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 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 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.

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.

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.

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.

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.

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.

Pharmacological blockade of the cannabinoid type 1 receptor, a G protein-coupled receptor expressed in the central nervous system and various peripheral tissues, reverses diet-induced obesity and dyslipidemia through the reduction of food intake and altered nutrient partitioning. This strategy is being explored for a number of therapeutic applications; however, its potency for the treatment of atherosclerotic cardiovascular disease via improvements in lipid metabolism remains unclear. Therefore, here, we aimed to investigate whether inhibition of the endocannabinoid system can attenuate atherosclerosis development through improvement of dyslipidemia. Lean, dyslipidemic female APOE∗3-Leiden.CETP transgenic mice were fed a Western-type diet supplemented with or without the cannabinoid type 1 receptor inverse agonist rimonabant (20 mg·kg body weight-1 day-1) for up to 20 weeks. Plasma lipids and bile acids were determined, and atherosclerotic lesions were scored in the aortic valve region. Rimonabant lowered plasma levels of triglyceride (TG) (-56%) and non-HDL-C (-19%) and increased HDL-C (+57%). These effects were explained by decreased VLDL-TG production (-52%) and accelerated VLDL-TG turnover accompanied by pronounced browning of white adipose tissue. In addition, rimonabant attenuated reverse cholesterol transport (-30%), increased plasma bile acid levels (+160%), and increased hepatic cholesterol accumulation (+88%). Importantly, rimonabant markedly lowered atherosclerotic lesion size (-64%), which coincided with decreased lesion severity (28% vs. 56% severe lesions) and which strongly correlated with non-HDL-C exposure (R2 = 0.60). Taken together, inhibition of the endocannabinoid system potently reverses dyslipidemia and prevents atherogenesis, even in the absence of obesity.

Glucagon-like peptide-1 receptor agonists (GLP-1RAs) are effective medications to reduce appetite and body weight. These actions are centrally mediated; however, the neuronal substrates involved are poorly understood.

Recent studies have found that glucose-dependent insulinotropic polypeptide receptor (GIPR) agonism can enhance the metabolic efficacy of glucagon-like peptide-1 receptor agonist treatment by promoting both weight-dependent and -independent improvements on systemic insulin sensitivity. These findings have prompted new investigations aimed at better understanding the broad metabolic benefit of GIPR activation. Herein, we determined whether GIPR agonism favorably influenced the pharmacologic efficacy of the insulin-sensitizing thiazolidinedione (TZD) rosiglitazone in obese insulin-resistant (IR) mice. Genetic and pharmacological approaches were used to examine the role of GIPR signaling on rosiglitazone-induced weight gain, hyperphagia, and glycemic control. RNA sequencing was conducted to uncover potential mechanisms by which GIPR activation influences energy balance and insulin sensitivity. In line with previous findings, treatment with rosiglitazone induced the mRNA expression of the GIPR in white and brown fat. However, obese GIPR-null mice dosed with rosiglitazone had equivalent weight gain to that of wild-type (WT) animals. Strikingly, chronic treatment of obese IR WT animals with a long-acting GIPR agonist prevented rosiglitazone-induced weight-gain and hyperphagia, and it enhanced the insulin-sensitivity effect of this TZD. The systemic insulin sensitization was accompanied by increased glucose disposal in brown adipose tissue, which was underlined by the recruitment of metabolic and thermogenic genes. These findings suggest that GIPR agonism can counter the negative consequences of rosiglitazone treatment on body weight and adiposity, while improving its insulin-sensitizing efficacy at the same time.

Combined glucose-dependent insulinotropic polypeptide receptor (GIPR) and glucagon-like peptide-1 receptor (GLP1R) agonism is superior to single GLP1R agonism with respect to glycemic control and weight loss in obese patients with or without type 2 diabetes. As insulin resistance and obesity are strong risk factors for nonalcoholic fatty liver disease (NAFLD), in the current study we investigated the effects of combined GIPR/GLP1R agonism on NAFLD development.

