We have recently shown that the trichodysplasia spinulosa-associated polyomavirus (TSPyV) belongs to a large monophyletic group of mammalian polyomaviruses that experienced accelerated codon-constrained Val-Ala (COCO-VA) toggling at a protein site common to both Middle and Alternative T-antigens (MT/ALTO). Here we analyzed thirteen, mostly newly sequenced TSPyV genomes, representing ~40% of reported TS disease cases world-wide. We found two deletions and 30 variable sites (≤0.6%) that included only four sites with non-synonymous substitutions (NSS). One NSS site was under positive selection in the exon shared by Small and Middle T antigens, while three others were segregated in MT/ALTO. Two MT/ALTO sites covaried with five sites elsewhere in the genome and determined separation of twelve TSPyVs into two most populous phylogenetic lineages. The other, most distant TSPyV was distinguished by NSS at the COCO-VA site, observed for the first time during intra-species evolution. Our findings reveal a connection between micro- and macro-evolution of polyomaviruses.

The FATZO/Pco mouse is the result of a cross of the C57BL/6J and AKR/J strains. The crossing of these two strains and the selective inbreeding for obesity, insulin resistance and hyperglycemia has resulted in an inbred strain exhibiting obesity in the presumed presence of an intact leptin pathway. Routinely used rodent models for obesity and diabetes research have a monogenic defect in leptin signaling that initiates obesity. Given that obesity and its sequelae in humans are polygenic in nature and not associated with leptin signaling defects, the FATZO mouse may represent a more translatable rodent model for study of obesity and its associated metabolic disturbances. The FATZO mouse develops obesity spontaneously when fed a normal chow diet. Glucose intolerance with increased insulin levels are apparent in FATZO mice as young as 6 weeks of age. These progress to hyperglycemia/pre-diabetes and frank diabetes with decreasing insulin levels as they age. The disease in these mice is multi-faceted, similar to the metabolic syndrome apparent in obese individuals, and thus provides a long pre-diabetic state for determining the preventive value of new interventions. We have assessed the utility of this new model for the pre-clinical screening of agents to stop or slow progression of the metabolic syndrome to severe diabetes. Our assessment included: 1) characterization of the spontaneous development of disease, 2) comparison of metabolic disturbances of FATZO mice to control mice and 3) validation of the model with regard to the effectiveness of current and emerging anti-diabetic agents; rosiglitazone, metformin and semaglutide.

Obesity in many current pre-clinical animal models of obesity and diabetes is mediated by monogenic mutations; these are rarely associated with the development of human obesity. A new mouse model, the FATZO mouse, has been developed to provide polygenic obesity and a metabolic pattern of hyperglycemia and hyperinsulinemia, that support the presence of insulin resistance similar to metabolic disease in patients with insulin resistance/type 2 diabetes. The FATZO mouse resulted from a cross of C57BL/6J and AKR/J mice followed by selective inbreeding for obesity, increased insulin and hyperglycemia. Since many clinical studies have established a close link between higher body weight and the development of type 2 diabetes, we investigated whether time to progression to type 2 diabetes or disease severity in FATZO mice was dependent on weight gain in young animals. Our results indicate that lighter animals developed metabolic disturbances much slower and to a lesser magnitude than their heavier counterparts. Consumption of a diet containing high fat, accelerated weight gain in parallel with disease progression. A naturally occurring and significant variation in the body weight of FATZO offspring enables these mice to be identified as low, mid and high body weight groups at a young age. These weight groups remain into adulthood and correspond to slow, medium and accelerated development of type 2 diabetes. Thus, body weight inclusion criteria can optimize the FATZO model for studies of prevention, stabilization or treatment of type 2 diabetes.

Brown adipose tissue (BAT) activation stimulates energy expenditure in human adults, which makes it an attractive target to combat obesity and related disorders. Recent studies demonstrated a role for G protein-coupled receptor 120 (GPR120) in BAT thermogenesis. Here, we investigated the therapeutic potential of GPR120 agonism and addressed GPR120-mediated signaling in BAT We found that activation of GPR120 by the selective agonist TUG-891 acutely increases fat oxidation and reduces body weight and fat mass in C57Bl/6J mice. These effects coincided with decreased brown adipocyte lipid content and increased nutrient uptake by BAT, confirming increased BAT activity. Consistent with these observations, GPR120 deficiency reduced expression of genes involved in nutrient handling in BAT Stimulation of brown adipocytes in vitro with TUG-891 acutely induced O2 consumption, through GPR120-dependent and GPR120-independent mechanisms. TUG-891 not only stimulated GPR120 signaling resulting in intracellular calcium release, mitochondrial depolarization, and mitochondrial fission, but also activated UCP1. Collectively, these data suggest that activation of brown adipocytes with the GPR120 agonist TUG-891 is a promising strategy to increase lipid combustion and reduce obesity.

