Knockdown of NPY expression in the dorsomedial hypothalamus promotes development of brown adipocytes and prevents diet-induced obesity

Hypothalamic neuropeptide Y (NPY) has been implicated in control of energy balance, but the physiological importance of NPY in the dorsomedial hypothalamus (DMH) remains unclear. Here we report that knockdown of NPY expression in the DMH by adeno-associated virus-mediated RNAi reduced fat depots in rats fed regular chow and ameliorated high-fat diet-induced hyperphagia and obesity. DMH NPY knockdown resulted in development of brown adipocytes in inguinal white adipose tissue through the sympathetic nervous system. This knockdown increased uncoupling protein 1 expression in both inguinal fat and interscapular brown adipose tissue (BAT). Consistent with the activation of BAT, DMH NPY knockdown increased energy expenditure and enhanced the thermogenic response to a cold environment. This knockdown also increased locomotor activity, improved glucose homeostasis, and enhanced insulin sensitivity. Together, these results demonstrate critical roles of DMH NPY in body weight regulation through affecting food intake, body adiposity, thermogenesis, energy expenditure, and physical activity.     

Implications of nonshivering thermogenesis for energy balance regulation in humans

The incidence of the metabolic syndrome has reached epidemic levels in the Western world. With respect to the energy balance, most attention has been given to reducing energy (food) intake. Increasing energy expenditure is an important alternative strategy. Facultative thermogenesis, which is the increase in energy expenditure in response to cold or diet, may be an effective way to affect the energy balance. The recent identification of functional brown adipose tissue (BAT) in adult humans promoted a renewed interest in nonshivering thermogenesis (NST). The purpose of this review is to highlight the recent insight in NST, general aspects of its regulation, the major tissues involved, and its metabolic consequences. Sustainable NST in adult humans amounts to 15% of the average daily energy expenditure. Calculations based on the limited available literature show that BAT thermogenesis can amount to 5% of the basal metabolic rate. It is likely that at least a substantial part of NST can be attributed to BAT, but it is possible that other tissues contribute to NST. Several studies on mitochondrial uncoupling indicate that skeletal muscle is another potential contributor to facultative thermogenesis in humans. The general and synergistic role of the sympathetic nervous system and the thyroid axis in relation to NST is discussed. Finally, perspectives on BAT and skeletal muscle NST are given.     

3,5-Diiodo-L-Thyronine Activates Brown Adipose Tissue Thermogenesis in Hypothyroid Rats

3,5-diiodo-l-thyronine (T2), a thyroid hormone derivative, is capable of increasing energy expenditure, as well as preventing high fat diet-induced overweight and related metabolic dysfunction. Most studies to date on T2 have been carried out on liver and skeletal muscle. Considering the role of brown adipose tissue (BAT) in energy and metabolic homeostasis, we explored whether T2 could activate BAT thermogenesis. Using euthyroid, hypothyroid, and T2-treated hypothyroid rats (all maintained at thermoneutrality) in morphological and functional studies, we found that hypothyroidism suppresses the maximal oxidative capacity of BAT and thermogenesis, as revealed by reduced mitochondrial content and respiration, enlarged cells and lipid droplets, and increased number of unilocular cells within the tissue. In vivo administration of T2 to hypothyroid rats activated BAT thermogenesis and increased the sympathetic innervation and vascularization of tissue. Likewise, T2 increased BAT oxidative capacity in vitro when added to BAT homogenates from hypothyroid rats. In vivo administration of T2 to hypothyroid rats enhanced mitochondrial respiration. Moreover, UCP1 seems to be a molecular determinant underlying the effect of T2 on mitochondrial thermogenesis. In fact, inhibition of mitochondrial respiration by GDP and its reactivation by fatty acids were greater in mitochondria from T2-treated hypothyroid rats than untreated hypothyroid rats. In vivo administration of T2 led to an increase in PGC-1α protein levels in nuclei (transient) and mitochondria (longer lasting), suggesting a coordinate effect of T2 in these organelles that ultimately promotes net activation of mitochondrial biogenesis and BAT thermogenesis. The effect of T2 on PGC-1α is similar to that elicited by triiodothyronine. As a whole, the data reported here indicate T2 is a thyroid hormone derivative able to activate BAT thermogenesis.     

