Obesity is one of the primary healthcare challenges of the 21st century. Signals relaying information regarding energy needs are integrated within the brain to influence body weight. Central among these integration nodes are the brain pro-opiomelanocortin (POMC) peptides, perturbations of which disrupt energy balance and promote severe obesity. However, POMC neurons are neurochemically diverse and the crucial source of POMC peptides that regulate energy homeostasis and body weight remains to be fully clarified.

Food intake and circadian rhythms are regulated by hypothalamic neuropeptides and circulating hormones, which could mediate the anti-ageing effect of calorie restriction (CR). We tested whether these two signaling pathways mediate CR by quantifying hypothalamic transcripts of male C57BL/6 mice exposed to graded levels of CR (10 % to 40 %) for 3 months. We found that the graded CR manipulation resulted in upregulation of core circadian rhythm genes, which correlated negatively with circulating levels of leptin, insulin-like growth factor 1 (IGF-1), insulin, and tumor necrosis factor alpha (TNF-α). In addition, key components in the hunger signaling pathway were expressed in a manner reflecting elevated hunger at greater levels of restriction, and which also correlated negatively with circulating levels of insulin, TNF-α, leptin and IGF-1. Lastly, phenotypes, such as food anticipatory activity and body temperature, were associated with expression levels of both hunger genes and core clock genes. Our results suggest modulation of the hunger and circadian signaling pathways in response to altered levels of circulating hormones, that are themselves downstream of morphological changes resulting from CR treatment, may be important elements in the response to CR, driving some of the key phenotypic outcomes.

Increasing evidence indicates that brown adipose tissue (BAT) transplantation enhances whole-body energy metabolism in a mouse model of diet-induced obesity. However, it remains unclear whether BAT also has such beneficial effects on genetically obese mice. To address this issue, we transplanted BAT from C57/BL6 mice into the dorsal subcutaneous region of age- and sex-matched leptin deficient Ob/Ob mice. Interestingly, BAT transplantation led to a significant reduction of body weight gain with increased oxygen consumption and decreased total body fat mass, resulting in improvement of insulin resistance and liver steatosis. In addition, BAT transplantation increased the level of circulating adiponectin, whereas it reduced the levels of circulating free T3 and T4, which regulate thyroid hormone sensitivity in peripheral tissues. BAT transplantation also increased β3-adrenergic receptor and fatty acid oxidation related gene expression in subcutaneous and epididymal (EP) white adipose tissue. Accordingly, BAT transplantation increased whole-body thermogenesis. Taken together our results demonstrate that BAT transplantation may reduce obesity and its related diseases by activating endogenous BAT.

Some large herbivores exhibit seasonal adjustments in their energy metabolism. Therefore, our aim was to determine if the llama (one of the most extensively kept livestock breeds) exhibits seasonal adjustment of their energy expenditure, body temperature and locomotion, under its natural high altitude Andean habitat. For this purpose, energy expenditure, body temperature and locomotion were measured in seven non-pregnant llama dams for ten months on the Andean High Plateau (4400 m above sea level). Daily energy expenditure was measured as field metabolic rate using the doubly labelled water method at four different measurement times. Additionally, a telemetry system was used to continuously record activity, body temperature (3 min intervals) as well as the position (hourly) of each animal. The results show that llamas adjusted their body temperature and daily energy expenditure according to environmental conditions. Furthermore, llamas under high altitude Andean climatic conditions exhibited a pronounced daily rhythm in body temperature and activity, with low values at sunrise and increasing values towards sunset. Llamas also had remarkably low energy expenditure compared to other herbivores. Thus, despite the domestication process, llamas have not lost the ability to adjust their body temperature and daily energy expenditure under adverse environmental conditions, similar to some wild herbivores.