The cytokine, GDF15, is produced in pathological states which cause cellular stress, including cancer. When over expressed, it causes dramatic weight reduction, suggesting a role in disease-related anorexia. Here, we demonstrate that the GDF15 receptor, GFRAL, is located in a subset of cholecystokinin neurons which span the area postrema and the nucleus of the tractus solitarius of the mouse. GDF15 activates GFRALAP/NTS neurons and supports conditioned taste and place aversions, while the anorexia it causes can be blocked by a monoclonal antibody directed at GFRAL or by disrupting CCK neuronal signalling. The cancer-therapeutic drug, cisplatin, induces the release of GDF15 and activates GFRALAP/NTS neurons, as well as causing significant reductions in food intake and body weight in mice. These metabolic effects of cisplatin are abolished by pre-treatment with the GFRAL monoclonal antibody. Our results suggest that GFRAL neutralising antibodies or antagonists may provide a co-treatment opportunity for patients undergoing chemotherapy.

Brown fat activation accelerates the uptake of cholesterol-enriched remnants by the liver and thereby lowers plasma cholesterol, consequently protecting against atherosclerosis development. Hepatic cholesterol is then converted into bile acids (BAs) that are secreted into the intestine and largely maintained within the enterohepatic circulation. We now aimed to evaluate the effects of prolonged brown fat activation combined with inhibition of intestinal BA reabsorption on plasma cholesterol metabolism and atherosclerosis development.

Synthetic glucocorticoids are clinically used to treat auto-immune and inflammatory disease. Despite the high efficacy, glucocorticoid treatments causes side effects such as obesity and insulin resistance in many patients. Via their pharmacological target, the glucocorticoid receptor (GR), glucocorticoids suppress endogenous glucocorticoid secretion. Endogenous, but not synthetic, glucocorticoids activate the mineralocorticoid receptor (MR) and side effects of synthetic glucocorticoids may thus not only result from GR hyperactivation but also from MR hypoactivation. Here, we tested the hypothesis that reactivation of MR with corticosterone add-on treatment can attenuate the metabolic effects of the synthetic glucocorticoid dexamethasone. Male 8-week-old C57Bl/6J mice received a high-fat diet supplemented with dexamethasone or vehicle, and were subcutaneously implanted with low-dose corticosterone- or vehicle-containing pellets. Dexamethasone strongly reduced body weight and fat mass gain, while corticosterone add-on partially normalized this. Dexamethasone-induced hyperglycemia and hyperinsulinemia were exacerbated by corticosterone add-on, which was prevented by MR antagonism. In subcutaneous white adipose tissue, corticosterone add-on prevented the dexamethasone-induced expression of intracellular lipolysis genes. In brown adipose tissue, dexamethasone also upregulated gene expression of brown adipose tissue identity markers, lipid transporters and lipolysis enzymes, which was prevented by corticosterone add-on. In conclusion, corticosterone add-on treatment prevents several, while exacerbating other metabolic effects of dexamethasone. While the exact role of MR remains elusive, this study suggests that corticosterone suppression by dexamethasone contributes to its effects in mice.

Thermogenic adipocytes possess a therapeutically appealing, energy-expending capacity, which is canonically cold-induced by ligand-dependent activation of β-adrenergic G protein-coupled receptors (GPCRs). Here, we uncover an alternate paradigm of GPCR-mediated adipose thermogenesis through the constitutively active receptor, GPR3. We show that the N terminus of GPR3 confers intrinsic signaling activity, resulting in continuous Gs-coupling and cAMP production without an exogenous ligand. Thus, transcriptional induction of Gpr3 represents the regulatory parallel to ligand-binding of conventional GPCRs. Consequently, increasing Gpr3 expression in thermogenic adipocytes is alone sufficient to drive energy expenditure and counteract metabolic disease in mice. Gpr3 transcription is cold-stimulated by a lipolytic signal, and dietary fat potentiates GPR3-dependent thermogenesis to amplify the response to caloric excess. Moreover, we find GPR3 to be an essential, adrenergic-independent regulator of human brown adipocytes. Taken together, our findings reveal a noncanonical mechanism of GPCR control and thermogenic activation through the lipolysis-induced expression of constitutively active GPR3.