Deficiency of PTP1B in POMC neurons leads to alterations in energy balance and homeostatic response to cold exposure

The adipose tissue-derived hormone leptin regulates energy balance through catabolic effects on central circuits, including proopiomelanocortin (POMC) neurons. Leptin activation of POMC neurons increases thermogenesis and locomotor activity. Protein tyrosine phosphatase 1B (PTP1B) is an important negative regulator of leptin signaling. POMC neuron-specific deletion of PTP1B in mice results in reduced high-fat diet-induced body weight and adiposity gain due to increased energy expenditure and greater leptin sensitivity. Mice lacking the leptin gene (ob/ob mice) are hypothermic and cold intolerant, whereas leptin delivery to ob/ob mice induces thermogenesis via increased sympathetic activity to brown adipose tissue (BAT). Here, we examined whether POMC PTP1B mediates the thermoregulatory response of CNS leptin signaling by evaluating food intake, body weight, core temperature (T(C)), and spontaneous physical activity (SPA) in response to either exogenous leptin or 4-day cold exposure (4°C) in male POMC-Ptp1b-deficient mice compared with wild-type controls. POMC-Ptp1b(-/-) mice were hypersensitive to leptin-induced food intake and body weight suppression compared with wild types, yet they displayed similar leptin-induced increases in T(C). Interestingly, POMC-Ptp1b(-/-) mice had increased BAT weight and elevated plasma triiodothyronine (T(3)) levels in response to a 4-day cold challenge, as well as reduced SPA 24 h after cold exposure, relative to controls. These data show that PTP1B in POMC neurons plays a role in short-term cold-induced reduction of SPA and may influence cold-induced thermogenesis via enhanced activation of the thyroid axis.     

Adipocyte lineages: Tracing back the origins of fat

The obesity epidemic has intensified efforts to understand the mechanisms controlling adipose tissue development. Adipose tissue is generally classified as white adipose tissue (WAT), the major energy storing tissue, or brown adipose tissue (BAT), which mediates non-shivering thermogenesis. It is hypothesized that brite adipocytes (brown in white) may represent a third adipocyte class. The recent realization that brown fat exist in adult humans suggests increasing brown fat energy expenditure could be a therapeutic strategy to combat obesity. To understand adipose tissue development, several groups are tracing the origins of mature adipocytes back to their adult precursor and embryonic ancestors. From these studies emerged a model that brown adipocytes originate from a precursor shared with skeletal muscle that expresses Myf5-Cre, while all white adipocytes originate from a Myf5-negative precursors. While this provided a rational explanation to why BAT is more metabolically favorable than WAT, recent work indicates the situation is more complex because subsets of white adipocytes also arise from Myf5-Cre expressing precursors. Lineage tracing studies further suggest that the vasculature may provide a niche supporting both brown and white adipocyte progenitors; however, the identity of the adipocyte progenitor cell is under debate. Differences in origin between adipocytes could explain metabolic heterogeneity between depots and/or influence body fat patterning particularly in lipodystrophy disorders. Here, we discuss recent insights into adipose tissue origins highlighting lineage-tracing studies in mice, how variations in metabolism or signaling between lineages could affect body fat distribution, and the questions that remain unresolved. This article is part of a Special Issue entitled: Modulation of Adipose Tissue in Health and Disease.     

Inhibition of in vitro and in vivo brown fat differentiation program by myostatin

Objective: Obesity arises mainly due to the imbalance between energy storage and its expenditure. Metabolically active brown adipose tissue (BAT) has recently been detected in humans and has been proposed as a new target for anti‐obesity therapy because of its unique capacity to regulate energy expenditure. Myostatin (Mst), a negative regulator of muscle mass, has been identified as a potential target to regulate overall body composition. Although the beneficial effects of Mst inhibition on muscle mass are well known, its role in the regulation of lipid metabolism, and energy expenditure is not very clear. Design and Methods: We tested the effects of Mst inhibition on the gene regulatory networks that control BAT differentiation using both in vivo and in vitro model systems. PRDM16 and UCP1, two key regulators of brown fat differentiation were significantly up regulated in levator‐ani (LA) and gastrocnemius (Gastroc) muscles as well as in epididymal (Epi) and subcutaneous (SC) fat pads isolated from Mst knock out (Mst KO) male mice compared with wild type (WT) mice. Results: Using mouse embryonic fibroblast (MEFs) primary cultures obtained from Mst KO group compared to the WT group undergoing adipogenic differentiation, we also demonstrate a significant increase in select genes and proteins that improve lipid metabolism and energy expenditure. Conclusion: Treatment of Mst KO MEFs with recombinant Mst protein significantly inhibited the gene expression levels of UCP1, PRDM16, PGC1‐α/β as well as BMP7. Future studies to extend these findings and explore the therapeutic potential of Mst inhibition on metabolic disorders are warranted.     

