Mitochondrial CypD Acetylation Promotes Endothelial Dysfunction and Hypertension.

BACKGROUND: Nearly half of adults have hypertension, a major risk factor for cardiovascular disease. Mitochondrial hyperacetylation is linked to hypertension, but the role of acetylation of specific proteins is not clear. We hypothesized that acetylation of mitochondrial CypD (cyclophilin D) at K166 contributes to endothelial dysfunction and hypertension. METHODS: To test this hypothesis, we studied CypD acetylation in patients with essential hypertension, defined a pathogenic role of CypD acetylation in deacetylation mimetic CypD-K166R mutant mice and endothelial-specific GCN5L1 (general control of amino acid synthesis 5 like 1)-deficient mice using an Ang II (angiotensin II) model of hypertension. RESULTS: Arterioles from hypertensive patients had 280% higher CypD acetylation coupled with reduced Sirt3 (sirtuin 3) and increased GCN5L1 levels. GCN5L1 regulates mitochondrial protein acetylation and promotes CypD acetylation, which is counteracted by mitochondrial deacetylase Sirt3. In human aortic endothelial cells, GCN5L1 depletion prevents superoxide overproduction. Deacetylation mimetic CypD-K166R mice were protected from vascular oxidative stress, endothelial dysfunction, and Ang II-induced hypertension. Ang II-induced hypertension increased mitochondrial GCN5L1 and reduced Sirt3 levels resulting in a 250% increase in GCN5L1/Sirt3 ratio promoting CypD acetylation. Treatment with mitochondria-targeted scavenger of cytotoxic isolevuglandins normalized GCN5L1/Sirt3 ratio, reduced CypD acetylation, and attenuated hypertension. The role of mitochondrial acetyltransferase GCN5L1 in the endothelial function was tested in endothelial-specific GCN5L1 knockout mice. Depletion of endothelial GCN5L1 prevented Ang II-induced mitochondrial oxidative stress, reduced the maladaptive switch of vascular metabolism to glycolysis, prevented inactivation of endothelial nitric oxide, preserved endothelial-dependent relaxation, and attenuated hypertension. CONCLUSIONS: These data support the pathogenic role of CypD acetylation in endothelial dysfunction and hypertension. We suggest that targeting cytotoxic mitochondrial isolevuglandins and GCN5L1 reduces CypD acetylation, which may be beneficial in cardiovascular disease.

Tubular CPT1A deletion minimally affects aging and chronic kidney injury.

Kidney tubules use fatty acid oxidation (FAO) to support their high energetic requirements. Carnitine palmitoyltransferase 1A (CPT1A) is the rate-limiting enzyme for FAO, and it is necessary to transport long-chain fatty acids into mitochondria. To define the role of tubular CPT1A in aging and injury, we generated mice with tubule-specific deletion of Cpt1a (Cpt1aCKO mice), and the mice were either aged for 2 years or injured by aristolochic acid or unilateral ureteral obstruction. Surprisingly, Cpt1aCKO mice had no significant differences in kidney function or fibrosis compared with wild-type mice after aging or chronic injury. Primary tubule cells from aged Cpt1aCKO mice had a modest decrease in palmitate oxidation but retained the ability to metabolize long-chain fatty acids. Very-long-chain fatty acids, exclusively oxidized by peroxisomes, were reduced in kidneys lacking tubular CPT1A, consistent with increased peroxisomal activity. Single-nuclear RNA-Seq showed significantly increased expression of peroxisomal FAO enzymes in proximal tubules of mice lacking tubular CPT1A. These data suggest that peroxisomal FAO may compensate in the absence of CPT1A, and future genetic studies are needed to confirm the role of peroxisomal ß-oxidation when mitochondrial FAO is impaired.

Insulin at the intersection of thermoregulation and glucose homeostasis.