Microglia play a critical role in maintaining neural function. While microglial activity follows a circadian rhythm, it is not clear how this intrinsic clock relates to their function, especially in stimulated conditions such as in the control of systemic energy homeostasis or memory formation. In this study, we found that microglia-specific knock-down of the core clock gene, Bmal1, resulted in increased microglial phagocytosis in mice subjected to high-fat diet (HFD)-induced metabolic stress and likewise among mice engaged in critical cognitive processes. Enhanced microglial phagocytosis was associated with significant retention of pro-opiomelanocortin (POMC)-immunoreactivity in the mediobasal hypothalamus in mice on a HFD as well as the formation of mature spines in the hippocampus during the learning process. This response ultimately protected mice from HFD-induced obesity and resulted in improved performance on memory tests. We conclude that loss of the rigorous control implemented by the intrinsic clock machinery increases the extent to which microglial phagocytosis can be triggered by neighboring neurons under metabolic stress or during memory formation. Taken together, microglial responses associated with loss of Bmal1 serve to ensure a healthier microenvironment for neighboring neurons in the setting of an adaptive response. Thus, microglial Bmal1 may be an important therapeutic target for metabolic and cognitive disorders with relevance to psychiatric disease.

To evaluate the effects of tirzepatide on body composition, appetite, and energy intake to address the potential mechanisms involved in body weight loss with tirzepatide.

Disturbance of the circadian clock has been associated with increased risk of cardio-metabolic disorders. Previous studies showed that optimal timing of food intake can improve metabolic health. We hypothesized that time-restricted feeding could be a strategy to minimize long term adverse metabolic health effects of shift work and jetlag. In this study, we exposed female FVB mice to weekly alternating light-dark cycles (i.e. 12 h shifts) combined with ad libitum feeding, dark phase feeding or feeding at a fixed clock time, in the original dark phase. In contrast to our expectations, long-term disturbance of the circadian clock had only modest effects on metabolic parameters. Mice fed at a fixed time showed a delayed adaptation compared to ad libitum fed animals, in terms of the similarity in 24 h rhythm of core body temperature, in weeks when food was only available in the light phase. This was accompanied by increased plasma triglyceride levels and decreased energy expenditure, indicating a less favorable metabolic state. On the other hand, dark phase feeding accelerated adaptation of core body temperature and activity rhythms, however, did not improve the metabolic state of animals compared to ad libitum feeding. Taken together, restricting food intake to the active dark phase enhanced adaptation to shifts in the light-dark schedule, without significantly affecting metabolic parameters.

Oxytocin (OXT) has been shown to suppress appetite, induce weight loss, and improve glycemic control and lipid metabolism in several species, including humans, monkeys, and rodents. However, OXT's short half-life in circulation and lack of receptor selectivity limit its application and efficacy. In this study, we report an OXT peptide analog (OXTGly) that is potent and selective for the OXT receptor (OXTR). OXT, but not OXTGly, activated vasopressin receptors in vitro and acutely increased blood pressure in vivo when administered IP. OXT suppressed food intake in mice, whereas OXTGly had a moderate effect on food intake when administered IP or intracerebroventricularly. Both OXT (IP) and OXTGly (IP) improved glycemic control in glucose tolerance tests. Additionally, both OXT (IP) and OXTGly (IP) stimulated insulin, glucagon-like peptide 1, and glucagon secretion in mice. We generated lipid-conjugated OXT (acylated-OXT) and OXTGly (acylated-OXTGly) and demonstrated that these molecules have significantly extended half-lives in vivo. Compared with OXT, 2-week treatment of diet-induced obese mice with acylated-OXT [subcutaneous(ly) (SC)] resulted in enhanced body weight reduction, an improved lipid profile, and gene expression changes consistent with increased lipolysis and decreased gluconeogenesis. Treatment with acylated-OXTGly (SC) also resulted in a statistically significant weight loss, albeit to a lesser degree compared with acylated-OXT treatment. In conclusion, we demonstrate that selective activation of the OXTR pathway results in both acute and chronic metabolic benefits, whereas potential activation of vasopressin receptors by nonselective OXT analogs causes physiological stress that contributes to additional weight loss.

