Chronic UCN2 treatment desensitizes CRHR2 and improves insulin sensitivity.

Urocortin 2 (UCN2) acts as a ligand for the G protein-coupled receptor corticotropin-releasing hormone receptor 2 (CRHR2). UCN2 has been reported to improve or worsen insulin sensitivity and glucose tolerance in vivo. Here we show that acute dosing of UCN2 induces systemic insulin resistance in male mice and skeletal muscle. Inversely, chronic elevation of UCN2 by injection with adenovirus encoding UCN2 resolves metabolic complications, improving glucose tolerance. CRHR2 recruits Gs in response to low concentrations of UCN2, as well as Gi and ß-Arrestin at high concentrations of UCN2. Pre-treating cells and skeletal muscle ex vivo with UCN2 leads to internalization of CRHR2, dampened ligand-dependent increases in cAMP, and blunted reductions in insulin signaling. These results provide mechanistic insights into how UCN2 regulates insulin sensitivity and glucose metabolism in skeletal muscle and in vivo. Importantly, a working model was derived from these results that unifies the contradictory metabolic effects of UCN2.

AMPK activation is sufficient to increase skeletal muscle glucose uptake and glycogen synthesis but is not required for contraction-mediated increases in glucose metabolism.

The AMP-activated protein kinase (AMPK) is a cellular sensor of energetics and when activated in skeletal muscle during contraction can impart changes in skeletal muscle metabolism. Therapeutics that selectively activate AMPK have been developed to lower glucose levels through increased glucose disposal rates as an approach to abrogate the hyperglycemic state of diabetes; however, the metabolic fate of glucose following AMPK activation remains unclear. We have used a combination of in vivo evaluation of glucose homeostasis and ex vivo skeletal muscle incubation to systematically evaluate metabolism following pharmacological activation of AMPK with PF-739, comparing this with AMPK activation through sustained intermittent electrical stimulation of contraction. These methods to activate AMPK result in increased glucose uptake but divergent metabolism of glucose: pharmacological activation results in increased glycogen accumulation while contraction-induced glucose uptake results in increased lactate formation and glucose oxidation. These results provide additional evidence to support a role for AMPK in control of skeletal muscle metabolism and additional insight into the potential for AMPK stimulation with small molecule direct activators.

Danuglipron (PF-06882961) in type 2 diabetes: a randomized, placebo-controlled, multiple ascending-dose phase 1 trial.

Agonism of the glucagon-like peptide-1 receptor (GLP-1R) results in glycemic lowering and body weight loss and is a therapeutic strategy to treat type 2 diabetes (T2D) and obesity. We developed danuglipron (PF-06882961), an oral small-molecule GLP-1R agonist and found it had comparable efficacy to injectable peptidic GLP-1R agonists in a humanized mouse model. We then completed a placebo-controlled, randomized, double-blind, multiple ascending-dose phase 1 study ( NCT03538743 ), in which we enrolled 98 patients with T2D on background metformin and randomized them to receive multiple ascending doses of danuglipron or placebo for 28 d, across eight cohorts. The primary outcomes were assessment of adverse events (AEs), safety laboratory tests, vital signs and 12-lead electrocardiograms. Most AEs were mild, with nausea, dyspepsia and vomiting most commonly reported. There were no clinically meaningful AEs in laboratory values across groups. Heart rate generally increased with danuglipron treatment at day 28, but no heart-rate AEs were reported. Systolic blood pressure was slightly decreased and changes in diastolic blood pressure were similar with danuglipron treatment at day 28, compared with placebo. There were no clinically meaningful electrocardiogram findings. In this study in T2D, danuglipron was generally well tolerated, with a safety profile consistent with the mechanism of action of GLP-1R agonism.

Dipyridamole Inhibits Lipogenic Gene Expression by Retaining SCAP-SREBP in the Endoplasmic Reticulum.

Sterol regulatory element-binding proteins (SREBPs) are master transcriptional regulators of the mevalonate pathway and lipid metabolism and represent an attractive therapeutic target for lipid metabolic disorders. SREBPs are maintained in the endoplasmic reticulum (ER) in a tripartite complex with SREBP cleavage-activating protein (SCAP) and insulin-induced gene protein (INSIG). When new lipid synthesis is required, the SCAP-SREBP complex dissociates from INSIG and undergoes ER-to-Golgi transport where the N-terminal transcription factor domain is released by proteolysis. The mature transcription factor translocates to the nucleus and stimulates expression of the SREBP gene program. Previous studies showed that dipyridamole, a clinically prescribed phosphodiesterase (PDE) inhibitor, potentiated statin-induced tumor growth inhibition. Dipyridamole limited nuclear accumulation of SREBP, but the mechanism was not well resolved. In this study, we show that dipyridamole selectively blocks ER-to-Golgi movement of the SCAP-SREBP complex and that this is independent of its PDE inhibitory activity.