Mammals are protected from changes in environmental temperature by altering energetic processes that modify heat production. Insulin is the dominant stimulus of glucose uptake and metabolism, which are fundamental for thermogenic processes. The purpose of this work was to determine the interaction of ambient temperature induced changes in energy expenditure (EE) on the insulin sensitivity of glucose fluxes. Short-term and adaptive responses to thermoneutral temperature (TN, ∼28 °C) and room (laboratory) temperature (RT, ∼22 °C) were studied in mice. This range of temperature does not cause detectable changes in circulating catecholamines or shivering and postabsorptive glucose homeostasis is maintained. We tested the hypothesis that a decrease in EE that occurs with TN causes insulin resistance and that this reduction in insulin action and EE is reversed upon short term (<12h) transition to RT. Insulin-stimulated glucose disposal (Rd) and tissue-specific glucose metabolic index were assessed combining isotopic tracers with hyperinsulinemic-euglycemic clamps. EE and insulin-stimulated Rd are both decreased (∼50%) in TN-adapted vs RT-adapted mice. When RT-adapted mice are switched to TN, EE rapidly decreases and Rd is reduced by ∼50%. TN-adapted mice switched to RT exhibit a rapid increase in EE, but whole-body insulin-stimulated Rd remains at the low rates of TN-adapted mice. In contrast, whole body glycolytic flux rose with EE. This higher EE occurs without increasing glucose uptake from the blood, but rather by diverting glucose from glucose storage to glycolysis. In addition to adaptations in insulin action, 'insulin-independent' glucose uptake in brown fat is exquisitely sensitive to thermoregulation. These results show that insulin action adjusts to non-stressful changes in ambient temperature to contribute to the support of body temperature homeostasis without compromising glucose homeostasis.

Disruption of hepatic mitochondrial pyruvate and amino acid metabolism impairs gluconeogenesis and endurance exercise capacity in mice.

Exercise robustly increases the glucose demands of skeletal muscle. This demand is met by not only muscle glycogenolysis but also accelerated liver glucose production from hepatic glycogenolysis and gluconeogenesis to fuel mechanical work and prevent hypoglycemia during exercise. Hepatic gluconeogenesis during exercise is dependent on highly coordinated responses within and between muscle and liver. Specifically, exercise increases the rate at which gluconeogenic precursors such as pyruvate/lactate or amino acids are delivered from muscle to the liver, extracted by the liver, and channeled into glucose. Herein, we examined the effects of interrupting hepatic gluconeogenic efficiency and capacity on exercise performance by deleting mitochondrial pyruvate carrier 2 (MPC2) and/or alanine transaminase 2 (ALT2) in the liver of mice. We found that deletion of MPC2 or ALT2 alone did not significantly affect time to exhaustion or postexercise glucose concentrations in treadmill exercise tests, but mice lacking both MPC2 and ALT2 in hepatocytes (double knockout, DKO) reached exhaustion faster and exhibited lower circulating glucose during and after exercise. Use of 2H/1³C metabolic flux analyses demonstrated that DKO mice exhibited lower endogenous glucose production owing to decreased glycogenolysis and gluconeogenesis at rest and during exercise. Decreased gluconeogenesis was accompanied by lower anaplerotic, cataplerotic, and TCA cycle fluxes. Collectively, these findings demonstrate that the transition of the liver to the gluconeogenic mode is critical for preventing hypoglycemia and sustaining performance during exercise. The results also illustrate the need for interorgan cross talk during exercise as described by the Cahill and Cori cycles.NEW & NOTEWORTHY Martino and colleagues examined the effects of inhibiting hepatic gluconeogenesis on exercise performance and systemic metabolism during treadmill exercise in mice. Combined inhibition of gluconeogenesis from lactate/pyruvate and alanine impaired exercise endurance and led to hypoglycemia during and after exercise. In contrast, suppressing either pyruvate-mediated or alanine-mediated gluconeogenesis alone had no effect on these parameters. These findings provide new insight into the molecular nodes that coordinate the metabolic responses of muscle and liver during exercise.

A Structure-function Analysis of Hepatocyte Arginase 2 Reveals Mitochondrial Ureahydrolysis as a Determinant of Glucose Oxidation.

