Medium-Chain Fatty Acid Feeding Reduces Oxidation and Causes Panacinar Steatosis in Livers of Neonatal Pigs.

BACKGROUND: Medium-chain fatty acids (MCFAs) are commonly used to enhance the caloric content of infant formulas. We previously reported that pigs fed MCFA developed hepatic steatosis when compared to those fed isocaloric long-chain fatty acid (LCFA) rich formula. OBJECTIVES: The objectives of this study were to investigate: 1) whether MCFA and LCFA feeding affect hepatic fatty acid oxidation, and 2) how fat type alters the expression of hepatic fatty acid metabolic genes. METHODS: Twenty-six, 7-d-old pigs were fed a low-energy control (CONT) formula, or 2 isocaloric high-energy formulas rich in LCFA or MCFA for 22 days. Livers were collected for examining ex vivo fatty acid oxidation, fatty acid content, and mRNA expression of fatty acid metabolic genes. RESULTS: Liver fat was 20% for pigs in the MCFA compared with 2.9% and 4.6% for those in the CONT and LCFA groups (P < 0.05). MCFA-fed pigs had greater amounts of hepatic laurate, myristate, palmitate, and palmitoleate (14, 34, 49, and 9.3 mg · g-1) than those fed LCFA and CONT (1.8, 1.9, 19, 1.5 mg · g-1) formulas (P ≤ 0.05). Hepatic laurate and palmitate oxidation was reduced for pigs fed MCFA (29 mmol · mg-1 · h-1) compared with those fed CONT (54 mmol · mg-1 · h-1) and LCFA (51 mmol · mg-1 · h-1) formulas (P < 0.05). Expression of fatty acid synthase 3 (FASN-3), fatty acid binding protein 1 (FABP-1), and acetyl-CoA carboxylase 1 (ACACA-1) were 8-, 6-, and 2-fold greater for pigs in the MCFA than those in the LCFA and CONT groups (P < 0.05). CONCLUSIONS: Feeding MCFA resulted in hepatic steatosis compared with an isocaloric formula rich in LCFA. Steatosis occurred concomitantly with reduced fatty acid oxidation but greater mRNA expression of fatty acid synthetic and catabolic genes.

Bioenergetics underlying single-cell migration on aligned nanofiber scaffolds.

Cell migration is centrally involved in a myriad of physiological processes, including morphogenesis, wound healing, tissue repair, and metastatic growth. The bioenergetics that underlie migratory behavior are not fully understood, in part because of variations in cell culture media and utilization of experimental cell culture systems that do not model physiological connective extracellular fibrous networks. In this study, we evaluated the bioenergetics of C2C12 myoblast migration and force production on fibronectin-coated nanofiber scaffolds of controlled diameter and alignment, fabricated using a nonelectrospinning spinneret-based tunable engineered parameters (STEP) platform. The contribution of various metabolic pathways to cellular migration was determined using inhibitors of cellular respiration, ATP synthesis, glycolysis, or glucose uptake. Despite immediate effects on oxygen consumption, mitochondrial inhibition only modestly reduced cell migration velocity, whereas inhibitors of glycolysis and cellular glucose uptake led to striking decreases in migration. The migratory metabolic sensitivity was modifiable based on the substrates present in cell culture media. Cells cultured in galactose (instead of glucose) showed substantial migratory sensitivity to mitochondrial inhibition. We used nanonet force microscopy to determine the bioenergetic factors responsible for single-cell force production and observed that neither mitochondrial nor glycolytic inhibition altered single-cell force production. These data suggest that myoblast migration is heavily reliant on glycolysis in cells grown in conventional media. These studies have wide-ranging implications for the causes, consequences, and putative therapeutic treatments aimed at cellular migration.

Effects of Seasonal Vitamin D3 Supplementation on Strength, Power, and Body Composition in College Swimmers.