The endocannabinoid system (ECS) controls energy balance by regulating both energy intake and energy expenditure. Endocannabinoid levels are elevated in obesity suggesting a potential causal relationship. This study aimed to elucidate the rate of dysregulation of the ECS, and the metabolic organs involved, in diet-induced obesity. Eight groups of age-matched male C57Bl/6J mice were randomized to receive a chow diet (control) or receive a high fat diet (HFD, 45% of calories derived from fat) ranging from 1 day up to 18 weeks before euthanasia. Plasma levels of the endocannabinoids 2-arachidonoylglycerol (2-AG) and anandamide (N-arachidonoylethanolamine, AEA), and related N-acylethanolamines, were quantified by UPLC-MS/MS and gene expression of components of the ECS was determined in liver, muscle, white adipose tissue (WAT) and brown adipose tissue (BAT) during the course of diet-induced obesity development. HFD feeding gradually increased 2-AG (+132% within 4 weeks, P < 0.05), accompanied by upregulated expression of its synthesizing enzymes Daglα and β in WAT and BAT. HFD also rapidly increased AEA (+81% within 1 week, P < 0.01), accompanied by increased expression of its synthesizing enzyme Nape-pld, specifically in BAT. Interestingly, Nape-pld expression in BAT correlated with plasma AEA levels (R 2 = 0.171, β = 0.276, P < 0.001). We conclude that a HFD rapidly activates adipose tissue depots to increase the synthesis pathways of endocannabinoids that may aggravate the development of HFD-induced obesity.

Chronic low-grade inflammation and increased serum levels of the cytokine IL-6 accompany obesity. For brain-produced IL-6, the mechanisms by which it controls energy balance and its role in obesity remain unclear. Here, we show that brain-produced IL-6 is decreased in obese mice and rats in a neuroanatomically and sex-specific manner. Reduced IL-6 mRNA localized to lateral parabrachial nucleus (lPBN) astrocytes, microglia, and neurons, including paraventricular hypothalamus-innervating lPBN neurons. IL-6 microinjection into lPBN reduced food intake and increased brown adipose tissue (BAT) thermogenesis in male lean and obese rats by increasing thyroid and sympathetic outflow to BAT. Parabrachial IL-6 interacted with leptin to reduce feeding. siRNA-mediated reduction of lPBN IL-6 leads to increased weight gain and adiposity, reduced BAT thermogenesis, and increased food intake. Ambient cold exposure partly normalizes the obesity-induced suppression of lPBN IL-6. These results indicate that lPBN-produced IL-6 regulates feeding and metabolism and pinpoints (patho)physiological contexts interacting with lPBN IL-6.

Brown adipose tissue (BAT) combusts high amounts of fatty acids, thereby lowering plasma triglyceride levels and reducing obesity. However, the precise role of BAT in plasma cholesterol metabolism and atherosclerosis development remains unclear. Here we show that BAT activation by β3-adrenergic receptor stimulation protects from atherosclerosis in hyperlipidemic APOE*3-Leiden.CETP mice, a well-established model for human-like lipoprotein metabolism that unlike hyperlipidemic Apoe(-/-) and Ldlr(-/-) mice expresses functional apoE and LDLR. BAT activation increases energy expenditure and decreases plasma triglyceride and cholesterol levels. Mechanistically, we demonstrate that BAT activation enhances the selective uptake of fatty acids from triglyceride-rich lipoproteins into BAT, subsequently accelerating the hepatic clearance of the cholesterol-enriched remnants. These effects depend on a functional hepatic apoE-LDLR clearance pathway as BAT activation in Apoe(-/-) and Ldlr(-/-) mice does not attenuate hypercholesterolaemia and atherosclerosis. We conclude that activation of BAT is a powerful therapeutic avenue to ameliorate hyperlipidaemia and protect from atherosclerosis.