BACKGROUND & AIMS: Restoring hepatic and peripheral insulin sensitivity is critical to prevent or reverse metabolic syndrome and type 2 diabetes. Glucose homeostasis comprises in part the complex regulation of hepatic glucose production and insulin-mediated glucose uptake and oxidation in peripheral tissues. We previously identified hepatocyte arginase 2 (Arg2) as an inducible ureahydrolase that improves glucose homeostasis and enhances glucose oxidation in multiple obese, insulin-resistant models. We therefore examined structure-function determinants through which hepatocyte Arg2 governs systemic insulin action and glucose oxidation. METHODS: To do this, we generated mice expressing wild-type murine Arg2, enzymatically inactive Arg2 (Arg2H160F) and Arg2 lacking its putative mitochondrial targeting sequence (Arg2Δ1-22). We expressed these hepatocyte-specific constructs in obese, diabetic (db/db) mice and performed genetic complementation analyses in hepatocyte-specific Arg2-deficent (Arg2LKO) mice. RESULTS: We show that Arg2 attenuates hepatic steatosis, independent of mitochondrial localization or ureahydrolase activity, and that enzymatic arginase activity is dispensable for Arg2 to augment total body energy expenditure. In contrast, mitochondrial localization and ureahydrolase activity were required for Arg2-mediated reductions in fasting glucose and insulin resistance indices. Mechanistically, Arg2Δ1-22 and Arg2H160F failed to suppress glucose appearance during hyperinsulinemic-euglycemic clamping. Quantification of heavy-isotope-labeled glucose oxidation further revealed that mistargeting or ablating Arg2 enzymatic function abrogates Arg2-induced peripheral glucose oxidation. CONCLUSION: We conclude that the metabolic effects of Arg2 extend beyond its enzymatic activity, yet hepatocyte mitochondrial ureahydrolysis drives hepatic and peripheral oxidative metabolism. The data define a structure-based mechanism mediating hepatocyte Arg2 function and nominate hepatocyte mitochondrial ureahydrolysis as a key determinant of glucose oxidative capacity in mammals.

Repression of latent NF-κB enhancers by PDX1 regulates ß cell functional heterogeneity.

Interactions between lineage-determining and activity-dependent transcription factors determine single-cell identity and function within multicellular tissues through incompletely known mechanisms. By assembling a single-cell atlas of chromatin state within human islets, we identified ß cell subtypes governed by either high or low activity of the lineage-determining factor pancreatic duodenal homeobox-1 (PDX1). ß cells with reduced PDX1 activity displayed increased chromatin accessibility at latent nuclear factor κB (NF-κB) enhancers. Pdx1 hypomorphic mice exhibited de-repression of NF-κB and impaired glucose tolerance at night. Three-dimensional analyses in tandem with chromatin immunoprecipitation (ChIP) sequencing revealed that PDX1 silences NF-κB at circadian and inflammatory enhancers through long-range chromatin contacts involving SIN3A. Conversely, Bmal1 ablation in ß cells disrupted genome-wide PDX1 and NF-κB DNA binding. Finally, antagonizing the interleukin (IL)-1ß receptor, an NF-κB target, improved insulin secretion in Pdx1 hypomorphic islets. Our studies reveal functional subtypes of single ß cells defined by a gradient in PDX1 activity and identify NF-κB as a target for insulinotropic therapy.

Epinephrine inhibits PI3Kα via the Hippo kinases.

The phosphoinositide 3-kinase p110α is an essential mediator of insulin signaling and glucose homeostasis. We interrogated the human serine, threonine, and tyrosine kinome to search for novel regulators of p110α and found that the Hippo kinases phosphorylate p110α at T1061, which inhibits its activity. This inhibitory state corresponds to a conformational change of a membrane-binding domain on p110α, which impairs its ability to engage membranes. In human primary hepatocytes, cancer cell lines, and rodent tissues, activation of the Hippo kinases MST1/2 using forskolin or epinephrine is associated with phosphorylation of T1061 and inhibition of p110α, impairment of downstream insulin signaling, and suppression of glycolysis and glycogen synthesis. These changes are abrogated when MST1/2 are genetically deleted or inhibited with small molecules or if the T1061 is mutated to alanine. Our study defines an inhibitory pathway of PI3K signaling and a link between epinephrine and insulin signaling.

Insulin at the Intersection of Thermoregulation and Glucose Homeostasis.

