Browsing by Author "McMillan, Ryan P."

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    Dietary Supplementation of Chinese Ginseng Prevents Obesity and Metabolic Syndrome in High-Fat Diet-Fed Mice
    Li, Xiaoxiao; Luo, Jing; Babu, Pon Velayutham Anandh; Zhang, Wei; Gilbert, Elizabeth R.; Cline, Mark A.; McMillan, Ryan P.; Hulver, Matthew W.; Alkhalidy, Hana; Zhen, Wei; Zhang, Haiyan; Liu, Dongmin (Mary Ann Liebert, 2014-12-01)
    Obesity and diabetes are growing health problems worldwide. In this study, dietary provision of Chinese ginseng (0.5 g/kg diet) prevented body weight gain in high-fat (HF) diet-fed mice. Dietary ginseng supplementation reduced body fat mass gain, improved glucose tolerance and whole body insulin sensitivity, and prevented hypertension in HF diet-induced obese mice. Ginseng consumption led to reduced concentrations of plasma insulin and leptin, but had no effect on plasma adiponectin levels in HF diet-fed mice. Body temperature was higher in mice fed the ginseng-supplemented diet but energy expenditure, respiration rate, and locomotive activity were not significantly altered. Dietary intake of ginseng increased fatty acid oxidation in the liver but not in skeletal muscle. Expression of several transcription factors associated with adipogenesis (C/EBP alpha and PPAR gamma) were decreased in the adipose tissue of HF diet-fed mice, effects that were mitigated in mice that consumed the HF diet supplemented with ginseng. Abundance of fatty acid synthase (FASN) mRNA was greater in the adipose tissue of mice that consumed the ginseng-supplemented HF diet as compared with control or un-supplemented HF diet-fed mice. Ginseng treatment had no effect on the expression of genes involved in the regulation of food intake in the hypothalamus. These data suggest that Chinese ginseng can potently prevent the development of obesity and insulin resistance in HF diet-fed mice.
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    Early Skeletal Muscle Adaptations to Short-Term High-Fat Diet in Humans Before Changes in Insulin Sensitivity
    Anderson, Angela S.; Haynie, Kimberly R.; McMillan, Ryan P.; Osterberg, Kristin L.; Boutagy, Nabil E.; Frisard, Madlyn I.; Davy, Brenda M.; Davy, Kevin P.; Hulver, Matthew W. (Wiley-Blackwell, 2015-04-01)
    Objective—The purpose of this investigation was to understand the metabolic adaptations to a short-term (5 days), isocaloric, high fat diet (HFD) in healthy, young males. Methods—Two studies were undertaken with 12 subjects. Study 1 investigated the effect of the HFD on skeletal muscle substrate metabolism and insulin sensitivity. Study 2 assessed the metabolic and transcriptional response in skeletal muscle to the transition from a fasted-to-fed state using a high fat meal challenge prior to and following 5 days of HFD. Results—Study 1 showed no effect of a HFD on skeletal muscle metabolism or insulin sensitivity in fasting samples. Study 2 showed that a HFD elicits significant increases in fasting serum endotoxin, and disrupts the normal postprandial excursions of serum endotoxin, and metabolic and transcriptional responses in skeletal muscle. These effects following 5 days of HFD were accompanied by an altered fasting and postprandial response in the ratio of phosphorylated to total p38 protein. These changes all occurred in the absence of alterations in insulin sensitivity. Conclusions—Our findings provide evidence for early biological adaptations to high fat feeding that proceed and possibly lead to insulin resistance.
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    Fish oil and indomethacin in combination potently reduce dyslipidemia and hepatic steatosis in LDLR⁻/⁻ mice
    Murali, Ganesan; Milne, Ginger L.; Webb, Corey D.; Stewart, Ann B.; McMillan, Ryan P.; Lyle, Brandon C.; Hulver, Matthew W.; Saraswathi, Viswanathan (ASBMB, 2012-07-29)
    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-infl ammatory drugs, can enhance the antiinfl ammatory 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. Moreover, 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.
