A Brain-Melanocortin-Vagus Axis Mediates Adipose Tissue Expansion Independently of Energy Intake.

The melanocortin system is a brain circuit that influences energy balance by regulating energy intake and expenditure. In addition, the brain-melanocortin system controls adipose tissue metabolism to optimize fuel mobilization and storage. Specifically, increased brain-melanocortin signaling or negative energy balance promotes lipid mobilization by increasing sympathetic nervous system input to adipose tissue. In contrast, calorie-independent mechanisms favoring energy storage are less understood. Here, we demonstrate that reduction of brain-melanocortin signaling actively promotes fat mass gain by activating the lipogenic program and adipocyte and endothelial cell proliferation in white fat depots independently of caloric intake via efferent nerve fibers conveyed by the common hepatic branch of the vagus nerve. Those vagally regulated obesogenic signals also contribute to the fat mass gain following chronic high-fat diet feeding. These data reveal a physiological mechanism whereby the brain controls energy stores that may contribute to increased susceptibility to obesity.

Deletion of the glucagon receptor gene before and after experimental diabetes reveals differential protection from hyperglycemia.

OBJECTIVE: Mice with congenital loss of the glucagon receptor gene (Gcgr-/- mice) remain normoglycemic in insulinopenic conditions, suggesting that unopposed glucagon action is the driving force for hyperglycemia in Type-1 Diabetes Mellitus (T1DM). However, chronic loss of GCGR results in a neomorphic phenotype that includes hormonal signals with hypoglycemic activity. We combined temporally-controlled GCGR deletion with pharmacological treatments to dissect the direct contribution of GCGR signaling to glucose control in a common mouse model of T1DM. METHODS: We induced experimental T1DM by injecting the beta-cell cytotoxin streptozotocin (STZ) in mice with congenital or temporally-controlled Gcgr loss-of-function using tamoxifen (TMX). RESULTS: Disruption of Gcgr expression, using either an inducible approach in adult mice or animals with congenital knockout, abolished the response to a long-acting Gcgr agonist. Mice with either developmental Gcgr disruption or inducible deletion several weeks before STZ treatment maintained normoglycemia. However, mice with inducible knockout of the Gcgr one week after the onset of STZ diabetes had only partial correction of hyperglycemia, an effect that was reversed by GLP-1 receptor blockade. Mice with Gcgr deletion for either 2 or 6 weeks had similar patterns of gene expression, although the changes were generally larger with longer GCGR knockout. CONCLUSIONS: These findings demonstrate that the effects of glucagon to mitigate diabetic hyperglycemia are not through acute signaling but require compensations that take weeks to develop.

An Hsp20-FBXO4 Axis Regulates Adipocyte Function through Modulating PPARγ Ubiquitination.

Exposure to cold temperature is well known to upregulate heat shock protein (Hsp) expression and recruit and/or activate brown adipose tissue and beige adipocytes in humans and animals. However, whether and how Hsps regulate adipocyte function for energy homeostatic responses is poorly understood. Here, we demonstrate a critical role of Hsp20 as a negative regulator of adipocyte function. Deletion of Hsp20 enhances non-shivering thermogenesis and suppresses inflammatory responses, leading to improvement of glucose and lipid metabolism under both chow diet and high-fat diet conditions. Mechanistically, Hsp20 controls adipocyte function by interacting with the subunit of the ubiquitin ligase complex, F-box only protein 4 (FBXO4), and regulating the ubiquitin-dependent degradation of peroxisome proliferation activated receptor gamma (PPARγ). Indeed, Hsp20 deficiency mimics and enhances the pharmacological effects of the PPARγ agonist rosiglitazone. Together, our findings suggest a role of Hsp20 in mediating adipocyte function by linking ß-adrenergic signaling to PPARγ activity.

Contribution of brown adipose tissue activity to the control of energy balance by GLP-1 receptor signalling in mice.