Mammals are protected from changes in environmental temperature by altering energetic processes that modify heat production. Insulin is the dominant stimulus of glucose uptake and metabolism, which are fundamental for thermogenic processes. The purpose of this work was to determine the interaction of ambient temperature induced changes in energy expenditure (EE) on the insulin sensitivity of glucose fluxes. Short-term and adaptive responses to thermoneutral temperature (TN, ~28°C) and room (laboratory) temperature (RT, ~22°C) were studied in mice. This range of temperature does not cause detectable changes in circulating catecholamines or shivering and postabsorptive glucose homeostasis is maintained. We tested the hypothesis that a decrease in EE that occurs with TN causes insulin resistance and that this reduction in insulin action and EE is reversed upon short term (<12h) transition to RT. Insulin-stimulated glucose disposal (Rd) and tissue specific glucose uptake were assessed combining isotopic tracers with hyperinsulinemic-euglycemic clamps. EE and insulin-stimulated Rd are both decreased (~50%) in TN-adapted vs RT-adapted mice. When RT-adapted mice are switched to TN, EE rapidly decreases and Rd is reduced by ~50%. TN-adapted mice switched to RT exhibit a rapid increase in EE, but whole body insulin-stimulated Rd remains at the low rates of TN-adapted mice. In contrast, whole body glycolytic flux rose with EE. This higher EE occurs without increasing glucose uptake from the blood, but rather by diverting glucose from glucose storage to glycolysis. In addition to adaptations in insulin action, 'insulin-independent' glucose uptake in brown fat is exquisitely sensitive to thermoregulation. These results show that insulin action adjusts to non-stressful changes in ambient temperature to contribute to the support of body temperature homeostasis without compromising glucose homeostasis.

Disruption of Hepatic Mitochondrial Pyruvate and Amino Acid Metabolism Impairs Gluconeogenesis and Endurance Exercise Capacity in Mice.

Exercise robustly increases the glucose demands of skeletal muscle. This demand is met not only by muscle glycogenolysis, but also by accelerated liver glucose production from hepatic glycogenolysis and gluconeogenesis to fuel mechanical work and prevent hypoglycemia during exercise. Hepatic gluconeogenesis during exercise is dependent on highly coordinated responses within and between muscle and liver. Specifically, exercise increases the rate at which gluconeogenic precursors such as pyruvate/lactate or amino acids are delivered from muscle to the liver, extracted by the liver, and channeled into glucose. Herein, we examined the effects of interrupting gluconeogenic efficiency and capacity on exercise performance by deleting hepatic mitochondrial pyruvate carrier 2 (MPC2) and/or alanine transaminase 2 (ALT2) in mice. We found that deletion of MPC2 or ALT2 alone did not significantly affect time to exhaustion or post-exercise glucose concentrations in treadmill exercise tests, but mice lacking both MPC2 and ALT2 in liver (DKO) reached exhaustion faster and exhibited lower circulating glucose during and after exercise. Use of ²H/¹³C metabolic flux analyses demonstrated that DKO mice exhibited lower endogenous glucose production owing to decreased glycogenolysis and gluconeogenesis at rest and during exercise. The decreased gluconeogenesis was accompanied by lower anaplerotic, cataplerotic, and TCA cycle fluxes. Collectively, these findings demonstrate that the transition of the liver to the gluconeogenic mode is critical for preventing hypoglycemia and sustaining performance during exercise. The results also illustrate the need for interorgan crosstalk during exercise as described by the Cahill and Cori cycles.

Improvement in insulin sensitivity and prevention of high fat diet-induced liver pathology using a CXCR2 antagonist.

