Lipid-encapsulated mRNA encoding an extended serum half-life interleukin-22 ameliorates metabolic disease in mice.

OBJECTIVE: Interleukin (IL)-22 is a potential therapeutic protein for the treatment of metabolic diseases such as obesity, type 2 diabetes, and metabolic dysfunction-associated steatotic liver disease due to its involvement in multiple cellular pathways and observed hepatoprotective effects. The short serum half-life of IL-22 has previously limited its use in clinical applications; however, the development of mRNA-lipid nanoparticle (LNP) technology offers a novel therapeutic approach that uses a host-generated IL-22 fusion protein. In the present study, the effects of administration of an mRNA-LNP encoding IL-22 on metabolic disease parameters was investigated in various mouse models. METHODS: C57BL/6NCrl mice were used to confirm mouse serum albumin (MSA)-IL-22 protein expression prior to assessments in C57BL/6NTac and CETP/ApoB transgenic mouse models of metabolic disease. Mice were fed either regular chow or a modified amylin liver nonalcoholic steatohepatitis-inducing diet prior to receiving either LNP-encapsulated MSA-IL-22 or MSA mRNA via intravenous or intramuscular injection. Metabolic markers were monitored for the duration of the experiments, and postmortem histology assessment and analysis of metabolic gene expression pathways were performed. RESULTS: MSA-IL-22 was detectable for ≥8 days following administration. Improvements in body weight, lipid metabolism, glucose metabolism, and lipogenic and fibrotic marker gene expression in the liver were observed in the MSA-IL-22-treated mice, and these effects were shown to be durable. CONCLUSIONS: These results support the application of mRNA-encoded IL-22 as a promising treatment strategy for metabolic syndrome and associated comorbidities in human populations.

Fatty acid synthesis suppresses dietary polyunsaturated fatty acid use.

Dietary polyunsaturated fatty acids (PUFA) are increasingly recognized for their health benefits, whereas a high production of endogenous fatty acids - a process called de novo lipogenesis (DNL) - is closely linked to metabolic diseases. Determinants of PUFA incorporation into complex lipids are insufficiently understood and may influence the onset and progression of metabolic diseases. Here we show that fatty acid synthase (FASN), the key enzyme of DNL, critically determines the use of dietary PUFA in mice and humans. Moreover, the combination of FASN inhibition and PUFA-supplementation decreases liver triacylglycerols (TAG) in mice fed with high-fat diet. Mechanistically, FASN inhibition causes higher PUFA uptake via the lysophosphatidylcholine transporter MFSD2A, and a diacylglycerol O-acyltransferase 2 (DGAT2)-dependent incorporation of PUFA into TAG. Overall, the outcome of PUFA supplementation may depend on the degree of endogenous DNL and combining PUFA supplementation and FASN inhibition might be a promising approach to target metabolic disease.

Mice lacking triglyceride synthesis enzymes in adipose tissue are resistant to diet-induced obesity.

Triglycerides (TGs) in adipocytes provide the major stores of metabolic energy in the body. Optimal amounts of TG stores are desirable as insufficient capacity to store TG, as in lipodystrophy, or exceeding the capacity for storage, as in obesity, results in metabolic disease. We hypothesized that mice lacking TG storage in adipocytes would result in excess TG storage in cell types other than adipocytes and severe lipotoxicity accompanied by metabolic disease. To test this hypothesis, we selectively deleted both TG synthesis enzymes, DGAT1 and DGAT2, in adipocytes (ADGAT DKO mice). As expected with depleted energy stores, ADGAT DKO mice did not tolerate fasting well and, with prolonged fasting, entered torpor. However, ADGAT DKO mice were unexpectedly otherwise metabolically healthy and did not accumulate TGs ectopically or develop associated metabolic perturbations, even when fed a high-fat diet. The favorable metabolic phenotype resulted from activation of energy expenditure, in part via BAT (brown adipose tissue) activation and beiging of white adipose tissue. Thus, the ADGAT DKO mice provide a fascinating new model to study the coupling of metabolic energy storage to energy expenditure.

Short-term dietary changes can result in mucosal and systemic immune depression.

