NOD mice have distinct metabolic and immunologic profiles when compared with genetically similar MHC-matched ICR mice.

Nonobese diabetic (NOD) mice are the most commonly used rodent model to study mechanisms relevant to the autoimmunity and immunology of type 1 diabetes. Although many different strains of mice have been used as controls for studies comparing nondiabetic lines to the NOD strain, we hypothesized that the parental strain that gave rise to the NOD line might be one of the best options. Therefore, we compared female ICR and NOD mice, which are matched at key major histocompatibility complex (MHC) loci, to understand their metabolic and immunologic similarities and differences. Several novel observations emerged: 1) NOD mice have greater circulating proinsulin when compared with ICR mice. 2) NOD mice display CD3+ and IBA1+ cell infiltration into and near pancreatic islets before hyperglycemia. 3) NOD mice show increased expression of the Il1b and Cxcl11 genes in islets when compared with islets from age-matched ICR mice. 4) NOD mice have a greater abundance of STAT1 and ICAM-1 protein in islets when compared with ICR mice. These data show that ICR mice, which are genetically similar to NOD mice, do not retain the same immunologic outcomes. Thus, ICR mice are an excellent choice as a genetically similar and MHC-matched control for NOD mice in studies designed to understand mechanisms relevant to autoimmune-mediated diabetes onset as well as novel therapeutic interventions.NEW & NOTEWORTHY Nonobese diabetic (NOD) mice have more proinsulin in circulation and STAT1 protein in islets compared with the major histocompatibility complex (MHC)-matched ICR line. NOD mice also display greater expression of cytokines and chemokines in pancreatic islets consistent with immune cell infiltration before hyperglycemia when compared with age-matched ICR mice. Thus, ICR mice represent an excellent control for autoimmunity and inflammation studies using the NOD line of mice.

Dynamin-related protein 1 regulates substrate oxidation in skeletal muscle by stabilizing cellular and mitochondrial calcium dynamics.

Mitochondria undergo continuous cycles of fission and fusion to promote inheritance, regulate quality control, and mitigate organelle stress. More recently, this process of mitochondrial dynamics has been demonstrated to be highly sensitive to nutrient supply, ultimately conferring bioenergetic plasticity to the organelle. However, whether regulators of mitochondrial dynamics play a causative role in nutrient regulation remains unclear. In this study, we generated a cellular loss-of-function model for dynamin-related protein 1 (DRP1), the primary regulator of outer membrane mitochondrial fission. Loss of DRP1 (shDRP1) resulted in extensive ultrastructural and functional remodeling of mitochondria, characterized by pleomorphic enlargement, increased electron density of the matrix, and defective NADH and succinate oxidation. Despite increased mitochondrial size and volume, shDRP1 cells exhibited reduced cellular glucose uptake and mitochondrial fatty acid oxidation. Untargeted transcriptomic profiling revealed severe downregulation of genes required for cellular and mitochondrial calcium homeostasis, which was coupled to loss of ATP-stimulated calcium flux and impaired substrate oxidation stimulated by exogenous calcium. The insights obtained herein suggest that DRP1 regulates substrate oxidation by altering whole-cell and mitochondrial calcium dynamics. These findings are relevant to the targetability of mitochondrial fission and have clinical relevance in the identification of treatments for fission-related pathologies such as hereditary neuropathies, inborn errors in metabolism, cancer, and chronic diseases.

Rickettsia conorii survival in THP-1 macrophages involves host lipid droplet alterations and active rickettsial protein production.