Role of thermogenesis in the regulation of energy balance in relation to obesity

Obligatory thermogenesis is a necessary accompaniment of all metabolic processes involved in maintenance of the body in the living state, and occurs in all organs. It includes energy expenditure involved in ingesting, digesting, and processing food (thermic effect of food (TEF]. At certain life stages extra energy expenditure for growth, pregnancy, or lactation would also be obligatory. Facultative thermogenesis is superimposed on obligatory thermogenesis and can be rapidly switched on and rapidly suppressed by the nervous system. Facultative thermogenesis is important in both thermal balance, in which control of thermoregulatory thermogenesis (shivering in muscle, nonshivering in brown adipose tissue (BAT] balances neural control of heat loss mechanisms, and in energy balance, in which control of facultative thermogenesis (exercise-induced in muscle, diet-induced thermogenesis (DIT) in BAT) balances control of energy intake. Thermal balance (i.e., body temperature) is much more stringently controlled than energy balance (i.e., body energy stores). Reduced energy expenditure for thermogenesis is important in two types of obesity in laboratory animals. In the first type, deficient DIT in BAT is a prominent feature of altered energy balance. It may or may not be associated with hyperphagia. In a second type, reduced cold-induced thermogenesis in BAT as well as in other organs is a prominent feature of altered thermal balance. This in turn results in altered energy balance and obesity, exacerbated in some examples by hyperphagia. In some of the hyperphagic obese animals it is likely that the exaggerated obligatory thermic effect of food so alters thermal balance that BAT thermogenesis is suppressed. In all obese animals, deficient hypothalamic control of facultative thermogenesis and (or) food intake is implicated.     

The orphan nuclear receptor SHP regulates PGC-1alpha expression and energy production in brown adipocytes

Brown adipocytes increase energy production in response to induction of PGC-1alpha, a dominant regulator of energy metabolism. We have found that the orphan nuclear receptor SHP (NR0B2) is a negative regulator of PGC-1alpha expression in brown adipocytes. Mice lacking SHP show increased basal expression of PGC-1alpha, increased energy expenditure, and resistance to diet-induced obesity. Increased PGC-1alpha expression in SHP null brown adipose tissue is not due to beta-adrenergic activation, since it is also observed in primary cultures of SHP(-/-) brown adipocytes that are not exposed to such stimuli. In addition, acute inhibition of SHP expression in cultured wild-type brown adipocytes increases basal PGC-1alpha expression, and SHP overexpression in SHP null brown adipocytes decreases it. The orphan nuclear receptor ERRgamma is expressed in BAT and its transactivation of the PGC-1alpha promoter is potently inhibited by SHP. We conclude that SHP functions as a negative regulator of energy production in BAT.     

Adaptive Activation of Thyroid Hormone and Energy Expenditure

The mechanisms by which thyroid hormone accelerates energy expenditure are poorly understood. In the brown adipose tissue (BAT), activation of thyroid hormone by type 2 iodothyronine deiodinase (D2) has been known to play a role in adaptive energy expenditure during cold exposure in human newborns and other small mammals. Although BAT is not present in significant amounts in normal adult humans, recent studies have found substantial amounts of D2 in skeletal muscle, a metabolically relevant tissue in humans. This article reviews current biological knowledge about D2 and adaptive T3 production and their roles in energy expenditure.     

Thermogenic Capacity Is Antagonistically Regulated in Classical Brown and White Subcutaneous Fat Depots by High Fat Diet and Endurance Training in Rats: IMPACT ON WHOLE-BODY ENERGY EXPENDITURE*

This study investigated the regulation of thermogenic capacity in classical brown adipose tissue (BAT) and subcutaneous inguinal (SC Ing) white adipose tissue (WAT) and how it affects whole-body energy expenditure in sedentary and endurance-trained rats fed ad libitum either low fat or high fat (HF) diets. Analysis of tissue mass, PGC-1α and UCP-1 content, the presence of multilocular adipocytes, and palmitate oxidation revealed that a HF diet increased the thermogenic capacity of the interscapular and aortic brown adipose tissues, whereas exercise markedly suppressed it. Conversely, exercise induced browning of the SC Ing WAT. This effect was attenuated by a HF diet. Endurance training neither affected skeletal muscle FNDC5 content nor circulating irisin, but it increased FNDC5 content in SC Ing WAT. This suggests that locally produced FNDC5 rather than circulating irisin mediated the exercise-induced browning effect on this fat tissue. Importantly, despite reducing the thermogenic capacity of classical BAT, exercise increased whole-body energy expenditure during the dark cycle. Therefore, browning of subcutaneous WAT likely exerted a compensatory effect and raised whole-body energy expenditure in endurance-trained rats. Based on these novel findings, we propose that exercise-induced browning of the subcutaneous WAT provides an alternative mechanism that reduces thermogenic capacity in core areas and increases it in peripheral body regions. This could allow the organism to adjust its metabolic rate to accommodate diet-induced thermogenesis while simultaneously coping with the stress of chronically increased heat production through exercise.     