AIMS/HYPOTHESIS: We assessed the contribution of glucagon-like peptide-1 (GLP-1) receptor (GLP-1R) signalling to thermogenesis induced by high-fat diet (HFD) consumption. Furthermore, we determined whether brown adipose tissue (BAT) activity contributes to weight loss induced by chronic subcutaneous treatment with the GLP-1R agonist, liraglutide, in a model of diet-induced obesity. METHODS: Metabolic phenotyping was performed using indirect calorimetry in wild-type (WT) and Glp1r-knockout (KO) mice during chow and HFD feeding at room temperature and at thermoneutrality. In a separate study, we investigated the contribution of BAT thermogenic capacity to the weight lowering effect induced by GLP-1 mimetics by administering liraglutide (10 or 30 nmol kg(-1) day(-1) s.c.) to diet-induced obese (DIO) mice for 6 or 4 weeks, respectively. In both studies, animals were subjected to a noradrenaline (norepinephrine)-stimulated oxygen consumption [Formula: see text] test. RESULTS: At thermoneutrality, HFD-fed Glp1r-KO mice had similar energy expenditure (EE) compared with HFD-fed WT controls. However, HFD-fed Glp1r-KO mice exhibited relatively less EE when housed at a cooler standard room temperature, and had relatively lower [Formula: see text] in response to a noradrenaline challenge, which is consistent with impaired BAT thermogenic capacity. In contrast to the loss of function model, chronic peripheral liraglutide treatment did not increase BAT activity as determined by noradrenaline-stimulated [Formula: see text] and BAT gene expression. CONCLUSIONS/INTERPRETATION: These data suggest that although endogenous GLP-1R signalling contributes to increased BAT thermogenesis, this mechanism does not play a significant role in the food intake-independent body weight lowering effect of the GLP-1 mimetic liraglutide in DIO mice.

Tachykinin-1 in the central nervous system regulates adiposity in rodents.

Ghrelin is a circulating hormone that targets the central nervous system to regulate feeding and adiposity. The best-characterized neural system that mediates the effects of ghrelin on energy balance involves the activation of neuropeptide Y/agouti-related peptide neurons, expressed exclusively in the arcuate nucleus of the hypothalamus. However, ghrelin receptors are expressed in other neuronal populations involved in the control of energy balance. We combined laser capture microdissection of several nuclei of the central nervous system expressing the ghrelin receptor (GH secretagoge receptor) with microarray gene expression analysis to identify additional neuronal systems involved in the control of central nervous system-ghrelin action. We identified tachykinin-1 (Tac1) as a gene negatively regulated by ghrelin in the hypothalamus. Furthermore, we identified neuropeptide k as the TAC1-derived peptide with more prominent activity, inducing negative energy balance when delivered directly into the brain. Conversely, loss of Tac1 expression enhances the effectiveness of ghrelin promoting fat mass gain both in male and in female mice and increases the susceptibility to diet-induced obesity in ovariectomized mice. Taken together, our data demonstrate a role TAC1 in the control energy balance by regulating the levels of adiposity in response to ghrelin administration and to changes in the status of the gonadal function.

The melanocortin-4 receptor integrates circadian light cues and metabolism.

The melanocortin system directs diverse physiological functions from coat color to body weight homoeostasis. A commonality among melanocortin-mediated processes is that many animals modulate similar processes on a circannual basis in response to longer, summer days, suggesting an underlying link between circadian biology and the melanocortin system. Despite key neuroanatomical substrates shared by both circadian and melanocortin-signaling pathways, little is known about the relationship between the two. Here we identify a link between circadian disruption and the control of glucose homeostasis mediated through the melanocortin-4 receptor (Mc4r). Mc4r-deficient mice exhibit exaggerated circadian fluctuations in baseline blood glucose and glucose tolerance. Interestingly, exposure to lighting conditions that disrupt circadian rhythms improve their glucose tolerance. This improvement occurs through an increase in glucose clearance by skeletal muscle and is food intake and body weight independent. Restoring Mc4r expression to the paraventricular nucleus prevents the improvement in glucose tolerance, supporting a role for the paraventricular nucleus in the integration of circadian light cues and metabolism. Altogether these data suggest that Mc4r signaling plays a protective role in minimizing glucose fluctuations due to circadian rhythms and environmental light cues and demonstrate a previously undiscovered connection between circadian biology and glucose metabolism mediated through the melanocortin system.

Duodenal nutrient exclusion improves metabolic syndrome and stimulates villus hyperplasia.