Fibroblast growth factor 21 is a novel hormonal regulator with the potential to treat a broad variety of metabolic abnormalities, such as type 2 diabetes, obesity, hepatic steatosis, and cardiovascular disease. Human recombinant wild type FGF21 (FGF21) has been shown to ameliorate metabolic disorders in rodents and non-human primates. However, development of FGF21 as a drug is challenging and requires re-engineering of its amino acid sequence to improve protein expression and formulation stability. Here we report the design and characterization of a novel FGF21 variant, LY2405319. To enable the development of a potential drug product with a once-daily dosing profile, in a preserved, multi-use formulation, an additional disulfide bond was introduced in FGF21 through Leu118Cys and Ala134Cys mutations. FGF21 was further optimized by deleting the four N-terminal amino acids, His-Pro-Ile-Pro (HPIP), which was subject to proteolytic cleavage. In addition, to eliminate an O-linked glycosylation site in yeast a Ser167Ala mutation was introduced, thus allowing large-scale, homogenous protein production in Pichia pastoris. Altogether re-engineering of FGF21 led to significant improvements in its biopharmaceutical properties. The impact of these changes was assessed in a panel of in vitro and in vivo assays, which confirmed that biological properties of LY2405319 were essentially identical to FGF21. Specifically, subcutaneous administration of LY2405319 in ob/ob and diet-induced obese (DIO) mice over 7-14 days resulted in a 25-50% lowering of plasma glucose coupled with a 10-30% reduction in body weight. Thus, LY2405319 exhibited all the biopharmaceutical and biological properties required for initiation of a clinical program designed to test the hypothesis that administration of exogenous FGF21 would result in effects on disease-related metabolic parameters in humans.

Fibroblast growth factors 19 (FGF19) and 21 (FGF21) have emerged as key regulators of energy metabolism. Several studies have been conducted to understand the mechanism of FGF19 and FGF21 action, however, the data presented has often been inconsistent and at times contradictory. Here in a single study we compare the mechanisms mediating FGF19/FGF21 actions, and how similarities/differences in actions at the cellular level between these two factors translate to common/divergent physiological outputs. Firstly, we show that in cell culture FGF19/FGF21 are very similar, however, key differences are still observed differentiating the two. In vitro we found that both FGF's activate FGFRs in the context of βKlotho (KLB) expression. Furthermore, both factors alter ERK phosphorylation and glucose uptake with comparable potency. Combination treatment of cells with both factors did not have additive effects and treatment with a competitive inhibitor, the FGF21 delta N17 mutant, also blocked FGF19's effects, suggestive of a shared receptor activation mechanism. The key differences between FGF21/FGF19 were noted at the receptor interaction level, specifically the unique ability of FGF19 to bind/signal directly via FGFR4. To determine if differential effects on energy homeostasis and hepatic mitogenicity exist we treated DIO and ob/ob mice with FGF19/FGF21. We find comparable efficacy of the two proteins to correct body weight and serum glucose in both DIO and ob/ob mice. Nevertheless, FGF21 and FGF19 had distinctly different effects on proliferation in the liver. Interestingly, in vivo blockade of FGF21 signaling in mice using ΔN17 caused profound changes in glycemia indicative of the critical role KLB and FGF21 play in the regulation of glucose homeostasis. Overall, our data demonstrate that while subtle differences exist in vitro the metabolic effects in vivo of FGF19/FGF21 are indistinguishable, supporting a shared mechanism of action for these two hormones in the regulation of energy balance.

The effect of dual glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide-1 (GLP-1) receptor agonist (RA) tirzepatide on gastric emptying (GE) was compared to that of GLP-1RAs in non-clinical and clinical studies. GE was assessed following acute and chronic treatment with tirzepatide in diet-induced obese mice versus semaglutide or long-acting GIP analogue alone. Participants [with and without type 2 diabetes (T2DM)] from a phase 1, 4-week multiple dose study received tirzepatide, dulaglutide or placebo. GE was assessed by acetaminophen absorption. In mice, tirzepatide delayed GE to a similar degree to that achieved with semaglutide; however, these acute inhibitory effects were abolished after 2 weeks of treatment. GIP analogue alone had no effect on GE or on GLP-1's effect on GE. In participants with and without T2DM, once-weekly tirzepatide (≥5 and ≥4.5 mg, respectively) delayed GE after a single dose. This effect diminished after multiple doses of tirzepatide or dulaglutide in healthy participants. In participants with T2DM treated with an escalation schedule of tirzepatide 5/5/10/10 or 5/5/10/15 mg, a residual GE delay was still observed after multiple doses. These data suggest that tirzepatide's activity on GE is comparable to that of selective GLP-1RAs.

Systemic exposure to high-dose corticosteroids effectively combats acute rejection after kidney transplantation, but at the cost of substantial side effects. In this study, a murine acute renal allograft rejection model was used to investigate whether liposomal-encapsulated prednisolone (LP) facilitates local exposure to enhance its therapeutic effect.

To compare the effects of cold exposure and the β3-adrenergic receptor agonist mirabegron on plasma lipids, energy expenditure and brown adipose tissue (BAT) activity in South Asians versus Europids.