ABSTRACT Background In a recent Phase 2 trial in patients with cancer cachexia, the anti‐GDF‐15 antibody ponsegromab resulted in increased body weight, appetite, muscle mass and physical activity. This study provides compelling evidence that targeting the GDF‐15 pathway may offer a viable therapeutic strategy, while raising new mechanistic questions about how GDF‐15 neutralization could be optimally integrated with other interventions to reverse the multifactorial cachexia syndrome. This series of experiments aimed to evaluate the effects of anti‐GDF‐15 antibody treatment in combination with muscle anabolic (anti‐myostatin antibody) or appetite stimulant (ghrelin receptor agonist anamorelin) modulators using mouse cancer cachexia models. Methods The effects of anti‐GDF‐15 monoclonal antibody alone and in combination with an anti‐myostatin antibody or anamorelin fumarate were examined in GDF‐15‐dependent (HT‐1080 and RENCA) and partially dependent (TOV21G) mouse tumour models. Comprehensive assessments included food intake, body weight, body composition (including fat, lean and muscle mass), muscle function and treadmill running. Circulating myostatin was measured in patient samples from an advanced NSCLC clinical study. Results Anti‐myostatin antibody treatment had limited efficacy in improving cachexia in mouse tumour models with high circulating GDF‐15 (HT‐1080 and RENCA), but improved cachexia (when combined with anti‐GDF‐15 antibody) in a tumour model with low circulating GDF‐15 levels (TOV21G). In the TOV21G model, combining anti‐myostatin and anti‐GDF‐15 antibodies led to even greater increases in body weight and hindlimb muscle mass compared with anti‐GDF‐15 antibody alone (p < 0.001 for muscle mass); however, the increase in muscle strength and treadmill running did not reach statistical significance over monotherapy. When anamorelin was combined with anti‐GDF‐15 antibody, body weight was elevated compared with the HT‐1080 tumour‐bearing vehicle group (p < 0.0001) but did not reach statistical significance over anti‐GDF‐15 antibody alone. Similar observations of the combination treatment were found for food intake, fat mass and gastrocnemius (p < 0.05). Circulating myostatin was negatively correlated with weight loss in patients with cancer (p < 0.01). Conclusion These data provide proof‐of‐principle that mechanistically distinct approaches targeting muscle anabolism and appetite may act additively with GDF‐15 neutralization, particularly in cancer cachexia settings with lower GDF‐15 dependence. Trial Registration: ClinicalTrials.gov identifier: NCT01360554.

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 β-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.

Objective: Branched chain amino acid (BCAA) catabolic defects are implicated to be causal determinates of multiple diseases. This work aimed to better understand how enhancing BCAA catabolism affected metabolic homeostasis as well as the mechanisms underlying these improvements. Methods: The rate limiting step of BCAA catabolism is the irreversible decarboxylation by the branched chain ketoacid dehydrogenase (BCKDH) enzyme complex, which is post-translationally controlled through phosphorylation by BCKDH kinase (BDK). This study utilized BT2, a small molecule allosteric inhibitor of BDK, in multiple mouse models of metabolic dysfunction and NAFLD including the high fat diet (HFD) model with acute and chronic treatment paradigms, the choline deficient and methionine minimal high fat diet (CDAHFD) model, and the low-density li-poprotein receptor null mouse model (Ldlr(-/-)). shRNA was additionally used to knock down BDK in liver to elucidate liver-specific effects of BDK inhibition in HFD-fed mice. Results: A rapid improvement in insulin sensitivity was observed in HFD-fed and lean mice after BT2 treatment. Resistance to steatosis was assessed in HFD-fed mice, CDAHFD-fed mice, and Ldlr(-/-) mice. In all cases, BT2 treatment reduced steatosis and/or inflammation. Fasting and refeeding demonstrated a lack of response to feeding-induced changes in plasma metabolites including insulin and beta-hydroxybutyrate and hepatic gene changes in BT2-treated mice. Mechanistically, BT2 treatment acutely altered the expression of genes involved in fatty acid oxidation and lipogenesis in liver, and upstream regulator analysis suggested that BT2 treatment activated PPARa. However, BT2 did not directly activate PPARa in vitro. Conversely, shRNA-AAV-mediated knockdown of BDK specifically in liver in vivo did not demonstrate any effects on glycemia, steatosis, or PPARa-mediated gene expression in mice. Conclusions: These data suggest that BT2 treatment acutely improves metabolism and liver steatosis in multiple mouse models. While many molecular changes occur in liver in BT2-treated mice, these changes were not observed in mice with AAV-mediated shRNA knockdown of BDK. All together, these data suggest that systemic BDK inhibition is required to improve metabolism and steatosis by prolonging a fasting signature in a paracrine manner. Therefore, BCAA may act as a "fed signal" to promote nutrient storage and reduced systemic BCAA levels as shown in this study via BDK inhibition may act as a "fasting signal" to prolong the catabolic state. (c) 2022 The Author(s). Published by Elsevier GmbH.