Rickettsia conorii is a Gram-negative, cytosolic intracellular bacterium that has classically been investigated in terms of endothelial cell infection. However, R. conorii and other human pathogenic Rickettsia species have evolved mechanisms to grow in various cell types, including macrophages, during mammalian infection. During infection of these phagocytes, R. conorii shifts the host cell's overall metabolism towards an anti-inflammatory M2 response, metabolically defined by an increase in host lipid metabolism and oxidative phosphorylation. Lipid metabolism has more recently been identified as a key regulator of host homeostasis through modulation of immune signalling and metabolism. Intracellular pathogens have adapted mechanisms of hijacking host metabolic pathways including host lipid catabolic pathways for various functions required for growth and survival. In the present study, we hypothesised that alterations of host lipid droplets initiated by lipid catabolic pathways during R. conorii infection is important for bacterial survival in macrophages. Herein, we determined that host lipid droplet modulation is initiated early during R. conorii infection, and these alterations rely on active bacteria and lipid catabolic pathways. We also find that these lipid catabolic pathways are essential for efficient bacterial survival. Unlike the mechanisms used by other intracellular pathogens, the catabolism of lipid droplets induced by R. conorii infection is independent of upstream host peroxisome proliferator-activated receptor-alpha (PPARα) signalling. Inhibition of PPARÉ£ signalling and lipid droplet accumulation in host cells cause a significant decrease in R. conorii survival suggesting a negative correlation with lipid droplet production and R. conorii survival. Together, these results strongly suggest that the modulation of lipid droplets in macrophage cells infected by R. conorii is an important and underappreciated aspect of the infection process. TAKE AWAYS: Host lipid droplets are differentially altered in early and replicative stages of THP-1 macrophage infection with R. conorii. Lipid droplet alterations are initiated in a bacterial-dependent manner and do not require host peroxisome proliferator-activated receptors α or É£ activation. Pharmacological inhibition of host lipid catabolic processes during R. conorii infection indicates a requirement of lipid catabolism for bacterial survival and initiation of lipid droplet modulation. A significant increase in host lipid droplets during infection has a negative impact on R. conorii survival in THP-1 macrophages.

Combined effects of a ketogenic diet and exercise training alter mitochondrial and peroxisomal substrate oxidative capacity in skeletal muscle.

Ketogenic diets (KDs) are reported to improve body weight, fat mass, and exercise performance in humans. Unfortunately, most rodent studies have used a low-protein KD, which does not recapitulate diets used by humans. Since skeletal muscle plays a critical role in responding to macronutrient perturbations induced by diet and exercise, the purpose of this study was to test if a normal-protein KD (NPKD) impacts shifts in skeletal muscle substrate oxidative capacity in response to exercise training (ExTr). A high fat, carbohydrate-deficient NPKD (16.1% protein, 83.9% fat, 0% carbohydrate) was given to C57BL/6J male mice for 6 wk, whereas controls (Con) received a low-fat diet with similar protein (15.9% protein, 11.9% fat, 72.2% carbohydrate). After 3 wk on the diet, mice began treadmill training 5 days/wk, 60 min/day for 3 wks. The NPKD increased body weight and fat mass, whereas ExTr negated a continued rise in adiposity. ExTr increased intramuscular glycogen, whereas the NPKD increased intramuscular triglycerides. Neither the NPKD nor ExTr alone altered mitochondrial content; however, in combination, the NPKD-ExTr group showed increases in PGC-1α and markers of mitochondrial fission/fusion. Pyruvate oxidative capacity was unchanged by either intervention, whereas ExTr increased leucine oxidation in NPKD-fed mice. Lipid metabolism pathways had the most notable changes as the NPKD and ExTr interventions both enhanced mitochondrial and peroxisomal lipid oxidation and many adaptations were additive or synergistic. Overall, these results suggest that a combination of a NPKD and ExTr induces additive and/or synergistic adaptations in skeletal muscle oxidative capacity.NEW & NOTEWORTHY A ketogenic diet with normal protein content (NPKD) increases body weight and fat mass, increases intramuscular triglyceride storage, and upregulates pathways related to protein metabolism. In combination with exercise training, a NPKD induces additive and/or synergistic activation of AMPK, PGC-1α, mitochondrial fission/fusion genes, mitochondrial fatty acid oxidation, and peroxisomal adaptations in skeletal muscle. Collectively, results from this study provide mechanistic insight into adaptations in skeletal muscle relevant to keto-adaptation.