BACKGROUND: Liver pathology (LP) characteristic of non-alcoholic fatty acid disease (NAFLD)/non-alcoholic steatohepatitis (NASH) is a prevalent co-morbidity of type 2 diabetes (T2D). Accumulating evidence indicates that neutrophils driving insulin resistance (IR), including hepatic IR, precipitate T2D-associated NAFLD/NASH. We hypothesized that targeting neutrophil accumulation into insulin-sensitive tissues in mice using a CXCR2 antagonist under T2D-precipitating high fat diet (HFD) could improve insulin sensitivity and prevent the progression towards liver pathology reminiscent of NAFLD/NASH. METHODS: Mice were age-matched and on standard rodent chow prior to 1:1 randomization into control and HFD formulated with the CXCR2 antagonist AZD5069 or with biologically inactive substitute. They were monitored for metabolic changes including insulin sensitivity using the hyperinsulinemic-euglycemic clamp and hepatic histopathologic evaluation in H&E-stained sections as well as via immunofluorescence microscopy of liver sections for leukocyte markers, collagen 1A1 formation, α-smooth muscle actin (SMA), and galectin-3 expression, for 16 weeks. Statistical tests used to determine significant differences among study groups and outcomes include Student's t-test, one-way ANOVA, repeated measures two-way ANOVA, and Fisher's exact test, depending on the analytical question. RESULTS: Compared to mice on HFD, mice in the AZD5069-formulated HFD exhibited improved insulin sensitivity, a modest reduction in weight gain, and a significant improvement in LP and markers related to NAFLD/NASH. Mice in the AZD5069-formulated HFD also exhibited reduced neutrophil accumulation into the liver at the end of the 16 week study period. CONCLUSIONS: These results show, for the first time, the effectiveness of a selective CXCR2 antagonist to improve insulin sensitivity, concomitantly preventing the progression towards LP characteristic of NAFLD/NASH. This represents a novel approach to target IR and developing LP under T2D-susceptible conditions using a single agent. Furthermore, our data extend the growing evidence in support of neutrophils as a leukocyte population that imprints and maintains a chronic inflammatory state in the progression of dysregulated metabolism in liver-specific co-morbid conditions.

The impact of intermittent exercise on mouse ethanol drinking and abstinence-associated affective behavior and physiology.

BACKGROUND: Negative emotional states are associated with the initiation and maintenance of alcohol use and drive relapse to drinking during withdrawal and protracted abstinence. Physical exercise is correlated with decreased negative affective symptoms, although a direct relationship between drinking patterns and exercise level has not been fully elucidated. METHODS: We incorporated intermittent running wheel access into a chronic continuous access, two-bottle choice alcohol drinking model in female C57BL/6J mice. Wheel access was granted intermittently once mice established a preference for alcohol over water. After 6 weeks, alcohol was removed (forced abstinence) and mice were given continuous access to unlocked or locked wheels. Negative affect-like behavior, home cage behavior, and metabolic activity were measured during protracted abstinence. RESULTS: Wheel access shifted drinking patterns in the mice, increasing drinking when the wheel was locked, and decreasing drinking when unlocked. Moreover, alcohol preference and consumption were strongly negatively correlated with the amount of running. An assessment of negative affect-like behavior in abstinence via the novelty suppressed feeding and saccharin preference tests (SPT) showed that unlimited wheel access mitigated abstinence-induced latency increases. Mice in abstinence also spent more time sleeping during the active dark cycle than control mice, providing additional evidence for abstinence-induced anhedonia- and depression-like behavior. Furthermore, running wheel access in abstinence decreased dark cycle sleep to comparable alcohol- and wheel-naïve mice. Given the positive impact of exercise and the negative impact of alcohol on metabolic health, we compared metabolic phenotypes of alcohol-abstinent mice with and without wheel access. Wheel access increased energy expenditure, carbon dioxide production, and oxygen consumption, providing a potential metabolic mechanism through which wheel access improves affective state. CONCLUSIONS: This study suggests that including exercise in AUD treatment regimens has the potential to reduce drinking, improve affective state during abstinence and could serve as a non-pharmacological approach to prevent the development of an AUD in high-risk individuals.

Peptide-YY3-36/glucagon-like peptide-1 combination treatment of obese diabetic mice improves insulin sensitivity associated with recovered pancreatic ß-cell function and synergistic activation of discrete hypothalamic and brainstem neuronal circuitries.