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.

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.

e15633 Background: Almost half of cancer deaths are attributed to cancers most frequently associated with cachexia. Cachexia is a complex metabolic disease characterized by anorexia and unintentional weight loss. Skeletal muscle depletion has been recognized as a key feature of the disease, however muscle anabolic therapies have not been successful, suggesting that treatments that target multiple aspects of the disease will be most effective. Growth differentiation factor 15 (GDF-15) is a cytokine that induces anorexia and weight loss and is associated with cachexia in cancer patients. In preclinical cancer cachexia models, GDF-15 inhibition is sufficient to normalize food intake and body weight, including skeletal muscle mass. However, it remains to be determined whether the increased skeletal muscle mass also results in restoration of muscle function. Therefore, we examined the effect of GDF-15 inhibition on muscle mass and function in mouse models of cancer cachexia in comparison with myostatin inhibition, an established muscle anabolic pathway. Methods: Cachectic mouse tumor models were established with subcutaneous implantation of tumor cell lines reported to be GDF-15-dependent; mouse renal cell carcinoma (RENCA) and human ovarian cancer (TOV-21G) cell lines. Mice were treated with anti-GDF-15 (mAB2) or anti-myostatin (RK35) monoclonal antibodies and skeletal muscle function was assessed in vivo via maximum force, maximum rate of contraction and half relax time. In the RENCA tumor model, GDF-15 inhibition fully restored body weight and skeletal muscle mass whereas myostatin inhibition showed only a modest effect. Results: Consistent with the muscle mass improvement, GDF-15 inhibition dramatically increased functional muscle endpoints compared to the partial effect of myostatin inhibition. Interestingly, in the TOV-21G tumor model GDF-15 inhibition only partially restored body weight, however skeletal muscle mass and muscle function were completely normalized. Consistent with the functional assessment, GDF-15 inhibition in the RENCA tumor model decreased the expression of several catabolic genes (i.e. Trim63, Fbxo32, Myh7 and Myh2). The GDF-15 effect is likely to be secondary to the reversal of anorexia since wildtype mice pair-fed to Fc-GDF-15-treated mice demonstrated equivalent muscle mass loss. Conclusions: Taken together these data suggest that GDF-15 inhibition holds potential as an effective therapeutic approach to alleviate multiple aspects of cachexia.

Circulating increases in branched chain amino acid (BCAA) levels have long been associated with type II diabetes and metabolic syndrome. Emerging data also suggest that impaired BCAA catabolism may play a role in heart failure progression. BCAA are catabolized via the branched chain ketoacid (BCKA) dehydrogenase enzyme complex (BCKDH). BCKD kinase (BCKDK) is a negative regulator of BCAA catabolism through its inhibitory phosphorylation of the BCKDHE1a subunit, and the phosphatase PPM1k dephosphorylates this same site to activate BCAA catabolism. Using an inhibitor of BCKDK (BT2), BCAA catabolism is increased in vivo. Here, we utilized metabolomics to evaluate the contribution of BCAA catabolism to substrate preference in heart and skeletal muscle. Surprisingly, BCKDK inhibition with BT2 had no effect on incorporation of glucose into TCA cycle intermediates in heart or skeletal muscle. Because others have recently shown that the primary site of BCAA catabolism is skeletal muscle, we knocked down BCKDK and PPM1k in human skeletal myocytes to further investigate how BCKDK loss or inhibition affects substrate utilization. Similar to our in vivo observations, knockdown of BCKDK and PPM1k had no effect on glucose and pyruvate utilization in a mitochondrial function assay. However, an increase in maximal respiration was observed after BCKDK knockdown when fatty acids were used. To evaluate the mechanisms underlying this increase we then performed RNAseq in these cells after BCKDK and PPM1K knockdown and observed changes in a number of genes that may explain these alterations in substrate utilization. Finally, we performed C13 BCAA metabolomics in human skeletal myocytes after BT2 treatment or knockdown of BCKDK and PPM1k. Using BT2, we observed a dose-responsive reduction in BCKA production from C13 BCAA by the muscle cells as expected; however, though BCKA production was increased after PPM1k was knocked down, we surprisingly did not observe a decrease in BCKA production after BCKDK knockdown. Collectively these data suggest that BCKDK inhibition may improve metabolism and cardiac function by altering substrate preference in skeletal myocytes.