Extensive metabolic remodeling after limiting mitochondrial lipid burden is consistent with an improved metabolic health profile.

Mitochondrial lipid overload in skeletal muscle contributes to insulin resistance, and strategies limiting this lipid pressure improve glucose homeostasis; however, comprehensive cellular adaptations that occur in response to such an intervention have not been reported. Herein, mice with skeletal muscle-specific deletion of carnitine palmitoyltransferase 1b (Cpt1bM-/-), which limits mitochondrial lipid entry, were fed a moderate fat (25%) diet, and samples were subjected to a multimodal analysis merging transcriptomics, proteomics, and nontargeted metabolomics to characterize the coordinated multilevel cellular responses that occur when mitochondrial lipid burden is mitigated. Limiting mitochondrial fat entry predictably improves glucose homeostasis; however, remodeling of glucose metabolism pathways pales compared with adaptations in amino acid and lipid metabolism pathways, shifts in nucleotide metabolites, and biogenesis of mitochondria and peroxisomes. Despite impaired fat utilization, Cpt1bM-/- mice have increased acetyl-CoA (14-fold) and NADH (2-fold), indicating metabolic shifts yield sufficient precursors to meet energy demand; however, this does not translate to enhance energy status as Cpt1bM-/- mice have low ATP and high AMP levels, signifying energy deficit. Comparative analysis of transcriptomic data with disease-associated gene-sets not only predicted reduced risk of glucose metabolism disorders but was also consistent with lower risk for hepatic steatosis, cardiac hypertrophy, and premature death. Collectively, these results suggest induction of metabolic inefficiency under conditions of energy surfeit likely contributes to improvements in metabolic health when mitochondrial lipid burden is mitigated. Moreover, the breadth of disease states to which mechanisms induced by muscle-specific Cpt1b inhibition may mediate health benefits could be more extensive than previously predicted.

Precision exercise medicine: understanding exercise response variability.

There is evidence from human twin and family studies as well as mouse and rat selection experiments that there are considerable interindividual differences in the response of cardiorespiratory fitness (CRF) and other cardiometabolic traits to a given exercise programme dose. We developed this consensus statement on exercise response variability following a symposium dedicated to this topic. There is strong evidence from both animal and human studies that exercise training doses lead to variable responses. A genetic component contributes to exercise training response variability.In this consensus statement, we (1) briefly review the literature on exercise response variability and the various sources of variations in CRF response to an exercise programme, (2) introduce the key research designs and corresponding statistical models with an emphasis on randomised controlled designs with or without multiple pretests and post-tests, crossover designs and repeated measures designs, (3) discuss advantages and disadvantages of multiple methods of categorising exercise response levels-a topic that is of particular interest for personalised exercise medicine and (4) outline approaches that may identify determinants and modifiers of CRF exercise response. We also summarise gaps in knowledge and recommend future research to better understand exercise response variability.

Oral Corticosterone Administration Reduces Insulitis but Promotes Insulin Resistance and Hyperglycemia in Male Nonobese Diabetic Mice.

Steroid-induced diabetes is the most common form of drug-induced hyperglycemia. Therefore, metabolic and immunological alterations associated with chronic oral corticosterone were investigated using male nonobese diabetic mice. Three weeks after corticosterone delivery, there was reduced sensitivity to insulin action measured by insulin tolerance test. Body composition measurements revealed increased fat mass and decreased lean mass. Overt hyperglycemia (>250 mg/dL) manifested 6 weeks after the start of glucocorticoid administration, whereas 100% of the mice receiving the vehicle control remained normoglycemic. This phenotype was fully reversed during the washout phase and readily reproducible across institutions. Relative to the vehicle control group, mice receiving corticosterone had a significant enhancement in pancreatic insulin-positive area, but a marked decrease in CD3+ cell infiltration. In addition, there were striking increases in both citrate synthase gene expression and enzymatic activity in skeletal muscle of mice in the corticosterone group relative to vehicle control. Moreover, glycogen synthase expression was greatly enhanced, consistent with elevations in muscle glycogen storage in mice receiving corticosterone. Corticosterone-induced hyperglycemia, insulin resistance, and changes in muscle gene expression were all reversed by the end of the washout phase, indicating that the metabolic alterations were not permanent. Thus, male nonobese diabetic mice allow for translational studies on the metabolic and immunological consequences of glucocorticoid-associated interventions in a mouse model with genetic susceptibility to autoimmune disease.