OBJECTIVE: Surgical interventions that prevent nutrient exposure to the duodenum are among the most successful treatments for obesity and diabetes. However, these interventions are highly invasive, irreversible and often carry significant risk. The duodenal-endoluminal sleeve (DES) is a flexible tube that acts as a barrier to nutrient-tissue interaction along the duodenum. We implanted this device in Zucker Diabetic Fatty (ZDF) rats to gain greater understanding of duodenal nutrient exclusion on glucose homeostasis. DESIGN: ZDF rats were randomised to four groups: Naive, sham ad libitum, sham pair-fed, and DES implanted. Food intake, body weight (BW) and body composition were measured for 28 days postoperatively. Glucose, lipid and bile acid metabolism were evaluated, as well as histological assessment of the upper intestine. RESULTS: DES implantation induced a sustained decrease in BW throughout the study that was matched by pair-fed sham animals. Decreased BW resulted from loss of fat, but not lean mass. DES rats were also found to be more glucose tolerant than either ad libitum-fed or pair-fed sham controls, suggesting fat mass independent metabolic benefits. DES also reduced circulating triglyceride and glycerol levels while increasing circulating bile acids. Interestingly, DES stimulated a considerable increase in villus length throughout the upper intestine, which may contribute to metabolic improvements. CONCLUSIONS: Our preclinical results validate DES as a promising therapeutic approach to diabetes and obesity, which offers reversibility, low risk, low invasiveness and triple benefits including fat mass loss, glucose and lipid metabolism improvement which mechanistically may involve increased villus growth in the upper gut.

GLP-1R responsiveness predicts individual gastric bypass efficacy on glucose tolerance in rats.

Several bariatric operations are currently used to treat obesity and obesity-related comorbidities. These vary in efficacy, but most are more effective than current pharmaceutical treatments. Roux-en-Y gastric bypass (RYGB) produces substantial body weight (BW) loss and enhanced glucose tolerance, and is associated with increased secretion of the gut hormone glucagon-like peptide 1 (GLP-1). Given the success of GLP-1-based agents in lowering blood glucose levels and BW, we hypothesized that an individual sensitivity to GLP-1 receptor agonism could predict metabolic benefits of surgeries associated with increased GLP-1 secretion. One hundred ninety-seven high-fat diet-induced obese male Long-Evans rats were monitored for BW loss during exendin-4 (Ex4) administration. Stable populations of responders and nonresponders were identified based on Ex4-induced BW loss and GLP-1-induced improvements in glucose tolerance. Subpopulations of Ex4 extreme responders and nonresponders underwent RYGB surgery. After RYGB, responders and nonresponders showed similar BW loss compared with sham, but nonresponders retained impaired glucose tolerance. These data indicate that the GLP-1 response tests may predict some but not all of the improvements observed after RYGB. These findings present an opportunity to optimize the use of bariatric surgery based on an improved understanding of GLP-1 biology and suggest an opportunity for a more personalized therapeutic approach to the metabolic syndrome.

Unimolecular dual incretins maximize metabolic benefits in rodents, monkeys, and humans.

We report the discovery and translational therapeutic efficacy of a peptide with potent, balanced co-agonism at both of the receptors for the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP). This unimolecular dual incretin is derived from an intermixed sequence of GLP-1 and GIP, and demonstrated enhanced antihyperglycemic and insulinotropic efficacy relative to selective GLP-1 agonists. Notably, this superior efficacy translated across rodent models of obesity and diabetes, including db/db mice and ZDF rats, to primates (cynomolgus monkeys and humans). Furthermore, this co-agonist exhibited synergism in reducing fat mass in obese rodents, whereas a selective GIP agonist demonstrated negligible weight-lowering efficacy. The unimolecular dual incretins corrected two causal mechanisms of diabesity, adiposity-induced insulin resistance and pancreatic insulin deficiency, more effectively than did selective mono-agonists. The duration of action of the unimolecular dual incretins was refined through site-specific lipidation or PEGylation to support less frequent administration. These peptides provide comparable pharmacology to the native peptides and enhanced efficacy relative to similarly modified selective GLP-1 agonists. The pharmacokinetic enhancement lessened peak drug exposure and, in combination with less dependence on GLP-1-mediated pharmacology, avoided the adverse gastrointestinal effects that typify selective GLP-1-based agonists. This discovery and validation of a balanced and high-potency dual incretin agonist enables a more physiological approach to management of diseases associated with impaired glucose tolerance.

GLP-1R agonism enhances adjustable gastric banding in diet-induced obese rats.