The purpose of this study was to evaluate the impact of fall season vitamin D3 supplementation on strength/power, body composition, and anabolic hormones in swimmers with optimal vitamin D status at summer's end. Male and female National Collegiate Athletic Association Division I swimmers (N = 19) with optimal 25-hydroxyvitamin D [25(OH)D] randomly received 5,000 IU of vitamin D3 (VITD) or placebo (PLA) daily for 12 weeks while participating in swimming and strength and conditioning training (August-November). Before and after the intervention, the participants underwent blood sampling for analysis of serum 25(OH)D, parathyroid hormone, total testosterone, free testosterone, sex hormone-binding globulin, and insulin-like growth factor 1, dual-energy X-ray absorptiometry, and strength/power testing (bench press, squat, dead lift, standing broad jump, vertical jump, and dips and pull-ups). Sex was used as a covariate for analyses. The 25(OH)D was decreased by 44% in PLA (p < .05) and increased by 8% in VITD over the 12 weeks. Fat-free mass increased in VITD (56.4-59.1 kg; p < .05), but not PLA (59.4-59.7 kg; p < .01). Significant Group × Time interaction effects were observed for dead lift (F = 21.577, p < .01) and vertical jump (F = 11.219, p < .01), but no other strength/power tests. Total testosterone decreased similarly in both groups, but free testosterone decreased and sex hormone-binding globulin increased only in PLA (p < .01). There were no group differences or changes in insulin-like growth factor 1 with the intervention. The findings suggest that vitamin D supplementation is an efficacious strategy to maintain 25(OH)D during the fall season training and to enhance some aspects of strength/power and fat-free mass in swimmers. Further research on the relationship between vitamin D and anabolic hormones is needed.

Heat stress decreases metabolic flexibility in skeletal muscle of growing pigs.

Heat-stressed pigs experience metabolic alterations, including altered insulin profiles, reduced lipid mobilization, and compromised intestinal integrity. This is bioenergetically distinct from thermal neutral pigs on a similar nutritional plane. To delineate differences in substrate preferences between direct and indirect (via reduced feed intake) heat stress effects, skeletal muscle fuel metabolism was assessed. Pigs (35.3 ± 0.8 kg) were randomly assigned to three treatments: thermal neutral fed ad libitum (TN; 21°C, n = 8), heat stress fed ad libitum (HS; 35°C, n = 8), and TN, pair-fed/HS intake (PF; n = 8) for 7 days. Body temperature (TB) and feed intake (FI) were recorded daily. Longissimus dorsi muscle was biopsied for metabolic assays on days -2, 3, and 7 relative to initiation of environmental treatments. Heat stress increased TB and decreased FI ( P < 0.05). Heat stress inhibited incomplete fatty acid oxidation and glucose oxidation ( P < 0.05). Metabolic flexibility decreased in HS pigs compared with TN and PF controls ( P < 0.05). Both phosphofructokinase and pyruvate dehydrogenase (PDH) activities increased in PF ( P < 0.05); however, TN and HS did not differ. Heat stress inhibited citrate synthase and ß-hydroxyacyl-CoA dehydrogenase (ß-HAD) activities ( P < 0.05). Heat stress did not alter PDH phosphorylation or carnitine palmitoyltransferase 1 abundance but reduced acetyl-CoA carboxylase 1 (ACC1) protein abundance ( P < 0.05). In conclusion, HS decreased skeletal muscle fatty acid oxidation and metabolic flexibility, likely involving ß-HAD and ACC regulation.

The Flavonoid Kaempferol Ameliorates Streptozotocin-Induced Diabetes by Suppressing Hepatic Glucose Production.

In diabetes mellitus, the excessive rate of glucose production from the liver is considered a primary contributor for the development of hyperglycemia, in particular, fasting hyperglycemia. In this study, we investigated whether kaempferol, a flavonol present in several medicinal herbs and foods, can be used to ameliorate diabetes in an animal model of insulin deficiency and further explored the mechanism underlying the anti-diabetic effect of this flavonol. We demonstrate that oral administration of kaempferol (50 mg/kg/day) to streptozotocin-induced diabetic mice significantly improved hyperglycemia and reduced the incidence of overt diabetes from 100% to 77.8%. This outcome was accompanied by a reduction in hepatic glucose production and an increase in glucose oxidation in the muscle of the diabetic mice, whereas body weight, calorie intake, body composition, and plasma insulin and glucagon levels were not altered. Consistently, treatment with kaempferol restored hexokinase activity in the liver and skeletal muscle of diabetic mice while suppressed hepatic pyruvate carboxylase activity and gluconeogenesis. These results suggest that kaempferol may exert antidiabetic action via promoting glucose metabolism in skeletal muscle and inhibiting gluconeogenesis in the liver.

Selective overexpression of Toll-like receptor-4 in skeletal muscle impairs metabolic adaptation to high-fat feeding.

