXPHOS capacity, β-oxidation, and energy expenditure; (ii) endogenous lipid mediators (namely endocannabinoids, eicosanoids, prostanoids, resolvins, and protectins) and their cognate receptors; and (iii) AMP-activated protein kinase in WAT. Quantitatively, the strong induction of FA oxidation in WAT in response to the combined intervention is similar to that observed in the transgenic mice rendered resistant to obesity by ectopic expression of UCP1 in WAT. The induction of UCP1-independent FA oxidation and energy expenditure in WAT in response to the above physiological stimuli could underlie the amelioration of obesity and low-grade WAT inflammation, and it could reduce the release of FA from adipose tissue and counteract harmful consequences of lipid accumulation in other tissues. In this respect, new combination treatments may be designed using naturally occurring micronutrients (e.g. omega 3), reduced calorie intake or pharmaceuticals, exerting synergism in the induction of the mitochondrial OXPHOS capacity and stimulation of lipid catabolism in white adipocytes, and improving metabolic flexibility of WAT. The role of mutual interactions between adipocytes and immune cells contained in WAT in tissue metabolism should be better characterised. This article is part of a Special Issue entitled Brown and White Fat: From Signaling to Disease.     

Thyroid-hormone effects on putative biochemical pathways involved in UCP3 activation in rat skeletal muscle mitochondria

In vitro, uncoupling protein 3 (UCP3)-mediated uncoupling requires cofactors [e.g., superoxides, coenzyme Q (CoQ) and fatty acids (FA)] or their derivatives, but it is not yet clear whether or how such activators interact with each other under given physiological or pathophysiological conditions. Since triiodothyronine (T3) stimulates lipid metabolism, UCP3 expression and mitochondrial uncoupling, we examined its effects on some biochemical pathways that may underlie UCP3-mediated uncoupling. T3-treated rats (Hyper) showed increased mitochondrial lipid-oxidation rates, increased expression and activity of enzymes involved in lipid handling and increased mitochondrial superoxide production and CoQ levels. Despite the higher mitochondrial superoxide production in Hyper, euthyroid and hyperthyroid mitochondria showed no differences in proton-conductance when FA were chelated by bovine serum albumin. However, mitochondria from Hyper showed a palmitoyl-carnitine-induced and GDP-inhibited increased proton-conductance in the presence of carboxyatractylate. We suggest that T3 stimulates the UCP3 activity in vivo by affecting the complex network of biochemical pathways underlying the UCP3 activation.     

Inhibition of uncoupling protein 2 by genipin reduces insulin-stimulated glucose uptake in 3T3-L1 adipocytes

Uncoupling protein 2 (UCP2) was reported to be involved in insulin-glucose homeostasis, based on well established event that inhibition of UCP2 stimulates insulin secretion in pancreatic β-cells. However, the role of UCP2 on insulin-stimulated glucose uptake in adipose tissue, which is an indispensable process in insulin-glucose homeostasis, remains unknown. In this study, UCP2 was inhibited by genipin in 3T3-L1 adipocytes, which increased mitochondrial membrane potential, intracellular ATP level and production of reactive oxygen species (ROS). Importantly, insulin-stimulated glucose uptake in 3T3-L1 adipocytes was largely impaired in the presence of genipin, and recovered by CCCP, a mitochondrial uncoupler. Furthermore, genipin leaded to suppression of insulin signal transduction through hyperactivation of c-Jun N-terminal kinase (JNK) and subsequent serine phosphorylation of insulin receptor substrate-1 (IRS-1). These results suggest that mitochondrial uncoupling in adipocytes positively regulates insulin-stimulated glucose uptake in adipocytes, and UCP2 may play an important role in insulin resistance.     

Beta 3-adrenoceptor knockout in C57BL/6J mice depresses the occurrence of brown adipocytes in white fat.

White and brown adipocytes are usually located in distinct depots; however, in response to cold, brown adipocytes appear in white fat. This response is mediated by beta-adrenoceptors but there is a controversy about the subtype(s) involved. In the present study, we exposed to cold beta 3-adrenoceptor knockout mice (beta 3KO) on a C57BL/6J genetic background and measured in white adipose tissue the density of multilocular cells and the expression of the brown adipocyte marker uncoupling protein-1 (UCP1). In brown fat of beta 3KO mice, UCP1 expression levels were normal at 24 degrees C as well as after a 10-day cold exposure. Strikingly, under both conditions, in the white fat of beta 3KO mice the levels of UCP1 mRNA and protein as well as the density of multilocular cells were decreased. These results indicate that beta 3-adrenoceptors play a major role in the appearance of brown adipocytes in white fat and suggest that the brown adipocytes present in white fat differ from those in brown fat.     