Tirzepatide, a dual GIP and GLP-1 receptor agonist, delivered superior glycemic control and weight loss compared to selective GLP-1 receptor (GLP-1R) agonism in patients with type 2 diabetes (T2D). These results have fueled mechanistic studies focused on understanding how tirzepatide achieves its therapeutic efficacy. Recently, we found that treatment with tirzepatide improves insulin sensitivity in humans with T2D and obese mice in concert with a reduction in circulating levels of branched-chain amino (BCAAs) and keto (BCKAs) acids, metabolites associated with development of systemic insulin resistance (IR) and T2D. Importantly, these systemic effects were found to be coupled to increased expression of BCAA catabolic genes in thermogenic brown adipose tissue (BAT) in mice. These findings led us to hypothesize that tirzepatide may lower circulating BCAAs/BCKAs by promoting their catabolism in BAT.

The induction of nausea and emesis is a major barrier to maximizing the weight loss profile of obesity medications, and therefore, identifying mechanisms that improve tolerability could result in added therapeutic benefit. The development of peptide YY (PYY)-based approaches to treat obesity are no exception, as PYY receptor agonism is often accompanied by nausea and vomiting. Here, we sought to determine whether glucose-dependent insulinotropic polypeptide (GIP) receptor (GIPR) agonism reduces PYY-induced nausea-like behavior in mice. We found that central and peripheral administration of a GIPR agonist reduced conditioned taste avoidance (CTA) without affecting hypophagia mediated by a PYY analog. The receptors for GIP and PYY (Gipr and Npy2r) were found to be expressed by the same neurons in the area postrema (AP), a brainstem nucleus involved in detecting aversive stimuli. Peripheral administration of a GIPR agonist induced neuronal activation (cFos) in the AP. Further, whole-brain cFos analyses indicated that PYY-induced CTA was associated with augmented neuronal activity in the parabrachial nucleus (PBN), a brainstem nucleus that relays aversive/emetic signals to brain regions that control feeding behavior. Importantly, GIPR agonism reduced PYY-mediated neuronal activity in the PBN, providing a potential mechanistic explanation for how GIPR agonist treatment reduces PYY-induced nausea-like behavior. Together, the results of our study indicate a novel mechanism by which GIP-based therapeutics may have benefit in improving the tolerability of weight loss agents.

FGF21 has gradually become a focal point in metabolic research given its intriguing and complex biology and relevance to drug discovery. Despite the large amount of accumulated data, there remains a dearth of understanding of FGF21 physiology at the molecular/whole organism level. The scaffold protein βklotho (KLB) has previously been demonstrated in vitro to function as a co-factor permitting FGF21 mediated FGF receptor activation. However, the requirement for KLB in the propagation of FGF21 action in living animals has yet to be evaluated. To answer this question, we tested FGF21 in mice with total body ablation of KLB (KLBKO) and found no detectable activity. Firstly, we demonstrate that the disruption of KLB entirely abrogates acute FGF21 signaling in adipose tissue. We go on to show that this signaling defect translates to the absence of FGF21 mediated metabolic improvements in DIO mice. Indeed, KLBKO mice are totally refractory to FGF21-induced normalization of glucose homeostasis, attenuation of dyslipidemia, elevation of energy expenditure and weight loss. The lack of FGF21-driven effects was further substantiated at the transcriptional level with no FGF21 target gene signature detectable in adipose tissue and liver of KLBKO animals. Taken together our data show that KLB is a vital component of the FGF21 in vivo signaling machinery and is critically required for FGF21 action at the whole organism level.

The molecular mediator and functional significance of meal-associated brown fat (BAT) thermogenesis remains elusive. Here, we identified the gut hormone secretin as a non-sympathetic BAT activator mediating prandial thermogenesis, which consequentially induces satiation, thereby establishing a gut-secretin-BAT-brain axis in mammals with a physiological role of prandial thermogenesis in the control of satiation. Mechanistically, meal-associated rise in circulating secretin activates BAT thermogenesis by stimulating lipolysis upon binding to secretin receptors in brown adipocytes, which is sensed in the brain and promotes satiation. Chronic infusion of a modified human secretin transiently elevates energy expenditure in diet-induced obese mice. Clinical trials with human subjects showed that thermogenesis after a single-meal ingestion correlated with postprandial secretin levels and that secretin infusions increased glucose uptake in BAT. Collectively, our findings highlight the largely unappreciated function of BAT in the control of satiation and qualify BAT as an even more attractive target for treating obesity.