Impaired mitochondrial fat oxidation induces adaptive remodeling of muscle metabolism.

The correlations between intramyocellular lipid (IMCL), decreased fatty acid oxidation (FAO), and insulin resistance have led to the hypothesis that impaired FAO causes accumulation of lipotoxic intermediates that inhibit muscle insulin signaling. Using a skeletal muscle-specific carnitine palmitoyltransferase-1 KO model, we show that prolonged and severe mitochondrial FAO inhibition results in increased carbohydrate utilization, along with reduced physical activity; increased circulating nonesterified fatty acids; and increased IMCLs, diacylglycerols, and ceramides. Perhaps more importantly, inhibition of mitochondrial FAO also initiates a local, adaptive response in muscle that invokes mitochondrial biogenesis, compensatory peroxisomal fat oxidation, and amino acid catabolism. Loss of its major fuel source (lipid) induces an energy deprivation response in muscle coordinated by signaling through AMP-activated protein kinase (AMPK) and peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) to maintain energy supply for locomotion and survival. At the whole-body level, these adaptations result in resistance to obesity.

Thiobenzothiazole-modified Hydrocortisones Display Anti-inflammatory Activity with Reduced Impact on Islet ß-Cell Function.

Glucocorticoids signal through the glucocorticoid receptor (GR) and are administered clinically for a variety of situations, including inflammatory disorders, specific cancers, rheumatoid arthritis, and organ/tissue transplantation. However, glucocorticoid therapy is also associated with additional complications, including steroid-induced diabetes. We hypothesized that modification of the steroid backbone is one strategy to enhance the therapeutic potential of GR activation. Toward this goal, two commercially unavailable, thiobenzothiazole-containing derivatives of hydrocortisone (termed MS4 and MS6) were examined using 832/13 rat insulinoma cells as well as rodent and human islets. We found that MS4 had transrepression properties but lacked transactivation ability, whereas MS6 retained both transactivation and transrepression activities. In addition, MS4 and MS6 both displayed anti-inflammatory activity. Furthermore, MS4 displayed reduced impact on islet ß-cell function in both rodent and human islets. Similar to dexamethasone, MS6 promoted adipocyte development in vitro, whereas MS4 did not. Moreover, neither MS4 nor MS6 activated the Pck1 (Pepck) gene in primary rat hepatocytes. We conclude that modification of the functional groups attached to the D-ring of the hydrocortisone steroid molecule produces compounds with altered structure-function GR agonist activity with decreased impact on insulin secretion and reduced adipogenic potential but with preservation of anti-inflammatory activity.

Obesity and lipid stress inhibit carnitine acetyltransferase activity.