GDF-15 Neutralization Alleviates Platinum-Based Chemotherapy-Induced Emesis, Anorexia, and Weight Loss in Mice and Nonhuman Primates.

Platinum-based cancer therapy is restricted by dose-limiting side effects and is associated with elevation of growth differentiation factor 15 (GDF-15). But whether this elevation contributes to such side effects has been unclear. Here, we explored the effects of GDF-15 blockade on platinum-based chemotherapy-induced emesis, anorexia, and weight loss in mice and/or nonhuman primate models. We found that circulating GDF-15 is higher in subjects with cancer receiving platinum-based chemotherapy and is positively associated with weight loss in colorectal cancer (NCT00609622). Further, chemotherapy agents associated with high clinical emetic score induce circulating GDF-15 and weight loss in mice. Platinum-based treatment-induced anorexia and weight loss are attenuated in GDF-15 knockout mice, while GDF-15 neutralization with the monoclonal antibody mAB1 improves survival. In nonhuman primates, mAB1 treatment attenuates anorexia and emesis. These results suggest that GDF-15 neutralization is a potential therapeutic approach to alleviate chemotherapy-induced side effects and improve the quality of life.

Characterization of cachexia in the human fibrosarcoma HT-1080 mouse tumour model.

BACKGROUND: Cancer cachexia is a complex metabolic disease with unmet medical need. Although many rodent models are available, none are identical to the human disease. Therefore, the development of new preclinical models that simulate some of the physiological, biochemical, and clinical characteristics of the human disease is valuable. The HT-1080 human fibrosarcoma tumour cell line was reported to induce cachexia in mice. Therefore, the purpose of this work was to determine how well the HT-1080 tumour model could recapitulate human cachexia and to examine its technical performance. Furthermore, the efficacy of ghrelin receptor activation via anamorelin treatment was evaluated, because it is one of few clinically validated mechanisms. METHODS: Female severe combined immunodeficient mice were implanted subcutaneously or heterotopically (renal capsule) with HT-1080 tumour cells. The cachectic phenotype was evaluated during tumour development, including body weight, body composition, food intake, muscle function (force and fatigue), grip strength, and physical activity measurements. Heterotopic and subcutaneous tumour histology was also compared. Energy balance was evaluated at standard and thermoneutral housing temperatures in the subcutaneous model. The effect of anamorelin (ghrelin analogue) treatment was also examined. RESULTS: The HT-1080 tumour model had excellent technical performance and was reproducible across multiple experimental conditions. Heterotopic and subcutaneous tumour cell implantation resulted in similar cachexia phenotypes independent of housing temperature. Tumour weight and histology was comparable between both routes of administration with minimal inflammation. Subcutaneous HT-1080 tumour-bearing mice presented with weight loss (decreased fat mass and skeletal muscle mass/fibre cross-sectional area), reduced food intake, impaired muscle function (reduced force and grip strength), and decreased spontaneous activity and voluntary wheel running. Key circulating inflammatory biomarkers were produced by the tumour, including growth differentiation factor 15, Activin A, interleukin 6, and TNF alpha. Anamorelin prevented but did not reverse anorexia and weight loss in the subcutaneous model. CONCLUSIONS: The subcutaneous HT-1080 tumour model displays many of the perturbations of energy balance and physical performance described in human cachexia, consistent with the production of key inflammatory factors. Anamorelin was most effective when administered early in disease progression. The HT-1080 tumour model is valuable for studying potential therapeutic targets for the treatment of cachexia.

Activation of Liver AMPK with PF-06409577 Corrects NAFLD and Lowers Cholesterol in Rodent and Primate Preclinical Models.

Dysregulation of hepatic lipid and cholesterol metabolism is a significant contributor to cardiometabolic health, resulting in excessive liver lipid accumulation and ultimately non-alcoholic steatohepatitis (NASH). Therapeutic activators of the AMP-Activated Protein Kinase (AMPK) have been proposed as a treatment for metabolic diseases; we show that the AMPK ß1-biased activator PF-06409577 is capable of lowering hepatic and systemic lipid and cholesterol levels in both rodent and monkey preclinical models. PF-06409577 is able to inhibit de novo lipid and cholesterol synthesis pathways, and causes a reduction in hepatic lipids and mRNA expression of markers of hepatic fibrosis. These effects require AMPK activity in the hepatocytes. Treatment of hyperlipidemic rats or cynomolgus monkeys with PF-06409577 for 6weeks resulted in a reduction in circulating cholesterol. Together these data suggest that activation of AMPK ß1 complexes with PF-06409577 is capable of impacting multiple facets of liver disease and represents a promising strategy for the treatment of NAFLD and NASH in humans.

