Saturated fatty acid concentrations are predictive of insulin sensitivity and beta cell compensation in dogs.

Chronic feeding of a high fat diet (HFD) in preclinical species induces broad metabolic dysfunction characterized by body weight gain, hyperinsulinemia, dyslipidemia and impaired insulin sensitivity. The plasma lipidome is not well characterized in dogs with HFD-induced metabolic dysfunction. We therefore aimed to describe the alterations that occur in the plasma lipid composition of dogs that are fed a HFD and examine the association of these changes with the clinical signs of metabolic dysfunction. Dogs were fed a normal diet (ND) or HFD for 12 weeks. Insulin sensitivity (SI) and beta cell compensation (AIRG) were assessed through an intravenous glucose tolerance test (IVGTT) and serum biochemistry was analyzed before the introduction of HFD and again after 12 weeks of continued ND or HFD feeding. Plasma lipidomics were conducted prior to the introduction of HFD and again at week 8 in both ND and HFD-fed dogs. 12 weeks of HFD feeding resulted in impaired insulin sensitivity and increased beta cell compensation measured by SI (ND mean: 11.5 [mU/l]-1 min-1, HFD mean: 4.7 [mU/l]-1 min-1) and AIRG (ND mean: 167.0 [mU/l]min, HFD mean: 260.2 [mU/l]min), respectively, compared to dogs fed ND over the same duration. Chronic HFD feeding increased concentrations of plasma lipid species and deleterious fatty acids compared to dogs fed a ND. Saturated fatty acid (SFA) concentrations were significantly associated with fasting insulin (R2 = 0.29), SI (R2 = 0.49) and AIRG (R2 = 0.37) in all dogs after 12 weeks, irrespective of diet. Our results demonstrate that chronic HFD feeding leads to significant changes in plasma lipid composition and fatty acid concentrations associated with metabolic dysfunction. High SFA concentrations may be predictive of deteriorated insulin sensitivity in dogs.

Feeding dogs a high-fat diet induces metabolic changes similar to natural aging, including dyslipidemia, hyperinsulinemia, and peripheral insulin resistance.

OBJECTIVE: The goal of this study was to characterize changes induced by a high-fat diet in body composition, insulin levels and sensitivity, blood lipids, and other key biomarkers also associated with the metabolic dysfunction that occurs with natural aging. ANIMALS: 24 male Beagle dogs, 3 to 7 years of age, of mixed castration status. METHODS: Dogs were randomly assigned to continue twice daily feeding of the commercial adult maintenance diet (n = 12, including 2 intact) that they were previously fed or to a high-fat diet (12, including 2 intact) for 17 weeks between December 1, 2021, and April 28, 2022. Assessments included body composition (weight, body condition score, and adipose mass determined by deuterium enrichment), clinical chemistries, plasma fatty acid quantification, oral glucose tolerance test, and histology of subcutaneous and visceral adipose biopsy samples. RESULTS: The high-fat diet led to increased body weight, body condition score, fat mass and adipocyte size, hyperinsulinemia and peripheral insulin resistance, and elevations in serum lipids, including cholesterol, triglycerides, and several species of free fatty acids. Leptin levels increased in dogs fed a high-fat diet but not in control dogs. There were no significant changes in routine clinical chemistry values in either group. CLINICAL RELEVANCE: Feeding a high-fat diet for 17 weeks led to potentially deleterious changes in metabolism similar to those seen in natural aging in dogs, including hyperinsulinemia, insulin resistance, and dyslipidemia. A high-fat diet model may provide insights into the similar metabolic dysfunction that occurs during natural aging.

The Oncolytic Activity of Zika Viral Therapy in Human Neuroblastoma In Vivo Models Confers a Major Survival Advantage in a CD24-dependent Manner.

Neuroblastoma is the most common extracranial tumor, accounting for 15% of all childhood cancer-related deaths. The long-term survival of patients with high-risk tumors is less than 40%, and MYCN amplification is one of the most common indicators of poor outcomes. Zika virus (ZIKV) is a mosquito-borne flavivirus associated with mild constitutional symptoms outside the fetal period. Our published data showed that high-risk and recurrent neuroblastoma cells are permissive to ZIKV infection, resulting in cell type-specific lysis. In this study, we assessed the efficacy of ZIKV as an oncolytic treatment for high-risk neuroblastoma using in vivo tumor models. Utilizing both MYCN-amplified and non-amplified models, we demonstrated that the application of ZIKV had a rapid tumoricidal effect. This led to a nearly total loss of the tumor mass without evidence of recurrence, offering a robust survival advantage to the host. Detection of the viral NS1 protein within the tumors confirmed that a permissive infection preceded tissue necrosis. Despite robust titers within the tumor, viral shedding to the host was poor and diminished rapidly, correlating with no detectable side effects to the murine host. Assessments from both primary pretreatment and recurrent posttreatment isolates confirmed that permissive sensitivity to ZIKV killing was dependent on the expression of CD24, which was highly expressed in neuroblastomas and conferred a proliferative advantage to tumor growth. Exploiting this viral sensitivity to CD24 offers the possibility of its use as a prognostic target for a broad population of expressing cancers, many of which have shown resistance to current clinical therapies. SIGNIFICANCE: Sensitivity to the tumoricidal effect of ZIKV on high-risk neuroblastoma tumors is dependent on CD24 expression, offering a prognostic marker for this oncolytic therapy in an extensive array of CD24-expressing cancers.

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.

BDK inhibition acts as a catabolic switch to mimic fasting and improve metabolism in mice.

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 lipoprotein 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 PPARα. However, BT2 did not directly activate PPARα in vitro. Conversely, shRNA-AAV-mediated knockdown of BDK specifically in liver in vivo did not demonstrate any effects on glycemia, steatosis, or PPARα-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.

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.

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.

In vivo GDF3 administration abrogates aging related muscle regeneration delay following acute sterile injury.

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.

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.

The Transcription Factor STAT6 Mediates Direct Repression of Inflammatory Enhancers and Limits Activation of Alternatively Polarized Macrophages.

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-1ß 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.

Macrophage PPARγ, a Lipid Activated Transcription Factor Controls the Growth Factor GDF3 and Skeletal Muscle Regeneration.

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-ß (TGF-ß) 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.

Is the Mouse a Good Model of Human PPARγ-Related Metabolic Diseases?

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.

Exposure to environmentally persistent free radicals during gestation lowers energy expenditure and impairs skeletal muscle mitochondrial function in adult mice.

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.