Carnitine acetyltransferase (CrAT) is a mitochondrial matrix enzyme that catalyzes the interconversion of acetyl-CoA and acetylcarnitine. Emerging evidence suggests that this enzyme functions as a positive regulator of total body glucose tolerance and muscle activity of pyruvate dehydrogenase (PDH), a mitochondrial enzyme complex that promotes glucose oxidation and is feedback inhibited by acetyl-CoA. Here, we used tandem mass spectrometry-based metabolic profiling to identify a negative relationship between CrAT activity and muscle content of lipid intermediates. CrAT specific activity was diminished in muscles from obese and diabetic rodents despite increased protein abundance. This reduction in enzyme activity was accompanied by muscle accumulation of long-chain acylcarnitines (LCACs) and acyl-CoAs and a decline in the acetylcarnitine/acetyl-CoA ratio. In vitro assays demonstrated that palmitoyl-CoA acts as a direct mixed-model inhibitor of CrAT. Similarly, in primary human myocytes grown in culture, nutritional and genetic manipulations that promoted mitochondrial influx of fatty acids resulted in accumulation of LCACs but a pronounced decrease of CrAT-derived short-chain acylcarnitines. These results suggest that lipid-induced antagonism of CrAT might contribute to decreased PDH activity and glucose disposal in the context of obesity and diabetes.

Albumin-bound fatty acids but not albumin itself alter redox balance in tubular epithelial cells and induce a peroxide-mediated redox-sensitive apoptosis.

Albuminuria is associated with metabolic syndrome and diabetes. It correlates with the progression of chronic kidney disease, particularly with tubular atrophy. The fatty acid load on albumin significantly increases in obesity, presenting a proinflammatory environment to the proximal tubules. However, little is known about changes in the redox milieu during fatty acid overload and how redox-sensitive mechanisms mediate cell death. Here, we show that albumin with fatty acid impurities or conjugated with palmitate but not albumin itself compromised mitochondrial and cell viability, membrane potential and respiration. Fatty acid overload led to a redox imbalance which deactivated the antioxidant protein peroxiredoxin 2 and caused a peroxide-mediated apoptosis through the redox-sensitive pJNK/caspase-3 pathway. Transfection of tubular cells with peroxiredoxin 2 was protective and mitigated apoptosis. Mitochondrial fatty acid entry and ceramide synthesis modulators suggested that mitochondrial ß oxidation but not ceramide synthesis may modulate lipotoxic effects on tubular cell survival. These results suggest that albumin overloaded with fatty acids but not albumin itself changes the redox environment in the tubules, inducing a peroxide-mediated redox-sensitive apoptosis. Thus, mitigating circulating fatty acid levels may be an important factor in both preserving redox balance and preventing tubular cell damage in proteinuric diseases.

Mitochondrial overload and incomplete fatty acid oxidation contribute to skeletal muscle insulin resistance.

Previous studies have suggested that insulin resistance develops secondary to diminished fat oxidation and resultant accumulation of cytosolic lipid molecules that impair insulin signaling. Contrary to this model, the present study used targeted metabolomics to find that obesity-related insulin resistance in skeletal muscle is characterized by excessive beta-oxidation, impaired switching to carbohydrate substrate during the fasted-to-fed transition, and coincident depletion of organic acid intermediates of the tricarboxylic acid cycle. In cultured myotubes, lipid-induced insulin resistance was prevented by manipulations that restrict fatty acid uptake into mitochondria. These results were recapitulated in mice lacking malonyl-CoA decarboxylase (MCD), an enzyme that promotes mitochondrial beta-oxidation by relieving malonyl-CoA-mediated inhibition of carnitine palmitoyltransferase 1. Thus, mcd(-/-) mice exhibit reduced rates of fat catabolism and resist diet-induced glucose intolerance despite high intramuscular levels of long-chain acyl-CoAs. These findings reveal a strong connection between skeletal muscle insulin resistance and lipid-induced mitochondrial stress.

Lysosomal glucose sensing and glycophagy in metabolism.

Lysosomes are cellular organelles that function to catabolize both extra- and intracellular cargo, act as a platform for nutrient sensing, and represent a core signaling node integrating bioenergetic cues to changes in cellular metabolism. Although lysosomal amino acid and lipid sensing in metabolism has been well characterized, lysosomal glucose sensing and the role of lysosomes in glucose metabolism is unrefined. This review will highlight the role of the lysosome in glucose metabolism with a focus on lysosomal glucose and glycogen sensing, glycophagy, and lysosomal glucose transport and how these processes impact autophagy and energy metabolism. Additionally, the role of lysosomal glucose metabolism in genetic and metabolic diseases will be briefly discussed.