OBJECTIVE: Obesity-linked type 2 diabetes (T2D) is a worldwide health concern and many novel approaches are being considered for its treatment and subsequent prevention of serious comorbidities. Co-administration of glucagon like peptide 1 (GLP-1) and peptide YY3-36 (PYY3-36) renders a synergistic decrease in energy intake in obese men. However, mechanistic details of the synergy between these peptide agonists and their effects on metabolic homeostasis remain relatively scarce. METHODS: In this study, we utilized long-acting analogues of GLP-1 and PYY3-36 (via Fc-peptide conjugation) to better characterize the synergistic pharmacological benefits of their co-administration on body weight and glycaemic regulation in obese and diabetic mouse models. Hyperinsulinemic-euglycemic clamps were used to measure weight-independent effects of Fc-PYY3-36 + Fc-GLP-1 on insulin action. Fluorescent light sheet microscopy analysis of whole brain was performed to assess activation of brain regions. RESULTS: Co-administration of long-acting Fc-IgG/peptide conjugates of Fc-GLP-1 and Fc-PYY3-36 (specific for PYY receptor-2 (Y2R)) resulted in profound weight loss, restored glucose homeostasis, and recovered endogenous ß-cell function in two mouse models of obese T2D. Hyperinsulinemic-euglycemic clamps in C57BLKS/J db/db and diet-induced obese Y2R-deficient (Y2RKO) mice indicated Y2R is required for a weight-independent improvement in peripheral insulin sensitivity and enhanced hepatic glycogenesis. Brain cFos staining demonstrated distinct temporal activation of regions of the hypothalamus and hindbrain following Fc-PYY3-36 + Fc-GLP-1R agonist administration. CONCLUSIONS: These results reveal a therapeutic approach for obesity/T2D that improved insulin sensitivity and restored endogenous ß-cell function. These data also highlight the potential association between the gut-brain axis in control of metabolic homeostasis.

Effects of a novel polyphenol-rich plant extract on body composition, inflammation, insulin sensitivity, and glucose homeostasis in obese mice.

BACKGROUND/OBJECTIVES: The worldwide prevalence of obesity, metabolic syndrome and type 2 diabetes (T2D) is reaching epidemic proportions that urge the development of new management strategies. Totum-63 is a novel, plant-based polyphenol-rich active principle that has been shown to reduce body weight, fasting glycemia, glucose intolerance, and fatty liver index in obese subjects with prediabetes. Here, we investigated the effects and underlying mechanism(s) of Totum-63 on metabolic homeostasis in insulin-resistant obese mice. METHODS: Male C57Bl6/J mice were fed a high-fat diet for 12 weeks followed by supplementation with Totum-63 for 4 weeks. The effects on whole-body energy and metabolic homeostasis, as well as on tissue-specific inflammation and insulin sensitivity were assessed using a variety of immunometabolic phenotyping tools. RESULTS: Totum-63 decreased body weight and fat mass in obese mice, without affecting lean mass, food intake and locomotor activity, and increased fecal energy excretion and whole-body fatty acid oxidation. Totum-63 reduced fasting plasma glucose, insulin and leptin levels, and improved whole-body insulin sensitivity and peripheral glucose uptake. The expression of insulin receptor ß and the insulin-induced phosphorylation of Akt/PKB were increased in liver, skeletal muscle, white adipose tissue (WAT) and brown adipose tissue (BAT). Hepatic steatosis was also decreased by Totum-63 and associated with a lower expression of genes involved in fatty acid uptake, de novo lipogenesis, inflammation, and fibrosis. Furthermore, a significant reduction in pro-inflammatory macrophages was also observed in epidydimal WAT. Finally, a potent decrease in BAT mass associated with enhanced tissue expression of thermogenic genes was found, suggesting BAT activation by Totum-63. CONCLUSIONS: Our results show that Totum-63 reduces inflammation and improves insulin sensitivity and glucose homeostasis in obese mice through pleiotropic effects on various metabolic organs. Altogether, plant-derived Totum-63 might constitute a promising novel nutritional supplement for alleviating metabolic dysfunctions in obese people with or without T2D.

Resolution of NASH and hepatic fibrosis by the GLP-1R/GcgR dual-agonist Cotadutide via modulating mitochondrial function and lipogenesis.

Non-alcoholic fatty liver disease and steatohepatitis are highly associated with obesity and type 2 diabetes mellitus. Cotadutide, a GLP-1R/GcgR agonist, was shown to reduce blood glycemia, body weight and hepatic steatosis in patients with T2DM. Here, we demonstrate that the effects of Cotadutide to reduce body weight, food intake and improve glucose control are predominantly mediated through the GLP-1 signaling, while, its action on the liver to reduce lipid content, drive glycogen flux and improve mitochondrial turnover and function are directly mediated through Gcg signaling. This was confirmed by the identification of phosphorylation sites on key lipogenic and glucose metabolism enzymes in liver of mice treated with Cotadutide. Complementary metabolomic and transcriptomic analyses implicated lipogenic, fibrotic and inflammatory pathways, which are consistent with a unique therapeutic contribution of GcgR agonism by Cotadutide in vivo. Significantly, Cotadutide also alleviated fibrosis to a greater extent than Liraglutide or Obeticholic acid (OCA), despite adjusting dose to achieve similar weight loss in 2 preclinical mouse models of NASH. Thus Cotadutide, via direct hepatic (GcgR) and extra-hepatic (GLP-1R) effects, exerts multi-factorial improvement in liver function and is a promising therapeutic option for the treatment of steatohepatitis.