Emerging evidence indicates that G-protein coupled receptor 55 (GPR55), a nonclassic receptor of the endocannabinoid system that is activated by L-α-lysophosphatidylinositol and various cannabinoid ligands, may regulate endocrine function and energy metabolism. We examined how GPR55 deficiency and modulation affects insulin signaling in skeletal muscle, adipose tissue, and liver alongside expression analysis of proteins implicated in insulin action and energy metabolism. We show that GPR55-null mice display decreased insulin sensitivity in these tissues, as evidenced by reduced phosphorylation of PKB/Akt and its downstream targets, concomitant with increased adiposity and reduced physical activity relative to wild-type counterparts. Impaired tissue insulin sensitivity coincided with reduced insulin receptor substrate-1 abundance in skeletal muscle, whereas in liver and epididymal fat it was associated with increased expression of the 3-phosphoinoistide lipid phosphatase, phosphatase and tensin homolog. In contrast, GPR55 activation enhanced insulin signaling in cultured skeletal muscle cells, adipocytes, and hepatocytes; this response was negated by receptor antagonists and GPR55 gene silencing in L6 myotubes. Sustained GPR55 antagonism in 3T3-L1 adipocytes enhanced expression of proteins implicated in lipogenesis and promoted triglyceride accumulation. Our findings identify GPR55 as a positive regulator of insulin action and adipogenesis and as a potential therapeutic target for countering obesity-induced metabolic dysfunction and insulin resistance.-Lipina, C., Walsh, S. K., Mitchell, S. E., Speakman, J. R., Wainwright, C. L., Hundal, H. S. GPR55 deficiency is associated with increased adiposity and impaired insulin signaling in peripheral metabolic tissues.

Energy expenditure is a key variable in the study of ageing, and the fruit fly Drosophila melanogaster is a model organism that has been used to make step changes in our understanding of the ageing process. Standard methods for measurement of energy expenditure involve placing individuals in metabolic chambers where their oxygen consumption and CO2 production can be quantified. These measurements require separating individuals from any social context, and may only poorly reflect the environment in which the animals normally live. The doubly-labeled water (DLW) method is an isotope-based technique for measuring energy expenditure which overcomes these problems. However, technical challenges mean that the smallest animals this method has been previously applied to weighed 50-200 mg. We overcame these technical challenges to measure energy demands in Drosophila weighing 0.78 mg. Mass-specific energy expenditure varied between 43 and 65 mW·g(-1). These estimates are considerably higher than estimates using indirect calorimetry of Drosophila in small metabolic chambers (around 18 mW·g(-1)). The methodology we have established extends downwards by three orders of magnitude the size of animals that can be measured using DLW. This approach may be of considerable value in future ageing research attempting to understand the genetic and genomic basis of ageing.

Calorie restriction (CR) may extend longevity by modulating the mechanisms involved in aging. Different hypotheses have been proposed for its main mode of action. We quantified hepatic transcripts of male C57BL/6 mice exposed to graded levels of CR (0% to 40% CR) for three months, and evaluated the responses relative to these various hypotheses. Of the four main signaling pathways implied to be linked to the impact of CR on lifespan (insulin/insulin like growth factor 1 (IGF-1), nuclear factor-kappa beta (NF-ĸB), mechanistic target of rapamycin (mTOR) and sirtuins (SIRTs)), all the pathways except SIRT were altered in a manner consistent with increased lifespan. However, the expression levels of SIRT4 and SIRT7 were decreased with increasing levels of CR. Changes consistent with altered fuel utilization under CR may reduce reactive oxygen species production, which was paralleled by reduced protection. Downregulated major urinary protein (MUP) transcription suggested reduced reproductive investment. Graded CR had a positive effect on autophagy and xenobiotic metabolism, and was protective with respect to cancer signaling. CR had no significant effect on fibroblast growth factor-21 (FGF21) transcription but affected transcription in the hydrogen sulfide production pathway. Responses to CR were consistent with several different hypotheses, and the benefits of CR on lifespan likely reflect the combined impact on multiple aging related processes.