Toll-like receptor-4 (TLR-4) is elevated in skeletal muscle of obese humans, and data from our laboratory have shown that activation of TLR-4 in skeletal muscle via LPS results in decreased fatty acid oxidation (FAO). The purpose of this study was to determine whether overexpression of TLR-4 in skeletal muscle alters mitochondrial function and whole body metabolism in the context of a chow and high-fat diet. C57BL/6J mice (males, 6-8 mo of age) with skeletal muscle-specific overexpression of the TLR-4 (mTLR-4) gene were created and used for this study. Isolated mitochondria and whole muscle homogenates from rodent skeletal muscle (gastrocnemius and quadriceps) were investigated. TLR-4 overexpression resulted in a significant reduction in FAO in muscle homogenates; however, mitochondrial respiration and reactive oxygen species (ROS) production did not appear to be affected on a standard chow diet. To determine the role of TLR-4 overexpression in skeletal muscle in response to high-fat feeding, mTLR-4 mice and WT control mice were fed low- and high-fat diets for 16 wk. The high-fat diet significantly decreased FAO in mTLR-4 mice, which was observed in concert with elevated body weight and fat, greater glucose intolerance, and increase in production of ROS and cellular oxidative damage compared with WT littermates. These findings suggest that TLR-4 plays an important role in the metabolic response in skeletal muscle to high-fat feeding.

Metabolic changes during ovarian cancer progression as targets for sphingosine treatment.

Tumor cells often exhibit an altered metabolic phenotype. However, it is unclear as to when this switch takes place in ovarian cancer, and the potential for these changes to serve as therapeutic targets in clinical prevention and intervention trials. We used our recently developed and characterized mouse ovarian surface epithelial (MOSE) cancer progression model to study metabolic changes in distinct disease stages. As ovarian cancer progresses, complete oxidation of glucose and fatty acids were significantly decreased, concurrent with increases in lactate excretion and (3)H-deoxyglucose uptake by the late-stage cancer cells, shifting the cells towards a more glycolytic phenotype. These changes were accompanied by decreases in TCA flux but an increase in citrate synthase activity, providing substrates for de novo fatty acid and cholesterol synthesis. Also, uncoupled maximal respiration rates in mitochondria decreased as cancer progressed. Treatment of the MOSE cells with 1.5 µM sphingosine, a bioactive sphingolipid metabolite, decreased citrate synthase activity, increased TCA flux, decreased cholesterol synthesis and glycolysis. Together, our data confirm metabolic changes during ovarian cancer progression, indicate a stage specificity of these changes, and suggest that multiple events in cellular metabolism are targeted by exogenous sphingosine which may be critical for future prevention trials.

Tlr5 deficiency exacerbates lupus-like disease in the MRL/lpr mouse model.

Introduction: Leaky gut has been linked to autoimmune disorders including lupus. We previously reported upregulation of anti-flagellin antibodies in the blood of lupus patients and lupus-prone mice, which led to our hypothesis that a leaky gut drives lupus through bacterial flagellin-mediated activation of toll-like receptor 5 (TLR5). Methods: We created MRL/lpr mice with global Tlr5 deletion through CRISPR/Cas9 and investigated lupus-like disease in these mice. Result: Contrary to our hypothesis that the deletion of Tlr5 would attenuate lupus, our results showed exacerbation of lupus with Tlr5 deficiency in female MRL/lpr mice. Remarkably higher levels of proteinuria were observed in Tlr5 -/- MRL/lpr mice suggesting aggravated glomerulonephritis. Histopathological analysis confirmed this result, and Tlr5 deletion significantly increased the deposition of IgG and complement C3 in the glomeruli. In addition, Tlr5 deficiency significantly increased renal infiltration of Th17 and activated cDC1 cells. Splenomegaly and lymphadenopathy were also aggravated in Tlr5-/- MRL/lpr mice suggesting impact on lymphoproliferation. In the spleen, significant decreased frequencies of regulatory lymphocytes and increased germinal centers were observed with Tlr5 deletion. Notably, Tlr5 deficiency did not change host metabolism or the existing leaky gut; however, it significantly reshaped the fecal microbiota. Conclusion: Global deletion of Tlr5 exacerbates lupus-like disease in MRL/lpr mice. Future studies will elucidate the underlying mechanisms by which Tlr5 deficiency modulates host-microbiota interactions to exacerbate lupus.

Fish oil and indomethacin in combination potently reduce dyslipidemia and hepatic steatosis in LDLR(-/-) mice.