Rats lacking Ucp1 present a novel translational tool for the investigation of thermogenic adaptation during cold challenge.

AIM: Valuable studies have tested the role of UCP1 on body temperature maintenance in mice, and we sought to knockout Ucp1 in rats (Ucp1-/- ) to provide insight into thermogenic mechanisms in larger mammals. METHODS: We used CRISPR/Cas9 technology to create Ucp1-/- rats. Body weight and adiposity were measured, and rats were subjected to indirect calorimetry. Rats were maintained at room temperature or exposed to 4°C for either 24 h or 14 days. Analyses of brown and white adipose tissue and skeletal muscle were conducted via histology, western blot comparison of oxidative phosphorylation proteins, and qPCR to compare mitochondrial DNA levels and mRNA expression profiles. RNA-seq was performed in skeletal muscle. RESULTS: Ucp1-/- rats withstood 4°C for 14 days, but core temperature steadily declined. All rats lost body weight after 14 days at 4°C, but controls increased food intake more robustly than Ucp1-/- rats. Brown adipose tissue showed signs of decreased activity in Ucp1-/- rats, while mitochondrial lipid metabolism markers in white adipose tissue and skeletal muscle were increased. Ucp1-/- rats displayed more visible shivering and energy expenditure than controls at 4°C. Skeletal muscle transcriptomics showed more differences between genotypes at 23°C than at 4°C. CONCLUSION: Room temperature presented sufficient cold stress to rats lacking UCP1 to activate compensatory thermogenic mechanisms in skeletal muscle, which were only activated in control rats following exposure to 4°C. These results provide novel insight into thermogenic responses to UCP1 deficiency; and highlight Ucp1-/- rats as an attractive translational model for the study of thermogenesis.

Pharmacological inhibition of lipolysis prevents adverse metabolic outcomes during glucocorticoid administration.

OBJECTIVE: Glucocorticoids are one of the most commonly prescribed classes of anti-inflammatory drugs; however, chronic treatment promotes iatrogenic (drug-induced) diabetes. As part of their physiological role, glucocorticoids stimulate lipolysis to spare glucose. We hypothesized that persistent stimulation of lipolysis during glucocorticoid therapy plays a causative role in the development of iatrogenic diabetes. METHODS: Male C57BL/6J mice were given 100 µg/mL corticosterone (Cort) in the drinking water for two weeks and were fed either normal chow (TekLad 8640) or the same diet supplemented with an adipose triglyceride lipase inhibitor (Atglistatin - 2  g/kg diet) to inhibit the first step of lipolysis. RESULTS: Herein, we report for the first time that glucocorticoid administration promotes a unique state of substrate excess and energetic overload in skeletal muscle that primarily results from the rampant mobilization of endogenous fuels. Inhibiting lipolysis protected mice from Cort-induced gains in fat mass, excess ectopic lipid accrual, hyperinsulinemia, and hyperglycemia. The role lipolysis plays in Cort-mediated pathology appears to differ between tissues. Within skeletal muscle, Cort-induced lipolysis facilitated diversion of glucose-derived carbons toward the pentose phosphate and hexosamine biosynthesis pathways but contributed to <3% of the Cort-induced genomic adaptations. In contrast, Cort stimulation of lipolysis accounted for ∼35% of the genomic changes in the liver but had minimal impact on hepatic metabolites reported. CONCLUSIONS: These data support the idea that activation of lipolysis plays a causal role in the progression toward iatrogenic diabetes during glucocorticoid therapy with differential impact on skeletal muscle and liver.

Effect of sleep restriction on insulin sensitivity and energy metabolism in postmenopausal women: A randomized crossover trial.