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    The Flavonoid Kaempferol Ameliorates Streptozotocin-Induced Diabetes by Suppressing Hepatic Glucose Production
    Alkhalidy, Hana; Moore, Will; Wang, Yao; Luo, Jing; McMillan, Ryan P.; Zhen, Wei; Zhou, Kequan; Liu, Dongmin (MDPI, 2018-09-13)
    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.
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    GPR30 regulates diet-induced adiposity in female mice and adipogenesis in vitro
    Wang, Aihua; Luo, Jing; Moore, William; Alkhalidy, Hana; Wu, Ling; Zhang, Jinhua; Zhen, Wei; Wang, Yao; Clegg, Deborah J.; Xu, Bin; Cheng, Zhiyong; McMillan, Ryan P.; Hulver, Matthew W.; Liu, Dongmin (Nature Publishing Group, 2016-10-04)
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    Heat Stress Reduces Metabolic Rate While Increasing Respiratory Exchange Ratio in Growing Pigs
    Fausnacht, Dane W.; Kroscher, Kellie A.; McMillan, Ryan P.; Martello, Luciane S.; Baumgard, Lance H.; Selsby, Joshua T.; Hulver, Matthew W.; Rhoads, Robert P. (MDPI, 2021-01-17)
    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.
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    Mitochondrial Dysregulation in Skeletal Muscle from Patients Diagnosed with Alzheimer’s Disease and Sporadic Inclusion Body Myositis
    Shabrokh, Elika; Kavanaugh, John; McMillan, Ryan P.; Pittman, Josh; Hulver, Matthew W.; Frisard, Madlyn I. (Scientific Research Publishing, 2014-05)
    Mitochondrial dysfunction is implicated in Alzheimer’s disease (AD) and disruption of mitochondrial dynamic pathways has been documented in brains from patients diagnosed with AD; although it is unclear whether other tissues are also affected. Much less is known about the mitochondria in patients diagnosed with sporadic Inclusion Body Myositis (sIBM). The current study examined mitochondrial biology in skeletal muscle from AD and sIBM patients compared to healthy, elderly individuals. Skeletal muscle samples were obtained from the National Disease Research Interchange and mRNA, protein content, and enzyme activity was used to assess mitochondrial parameters. Patients diagnosed with AD or sIBM demonstrated reduced mitofusin 2 and optic atrophy protein 1 protein. AD patients also displayed increased mRNA of superoxide dismutase 2, catalase, and uncoupling protein 3. Amyloid β precursor protein mRNA was higher in sIBM patients only compared to both AD patients and elderly individuals. Both total and phosphorylated AMPK protein content, an upstream regulator of mitochondrial dynamics and biogenesis, were also reduced in sIBM patients. The current study demonstrates a disruption in signaling pathways regulating mitochondrial dynamics in both AD and sIBM patients, although the underlying causes may differ.
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    Nox4 mediates skeletal muscle metabolic responses to exercise
    Specht, Kalyn S.; Kant, Shashi; Addington, Adele K.; McMillan, Ryan P.; Hulver, Matthew W.; Learnard, Heather; Campbell, Maura; Donnelly, Sarah R.; Caliz, Amada D.; Pei, Yongmei; Reif, Michaella M.; Bond, Jacob M.; DeMarco, Anthony; Craige, Branch; Keaney, John F. Jr.; Craige, Siobhan M. (2021-03)
    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 C-14-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-1 alpha). 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. (C) 2021 The Authors. Published by Elsevier GmbH.
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    Overfeeding and Substrate Availability, But Not Age or BMI, Alter Human Satellite Cell Function
    Fausnacht, Dane W.; McMillan, Ryan P.; Boutagy, Nabil E.; Lupi, Ryan A.; Harvey, Mordecai M.; Davy, Brenda M.; Davy, Kevin P.; Rhoads, Robert P.; Hulver, Matthew W. (MDPI, 2020-07-24)
    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.