The circadian rhythm allows for metabolic processes to be primed to anticipate the expected daily cycling of behaviors like food consumption and lipid storage. Previous genetic and epidemiological studies have implicated circadian disruption as a potential causal determinant for metabolic diseases such as obesity, diabetes and fatty liver. Here we leverage diet induced obese (DIO) mice to test the potential for circadian modulation by inhibition of casein kinase 1δ/ε (CK1 δ/ε) to improve endpoints in metabolic disease models. Our data reveal that the CK1 δ/ε inhibition with small molecule PF-05006739 was able to significantly lower fasted and fed insulin levels, fasted glucose and hepatic lipid content with a concomitant ∼9% decrease in body weight. PF-05006739 was also capable of shifting the gene expression of circadian oscillating transcripts in the liver, consistent with reported actions of this compound in previous preclinical evaluations. Furthermore, this shift in gene expression also correlated with a shift in respiratory exchange ratio (RER) and energy expenditure (EE) in vivo. Taken together these data suggest CK1 δ/ε inhibition as a potential therapeutic approach for metabolic diseases, specifically hyperinsulinemia and NAFLD/NASH. Disclosure M. Peloquin: Employee; Self; Pfizer Inc. J. Litchfield: Employee; Self; Pfizer Inc. B.B. Zhang: Employee; Spouse/Partner; Janssen Pharmaceuticals, Inc. Employee; Self; Pfizer Inc. Other Relationship; Self; Eli Lilly and Company. C. Chang: Employee; Self; Pfizer Inc. R.A. Miller: Employee; Self; Pfizer Inc. B. Bernardo: Employee; Self; Pfizer Inc. Employee; Spouse/Partner; Pfizer Inc. S. Luthra: None. C. Cortes: None. X. Li: Employee; Self; Pfizer Inc. C.J. Zou: None.

The mechanistic target of rapamycin (mTORC1) is a nutrient responsive protein kinase complex that helps co-ordinate anabolic processes across all tissues. There is evidence that signaling through mTORC1 in skeletal muscle may be a determinant of energy expenditure and aging and therefore components downstream of mTORC1 signaling may be potential targets for treating obesity and age-associated metabolic disease. Here, we generated mice with driven ablation of , which confers constitutive activation of mTORC1 in skeletal muscle and performed unbiased transcriptional analyses to identify pathways and candidate genes that may explain how skeletal muscle mTORC1 activity regulates energy balance and aging. Activation of skeletal muscle mTORC1 produced a striking resistance to diet-and age-induced obesity without inducing systemic insulin resistance. We found that increases in energy expenditure following a high fat diet were mTORC1-dependent and that elevated energy expenditure caused by ablation of coincided with the upregulation of skeletal muscle-specific thermogenic mechanisms that involve sarcolipin-driven futile cycling of Ca through SERCA2. Additionally, we report that constitutive activation of mTORC1 in skeletal muscle reduces lifespan. These findings support the hypothesis that activation of mTORC1 and its downstream targets, specifically in skeletal muscle, may play a role in nutrient-dependent thermogenesis and aging.

The molecular basis of signal-dependent transcriptional activation has been extensively studied in macrophage polarization, but our understanding remains limited regarding the molecular determinants of repression. Here we show that IL-4-activated STAT6 transcription factor is required for the direct transcriptional repression of a large number of genes during in vitro and in vivo alternative macrophage polarization. Repression results in decreased lineage-determining transcription factor, p300, and RNA polymerase II binding followed by reduced enhancer RNA expression, H3K27 acetylation, and chromatin accessibility. The repressor function of STAT6 is HDAC3 dependent on a subset of IL-4-repressed genes. In addition, STAT6-repressed enhancers show extensive overlap with the NF-κB p65 cistrome and exhibit decreased responsiveness to lipopolysaccharide after IL-4 stimulus on a subset of genes. As a consequence, macrophages exhibit diminished inflammasome activation, decreased IL-1β production, and pyroptosis. Thus, the IL-4-STAT6 signaling pathway establishes an alternative polarization-specific epigenenomic signature resulting in dampened macrophage responsiveness to inflammatory stimuli.

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 β1-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 6 weeks resulted in a reduction in circulating cholesterol. Together these data suggest that activation of AMPK β1 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.

Tissue regeneration is a highly coordinated process with sequential events including immune cell infiltration, clearance of damaged tissues, and immune-supported regrowth of the tissue. Aging has a well-documented negative impact on this process globally; however, whether changes in immune cells per se are contributing to the decline in the body's ability to regenerate tissues with aging is not clearly understood. Here, we set out to characterize the dynamics of macrophage infiltration and their functional contribution to muscle regeneration by comparing young and aged animals upon acute sterile injury. Injured muscle of old mice showed markedly elevated number of macrophages, with a predominance for Ly6Chigh pro-inflammatory macrophages and a lower ratio of the Ly6Clow repair macrophages. Of interest, a recently identified repair macrophage-derived cytokine, growth differentiation factor 3 (GDF3), was markedly downregulated in injured muscle of old relative to young mice. Supplementation of recombinant GDF3 in aged mice ameliorated the inefficient regenerative response. Together, these results uncover a deficiency in the quantity and quality of infiltrating macrophages during aging and suggest that in vivo administration of GDF3 could be an effective therapeutic approach.