OBJECTIVE: The aim of this study was to investigate the effect of sleep restriction (SR) on insulin sensitivity and energy metabolism in postmenopausal women. METHODS: In a randomized crossover trial, 14 women underwent four nights of habitual sleep (HS, 100% normal sleep) and SR (60% of HS) while following a eucaloric diet. Outcomes included the following: (1) insulin sensitivity by hyperinsulinemic-euglycemic clamp, defined as the glucose infusion rate (GIR); (2) resting metabolism and substrate oxidation by indirect calorimetry; and (3) glucose, insulin, and C-peptide concentrations following a standard meal test. RESULTS: Nine postmenopausal women (mean [SD], age 59 [4] years, BMI 28.0 [2.6] kg/m2 ) were analyzed. Accelerometer-determined total time in bed was 8.4 ± 0.6 hours during HS versus 5.0 ± 0.4 hours during SR (38% reduction, p < 0.0001). SR reduced low-dose insulin GIR by 20% (HS: 2.55 ± 0.22 vs. SR: 2.03 ± 0.20 mg/kg/min; p = 0.01) and high-dose insulin GIR by 12% (HS: 10.48 ± 0.72 vs. SR: 9.19 ± 0.72 mg/kg/min; p < 0.001). SR reduced fat oxidation during high-dose insulin infusion (p < 0.01), and it did not alter resting energy metabolism. CONCLUSIONS: Four nights of SR reduced insulin sensitivity and fat oxidation in postmenopausal women. These findings underscore the role of insufficient sleep in metabolic dysfunction following menopause. Larger trials investigating how sleep disturbances cause metabolic dysfunction during menopause are needed across all stages of menopause.

Intact mitochondrial substrate efflux is essential for prevention of tubular injury in a sex-dependent manner.

The importance of healthy mitochondrial function is implicated in the prevention of chronic kidney disease (CKD) and diabetic kidney disease (DKD). Sex differences also play important roles in DKD. Our previous studies revealed that mitochondrial substrate overload (modeled by homozygous deletion of carnitine acetyl-transferase [CrAT]) in proximal tubules causes renal injury. Here, we demonstrate the importance of intact mitochondrial substrate efflux by titrating the amount of overload through the generation of a heterozygous CrAT-KO model (PT-CrATHET mouse). Intriguingly, these animals developed renal injury similarly to their homozygous counterparts. Mitochondria were structurally and functionally impaired in both sexes. Transcriptomic analyses, however, revealed striking sex differences. Male mice shut down fatty acid oxidation and several other metabolism-related pathways. Female mice had a significantly weaker transcriptional response in metabolism, but activation of inflammatory pathways was prominent. Proximal tubular cells from PT-CrATHET mice of both sexes exhibited a shift toward a more glycolytic phenotype, but female mice were still able to oxidize fatty acid-based substrates. Our results demonstrate that maintaining mitochondrial substrate metabolism balance is crucial to satisfying proximal tubular energy demand. Our findings have potentially broad implications, as both the glycolytic shift and the sexual dimorphisms discovered herein offer potentially new modalities for future interventions for treating kidney disease.

Female Mice Are Protected from Metabolic Decline Associated with Lack of Skeletal Muscle HuR.

Male mice lacking HuR in skeletal muscle (HuRm-/-) have been shown to have decreased gastrocnemius lipid oxidation and increased adiposity and insulin resistance. The same consequences have not been documented in female HuRm-/- mice. Here we examine this sexually dimorphic phenotype. HuRm-/- mice have an increased fat mass to lean mass ratio (FM/LM) relative to controls where food intake is similar. Increased body weight for male mice correlates with increased blood glucose during glucose tolerance tests (GTT), suggesting increased fat mass in male HuRm-/- mice as a driver of decreased glucose clearance. However, HuRm-/- female mice show decreased blood glucose levels during GTT relative to controls. HuRm-/- mice display decreased palmitate oxidation in skeletal muscle relative to controls. This difference is more robust for male HuRm-/- mice and more exaggerated for both sexes at high dietary fat. A high-fat diet stimulates expression of Pgc1α in HuRm-/- male skeletal muscle, but not in females. However, the lipid oxidation Pparα pathway remains decreased in HuRm-/- male mice relative to controls regardless of diet. This pathway is only decreased in female HuRm-/- mice fed high fat diet. A decreased capacity for lipid oxidation in skeletal muscle in the absence of HuR may thus be linked to decreased glucose clearance in male but not female mice.