Bariatric procedures vary in efficacy, but overall are more effective than behavioral and pharmaceutical treatment. Roux-en-Y gastric bypass causes increased secretion of glucagon-like peptide 1 (GLP-1) and reduces body weight (BW) more than adjustable gastric banding (AGB), which does not trigger increased GLP-1 secretion. Since GLP-1-based drugs consistently reduce BW, we hypothesized that GLP-1 receptor (GLP-1R) agonists would augment the effects of AGB. Male Long-Evans rats with diet-induced obesity received AGB implantation or sham surgery. GLP-1R agonism, cannabinoid receptor-1 (CB1-R) antagonism, or vehicle was combined with inflation to evaluate interaction between AGB and pharmacological treatments. GLP1-R agonism reduced BW in both sham and AGB rats (left uninflated) compared with vehicle-treated animals. Subsequent band inflation was ineffective in vehicle-treated rats but enhanced weight loss stimulated by GLP1-R agonism. In contrast, there was no additional BW loss when CB1-R antagonism was given with AGB. We found band inflation to trigger neural activation in areas of the nucleus of the solitary tract known to be targeted by GLP-1R agonism, offering a potential mechanism for the interaction. These data show that GLP-1R agonism, but not CB1-R antagonism, improves weight loss achieved by AGB and suggest an opportunity to optimize bariatric surgery with adjunctive pharmacotherapy.

The orphan receptor Gpr83 regulates systemic energy metabolism via ghrelin-dependent and ghrelin-independent mechanisms.

The G protein-coupled receptor 83 (Gpr83) is widely expressed in brain regions regulating energy metabolism. Here we report that hypothalamic expression of Gpr83 is regulated in response to nutrient availability and is decreased in obese mice compared with lean mice. In the arcuate nucleus, Gpr83 colocalizes with the ghrelin receptor (Ghsr1a) and the agouti-related protein. In vitro analyses show heterodimerization of Gpr83 with Ghsr1a diminishes activation of Ghsr1a by acyl-ghrelin. The orexigenic and adipogenic effect of ghrelin is accordingly potentiated in Gpr83-deficient mice. Interestingly, Gpr83 knock-out mice have normal body weight and glucose tolerance when fed a regular chow diet, but are protected from obesity and glucose intolerance when challenged with a high-fat diet, despite hyperphagia and increased hypothalamic expression of agouti-related protein, Npy, Hcrt and Ghsr1a. Together, our data suggest that Gpr83 modulates ghrelin action but also indicate that Gpr83 regulates systemic metabolism through other ghrelin-independent pathways.

Ablation of ghrelin O-acyltransferase does not improve glucose intolerance or body adiposity in mice on a leptin-deficient ob/ob background.

Type 2 Diabetes is a global health burden and based on current estimates will become an even larger problem in the future. Developing new strategies to prevent and treat diabetes is a scientific challenge of high priority. The stomach hormone ghrelin has been associated with playing a role in the regulation of glucose homeostasis. However, its precise mechanism and impact on whole glucose metabolism remains to be elucidated. This study aims to clarify the role of the two ghrelin isoforms acyl- and desacyl ghrelin in regulating glucose homeostasis. Therefore ghrelin activating enzyme Ghrelin-O-acyltransferase (GOAT) was ablated in leptin-deficient ob/ob mice to study whether specific acyl ghrelin deficiency or desacyl ghrelin abundance modifies glucose tolerance on a massively obese background. As targeted deletion of acyl ghrelin does not improve glucose homeostasis in our GOAT-ob/ob mouse model we conclude that neither acyl ghrelin nor the increased ratio of desacyl/acyl ghrelin is crucial for controlling glucose homeostasis in the here presented model of massive obesity induced by leptin deficiency.

Fibroblast growth factor 21 mediates specific glucagon actions.