Previous work has suggested that white adipocytes may also show a mammary luminal secretory cell phenotype during lactation. The capacity of brown and beige/brite adipocytes to display a mammary cell phenotype and the levels at which they demonstrate such phenotypes in vivo is currently unknown.

Cold exposure imposes a metabolic challenge to mammals that is met by a coordinated response in different tissues to prevent hypothermia. This study reports a transcriptomic analysis in brown adipose tissue (BAT), white adipose (WAT) and liver of mice in response to 24 h cold exposure at 8°C. Expression of 1895 genes were significantly (P<0.05) up- or down-regulated more than two fold by cold exposure in all tissues but only 5 of these genes were shared by all three tissues, and only 19, 14 and 134 genes were common between WAT and BAT, WAT and liver, and BAT and liver, respectively. We confirmed using qRT-PCR, the increased expression of a number of characteristic BAT genes during cold exposure. In both BAT and the liver, the most common direction of change in gene expression was suppression (496 genes in BAT and 590 genes in liver). Gene ontology analysis revealed for the first time significant (P<0.05) down regulation in response to cold, of genes involved in oxidoreductase activity, lipid metabolic processes and protease inhibitor activity, in both BAT and liver, but not WAT. The results reveal an unexpected importance of down regulation of cytochrome P450 gene expression and apolipoprotein, in both BAT and liver, but not WAT, in response to cold exposure. Pathway analysis suggests a model in which down regulation of the nuclear transcription factors HNF4α and PPARα in both BAT and liver may orchestrate the down regulation of genes involved in lipoprotein and steroid metabolism as well as Phase I enzymes belonging to the cytochrome P450 group in response to cold stress in mice. We propose that the response to cold stress involves decreased gene expression in a range of cellular processes in order to maximise pathways involved in heat production.

Caloric restriction in mice can trigger gorging behaviour, which is characterized by periods of excessive food ingestion in a short time. Animals that gorge are thought to have a reduced metabolism compared to those that nibble their food over a longer period and might therefore be more able to compensate for reduced energy intake. We examined whether mice that gorged showed less weigh loss during restriction. We placed female mice (n=60) on a restriction of 75% of their ad libitum food intake (FI) for 22 days. FI and body mass (BM) were measured at 1, 2 and 24 h after food provision. Ten controls remained feeding ad lib and we selected the 10 strongest gorgers and 10 strongest non-gorgers for comparison. Mice had BM, FI, resting metabolic rate (RMR), body composition, body temperature, daily energy expenditure (DEE) and circulating levels of the regulatory hormones leptin and ghrelin measured. Gorgers had a significantly lower BM at the end of restriction than non-gorgers or controls, indicating that they were less able to compensate for the reduced energy. Both groups of restricted mice had reduced RMR, however reduced activity was only used as an energy saving mechanism in non-gorgers. Gorging mice had a significantly lower level of circulating leptin than controls and non-gorgers but no differences in ghrelin levels. Gorging mice were, in fact, less able to compensate for reduced energy intake, as they reduced RMR by a similar extent as non-gorgers, but did not reduce activity compared to non-gorgers on the same restriction level. The reduced leptin levels may drive the gorging behaviour.

Resistance exercise increases muscle mass and function in older adults, but responses are attenuated compared with younger people. Data suggest that long-chain n-3 polyunsaturated fatty acids (PUFAs) may enhance adaptations to resistance exercise in older women. To our knowledge, this possibility has not been investigated in men.

The carbohydrate-insulin model (CIM) predicts that increases in fasting and post-prandial insulin in response to dietary carbohydrates stimulate energy intake and lower energy expenditures, leading to positive energy balance and weight gain. The objective of the present study was to directly test the CIM's predictions using C57BL/6 mice.