Serum leptin, energy budget, and thermogenesis in striped hamsters exposed to consecutive decreases in ambient temperatures

Leptin has been found to be a direct participant in the regulation of both energy intake and energy expenditure in small mammals showing seasonal declines in body mass (M(b)) and fat mass, but its roles in an animal exhibiting seasonally increased thermogenesis and unchanged M(b) remain unclear. Serum leptin levels, energy budget, and thermogenesis were measured in striped hamsters exposed to consecutive decreases in ambient temperatures ranging from 23° to -23°C. Cold-exposed hamsters had significant increases in gross energy intake (GEI), the rate of basal metabolism, nonshivering thermogenesis, and activity of cytochrome c oxidase (COX) in brown adipose tissue (BAT), compared with control hamsters, indicating a cold-induced elevation of thermogenesis. Body mass and fat content were decreased in cold-exposed animals, and serum leptin levels were increased in hamsters exposed to temperatures of -8°C and below in inverse proportion to body fat content. Serum leptin levels were positively correlated with GEI and BAT COX activity in cold-exposed hamsters, but no such relationships were observed in control animals. These findings suggest that cold-exposed hamsters increase food consumption to meet the energy requirements for increased BAT thermogenesis. The increases in serum leptin levels are likely involved in increased thermogenesis in hamsters under cold stress. Cold-exposed hamsters may become leptin resistant, which is associated with impaired regulation of food intake. This new natural model of leptin resistance may also provide insight into the dynamic long-term control of energy homeostasis for animals that do not exhibit seasonal decline in M(b).     

Brown adipose tissue thermogenesis: interdisciplinary studies

Energy expenditure for thermogenesis in brown adipose tissue (BAT) serves either to maintain body temperature in the cold or to waste food energy. It has roles in thermal balance and energy balance, and when defective, is usually associated with obesity. BAT can grow or atrophy; it is usually atrophied in obese animals. Control of BAT thermogenesis and growth is by the sympathetic nervous system, with integration of signals in the hypothalamus. Sensory nerves may also be involved. Understanding the control of growth and differentiation of BAT is important for discovering how to reactivate it is obesity. Studies on control of gene expression in BAT are concentrating on thermogenically important components such as the uncoupling protein (which allows BAT mitochondria to operate in a thermogenic uncoupled mode), lipoprotein lipase (which allows BAT to compete with white adipose tissue for dietary lipid), and thyroxine 5'-deiodinase (which allows endogenous triiodothyronine generation, part of the control of differentiation and growth of BAT). Differentiation of BAT cell precursors in culture has recently been achieved. BAT is present in adult humans and some anti-obesity drugs are targeted to stimulation of BAT thermogenesis. However, extrapolation to humans of results of studies of BAT requires the development of novel approaches to the noninvasive assessment of amount and function of human BAT.     

NCLX pumps up the heat

Mitochondrial Ca²⁺ signaling is a well-appreciated regulator of cell metabolism and energy production. A major function of mitochondria in brown adipose tissue (BAT) is thermogenesis. Assali et al. offer new insights into how the mitochondrial Ca²⁺ extrusion mediator NCLX is crucial for BAT survival and thermogenesis.     

Cold exposure increases glucose utilization and glucose transporter expression in brown adipose tissue.

When rats were exposed to a cold environment (4 degrees C) for 10 days, tissue glucose utilization was increased in brown adipose tissue (BAT), a tissue specified for non-shivering thermogenesis, but not in skeletal muscle. Cold exposure also caused an increase in the amount of GLUT4, an isoform of glucose transporters expressed in insulin-sensitive tissues, in parallel with an increased cellular level of GLUT4 mRNA. In contrast to BAT, no significant effect of cold exposure was found in skeletal muscle. The results suggest the cold-induced increase in glucose utilization by BAT is attributable, at least in part, to the increased expression of GLUT4.     