Glucagon, an essential regulator of glucose homeostasis, also modulates lipid metabolism and promotes weight loss, as reflected by the wasting observed in glucagonoma patients. Recently, coagonist peptides that include glucagon agonism have emerged as promising therapeutic candidates for the treatment of obesity and diabetes. We developed a novel stable and soluble glucagon receptor (GcgR) agonist, which allowed for in vivo dissection of glucagon action. As expected, chronic GcgR agonism in mice resulted in hyperglycemia and lower body fat and plasma cholesterol. Notably, GcgR activation also raised hepatic expression and circulating levels of fibroblast growth factor 21 (FGF21). This effect was retained in isolated primary hepatocytes from wild-type (WT) mice, but not GcgR knockout mice. We confirmed this link in healthy human volunteers, where injection of natural glucagon increased plasma FGF21 within hours. Functional relevance was evidenced in mice with genetic deletion of FGF21, where GcgR activation failed to induce the body weight loss and lipid metabolism changes observed in WT mice. Taken together, these data reveal for the first time that glucagon controls glucose, energy, and lipid metabolism at least in part via FGF21-dependent pathways.

p62 links ß-adrenergic input to mitochondrial function and thermogenesis.

The scaffold protein p62 (sequestosome 1; SQSTM1) is an emerging key molecular link among the metabolic, immune, and proliferative processes of the cell. Here, we report that adipocyte-specific, but not CNS-, liver-, muscle-, or myeloid-specific p62-deficient mice are obese and exhibit a decreased metabolic rate caused by impaired nonshivering thermogenesis. Our results show that p62 regulates energy metabolism via control of mitochondrial function in brown adipose tissue (BAT). Accordingly, adipocyte-specific p62 deficiency led to impaired mitochondrial function, causing BAT to become unresponsive to ß-adrenergic stimuli. Ablation of p62 leads to decreased activation of p38 targets, affecting signaling molecules that control mitochondrial function, such as ATF2, CREB, PGC1α, DIO2, NRF1, CYTC, COX2, ATP5ß, and UCP1. p62 ablation in HIB1B and BAT primary cells demonstrated that p62 controls thermogenesis in a cell-autonomous manner, independently of brown adipocyte development or differentiation. Together, our data identify p62 as a novel regulator of mitochondrial function and brown fat thermogenesis.

Targeted estrogen delivery reverses the metabolic syndrome.

We report the development of a new combinatorial approach that allows for peptide-mediated selective tissue targeting of nuclear hormone pharmacology while eliminating adverse effects in other tissues. Specifically, we report the development of a glucagon-like peptide-1 (GLP-1)-estrogen conjugate that has superior sex-independent efficacy over either of the individual hormones alone to correct obesity, hyperglycemia and dyslipidemia in mice. The therapeutic benefits are driven by pleiotropic dual hormone action to improve energy, glucose and lipid metabolism, as shown by loss-of-function models and genetic action profiling. Notably, the peptide-based targeting strategy also prevents hallmark side effects of estrogen in male and female mice, such as reproductive endocrine toxicity and oncogenicity. Collectively, selective activation of estrogen receptors in GLP-1-targeted tissues produces unprecedented efficacy to enhance the metabolic benefits of GLP-1 agonism. This example of targeting the metabolic syndrome represents the discovery of a new class of therapeutics that enables synergistic co-agonism through peptide-based selective delivery of small molecules. Although our observations with the GLP-1-estrogen conjugate justify translational studies for diabetes and obesity, the multitude of other possible combinations of peptides and small molecules may offer equal promise for other diseases.

Restoration of leptin responsiveness in diet-induced obese mice using an optimized leptin analog in combination with exendin-4 or FGF21.

The identification of leptin as a mediator of body weight regulation provided much initial excitement for the treatment of obesity. Unfortunately, leptin monotherapy is insufficient in reversing obesity in rodents or humans. Recent findings suggest that amylin is able to restore leptin sensitivity and when used in combination with leptin enhances body weight loss in obese rodents and humans. However, as the uniqueness of this combination therapy remains unclear, we assessed whether co-administration of leptin with other weight loss-inducing hormones equally restores leptin responsiveness in diet-induced obese (DIO) mice. Accordingly, we report here the design and characterization of a series of site-specifically enhanced leptin analogs of high potency and sustained action that, when administered in combination with exendin-4 or fibroblast growth factor 21 (FGF21), restores leptin responsiveness in DIO mice after an initial body weight loss of 30%. Using either combination, body weight loss was enhanced compared with either exendin-4 or FGF21 monotherapy, and leptin alone was sufficient to maintain the reduced body weight. In contrast, leptin monotherapy proved ineffective when identical weight loss was induced by caloric restriction alone over a comparable time. Accordingly, we find that a hypothalamic counter-regulatory response to weight loss, assessed using changes in hypothalamic agouti related peptide (AgRP) levels, is triggered by caloric restriction, but blunted by treatment with exendin-4. We conclude that leptin re-sensitization requires pharmacotherapy but does not appear to be restricted to a unique signaling pathway. Our findings provide preclinical evidence that high activity, long-acting leptin analogs are additively efficacious when used in combination with other weight-lowering agents.