Erin J. Stephenson, Alyse Ragauskas, Sridhar Jaligama, JeAnna R. Redd, Jyothi Parvathareddy, Matthew J. Peloquin, Jordy Saravia, Joan C. Han, Stephania A. Cormier, and X Dave Bridges Department of Physiology, University of Tennessee Health Science Center, Memphis, Tennessee; Department of Pediatrics, University of Tennessee Health Science Center, Memphis, Tennessee; and Children’s Foundation Research Institute, Le Bonheur Children’s Hospital, Memphis, Tennessee

With the increasing number of patients affected with metabolic diseases such as type 2 diabetes, obesity, atherosclerosis and insulin resistance, academic researchers and pharmaceutical companies are eager to better understand metabolic syndrome and develop new drugs for its treatment. Many studies have focused on the nuclear receptor peroxisome proliferator-activated receptor gamma (PPARγ), which plays a crucial role in adipogenesis and lipid metabolism. These studies have been able to connect this transcription factor to several human metabolic diseases. Due to obvious limitations concerning experimentation in humans, animal models-mainly mouse models-have been generated to investigate the role of PPARγ in different tissues. This review focuses on the metabolic features of human and mouse PPARγ-related diseases and the utility of the mouse as a model.

We have investigated the effects of in utero exposure to environmentally persistent free radicals (EPFRs) on growth, metabolism, energy utilization, and skeletal muscle mitochondria in a mouse model of diet-induced obesity. Pregnant mice were treated with laboratory-generated, combustion-derived particular matter (MCP230). The adult offspring were placed on a high-fat diet for 12 wk, after which we observed a 9.8% increase in their body weight. The increase in body size observed in the MCP230-exposed mice was not associated with increases in food intake but was associated with a reduction in physical activity and lower energy expenditure. The reduced energy expenditure in mice indirectly exposed to MCP230 was associated with reductions in skeletal muscle mitochondrial DNA copy number, lower mRNA levels of electron transport genes, and reduced citrate synthase activity. Upregulation of key genes involved in ameliorating oxidative stress was also observed in the muscle of MCP230-exposed mice. These findings suggest that gestational exposure to MCP230 leads to a reduction in energy expenditure at least in part through alterations to mitochondrial metabolism in the skeletal muscle.

Tissue regeneration requires inflammatory and reparatory activity of macrophages. Macrophages detect and eliminate the damaged tissue and subsequently promote regeneration. This dichotomy requires the switch of effector functions of macrophages coordinated with other cell types inside the injured tissue. The gene regulatory events supporting the sensory and effector functions of macrophages involved in tissue repair are not well understood. Here we show that the lipid activated transcription factor, PPARγ, is required for proper skeletal muscle regeneration, acting in repair macrophages. PPARγ controls the expression of the transforming growth factor-β (TGF-β) family member, GDF3, which in turn regulates the restoration of skeletal muscle integrity by promoting muscle progenitor cell fusion. This work establishes PPARγ as a required metabolic sensor and transcriptional regulator of repair macrophages. Moreover, this work also establishes GDF3 as a secreted extrinsic effector protein acting on myoblasts and serving as an exclusively macrophage-derived regeneration factor in tissue repair.

This repository contains code and manuscripts for a project in the lab relating to predicting the amount of diet induced obesity. This PredictorsDietInducedObesity data is made available under the Open Data Commons Attribution License: http://opendatacommons.org/licenses/by/1.0. - See more at: http://opendatacommons.org/licenses/by/#sthash.PVeWmN1l.dpuf. For more information see the LICENSE.txt file in this directory. Raw Data--------------- All raw data is in the data/raw folder. This data is automatically obtained from our internal LIMS system and can only be updated from that point. The script files use this raw data to do the analysis. Data which has been processed is saved in the data/processed folder. Script files-------------- This code base includes the raw data and reproducible R code for these analyses within the scripts folder. All analysis file are R scripts as Rmarkdown (Rmd) files. These files can be run inside RStudio https://www.rstudio.com/ and will generate the md and html files which include the processed data. For more information on using R and using these files see http://cran.us.r-project.org/. Publications ------------------ All posters, manuscripts or external presentations are in the publications folder. This includes revisions, and where possible reviewer comments and responses.