Fish oil (FO) is a potent anti-inflammatory and lipid-lowering agent. Because inflammation can modulate lipid metabolism and vice versa, we hypothesized that combining FO with cyclooxygenase inhibitors (COXIBs), well-known anti-inflammatory drugs, can enhance the anti-inflammatory and lipid-lowering effect of FO. LDLR(-/-) mice were fed a high-fat diet supplemented with 6% olive oil or FO for 12 wk in the presence or absence of indomethacin (Indo, 6 mg/l drinking water). FO reduced plasma total cholesterol by 30% but, in combination with Indo, exerted a greater decrease (44%). The reduction of liver cholesterol ester (CE) and triglycerides (TG) by FO (63% and 41%, respectively) was enhanced by Indo (80% in CE and 64% in TG). FO + Indo greatly increased the expression of genes modulating lipid metabolism and reduced the expression of inflammatory genes compared with control. The mRNA and/or protein expression of pregnane X receptor (PXR) and cytochrome P450 isoforms that alter inflammation and/or lipid metabolism are increased to a greater extent in mice that received FO + Indo. Moroever, the nuclear level of PXR is significantly increased in FO + Indo group. Combining FO with COXIBs may exert their beneficial effects on inflammation and lipid metabolism via PXR and cytochrome P450.

An Association between Bioavailable 25-Hydroxyvitamin D and Bone Mineral Density in a Diverse Cohort of Collegiate Athletes.

INTRODUCTION: Although vitamin D is intimately involved in bone metabolism, the relationship between vitamin D status, as measured by serum total 25-hydroxyvitamin D [25(OH)D] concentration, and bone mineral density (BMD) is weak, particularly in non-White populations. Measurement of bioavailable 25(OH)D has been suggested as a better indicator of vitamin D status than total 25(OH)D concentration. To date, the bioavailable 25(OH)D biomarker has been explored minimally in athletic populations. The purpose of this study was to investigate the relationship between total and bioavailable 25(OH)D concentrations and BMD in collegiate athletes. METHODS: NCAA Division I basketball and swimming athletes served as study participants (n = 53; 28 females, 25 males; 28 basketball players, 25 swimmers). All participants completed dual-energy x-ray absorptiometry scans for analysis of BMD, blood draws for vitamin D measures, and diet/lifestyle questionnaires. RESULTS: Overall, total 25(OH)D was 80.0 + 13.9 nmol·L-1 and bioavailable 25(OH)D was 6.0 ± 1.9 nmol·L-1. There was strong disagreement between total 25(OH)D and bioavailable 25(OH)D concentrations (κ = -0.299, r = -0.129) (P = 0.100); 53% of total participants and 77% of Black participants were classified differently (low vs normal vitamin D status) based on total and bioavailable 25(OH)D criteria. Black participants had significantly lower total 25(OH)D and higher bioavailable 25(OH)D concentrations than White participants (59.5 vs 102.5 nmol·L-1 and 7.9 vs 5.4 nmol·L-1, respectively) (P < 0.001). Total 25(OH)D and total BMD were not correlated, but bioavailable 25(OH)D and total BMD demonstrated a positive correlation (r = 0.618, P < 0.01). CONCLUSIONS: These results suggest that bioavailable 25(OH)D concentration may be a better clinical measure of vitamin D status than total 25(OH)D as related to BMD in collegiate athletes, particularly in Black athletes. Further research on the utility of the bioavailable 25(OH)D biomarker in athletes is needed.

Supplementation with artificial sweetener and capsaicin alters metabolic flexibility and performance in heat-stressed and feed-restricted pigs.