Lack of TRPV2 impairs thermogenesis in mouse brown adipose tissue

Brown adipose tissue (BAT), a major site for mammalian non‐shivering thermogenesis, could be a target for prevention and treatment of human obesity. Transient receptor potential vanilloid 2 (TRPV2), a Ca²⁺‐permeable non‐selective cation channel, plays vital roles in the regulation of various cellular functions. Here, we show that TRPV2 is expressed in brown adipocytes and that mRNA levels of thermogenic genes are reduced in both cultured brown adipocytes and BAT from TRPV2 knockout (TRPV2KO) mice. The induction of thermogenic genes in response to β‐adrenergic receptor stimulation is also decreased in TRPV2KO brown adipocytes and suppressed by reduced intracellular Ca²⁺ concentrations in wild‐type brown adipocytes. In addition, TRPV2KO mice have more white adipose tissue and larger brown adipocytes and show cold intolerance, and lower BAT temperature increases in response to β‐adrenergic receptor stimulation. Furthermore, TRPV2KO mice have increased body weight and fat upon high‐fat‐diet treatment. Based on these findings, we conclude that TRPV2 has a role in BAT thermogenesis and could be a target for human obesity therapy.     

Different metabolic responses of human brown adipose tissue to activation by cold and insulin

We investigated the metabolism of human brown adipose tissue (BAT) in healthy subjects by determining its cold-induced and insulin-stimulated glucose uptake and blood flow (perfusion) using positron emission tomography (PET) combined with computed tomography (CT). Second, we assessed gene expression in human BAT and white adipose tissue (WAT). Glucose uptake was induced 12-fold in BAT by cold, accompanied by doubling of perfusion. We found a positive association between whole-body energy expenditure and BAT perfusion. Insulin enhanced glucose uptake 5-fold in BAT independently of its perfusion, while the effect on WAT was weaker. The gene expression level of insulin-sensitive glucose transporter GLUT4 was also higher in BAT as compared to WAT. In conclusion, BAT appears to be differently activated by insulin and cold; in response to insulin, BAT displays high glucose uptake without increased perfusion, but when activated by cold, it dissipates energy in a perfusion-dependent manner.     

N-butylidenephthalide ameliorates high-fat diet-induced obesity in mice and promotes browning through adrenergic response/AMPK activation in mouse beige adipocytes

Thermogenesis (non-exercise activity) in brown adipose tissue (BAT) promotes energy expenditure because of its higher number of mitochondria than white adipose tissue (WAT). The main function of thermogenesis in BAT can counteract obesity through the dissipation of calories as heat. N-butylidenephthalide (BP) is a natural derivative from Angelica sinensis, a Chinese herb that has been used for thousands of years. In this report, we demonstrated that BP improved the metabolic profiles of mice with high fat diet-induced obesity (DIO) by preventing weight gain, improving serum blood parameters, enhancing energy expenditure, stimulating white fat browning, and reversing hepatic steatosis. Further investigations demonstrated that BP administration upregulated the mRNA expression of beige (CD137, TMEM26) and brown fat selected genes (UCP1, PRDM16, PGC-1α, PPARγ) in white adipose tissues. In vitro studies, BP treatment increased multilocular lipid droplet levels, induced β-adrenergic receptor (cAMP/PKA) and AMP-activated protein kinase (AMPK) signaling (AMPK/acetyl-CoA carboxylase/SIRT1), and increased oxygen consumption in murine differentiated beige adipocytes, and the effects of BP were blocked by an AMPK inhibitor. BP promoted the interaction of AMPK with PGC-1α in beige adipocytes. Our findings provide novel insights into the application of BP in regulating energy metabolism and suggest its utility for clinical use in the treatment of obesity and related diseases.     

Regulatory networks of non-coding RNAs in brown/beige adipogenesis

BAT (brown adipose tissue) is specialized to burn fatty acids for heat generation and energy expenditure to defend against cold and obesity. Accumulating studies have demonstrated that manipulation of BAT activity through various strategies can regulate metabolic homoeostasis and lead to a healthy phenotype. Two classes of ncRNA (non-coding RNA), miRNA and lncRNA (long non-coding RNA), play crucial roles in gene regulation during tissue development and remodelling. In the present review, we summarize recent findings on regulatory role of distinct ncRNAs in brown/beige adipocytes, and discuss how these ncRNA regulatory networks contribute to brown/beige fat development, differentiation and function. We suggest that targeting ncRNAs could be an attractive approach to enhance BAT activity for protecting the body against obesity and its pathological consequences.