Activation of peroxisome proliferator-activated receptor-alpha stimulates both differentiation and fatty acid oxidation in adipocytes

Peroxisome proliferator-activated receptor-α (PPARα) is a dietary lipid sensor, whose activation results in hypolipidemic effects. In this study, we investigated whether PPARα activation affects energy metabolism in white adipose tissue (WAT). Activation of PPARα by its agonist (bezafibrate) markedly reduced adiposity in KK mice fed a high-fat diet. In 3T3-L1 adipocytes, addition of GW7647, a highly specific PPARα agonist, during adipocyte differentiation enhanced glycerol-3-phosphate dehydrogenase activity, insulin-stimulated glucose uptake, and adipogenic gene expression. However, triglyceride accumulation was not increased by PPARα activation. PPARα activation induced expression of target genes involved in FA oxidation and stimulated FA oxidation. In WAT of KK mice treated with bezafibrate, both adipogenic and FA oxidation-related genes were significantly upregulated. These changes in mRNA expression were not observed in PPARα-deficient mice. Bezafibrate treatment enhanced FA oxidation in isolated adipocytes, suppressing adipocyte hypertrophy. Chromatin immunoprecipitation (ChIP) assay revealed that PPARα was recruited to promoter regions of both adipogenic and FA oxidation-related genes in the presence of GW7647 in 3T3-L1 adipocytes. These findings indicate that the activation of PPARα affects energy metabolism in adipocytes, and PPARα activation in WAT may contribute to the clinical effects of fibrate drugs.     

Rhein Protects against Obesity and Related Metabolic Disorders through Liver X Receptor-Mediated Uncoupling Protein 1 Upregulation in Brown Adipose Tissue

Liver X receptors (LXRs) play important roles in regulating cholesterol homeostasis, and lipid and energy metabolism. Therefore, LXR ligands could be used for the management of metabolic disorders. We evaluated rhein, a natural compound from Rheum palmatum L., as an antagonist for LXRs and investigated its anti-obesity mechanism in high-fat diet-fed mice. Surface plasmon resonance assays were performed to examine the direct binding of rhein to LXRs. LXR target gene expression was assessed in 3T3-L1 adipocytes and HepG2 hepatic cells in vitro. C57BL/6J mice fed a high-fat diet were orally administered with rhein for 4 weeks, and then the expression levels of LXR-related genes were analyzed. Rhein bound directly to LXRs. The expression levels of LXR target genes were suppressed by rhein in 3T3-L1 and HepG2 cells. In white adipose tissue, muscle and liver, rhein reprogrammed the expression of LXR target genes related to adipogenesis and cholesterol metabolism. Rhein activated uncoupling protein 1 (UCP1) expression in brown adipose tissue (BAT) in wild-type mice, but did not affect UCP1 expression in LXR knockout mice. In HIB-1B brown adipocytes, rhein activated the UCP1 gene by antagonizing the repressive effect of LXR on UCP1 expression. This study suggests that rhein may protect against obesity and related metabolic disorders through LXR antagonism and regulation of UCP1 expression in BAT.     

Inactivation of UCP1 and the glycerol phosphate cycle synergistically increases energy expenditure to resist diet-induced obesity

Our current paradigm for obesity assumes that reduced thermogenic capacity increases susceptibility to obesity, whereas enhanced thermogenic capacity protects against obesity. Here we report that elimination of two major thermogenic pathways encoded by the mitochondrial uncoupling protein (Ucp1) and mitochondrial glycerol-3-phosphate dehydrogenase (Gdm) result in mice with increased resistance to diet-induced obesity when housed at 28 degrees C, provided prior adaptation occurred at 20 degrees C. Obesity resistant Gdm(-/-).Ucp1(-/-) mice maintained at 28 degrees C have increased energy expenditure, in part through conversion of white to brown adipocytes in inguinal fat. Increased oxygen consumption in inguinal fat cell suspensions and the up-regulation of genes of mitochondrial function and fat metabolism indicated increased thermogenic activity, despite the absence of UCP1, whereas liver and skeletal muscle showed no changes in gene expression. Additionally, comparisons of energy expenditure in UCP1-deficient and wild type mice fed an obesogenic diet indicates that UCP1-based brown fat-based thermogenesis plays no role in so-called diet-induced thermogenesis. Accordingly, a new paradigm for obesity emerges in which the inactivation of major thermogenic pathways force the induction of alternative pathways that increase metabolic inefficiency.     

Ablation of ghrelin receptor reduces adiposity and improves insulin sensitivity during aging by regulating fat metabolism in white and brown adipose tissues