Carnitine insufficiency caused by aging and overnutrition compromises mitochondrial performance and metabolic control.

In addition to its essential role in permitting mitochondrial import and oxidation of long chain fatty acids, carnitine also functions as an acyl group acceptor that facilitates mitochondrial export of excess carbons in the form of acylcarnitines. Recent evidence suggests carnitine requirements increase under conditions of sustained metabolic stress. Accordingly, we hypothesized that carnitine insufficiency might contribute to mitochondrial dysfunction and obesity-related impairments in glucose tolerance. Consistent with this prediction whole body carnitine diminution was identified as a common feature of insulin-resistant states such as advanced age, genetic diabetes, and diet-induced obesity. In rodents fed a lifelong (12 month) high fat diet, compromised carnitine status corresponded with increased skeletal muscle accumulation of acylcarnitine esters and diminished hepatic expression of carnitine biosynthetic genes. Diminished carnitine reserves in muscle of obese rats was accompanied by marked perturbations in mitochondrial fuel metabolism, including low rates of complete fatty acid oxidation, elevated incomplete beta-oxidation, and impaired substrate switching from fatty acid to pyruvate. These mitochondrial abnormalities were reversed by 8 weeks of oral carnitine supplementation, in concert with increased tissue efflux and urinary excretion of acetylcarnitine and improvement of whole body glucose tolerance. Acetylcarnitine is produced by the mitochondrial matrix enzyme, carnitine acetyltransferase (CrAT). A role for this enzyme in combating glucose intolerance was further supported by the finding that CrAT overexpression in primary human skeletal myocytes increased glucose uptake and attenuated lipid-induced suppression of glucose oxidation. These results implicate carnitine insufficiency and reduced CrAT activity as reversible components of the metabolic syndrome.

Metabolic profiling of PPARalpha-/- mice reveals defects in carnitine and amino acid homeostasis that are partially reversed by oral carnitine supplementation.

Peroxisome proliferator-activated receptor-alpha (PPARalpha) is a master transcriptional regulator of beta-oxidation and a prominent target of hypolipidemic drugs. To gain deeper insights into the systemic consequences of impaired fat catabolism, we used quantitative, mass spectrometry-based metabolic profiling to investigate the fed-to-fasted transition in PPARalpha(+/+) and PPARalpha(-/-) mice. Compared to PPARalpha(+/+) animals, acylcarnitine profiles of PPARalpha(-/-) mice revealed 2- to 4-fold accumulation of long-chain species in the plasma, whereas short-chain species were reduced by as much as 69% in plasma, liver, and skeletal muscle. These results reflect a metabolic bottleneck downstream of carnitine palmitoyltransferase-1, a mitochondrial enzyme that catalyzes the first step in beta-oxidation. Organic and amino acid profiles of starved PPARalpha(-/-) mice suggested compromised citric acid cycle flux, enhanced urea cycle activity, and increased amino acid catabolism. PPARalpha(-/-) mice had 40-50% lower plasma and tissue levels of free carnitine, corresponding with diminished hepatic expression of genes involved in carnitine biosynthesis and transport. One week of oral carnitine supplementation conferred partial metabolic recovery in the PPARalpha(-/-) mice. In summary, comprehensive metabolic profiling revealed novel biomarkers of defective fat oxidation, while also highlighting the potential value of supplemental carnitine as a therapy and diagnostic tool for metabolic disorders.