Omnivorous animals, including mice and humans, tend to prefer energy-dense nutrients rich in fat over plant-based diets, especially for short periods of time, but the health consequences of this short-term consumption of energy-dense nutrients are unclear. Here, we show that short-term reiterative switching to 'feast diets', mimicking our social eating behavior, breaches the potential buffering effect of the intestinal microbiota and reorganizes the immunological architecture of mucosa-associated lymphoid tissues. The first dietary switch was sufficient to induce transient mucosal immune depression and suppress systemic immunity, leading to higher susceptibility to Salmonella enterica serovar Typhimurium and Listeria monocytogenes infections. The ability to respond to antigenic challenges with a model antigen was also impaired. These observations could be explained by a reduction of CD4+ T cell metabolic fitness and cytokine production due to impaired mTOR activity in response to reduced microbial provision of fiber metabolites. Reintroducing dietary fiber rewired T cell metabolism and restored mucosal and systemic CD4+ T cell functions and immunity. Finally, dietary intervention with human volunteers confirmed the effect of short-term dietary switches on human CD4+ T cell functionality. Therefore, short-term nutritional changes cause a transient depression of mucosal and systemic immunity, creating a window of opportunity for pathogenic infection.

TREM2 Regulates the Removal of Apoptotic Cells and Inflammatory Processes during the Progression of NAFLD.

Nonalcoholic fatty liver disease (NAFLD) is the most common liver pathology worldwide. In mice and humans, NAFLD progression is characterized by the appearance of TREM2-expressing macrophages in the liver. However, their mechanistic contributions to disease progression have not been completely elucidated. Here, we show that TREM2+ macrophages prevent the generation of a pro-inflammatory response elicited by LPS-laden lipoproteins in vitro. Further, Trem2 expression regulates bone-marrow-derived macrophages (BMDMs) and Kupffer cell capacity to phagocyte apoptotic cells in vitro, which is dependent on CD14 activation. In line with this, loss of Trem2 resulted in an increased pro-inflammatory response, which ultimately aggravated liver fibrosis in murine models of NAFLD. Similarly, in a human NAFLD cohort, plasma levels of TREM2 were increased and hepatic TREM2 expression was correlated with higher levels of liver triglycerides and the acquisition of a fibrotic gene signature. Altogether, our results suggest that TREM2+ macrophages have a protective function during the progression of NAFLD, as they are involved in the processing of pro-inflammatory lipoproteins and phagocytosis of apoptotic cells and, thereby, are critical contributors for the re-establishment of liver homeostasis.

Leptin as an Antitorpor Hormone: An Explanation for the Increased Metabolic Efficiency and Cold Sensitivity of ob/ob Mice?

AbstractLeptin is recognized as an anorexigenic hormone. In its absence (e.g., in ob/ob mutant mice), mice become obese, primarily as a result of hyperphagia. A recurrent question is whether, additionally, leptin is thermogenic and thus also an antiobesity hormone in this way. We have earlier reviewed available data and have concluded that most articles implying a thermogenic effect of leptin have based this on a misconstrued division by body weight. Here, we have collected evidence that the remaining observations that imply that leptin is a thermogenic hormone are better understood as implying that leptin is an antitorpor hormone. Leptin levels increase in proportion to the body's energy reserves (i.e., stored lipids in the adipose tissue), and leptin thus serves as an indicator of energy availability. In the absence of leptin, ob/ob mice are exceedingly prone to enter daily torpor, since the absence of leptin causes them to perceive a lack of body energy reserves that, in combination with restricted or no food, induces them to enter the torpid state to save energy. This antitorpor effect of leptin probably explains the following earlier observations. First, ob/ob mice have the ability to gain weight even when pair fed with leptin-treated ob/ob mice. This is understood as follows: In the leptin-treated ob/ob mice, food intake is reduced. Untreated pair-fed mice enter daily torpor, and this markedly lowers total daily energy expenditure; the resulting surplus food energy is then accumulated as fat in these mice. However, ob/ob mice fed ad lib. do not enter torpor, so under normal conditions this mechanism does not contribute to the obesity found in the ob/ob mice. Second, neonatal ob/ob mice have the ability to become obese despite eating the same amount as wild-type mice: this is understood as these mice similarly entering daily torpor. Third, ob/ob mice on the C57BL/6J background have a lower metabolic rate: these mice were examined in the absence of food, and torpor was thus probably induced. Fourth, ob/ob mice have apparent high cold sensitivity: these mice experienced cold in the absence of food and would immediately enter deep torpor. It is suggested that this novel explanation of how the antitorpor effects of leptin affect mouse energy metabolism can open new avenues for leptin research.