Activation of Skeletal Muscle AMPK Promotes Glucose Disposal and Glucose Lowering in Non-human Primates and Mice.

The AMP-activated protein kinase (AMPK) is a potential therapeutic target for metabolic diseases based on its reported actions in the liver and skeletal muscle. We evaluated two distinct direct activators of AMPK: a non-selective activator of all AMPK complexes, PF-739, and an activator selective for AMPK ß1-containing complexes, PF-249. In cells and animals, both compounds were effective at activating AMPK in hepatocytes, but only PF-739 was capable of activating AMPK in skeletal muscle. In diabetic mice, PF-739, but not PF-249, caused a rapid lowering of plasma glucose levels that was diminished in the absence of skeletal muscle, but not liver, AMPK heterotrimers and was the result of an increase in systemic glucose disposal with no impact on hepatic glucose production. Studies of PF-739 in cynomolgus monkeys confirmed translation of the glucose lowering and established activation of AMPK in skeletal muscle as a potential therapeutic approach to treat diabetic patients.

SETDB2 Links Glucocorticoid to Lipid Metabolism through Insig2a Regulation.

Transcriptional and chromatin regulations mediate the liver response to nutrient availability. The role of chromatin factors involved in hormonal regulation in response to fasting is not fully understood. We have identified SETDB2, a glucocorticoid-induced putative epigenetic modifier, as a positive regulator of GR-mediated gene activation in liver. Insig2a increases during fasting to limit lipid synthesis, but the mechanism of induction is unknown. We show Insig2a induction is GR-SETDB2 dependent. SETDB2 facilitates GR chromatin enrichment and is key to glucocorticoid-dependent enhancer-promoter interactions. INSIG2 is a negative regulator of SREBP, and acute glucocorticoid treatment decreased active SREBP during refeeding or in livers of Ob/Ob mice, both systems of elevated SREBP-1c-driven lipogenesis. Knockdown of SETDB2 or INSIG2 reversed the inhibition of SREBP processing. Overall, these studies identify a GR-SETDB2 regulatory axis of hepatic transcriptional reprogramming and identify SETDB2 as a potential target for metabolic disorders with aberrant glucocorticoid actions.

Flavin-containing monooxygenase 3 as a potential player in diabetes-associated atherosclerosis.

Despite the well-documented association between insulin resistance and cardiovascular disease, the key targets of insulin relevant to the development of cardiovascular disease are not known. Here, using non-biased profiling methods, we identify the enzyme flavin-containing monooxygenase 3 (Fmo3) to be a target of insulin. FMO3 produces trimethylamine N-oxide (TMAO), which has recently been suggested to promote atherosclerosis in mice and humans. We show that FMO3 is suppressed by insulin in vitro, increased in obese/insulin resistant male mice and increased in obese/insulin-resistant humans. Knockdown of FMO3 in insulin-resistant mice suppresses FoxO1, a central node for metabolic control, and entirely prevents the development of hyperglycaemia, hyperlipidemia and atherosclerosis. Taken together, these data indicate that FMO3 is required for FoxO1 expression and the development of metabolic dysfunction.

An SREBP-responsive microRNA operon contributes to a regulatory loop for intracellular lipid homeostasis.

Sterol regulatory element-binding proteins (SREBPs) have evolved as a focal point for linking lipid synthesis with other pathways that regulate cell growth and survival. Here, we have uncovered a polycistrionic microRNA (miRNA) locus that is activated directly by SREBP-2. Two of the encoded miRNAs, miR-182 and miR-96, negatively regulate the expression of Fbxw7 and Insig-2, respectively, and both are known to negatively affect nuclear SREBP accumulation. Direct manipulation of this miRNA pathway alters nuclear SREBP levels and endogenous lipid synthesis. Thus, we have uncovered a mechanism for the regulation of intracellular lipid metabolism mediated by the concerted action of a pair of miRNAs that are expressed from the same SREBP-2-regulated miRNA locus, and each targets a different protein of the multistep pathway that regulates SREBP function. These studies reveal an miRNA "operon" analogous to the classic model for genetic control in bacterial regulatory systems.