A big-data approach to understanding metabolic rate and response to obesity in laboratory mice.

Maintaining a healthy body weight requires an exquisite balance between energy intake and energy expenditure. To understand the genetic and environmental factors that contribute to the regulation of body weight, an important first step is to establish the normal range of metabolic values and primary sources contributing to variability. Energy metabolism is measured by powerful and sensitive indirect calorimetry devices. Analysis of nearly 10,000 wild-type mice from two large-scale experiments revealed that the largest variation in energy expenditure is due to body composition, ambient temperature, and institutional site of experimentation. We also analyze variation in 2329 knockout strains and establish a reference for the magnitude of metabolic changes. Based on these findings, we provide suggestions for how best to design and conduct energy balance experiments in rodents. These recommendations will move us closer to the goal of a centralized physiological repository to foster transparency, rigor and reproducibility in metabolic physiology experimentation.

Maintaining a healthy weight requires the body to balance energy intake and expenditure. The body converts food to energy through a process called energy metabolism. Genetic and environmental factors can affect energy metabolism and energy balance contributing to conditions like obesity. To better understand metabolism, scientists often study mice in laboratories, but mice from different laboratories appear to convert food to energy at different rates. This makes it hard to determine what is 'normal' for mouse metabolism. These discrepancies could be due to small differences between how mice are kept in different laboratories. For example, the temperatures of the mouse cages or how active the mice are might differ depending on the laboratory. Identifying the effects of such differences is essential, but it requires looking at data from hundreds of mice. Corrigan et al. examined data from more than 30,000 mice at laboratories around the world to show that room temperatures and the amount of muscle and fat in a mouse's body have the biggest influence on energy balance. These two factors affected the metabolism of both typical mice and mice with mutations that affect their energy balance. These results suggest that it is important for scientists to report factors like room temperatures, the body make-up of the mice, or the animals' activity levels in metabolism studies. This can help scientists compare results and repeat experiments, which could speed up research into mouse metabolism. Corrigan et al. also found that other unknown factors also affect mouse metabolism in different laboratories. Further studies are needed to identify these factors.

Reciprocity Between Skeletal Muscle AMPK Deletion and Insulin Action in Diet-Induced Obese Mice.

Insulin resistance due to overnutrition places a burden on energy-producing pathways in skeletal muscle (SkM). Nevertheless, energy state is not compromised. The hypothesis that the energy sensor AMPK is necessary to offset the metabolic burden of overnutrition was tested using chow-fed and high-fat (HF)-fed SkM-specific AMPKα1α2 knockout (mdKO) mice and AMPKα1α2lox/lox littermates (wild-type [WT]). Lean mdKO and WT mice were phenotypically similar. HF-fed mice were equally obese and maintained lean mass regardless of genotype. Results did not support the hypothesis that AMPK is protective during overnutrition. Paradoxically, mdKO mice were more insulin sensitive. Insulin-stimulated SkM glucose uptake was approximately twofold greater in mdKO mice in vivo. Furthermore, insulin signaling, SkM GLUT4 translocation, hexokinase activity, and glycolysis were increased. AMPK and insulin signaling intersect at mammalian target of rapamycin (mTOR), a critical node for cell proliferation and survival. Basal mTOR activation was reduced by 50% in HF-fed mdKO mice, but was normalized by insulin stimulation. Mitochondrial function was impaired in mdKO mice, but energy charge was preserved by AMP deamination. Results show a surprising reciprocity between SkM AMPK signaling and insulin action that manifests with diet-induced obesity, as insulin action is preserved to protect fundamental energetic processes in the muscle.

Urocortin 2 Gene Transfer Improves Glycemic Control and Reduces Retinopathy and Mortality in Murine Insulin Deficiency.