Caloric restriction is a robust intervention to increase lifespan. Giving less food (calorie restriction [CR]) or allowing free access to a diluted diet with indigestible components (calorie dilution [CD]) are two methods to impose restriction. CD does not generate the same lifespan effect as CR. We compare responses of C57BL/6 mice with equivalent levels of CR and CD. The two groups have different responses in fat loss, circulating hormones, and metabolic rate. CR mice are hungrier, as assessed by behavioral assays. Although gene expression of Npy, Agrp, and Pomc do not differ between CR and CD groups, CR mice had a distinctive hypothalamic gene-expression profile with many genes related to starvation upregulated relative to CD. While both result in lower calorie intake, CR and CD are not equivalent procedures. Increased hunger under CR supports the hypothesis that hunger signaling is a key process mediating the benefits of CR.

Calorie restriction (CR) extends life span by modulating the mechanisms involved in aging. We quantified the hepatic proteome of male C57BL/6 mice exposed to graded levels of CR (0%-40% CR) for 3 months, and evaluated which signaling pathways were most affected. The metabolic pathways most significantly stimulated by the increase in CR, included the glycolysis/gluconeogenesis pathway, the pentose phosphate pathway, the fatty acid degradation pathway, the valine, leucine, and isoleucine degradation pathway, and the lysine degradation pathway. The metabolism of xenobiotics by cytochrome P450 pathway was activated and feminized by increased CR, while production in major urinary proteins (Mups) was strongly reduced, consistent with a reduced investment in reproduction as predicted by the disposable soma hypothesis. However, we found no evidence of increased somatic protection, and none of the 4 main pathways implied to be linked to the impact of CR on life span (insulin/insulin-like growth factor [IGF-1], nuclear factor-κB [NF-κB], mammalian Target of Rapamycin [mTOR], and sirtuins) as well as pathways in cancer, were significantly changed at the protein level in relation to the increase in CR level. This was despite previous work at the transcriptome level in the same individuals indicating such changes. On the other hand, we found Aldh2, Aldh3a2, and Aldh9a1 in carnitine biosynthesis and Acsl5 in carnitine shuttle system were up-regulated by increased CR, which are consistent with our previous work on metabolome of the same individuals. Overall, the patterns of protein expression were more consistent with a "clean cupboards" than a "disposable soma" interpretation.

The protein leverage hypothesis predicts that low dietary protein should increase energy intake and cause adiposity. We designed 10 diets varying from 1% to 20% protein combined with either 60% or 20% fat. Contrasting the expectation, very low protein did not cause increased food intake. Although these mice had activated hunger signaling, they ate less food, resulting in decreased body weight and improved glucose tolerance but not increased frailty, even under 60% fat. Moreover, they did not show hyperphagia when returned to a 20% protein diet, which could be mimicked by treatment with rapamycin. Intracerebroventricular injection of AAV-S6K1 significantly blunted the decrease in both food intake and body weight in mice fed 1% protein, an effect not observed with inhibition of eIF2a, TRPML1, and Fgf21 signaling. Hence, the 1% protein diet induced decreased food intake and body weight via a mechanism partially dependent on hypothalamic mTOR signaling.

Considerable attention is currently focused on the potential to switch on brown adipose tissue (BAT), or promote browning of white adipose tissue, to elevate energy expenditure and thereby reduce obesity levels. These processes are already known to be switched on by cold exposure. Yet humans living in colder regions do not show lower levels of obesity. This could be because humans shield themselves from external temperatures, or because the resultant changes in BAT and thermogenesis are offset by elevated food intake, or reductions in other components of expenditure.