Substantial economic losses in animal agriculture result from animals experiencing heat stress (HS). Pigs are especially susceptible to HS, resulting in reductions in growth, altered body composition, and compromised substrate metabolism. In this study, an artificial high-intensity sweetener and capsaicin (CAPS-SUC; Pancosma, Switzerland) were supplemented in combination to mitigate the adverse effects of HS on pig performance. Forty cross-bred barrows (16.2 ± 6 kg) were assigned to one of five treatments: thermal neutral controls (TN) (22 ± 1.2 °C; 38%-73% relative humidity) with ad libitum feed, HS conditions with ad libitum feed with (HS+) or without (HS-) supplementation, and pair-fed to HS with (PF+) or without supplementation (PF-). Pigs in heat-stressed treatments were exposed to a cyclical environmental temperature of 12 h at 35 ± 1.2 °C with 27%-45% relative humidity and 12 h at 30 ± 1.1 °C with 24%-35% relative humidity for 21 d. Supplementation (0.1 g/kg feed) began 7 d before and persisted through the duration of environmental or dietary treatments (HS/PF), which lasted for 21 d. Rectal temperatures and respiration rates (RR; breaths/minute) were recorded thrice daily, and feed intake (FI) was recorded daily. Before the start and at the termination of environmental treatments (HS/PF), a muscle biopsy of the longissimus dorsi was taken for metabolic analyses. Blood samples were collected weekly, and animals were weighed every 3 d during treatment. Core temperature (TN 39.2 ± 0.02 °C, HS- 39.6 ± 0.02 °C, and HS+ 39.6 ± 0.02 °C, P < 0.001) and RR (P < 0.001) were increased in both HS- and HS+ groups, but no difference was detected between HS- and HS+. PF- pigs exhibited reduced core temperature (39.1 ± 0.02 °C, P < 0.001), which was restored in PF+ pigs (39.3 ± 0.02 °C) to match TN. Weight gain and feed efficiency were reduced in PF- pigs (P < 0.05) but not in the PF+ or the HS- or HS+ groups. Metabolic flexibility was decreased in the HS- group (-48.4%, P < 0.05) but maintained in the HS+ group. CAPS-SUC did not influence core temperature or weight gain in HS pigs but did restore core temperature, weight gain, and feed efficiency in supplemented PF pigs. In addition, supplementation restored metabolic flexibility during HS and improved weight gain and feed efficiency during PF, highlighting CAPS-SUC's therapeutic metabolic effects.

Heat stress reduces pig performance due to metabolic responses to heat. During heat stress, pigs lose the ability to metabolize fatty acids for energy and rely on carbohydrates to fuel growth. Evidence has shown that capsaicin, the active ingredient in chili peppers, interacts with heat-sensing receptors to protect against heat stress by preventing changes to metabolism. Artificial sweeteners can also preserve fat metabolism by inducing the secretion of metabolic regulatory hormones from the gut. This study examined a combination of capsaicin and artificial sweetener to restore growth and maintain metabolism during 3 wk of heat stress. As pigs often reduce their feed intake during heat stress, a group of pigs was feed restricted to match the reduced feeding observed in the heat-stressed pigs. Pigs given the feed supplement during heat stress maintained their metabolic flexibility, a measure of metabolic health. In agreement with previous short-term studies, the capsaicin and artificial sweetener supplement improved feed efficiency and weight gain in feed-restricted pigs. This study demonstrated that supplementation with capsaicin and artificial sweetener may prevent metabolic dysfunction during heat stress. This study also confirmed that supplementation with capsaicin and artificial sweetener does improve feed-restricted pigs' growth and feed efficiency.

Central nervous system melanocortin-3 receptors are required for synchronizing metabolism during entrainment to restricted feeding during the light cycle.

Melanocortin-3 receptors (Mc3rs) in the central nervous system are involved in expression of anticipatory rhythms and synchronizing clocks maintaining circadian rhythms during restricted feeding (RF) [mice housed under a 12-h light-dark cycle with lights on between zeitgeber time (ZT) 0 to ZT12 fed 60% of normal calories between ZT7 and ZT11]. Because the systems governing circadian rhythms are important for adaptation to RF, we investigated whether Mc3rs are required for metabolic adaption to RF. Mc3r(-/-) mice subjected to RF exhibited normal weight loss; however, they developed hyperinsulinemia, glucose intolerance, increased expression of lipogenic genes, and increased ketogenesis relative to controls. Rhythmic expression of transcription factors regulating liver clock activity and energy metabolism (Bmal1, Rev-erbalpha, Pgc1, Foxo1, Hnf4alpha, and Pck1) was severely compromised in Mc3r(-/-) mice during RF. Inhibition of neural melanocortin receptors by agouti-related peptide also attenuated rhythmicity in the hepatic expression of these genes during RF. Collectively, these data suggest that neural Mc3rs are important for adapting metabolism and maintaining rhythms of liver metabolism during periods when feeding is restricted to the light cycle.-Sutton, G. M., Begriche, K., Kumar, K. G., Gimble, J. M., Perez-Tilve, D., Nogueiras, R., McMillan, R. P., Hulver, M. W., Tschöp, M. H., Butler, A. A. Central nervous system melanocortin-3 receptors are required for synchronizing metabolism during entrainment to restricted feeding during the light cycle.