Type 1 diabetes affects 20 million patients worldwide. Insulin is the primary and commonly the sole therapy for type 1 diabetes. However, only a minority of patients attain the targeted glucose control and reduced adverse events. We tested urocortin 2 gene transfer as single-agent therapy for insulin deficiency using two mouse models. Urocortin 2 gene transfer reduced blood glucose for months after a single intravenous injection, through increased skeletal muscle insulin sensitivity, increased insulin release in response to glucose stimulation, and increased plasma insulin levels before and during euglycemic clamp. The combined increases in both insulin availability and sensitivity resulted in improved glycemic indices-events that were not anticipated in these insulin-deficient models. In addition, urocortin 2 gene transfer reduced ocular manifestations of long-standing insulin deficiency such as vascular leak and improved retinal function. Finally, mortality was reduced by urocortin 2 gene transfer. The mechanisms for these beneficial effects included increased activities of AMP-activated protein kinase and Akt (protein kinase B) in skeletal muscle, increased skeletal muscle glucose uptake, and increased insulin release. These data suggest that urocortin 2 gene transfer may be a viable therapy for new onset type 1 diabetes and might reduce insulin needs in later stage disease.

Disruption of Acetyl-Lysine Turnover in Muscle Mitochondria Promotes Insulin Resistance and Redox Stress without Overt Respiratory Dysfunction.

This study sought to examine the functional significance of mitochondrial protein acetylation using a double knockout (DKO) mouse model harboring muscle-specific deficits in acetyl-CoA buffering and lysine deacetylation, due to genetic ablation of carnitine acetyltransferase and Sirtuin 3, respectively. DKO mice are highly susceptible to extreme hyperacetylation of the mitochondrial proteome and develop a more severe form of diet-induced insulin resistance than either single KO mouse line. However, the functional phenotype of hyperacetylated DKO mitochondria is largely normal. Of the >120 measures of respiratory function assayed, the most consistently observed traits of a markedly heightened acetyl-lysine landscape are enhanced oxygen flux in the context of fatty acid fuel and elevated rates of electron leak. In sum, the findings challenge the notion that lysine acetylation causes broad-ranging damage to mitochondrial quality and performance and raise the possibility that acetyl-lysine turnover, rather than acetyl-lysine stoichiometry, modulates redox balance and carbon flux.

CD44 contributes to hyaluronan-mediated insulin resistance in skeletal muscle of high-fat-fed C57BL/6 mice.

Extracellular matrix hyaluronan is increased in skeletal muscle of high-fat-fed insulin-resistant mice, and reduction of hyaluronan by PEGPH20 hyaluronidase ameliorates diet-induced insulin resistance (IR). CD44, the main hyaluronan receptor, is positively correlated with type 2 diabetes. This study determines the role of CD44 in skeletal muscle IR. Global CD44-deficient (cd44-/-) mice and wild-type littermates (cd44+/+) were fed a chow diet or 60% high-fat diet for 16 wk. High-fat-fed cd44-/- mice were also treated with PEGPH20 to evaluate its CD44-dependent action. Insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp (ICv). High-fat feeding increased muscle CD44 protein expression. In the absence of differences in body weight and composition, despite lower clamp insulin during ICv, the cd44-/- mice had sustained glucose infusion rate (GIR) regardless of diet. High-fat diet-induced muscle IR as evidenced by decreased muscle glucose uptake (Rg) was exhibited in cd44+/+ mice but absent in cd44-/- mice. Moreover, gastrocnemius Rg remained unchanged between genotypes on chow diet but was increased in high-fat-fed cd44-/- compared with cd44+/+ when normalized to clamp insulin concentrations. Ameliorated muscle IR in high-fat-fed cd44-/- mice was associated with increased vascularization. In contrast to previously observed increases in wild-type mice, PEGPH20 treatment in high-fat-fed cd44-/- mice did not change GIR or muscle Rg during ICv, suggesting a CD44-dependent action. In conclusion, genetic CD44 deletion improves muscle IR, and the beneficial effects of PEGPH20 are CD44-dependent. These results suggest a critical role of CD44 in promoting hyaluronan-mediated muscle IR, therefore representing a potential therapeutic target for diabetes.