Role of Liver CD38 in the Regulation of Metabolic Pathways during Cold-Induced Thermogenesis in Mice.

Boosting NAD+ levels are considered a promising means to promote healthy aging and ameliorate dysfunctional metabolism. The expression of CD38, the major NAD+-consuming enzyme, is downregulated during thermogenesis in both brown and white adipose tissues (BAT and WAT). Moreover, BAT activation and WAT "browning" were enhanced in Cd38-/- mice. In this study, the role of CD38 in the liver during thermogenesis was investigated, with the liver being the central organ controlling systemic energy metabolism. Wild-type mice and Cd38-/- mice were exposed to cold temperatures, and levels of metabolites and enzymes were measured in the livers and plasma. During cold exposure, CD38 expression was downregulated in the liver, as in BAT and WAT, with a concomitant increase in NAD(H) and a marked decrease in NADPH levels. Glucose-6-phosphate dehydrogenase and the malic enzyme, along with enzymes in the glycolytic pathway, were downregulated, which is in line with glucose-6-P being re-directed towards glucose release. In Cd38-/- mice, the cross-regulation between glycolysis and glucose release was lost, although this did not impair the glucose release from glycogen. Glycerol levels were decreased in the liver from Cd38-/- animals upon cold exposure, suggesting that glyceroneogenesis, as gluconeogenesis, was not properly activated in the absence of CD38. SIRT3 activity, regulating mitochondrial metabolism, was enhanced by cold exposure, whereas its activity was already high at a warm temperature in Cd38-/- mice and was not further increased by the cold. Notably, FGF21 and bile acid release was enhanced in the liver of Cd38-/- mice, which might contribute to enhanced BAT activation in Cd38-/- mice. These results demonstrate that CD38 inhibition can be suggested as a strategy to boost NAD+ and would not negatively affect hepatic functions during thermogenesis.

Deficiency of the frontotemporal dementia gene GRN results in gangliosidosis.

Haploinsufficiency of GRN causes frontotemporal dementia (FTD). The GRN locus produces progranulin (PGRN), which is cleaved to lysosomal granulin polypeptides. The function of lysosomal granulins and why their absence causes neurodegeneration are unclear. Here we discover that PGRN-deficient human cells and murine brains, as well as human frontal lobes from GRN-mutation FTD patients have increased levels of gangliosides, glycosphingolipids that contain sialic acid. In these cells and tissues, levels of lysosomal enzymes that catabolize gangliosides were normal, but levels of bis(monoacylglycero)phosphates (BMP), lipids required for ganglioside catabolism, were reduced with PGRN deficiency. Our findings indicate that granulins are required to maintain BMP levels to support ganglioside catabolism, and that PGRN deficiency in lysosomes leads to gangliosidosis. Lysosomal ganglioside accumulation may contribute to neuroinflammation and neurodegeneration susceptibility observed in FTD due to PGRN deficiency and other neurodegenerative diseases.

Lysosomal acid lipase promotes endothelial proliferation in cold-activated adipose tissue.

Oxidative tissues such as brown adipose tissue and muscle internalize large amounts of circulating lipids and glucose as energy source. Endothelial cells (ECs) provide a platform for regulated transport and processing of blood-borne nutrients. Next to this role, it has become recognized that intercellular crosstalk between ECs and underlying parenchymal cells is indispensable for maintenance of tissue homoeostasis. Here, we comment on our recent observation that capillary ECs in thermogenic adipose tissues take up and metabolize entire triglyceride-rich lipoprotein (TRL) particles in response to cold exposure. This process is dependent on CD36, lipoprotein lipase (LPL) and lysosomal acid lipase (LAL). Remarkably, loss of LAL specifically in endothelial cells results in impaired endothelial proliferation and diminished thermogenic adaptation. Mechanistically, cell culture experiments indicate that LAL-mediated TRL processing leads to the generation of reactive oxygen species, which in turn activate hypoxia-induced factor (HIF)-mediated proliferative responses. In the current manuscript, we provide in vivo evidence that LAL-deficiency impairs proliferation of endothelial cells in thermogenic adipose tissue. In addition, we show uptake of nanoparticle-labelled TRL and LAL expression in cardiac endothelial cells, suggesting a physiological function of endothelial lipoprotein processing not only in thermogenic adipose tissue but also in cardiac muscle.