Muscle-Specific Deletion of Toll-like Receptor 4 Impairs Metabolic Adaptation to Wheel Running in Mice.

PURPOSE: Toll-like receptor 4 (TLR4) is an inflammatory receptor expressed ubiquitously in immune cells as well as skeletal muscle and other metabolic tissues. Skeletal muscle develops favorable inflammation-mediated metabolic adaptations from exercise training. Multiple inflammatory myokines, downstream from TLR4, are proposed links to the metabolic benefits of exercise. In addition, activation of TLR4 alters skeletal muscle substrate preference. The role of skeletal muscle TLR4 (mTLR4) in exercise metabolism has not previously been investigated. Herein, we aimed to specifically test the significance of mTLR4 to exercise-induced metabolic adaptations. METHODS: We developed a novel muscle-specific TLR4 knockout (mTLR4-/-) mouse model on C57BL/6J background. Male mTLR4-/- mice and wild-type (WT) littermates were compared under sedentary (SED) and voluntary wheel running (WR) conditions for 4 wk. RESULTS: mTLR4 deletion revealed marked reductions in downstream interleukin-1 receptor-associated kinase-4 (IRAK4) phosphorylation. In addition, the disruption of mTLR4 signaling prominently blunted the metabolic adaptations in WR-mTLR4-/- mice as opposed to substantial improvements exhibited by the WT counterparts. Voluntary WR in WT mice, relative to SED, resulted in significant increases in skeletal muscle fatty acid oxidation, glucose oxidation, and associated mitochondrial enzyme activities, all of which were not significantly changed in mTLR4-/- mice. CONCLUSIONS: This study introduces a novel mTLR4-/- mouse model and identifies mTLR4 as an immunomodulatory effector of exercise-induced metabolic adaptations in skeletal muscle.

Heat Stress Reduces Metabolic Rate While Increasing Respiratory Exchange Ratio in Growing Pigs.

Heat stress (HS) diminishes animal production, reducing muscle growth and increasing adiposity, especially in swine. Excess heat creates a metabolic phenotype with limited lipid oxidation that relies on aerobic and anaerobic glycolysis as a predominant means of energy production, potentially reducing metabolic rate. To evaluate the effects of HS on substrate utilization and energy expenditure, crossbred barrows (15.2 ± 2.4 kg) were acclimatized for 5 days (22 °C), then treated with 5 days of TN (thermal neutral, 22 °C, n = 8) or HS (35 °C, n = 8). Pigs were fed ad libitum and monitored for respiratory rate (RR) and rectal temperature. Daily energy expenditure (DEE) and respiratory exchange ratio (RER, CO2:O2) were evaluated fasted in an enclosed chamber through indirect calorimetry. Muscle biopsies were obtained from the longissimus dorsi pre/post. HS increased temperature (39.2 ± 0.1 vs. 39.6 ± 0.1 °C, p < 0.01) and RER (0.91 ± 0.02 vs. 1.02 ± 0.02 VCO2:VO2, p < 0.01), but decreased DEE/BW (68.8 ± 1.7 vs. 49.7 ± 4.8 kcal/day/kg, p < 0.01) relative to TN. Weight gain (p = 0.80) and feed intake (p = 0.84) did not differ between HS and TN groups. HS decreased muscle metabolic flexibility (~33%, p = 0.01), but increased leucine oxidation (~35%, p = 0.02) compared to baseline values. These data demonstrate that HS disrupts substrate regulation and energy expenditure in growing pigs.

Prebiotic Inulin Supplementation and Peripheral Insulin Sensitivity in adults at Elevated Risk for Type 2 Diabetes: A Pilot Randomized Controlled Trial.

Prediabetes affects 84.1 million adults, and many will progress to type 2 diabetes (T2D). The objective of this proof-of-concept trial was to determine the efficacy of inulin supplementation to improve glucose metabolism and reduce T2D risk. Adults (n = 24; BMI: 31.3 ± 2.9 kg/m2; age: 54.4 ± 8.3 years) at risk for T2D were enrolled in this controlled feeding trial and consumed either inulin (10 g/day) or placebo (maltodextrin, 10 g/day) for six weeks. Assessments included peripheral insulin sensitivity, fasting glucose, and insulin, HOMA-IR, in vivo skeletal muscle substrate preference, Bifidobacteria copy number, intestinal permeability, and endotoxin concentrations. Participant retention was 92%. There were no baseline group differences except for fasting insulin (p = 0.003). The magnitude of reduction in fasting insulin concentrations with inulin (p = 0.003, inulin = Δ-2.9, placebo = Δ2.3) was attenuated after adjustment for baseline concentrations (p = 0.04). After adjusting for baseline values, reduction in HOMA-IR with inulin (inulin = Δ-0.40, placebo=Δ0.27; p = 0.004) remained significant. Bifidobacteria 16s increased (p = 0.04; inulin = Δ3.1e9, placebo = Δ-8.9e8) with inulin supplementation. Despite increases in gut Bifidobacteria, inulin supplementation did not improve peripheral insulin sensitivity. These findings question the need for larger investigations of inulin and insulin sensitivity in this population.