Aging is associated with increased adiposity in white adipose tissues and impaired thermogenesis in brown adipose tissues; both contribute to increased incidences of obesity and type 2 diabetes. Ghrelin is the only known circulating orexigenic hormone that promotes adiposity. In this study, we show that ablation of the ghrelin receptor (growth hormone secretagogue receptor, GHS-R) improves insulin sensitivity during aging. Compared to wild-type (WT) mice, old Ghsr(-/-) mice have reduced fat and preserve a healthier lipid profile. Old Ghsr(-/-) mice also exhibit elevated energy expenditure and resting metabolic rate, yet have similar food intake and locomotor activity. While GHS-R expression in white and brown adipose tissues was below the detectable level in the young mice, GHS-R expression was readily detectable in visceral white fat and interscapular brown fat of the old mice. Gene expression profiles reveal that Ghsr ablation reduced glucose/lipid uptake and lipogenesis in white adipose tissues but increased thermogenic capacity in brown adipose tissues. Ghsr ablation prevents age-associated decline in thermogenic gene expression of uncoupling protein 1 (UCP1). Cell culture studies in brown adipocytes further demonstrate that ghrelin suppresses the expression of adipogenic and thermogenic genes, while GHS-R antagonist abolishes ghrelin's effects and increases UCP1 expression. Hence, GHS-R plays an important role in thermogenic impairment during aging. Ghsr ablation improves aging-associated obesity and insulin resistance by reducing adiposity and increasing thermogenesis. Growth hormone secretagogue receptor antagonists may be a new means of combating obesity by shifting the energy balance from obesogenesis to thermogenesis.     

Effects of 2-G exposure on temperature regulation, circadian rhythms, and adiposity in UCP2/3 transgenic mice.

Altered ambient force environments affect energy expenditure via changes in thermoregulation, metabolism, and body composition. Uncoupling proteins (UCPs) have been implicated as potential enhancers of energy expenditure and may participate in some of the adaptations to a hyperdynamic environment. To test this hypothesis, this study examined the homeostatic and circadian profiles of body temperature (T(b)) and activity and adiposity in wild-type and UCP2/3 transgenic mice exposed to 1 and 2 G. There were no significant differences between the groups in the means, amplitudes, or phases of T(b) and activity rhythms at either the 1- or 2-G level. Percent body fat was significantly lower in transgenic (5.2 +/- 0. 2%) relative to the wild-type mice (6.2 +/- 0.1%) after 2-G exposure; mass-adjusted mesenteric and epididymal fat pads in transgenic mice were also significantly lower (P < 0.05). The data suggest that 1) the actions of two UCPs (UCP2 and UCP3) do not contribute to an altered energy balance at 2 G, although 2) UCP2 and UCP3 do contribute to the utilization of lipids as a fuel substrate at 2 G.     

Beige adipocytes are a distinct type of thermogenic fat cell in mouse and human

Brown fat generates heat via the mitochondrial uncoupling protein UCP1, defending against hypothermia and obesity. Recent data suggest that there are two distinct types of brown fat: classical brown fat derived from a myf-5 cellular lineage and UCP1-positive cells that emerge in white fat from a non-myf-5 lineage. Here, we report the isolation of "beige" cells from murine white fat depots. Beige cells resemble white fat cells in having extremely low basal expression of UCP1, but, like classical brown fat, they respond to cyclic AMP stimulation with high UCP1 expression and respiration rates. Beige cells have a gene expression pattern distinct from either white or brown fat and are preferentially sensitive to the polypeptide hormone irisin. Finally, we provide evidence that previously identified brown fat deposits in adult humans are composed of beige adipocytes. These data provide a foundation for studying this mammalian cell type with therapeutic potential. PAPERCLIP:     

Milk fat globule membrane and its component phosphatidylcholine induce adipose browning both in vivo and in vitro

The functional induction of brown-like adipocytes in white adipose tissue (WAT) provides a defense against obesity. The aim of this study was to analyze the effects of milk fat globule membrane (MFGM) and its component phosphatidylcholine (PC) on the brown remodeling of WAT. Male C57BL/6 J mice were fed a high-fat diet (HFD) for 8 weeks and then fed HFD for another 8 weeks with MFGM. In vitro studies were performed in C3H10T1/2 pluripotent stem cells, 3T3-L1 pre-adipocytes and differentiated inguinal WAT stromal vascular cells (SVCs) to determine the role of MFGM and PC on the formation of brown-like adipocytes. MFGM decreased fasting glucose and serum insulin levels in HFD-fed mice. MFGM improved glucose tolerance and insulin sensitivity, and induced browning of inguinal WAT. MFGM and its component PC stimulated transformation of brown-like adipocytes in C3H10T1/2 pluripotent stem cells, 3T3-L1 adipocytes and SVCs by increasing the protein expression of UCP1, PGC-1α, PRDM16 as well as the mRNA expression of other thermogenic genes and beige cell markers. MFGM and PC also increased mitochondrial DNA (mtDNA) copy number, mitochondrial density and oxygen consumption rate and up-regulated the mRNA expression of mitochondria-biogenesis-related genes in vitro. PPARα inhibitor GW6471 treatment or knockdown of PPARα using lentivirus-expressing shRNA inhibited the PC-induced increase in the protein expression of UCP1, PGC-1α and PRDM16 in C3H10T1/2 pluripotent stem cells and 3T3-L1 adipocytes, indicating the potential role of PPARα in PC-mediated brown-like adipocyte formation. In conclusion, MFGM and milk PC induced adipose browning, which has major protective effects against obesity and metabolic dysfunction.