Endogenous Fatty Acid Synthesis Drives Brown Adipose Tissue Involution.

Thermoneutral conditions typical for standard human living environments result in brown adipose tissue (BAT) involution, characterized by decreased mitochondrial mass and increased lipid deposition. Low BAT activity is associated with poor metabolic health, and BAT reactivation may confer therapeutic potential. However, the molecular drivers of this BAT adaptive process in response to thermoneutrality remain enigmatic. Using metabolic and lipidomic approaches, we show that endogenous fatty acid synthesis, regulated by carbohydrate-response element-binding protein (ChREBP), is the central regulator of BAT involution. By transcriptional control of lipogenesis-related enzymes, ChREBP determines the abundance and composition of both storage and membrane lipids known to regulate organelle turnover and function. Notably, ChREBP deficiency and pharmacological inhibition of lipogenesis during thermoneutral adaptation preserved mitochondrial mass and thermogenic capacity of BAT independently of mitochondrial biogenesis. In conclusion, we establish lipogenesis as a potential therapeutic target to prevent loss of BAT thermogenic capacity as seen in adult humans.

TFEB deficiency attenuates mitochondrial degradation upon brown adipose tissue whitening at thermoneutrality.

OBJECTIVE: Brown adipose tissue (BAT) thermogenesis offers the potential to improve metabolic health in mice and humans. However, humans predominantly live under thermoneutral conditions, leading to BAT whitening, a reduction in BAT mitochondrial content and metabolic activity. Recent studies have established mitophagy as a major driver of mitochondrial degradation in the whitening of thermogenic brite/beige adipocytes, yet the pathways mediating mitochondrial breakdown in whitening of classical BAT remain largely elusive. The transcription factor EB (TFEB), a master regulator of lysosomal biogenesis and autophagy belonging to the MiT family of transcription factors, is the only member of this family that is upregulated during whitening, pointing toward a role of TFEB in whitening-associated mitochondrial breakdown. METHODS: We generated brown adipocyte-specific TFEB knockout mice, and induced BAT whitening by thermoneutral housing. We characterized gene and protein expression patterns, BAT metabolic activity, systemic metabolism, and mitochondrial localization using in vivo and in vitro approaches. RESULTS: Under low thermogenic activation conditions, deletion of TFEB preserves mitochondrial mass independently of mitochondriogenesis in BAT and primary brown adipocytes. However, this does not translate into elevated thermogenic capacity or protection from diet-induced obesity. Autophagosomal/lysosomal marker levels are altered in TFEB-deficient BAT and primary adipocytes, and lysosomal markers co-localize and co-purify with mitochondria in TFEB-deficient BAT, indicating trapping of mitochondria in late stages of mitophagy. CONCLUSION: We identify TFEB as a driver of BAT whitening, mediating mitochondrial degradation via the autophagosomal and lysosomal machinery. This study provides proof of concept that interfering with the mitochondrial degradation machinery can increase mitochondrial mass in classical BAT under human-relevant conditions. However, it must be considered that interfering with autophagy may result in accumulation of non-functional mitochondria. Future studies targeting earlier steps of mitophagy or target recognition are therefore warranted.

Lysosomal lipoprotein processing in endothelial cells stimulates adipose tissue thermogenic adaptation.

In response to cold exposure, thermogenic adipocytes internalize large amounts of fatty acids after lipoprotein lipase-mediated hydrolysis of triglyceride-rich lipoproteins (TRL) in the capillary lumen of brown adipose tissue (BAT) and white adipose tissue (WAT). Here, we show that in cold-exposed mice, vascular endothelial cells in adipose tissues endocytose substantial amounts of entire TRL particles. These lipoproteins subsequently follow the endosomal-lysosomal pathway, where they undergo lysosomal acid lipase (LAL)-mediated processing. Endothelial cell-specific LAL deficiency results in impaired thermogenic capacity as a consequence of reduced recruitment of brown and brite/beige adipocytes. Mechanistically, TRL processing by LAL induces proliferation of endothelial cells and adipocyte precursors via beta-oxidation-dependent production of reactive oxygen species, which in turn stimulates hypoxia-inducible factor-1α-dependent proliferative responses. In conclusion, this study demonstrates a physiological role for TRL particle uptake into BAT and WAT and establishes endothelial lipoprotein processing as an important determinant of adipose tissue remodeling during thermogenic adaptation.