Nox4 mediates skeletal muscle metabolic responses to exercise.

OBJECTIVE: The immediate signals that couple exercise to metabolic adaptations are incompletely understood. Nicotinamide adenine dinucleotide phosphate oxidase 4 (Nox4) produces reactive oxygen species (ROS) and plays a significant role in metabolic and vascular adaptation during stress conditions. Our objective was to determine the role of Nox4 in exercise-induced skeletal muscle metabolism. METHODS: Mice were subjected to acute exercise to assess their immediate responses. mRNA and protein expression responses to Nox4 and hydrogen peroxide (H2O2) were measured by qPCR and immunoblotting. Functional metabolic flux was measured via ex vivo fatty acid and glucose oxidation assays using 14C-labeled palmitate and glucose, respectively. A chronic exercise regimen was also utilized and the time to exhaustion along with key markers of exercise adaptation (skeletal muscle citrate synthase and beta-hydroxyacyl-coA-dehydrogenase activity) were measured. Endothelial-specific Nox4-deficient mice were then subjected to the same acute exercise regimen and their subsequent substrate oxidation was measured. RESULTS: We identified key exercise-responsive metabolic genes that depend on H2O2 and Nox4 using catalase and Nox4-deficient mice. Nox4 was required for the expression of uncoupling protein 3 (Ucp3), hexokinase 2 (Hk2), and pyruvate dehydrogenase kinase 4 (Pdk4), but not the expression of peroxisome proliferator-activated receptor gamma coactivator 1-alpha (Pgc-1α). Global Nox4 deletion resulted in decreased UCP3 protein expression and impaired glucose and fatty acid oxidization in response to acute exercise. Furthermore, Nox4-deficient mice demonstrated impaired adaptation to chronic exercise as measured by the time to exhaustion and activity of skeletal muscle citrate synthase and beta-hydroxyacyl-coA-dehydrogenase. Importantly, mice deficient in endothelial-Nox4 similarly demonstrated attenuated glucose and fatty acid oxidation following acute exercise. CONCLUSIONS: We report that H2O2 and Nox4 promote immediate responses to exercise in skeletal muscle. Glucose and fatty acid oxidation were blunted in the Nox4-deficient mice post-exercise, potentially through regulation of UCP3 expression. Our data demonstrate that endothelial-Nox4 is required for glucose and fatty acid oxidation, suggesting inter-tissue cross-talk between the endothelium and skeletal muscle in response to exercise.

Voluntary wheel running complements microdystrophin gene therapy to improve muscle function in mdx mice.

We tested the hypothesis that voluntary wheel running would complement microdystrophin gene therapy to improve muscle function in young mdx mice, a model of Duchenne muscular dystrophy. mdx mice injected with a single dose of AAV9-CK8-microdystrophin or vehicle at age 7 weeks were assigned to three groups: mdxRGT (run, gene therapy), mdxGT (no run, gene therapy), or mdx (no run, no gene therapy). Wild-type (WT) mice were assigned to WTR (run) and WT (no run) groups. WTR and mdxRGT performed voluntary wheel running for 21 weeks; remaining groups were cage active. Robust expression of microdystrophin occurred in heart and limb muscles of treated mice. mdxRGT versus mdxGT mice showed increased microdystrophin in quadriceps but decreased levels in diaphragm. mdx final treadmill fatigue time was depressed compared to all groups, improved in mdxGT, and highest in mdxRGT. Both weekly running distance (km) and final treadmill fatigue time for mdxRGT and WTR were similar. Remarkably, mdxRGT diaphragm power was only rescued to 60% of WT, suggesting a negative impact of running. However, potential changes in fiber type distribution in mdxRGT diaphragms could indicate an adaptation to trade power for endurance. Post-treatment in vivo maximal plantar flexor torque relative to baseline values was greater for mdxGT and mdxRGT versus all other groups. Mitochondrial respiration rates from red quadriceps fibers were significantly improved in mdxGT animals, but the greatest bioenergetic benefit was observed in the mdxRGT group. Additional assessments revealed partial to full functional restoration in mdxGT and mdxRGT muscles relative to WT. These data demonstrate that voluntary wheel running combined with microdystrophin gene therapy in young mdx mice improved whole-body performance, affected muscle function differentially, mitigated energetic deficits, but also revealed some detrimental effects of exercise. With microdystrophin gene therapy currently in clinical trials, these data may help us understand the potential impact of exercise in treated patients.