Carbohydrate content of post-operative diet influences the effect of vertical sleeve gastrectomy on body weight reduction in obese rats.

BACKGROUND: Vertical sleeve gastrectomy (VSG) effectively reduces body weight (BW) in obese rats and humans. However, post-surgical weight regain is frequently observed in subjects after VSG, but the underlying reasons remain poorly understood. We therefore investigated if post-surgical consumption of different diets can affect the outcome of VSG. METHODS: VSG or sham operation was performed in Long-Evans rats with diet-induced obesity (n = 37). After post-surgical recovery, rats were fed ad libitum either with standard chow (CH), high-fat (HF) or low-carbohydrate, high-fat (LCHF) diets. BW and food intake were measured every second day; serum leptin, cholesterol, HDL cholesterol, and triglycerides were analyzed 4 weeks after surgery. Energy expenditure and locomotor activity were determined by a combined indirect calorimetry system, lean and fat mass by nuclear magnetic resonance. RESULTS: After 4 weeks, BW gain, fat mass, and leptin were lower in VSG rats when compared to sham controls (p < 0.05). Energy expenditure and locomotor activity were not affected by VSG indicating that weight reduction derives from the significantly lower cumulative 4-week energy intake in VSG compared to sham. Sham rats fed LCHF consumed the most energy, followed by rats fed HF. In contrast, after VSG cumulative energy intake was highest in rats fed HF, but not different between CH and LCHF. Consistently, post-surgical BW and fat mass regain were highest in the HF-VSG group. Lipid profiles were improved by VSG but not differentially affected by diets. CONCLUSION: In conclusion, consumption of a HF diet but not the more energy-dense LCHF diet reduced the effectiveness of VSG in rats.

Role of adipose and hepatic atypical protein kinase C lambda (PKCλ) in the development of obesity and glucose intolerance.

PKCλ, an atypical member of the multifunctional protein kinase C family, has been implicated in the regulation of insulin-stimulated glucose transport and of the intracellular immune response. To further elucidate the role of this cellular regulator in diet-induced obesity and insulin resistance, we generated both liver (PKC-Alb) and adipose tissue (PKC-Ap2) specific knockout mice. Body weight, fat mass, food intake, glucose homeostasis and energy expenditure were evaluated in mice maintained on either chow or high fat diet (HFD). Ablation of PKCλ from the adipose tissue resulted in mice that were indistinguishable from their wild-type littermates. However, PKC-Alb mice were resistant to diet-induced obesity (DIO). Surprisingly this DIO resistance was not associated with either a reduction in caloric intake or an increase in energy expenditure as compared with their wild-type littermates. Furthermore, these mice displayed an improvement in glucose tolerance. When maintained on chow diet, these mice were similar to wild types in respect to body weight and fat mass, yet insulin sensitivity was impaired compared with wt littermates. Taken together these data suggest that hepatic PKCλ is modulating insulin-mediated glucose turnover and response to high fat diet feeding, thus offering a deeper understanding of an important target for anti-obesity therapeutics.

A new glucagon and GLP-1 co-agonist eliminates obesity in rodents.

We report the efficacy of a new peptide with agonism at the glucagon and GLP-1 receptors that has potent, sustained satiation-inducing and lipolytic effects. Selective chemical modification to glucagon resulted in a loss of specificity, with minimal change to inherent activity. The structural basis for the co-agonism appears to be a combination of local positional interactions and a change in secondary structure. Two co-agonist peptides differing from each other only in their level of glucagon receptor agonism were studied in rodent obesity models. Administration of PEGylated peptides once per week normalized adiposity and glucose tolerance in diet-induced obese mice. Reduction of body weight was achieved by a loss of body fat resulting from decreased food intake and increased energy expenditure. These preclinical studies indicate that when full GLP-1 agonism is augmented with an appropriate degree of glucagon receptor activation, body fat reduction can be substantially enhanced without any overt adverse effects.