The nucleoside analog N4-hydroxycytidine (NHC) is the active metabolite of the prodrug molnupiravir, which has been approved for the treatment of COVID-19. SARS-CoV-2 incorporates NHC into its RNA, resulting in defective virus genomes. Likewise, inhibitors of dihydroorotate dehydrogenase (DHODH) reduce virus yield upon infection, by suppressing the cellular synthesis of pyrimidines. Here, we show that NHC and DHODH inhibitors strongly synergize in the inhibition of SARS-CoV-2 replication in vitro. We propose that the lack of available pyrimidine nucleotides upon DHODH inhibition increases the incorporation of NHC into nascent viral RNA. This concept is supported by the rescue of virus replication upon addition of pyrimidine nucleosides to the media. DHODH inhibitors increased the antiviral efficiency of molnupiravir not only in organoids of human lung, but also in Syrian Gold hamsters and in K18-hACE2 mice. Combining molnupiravir with DHODH inhibitors may thus improve available therapy options for COVID-19.

As a major health issue, obesity is linked with elevated risk of type 2 diabetes. However, whether disrupted glucose homeostasis is due to altered body composition alone, or whether dietary macronutrients play an additional role, independent of their impact on body composition, remains unclear. We investigated the associations between macronutrients, body composition, blood hormones and glucose homeostasis. We fed C57BL/6N mice 29 different diets with variable macronutrients for 12 weeks. After 10 weeks, intraperitoneal glucose tolerance tests were performed. Generalized linear models were generated to evaluate the impacts of macronutrients, body composition and blood hormones on glucose homeostasis. The area under the glucose curve (AUC) was strongly associated with body fat mass, but not dietary macronutrients. AUC was significantly associated with fasting insulin levels. Six genes from transcriptomic analysis of epididymal white adipose tissue and subcutaneous white adipose tissue were significantly associated with AUC. These genes may encode secreted proteins that play important previously unanticipated roles in glucose homeostasis.

Seasonal energetic challenges may constrain an animal's ability to respond to changing individual and environmental conditions. Here, we investigated variation in heart rate, a well-established proxy for metabolic rate, in Svalbard reindeer (Rangifer tarandus platyrhynchus), a species with strong seasonal changes in foraging and metabolic activity. In 19 adult females, we recorded heart rate, subcutaneous temperature and activity using biologgers. Mean heart rate more than doubled from winter to summer. Typical drivers of energy expenditure, such as reproduction and activity, explained a relatively limited amount of variation (2-6% in winter and 16-24% in summer) compared to seasonality, which explained 75% of annual variation in heart rate. The relationship between heart rate and subcutaneous temperature depended on individual state via body mass, age and reproductive status, and the results suggested that peripheral heterothermy is an important pathway of energy management in both winter and summer. While the seasonal plasticity in energetics makes Svalbard reindeer well-adapted to their highly seasonal environment, intraseasonal constraints on modulation of their heart rate may limit their ability to respond to severe environmental change. This study emphasizes the importance of encompassing individual state and seasonal context when studying energetics in free-living animals. This article is part of the theme issue 'Measuring physiology in free-living animals (Part II)'.

Caloric restriction that promotes weight loss is an effective strategy for treating non-alcoholic fatty liver disease and improving insulin sensitivity in people with type 2 diabetes1. Despite its effectiveness, in most individuals, weight loss is usually not maintained partly due to physiological adaptations that suppress energy expenditure, a process known as adaptive thermogenesis, the mechanistic underpinnings of which are unclear2,3. Treatment of rodents fed a high-fat diet with recombinant growth differentiating factor 15 (GDF15) reduces obesity and improves glycaemic control through glial-cell-derived neurotrophic factor family receptor α-like (GFRAL)-dependent suppression of food intake4-7. Here we find that, in addition to suppressing appetite, GDF15 counteracts compensatory reductions in energy expenditure, eliciting greater weight loss and reductions in non-alcoholic fatty liver disease (NAFLD) compared to caloric restriction alone. This effect of GDF15 to maintain energy expenditure during calorie restriction requires a GFRAL-β-adrenergic-dependent signalling axis that increases fatty acid oxidation and calcium futile cycling in the skeletal muscle of mice. These data indicate that therapeutic targeting of the GDF15-GFRAL pathway may be useful for maintaining energy expenditure in skeletal muscle during caloric restriction.