Erratum: Voluntary wheel running complements microdystrophin gene therapy to improve muscle function in mdx mice.

[This corrects the article DOI: 10.1016/j.omtm.2021.02.024.].

Toll-like receptor 4 modulates skeletal muscle substrate metabolism.

Toll-like receptor 4 (TLR4), a protein integral to innate immunity, is elevated in skeletal muscle of obese and type 2 diabetic humans and has been implicated in the development of lipid-induced insulin resistance. The purpose of this study was to examine the role of TLR4 as a modulator of basal (non-insulin-stimulated) substrate metabolism in skeletal muscle with the hypothesis that its activation would result in reduced fatty acid oxidation and increased partitioning of fatty acids toward neutral lipid storage. Human skeletal muscle, rodent skeletal muscle, and skeletal muscle cell cultures were employed to study the functional consequences of TLR4 activation on glucose and fatty acid metabolism. Herein, we demonstrate that activation of TLR4 with low (metabolic endotoxemia) and high (septic conditions) doses of LPS results in increased glucose utilization and reduced fatty acid oxidation in skeletal muscle and that these changes in metabolism in vivo occur in concert with increased circulating triglycerides. Moreover, animals with a loss of TLR4 function possess increased oxidative capacity in skeletal muscle and present with lower fasting levels of triglycerides and nonesterified free fatty acids. Evidence is also presented to suggest that these changes in substrate metabolism under metabolic endotoxemic conditions are independent of skeletal muscle-derived proinflammatory cytokine production. This report illustrates that skeletal muscle is a target for circulating endotoxin and may provide critical insight into the link between a proinflammatory state and dysregulated metabolism as observed with obesity, type 2 diabetes, and metabolic syndrome.

Overfeeding and Substrate Availability, But Not Age or BMI, Alter Human Satellite Cell Function.

Satellite cells (SC) aid skeletal muscle growth and regeneration. SC-mediated skeletal muscle repair can both be influenced by and exacerbate several diseases linked to a fatty diet, obesity, and aging. The purpose of this study was to evaluate the effects of different lifestyle factors on SC function, including body mass index (BMI), age, and high-fat overfeeding. For this study, SCs were isolated from the vastus lateralis of sedentary young (18-30 years) and sedentary older (60-80 years) men with varying BMIs (18-32 kg/m2), as well as young sedentary men before and after four weeks of overfeeding (OVF) (55% fat/ + 1000 kcal, n = 4). The isolated SCs were then treated in vitro with a control (5 mM glucose, 10% fetal bovine serum (FBS)) or a high substrate growth media (HSM) (10% FBS, 25 mM glucose, and 400 µM 2:1 oleate-palmitate). Cells were assessed on their ability to proliferate, differentiate, and fuel substrate oxidation after differentiation. The effect of HSM was measured as the percentage difference between SCs exposed to HSM compared to control media. In vitro SC function was not affected by donor age. OVF reduced SC proliferation rates (-19% p < 0.05) but did not influence differentiation. Cellular proliferation in response to HSM was correlated to the donor's body mass index (BMI) (r2 = 0.6121, p < 0.01). When exposed to HSM, SCs from normal weight (BMI 18-25 kg/m2) participants exhibited reduced proliferation and fusion rates with increased fatty-acid oxidation (p < 0.05), while SCs from participants with higher BMIs (BMI 25-32 kg/m2) demonstrated enhanced proliferation in HSM. HSM reduced proliferation and fusion (p < 0.05) in SCs isolated from subjects before OVF, whereas HSM exposure accelerated proliferation and fusion in SCs collected following OVF. These results indicated that diet has a greater influence on SC function than age and BMI. Though age and BMI do not influence in vitro SC function when grown in controlled conditions, both factors influenced the response of SCs to substrate challenges, indicating age and BMI may mediate responses to diet.