CD38 downregulation modulates NAD+ and NADP(H) levels in thermogenic adipose tissues.

Different strategies to boost NAD+ levels are considered promising means to promote healthy aging and ameliorate dysfunctional metabolism. CD38 is a NAD+-dependent enzyme involved in the regulation of different cell functions. In the context of systemic energy metabolism, it has been demonstrated that brown adipocytes, the parenchymal cells of brown adipose tissue (BAT) as well as beige adipocytes that emerge in white adipose tissue (WAT) depots in response to catabolic conditions, are important to maintain metabolic homeostasis. In this study we aim to understand the functional relevance of CD38 for NAD+ and energy metabolism in BAT and WAT, also using a CD38-/- mouse model. During cold exposure, an increase in NAD+ levels occurred in BAT of wild type mice, together with a marked downregulation of CD38, as detected at the mRNA and protein level. CD38 downregulation was observed also in WAT of cold-exposed mice, where it was accompanied by a strong increase in NADP(H) levels. Accordingly, NAD kinase and glucose-6-phosphate dehydrogenase activities were enhanced in WAT (but not in BAT). Increased NAD+ levels were observed in BAT/WAT from CD38-/- compared with wild type mice, in line with CD38 being a major NAD+-consumer in AT. CD38-/- mice kept at 6 °C had higher levels of Ucp1 and Pgc-1α in BAT and WAT, and increased levels of phosphorylated hormone-sensitive lipase in BAT, compared with wild type mice. These results demonstrate that CD38, by modulating cellular NAD(P)+ levels, is involved in the regulation of thermogenic responses in cold-activated BAT and WAT.

Thermoneutrality-Induced Macrophage Accumulation in Brown Adipose Tissue Does Not Impair the Tissue's Competence for Cold-Induced Thermogenic Recruitment.

Brown adipose tissue from mice living under conditions approaching human thermal and nutritional conditions (prolonged exposure to thermoneutral temperature and to an energy-rich (high-fat, high-sugar) diet) - referred to as "physiologically humanized" mice, displays morphological and molecular characteristics significantly different from those observed in young, chow-fed mice maintained at room temperature - referred to as "standard" mice. Here, we further examined brown fat from physiologically humanized and standard mice, as well as from mice exposed to thermoneutrality for a long time but not to an energy-rich diet - referred to here as "long-term thermoneutral" mice. Global transcriptome analysis of brown fat revealed that genes that were the most upregulated in brown fat of thermoneutral mice (both physiologically humanized and long-term thermoneutral) were those related to inflammatory processes, including genes expressed selectively in macrophages. Cellular and molecular analyses confirmed that brown fat from thermoneutral mice was heavily infiltrated by macrophages, predominantly organized into crown-like structures. However, despite this, the brown fat of thermoneutral mice retained full competence to attain the greatest possible recruitment state and became macrophage-depleted during the process of cold acclimation. Thus, profound macrophage accumulation does not influence the thermogenic recruitment competence of brown fat.

Lipid Droplets in Brown Adipose Tissue Are Dispensable for Cold-Induced Thermogenesis.

Brown adipocytes store metabolic energy as triglycerides (TGs) in lipid droplets (LDs). Fatty acids released from brown adipocyte LDs by lipolysis are thought to activate and fuel UCP1-mediated thermogenesis. Here, we test this hypothesis by preventing fatty acid storage in murine brown adipocytes through brown adipose tissue (BAT)-specific deletions of the TG synthesis enzymes DGAT1 and DGAT2 (BA-DGAT KO). Despite the absence of TGs in brown adipocytes, BAT is functional, and BA-DGAT-KO mice maintain euthermia during acute or chronic cold exposure. As apparent adaptations to the lack of TG, brown adipocytes of BA-DGAT-KO mice appear to use circulating glucose and fatty acids, and stored glycogen, to fuel thermogenesis. Moreover, BA-DGAT-KO mice are resistant to diet-induced glucose intolerance, likely because of increased glucose disposal by BAT. We conclude that TGs in BAT are dispensable for its contribution to cold-induced thermogenesis, at least when other fuel sources are available.

Brown adipose tissue lipoprotein and glucose disposal is not determined by thermogenesis in uncoupling protein 1-deficient mice.

Adaptive thermogenesis is highly dependent on uncoupling protein 1 (UCP1), a protein expressed by thermogenic adipocytes present in brown adipose tissue (BAT) and white adipose tissue (WAT). Thermogenic capacity of human and mouse BAT can be measured by positron emission tomography-computed tomography quantifying the uptake of 18F-fluodeoxyglucose or lipid tracers. BAT activation is typically studied in response to cold exposure or treatment with ß-3-adrenergic receptor agonists such as CL316,243 (CL). Currently, it is unknown whether cold-stimulated uptake of glucose or lipid tracers is a good surrogate marker of UCP1-mediated thermogenesis. In metabolic studies using radiolabeled tracers, we found that glucose uptake is increased in mildly cold-activated BAT of Ucp1-/- versus WT mice kept at subthermoneutral temperature. Conversely, lower glucose disposal was detected after full thermogenic activation achieved by sustained cold exposure or CL treatment. In contrast, uptake of lipoprotein-derived fatty acids into chronically activated thermogenic adipose tissues was substantially increased in UCP1-deficient mice. This effect is linked to higher sympathetic tone in adipose tissues of Ucp1-/- mice, as indicated by elevated levels of thermogenic genes in BAT and WAT. Thus, glucose and lipoprotein handling does not necessarily reflect UCP1-dependent thermogenic activity, but especially lipid uptake rather mirrors sympathetic activation of adipose tissues.

Human brown adipose tissue: Classical brown rather than brite/beige?

NEW FINDINGS: What is the topic of this review? It has been suggested that human brown adipose tissue (BAT) is more similar to the brite/beige adipose tissue of mice than to classical BAT of mice. The basis of this is discussed in relationship to the physiological conditions of standard experimental mice. What advances does it highlight? We highlight that, provided mouse adipose tissues are examined under physiological conditions closer to those prevalent for most humans, the gene expression profile of mouse classical BAT is more similar to that of human BAT than is the profile of mouse brite/beige adipose tissue. Human BAT is therefore not different in nature from classical mouse BAT. ABSTRACT: Since the presence of brown adipose tissue (BAT) was established in adult humans some 13 years ago, its physiological significance and molecular characteristics have been discussed. In particular, it has been proposed that the mouse adipose tissue depot most closely resembling and molecularly parallel to human BAT is not classical mouse BAT. Instead, so-called brite or beige adipose tissue, which is characteristically observed in the inguinal 'white' adipose tissue depot of mice, has been proposed to be the closest mouse equivalent of human BAT. We summarize here the published evidence examining this question. We emphasize the differences in tissue appearance and tissue transcriptomes from 'standard' mice [young, chow fed and, in effect semi-cold exposed (20°C)] versus 'physiologically humanized' mice [middle-aged, high-fat diet-fed mice living at thermoneutrality (30°C)]. We find that in the physiologically humanized mice, classical BAT displays molecular and cellular characteristics that are more akin to human BAT than are those of brite/beige adipose tissues from either standard or physiologically humanized mice. We suggest, therefore, that mouse BAT is the more relevant tissue for translational studies. This is an invited summary of a presentation given at Physiology 2019 (Aberdeen).

Leptin: Is It Thermogenic?

Animals that lack the hormone leptin become grossly obese, purportedly for 2 reasons: increased food intake and decreased energy expenditure (thermogenesis). This review examines the experimental evidence for the thermogenesis component. Analysis of the data available led us to conclude that the reports indicating hypometabolism in the leptin-deficient ob/ob mice (as well as in the leptin-receptor-deficient db/db mice and fa/fa rats) derive from a misleading calculation artefact resulting from expression of energy expenditure per gram of body weight and not per intact organism. Correspondingly, the body weight-reducing effects of leptin are not augmented by enhanced thermogenesis. Congruent with this, there is no evidence that the ob/ob mouse demonstrates atrophied brown adipose tissue or diminished levels of total UCP1 mRNA or protein when the ob mutation is studied on the inbred C57BL/6 mouse background, but a reduced sympathetic nerve activity is observed. On the outbred "Aston" mouse background, brown adipose tissue atrophy is seen, but whether this is of quantitative significance for the development of obesity has not been demonstrated. We conclude that leptin is not a thermogenic hormone. Rather, leptin has effects on body temperature regulation, by opposing torpor bouts and by shifting thermoregulatory thresholds. The central pathways behind these effects are largely unexplored.