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    The pivotal role of pyruvate dehydrogenase kinases in metabolic flexibility
    Zhang, Shuai; Hulver, Matthew W.; McMillan, Ryan P.; Cline, Mark A.; Gilbert, Elizabeth R. (2014-02-12)
    Metabolic flexibility is the capacity of a system to adjust fuel (primarily glucose and fatty acids) oxidation based on nutrient availability. The ability to alter substrate oxidation in response to nutritional state depends on the genetically influenced balance between oxidation and storage capacities. Competition between fatty acids and glucose for oxidation occurs at the level of the pyruvate dehydrogenase complex (PDC). The PDC is normally active in most tissues in the fed state, and suppressing PDC activity by pyruvate dehydrogenase (PDH) kinase (PDK) is crucial to maintain energy homeostasis under some extreme nutritional conditions in mammals. Conversely, inappropriate suppression of PDC activity might promote the development of metabolic diseases. This review summarizes PDKs’ pivotal role in control of metabolic flexibility under various nutrient conditions and in different tissues, with emphasis on the best characterized PDK4. Understanding the regulation of PDC and PDKs and their roles in energy homeostasis could be beneficial to alleviate metabolic inflexibility and to provide possible therapies for metabolic diseases, including type 2 diabetes (T2D).
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    Prebiotic Inulin Supplementation and Peripheral Insulin Sensitivity in adults at Elevated Risk for Type 2 Diabetes: A Pilot Randomized Controlled Trial
    Mitchell, Cassie M.; Davy, Brenda M.; Ponder, Monica A.; McMillan, Ryan P.; Hughes, Michael D.; Hulver, Matthew W.; Neilson, Andrew P.; Davy, Kevin P. (MDPI, 2021-09-17)
    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.
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    Quantitative Variation in m.3243A > G Mutation Produce Discrete Changes in Energy Metabolism
    McMillan, Ryan P.; Stewart, Sidney; Budnick, James A.; Caswell, Clayton C.; Hulver, Matthew W.; Mukherjee, Konark; Srivastava, Sarika (Springer Nature, 2019-04-08)
    Mitochondrial DNA (mtDNA) 3243A > G tRNALeu((UUR)) heteroplasmic mutation (m.3243A > G) exhibits clinically heterogeneous phenotypes. While the high mtDNA heteroplasmy exceeding a critical threshold causes mitochondrial encephalomyopathy, lactic acidosis with stroke-like episodes (MELAS) syndrome, the low mtDNA heteroplasmy causes maternally inherited diabetes with or without deafness (MIDD) syndrome. How quantitative differences in mtDNA heteroplasmy produces distinct pathological states has remained elusive. Here we show that despite striking similarities in the energy metabolic gene expression signature, the mitochondrial bioenergetics, biogenesis and fuel catabolic functions are distinct in cells harboring low or high levels of the m.3243 A > G mutation compared to wild type cells. We further demonstrate that the low heteroplasmic mutant cells exhibit a coordinate induction of transcriptional regulators of the mitochondrial biogenesis, glucose and fatty acid metabolism pathways that lack in near homoplasmic mutant cells compared to wild type cells. Altogether, these results shed new biological insights on the potential mechanisms by which low mtDNA heteroplasmy may progressively cause diabetes mellitus.
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    Regulation of Insulin and Leptin Signaling by Muscle Suppressor of Cytokine Signaling 3 (SOCS3)
    Yang, Zhenggang; Hulver, Matthew W.; McMillan, Ryan P.; Cai, Lingzhi; Kershaw, Erin E.; Yu, Liqing; Xue, Bingzhong; Shi, Hang (PLOS, 2012-10-24)
    Skeletal muscle resistance to the key metabolic hormones, leptin and insulin, is an early defect in obesity. Suppressor of cytokine signaling 3 (SOCS3) is a major negative regulator of both leptin and insulin signaling, thereby implicating SOCS3 in the pathogenesis of obesity and associated metabolic abnormalities. Here, we demonstrate that SOCS3 mRNA expression is increased in murine skeletal muscle in the setting of diet-induced and genetic obesity, inflammation, and hyperlipidemia. To further evaluate the contribution of muscle SOCS3 to leptin and insulin resistance in obesity, we generated transgenic mice with muscle-specific overexpression of SOCS3 (MCK/SOCS3 mice). Despite similar body weight, MCK/SOCS3 mice develop impaired systemic and muscle-specific glucose homeostasis and insulin action based on glucose and insulin tolerance tests, hyperinsulinemic-euglycemic clamps, and insulin signaling studies. With regards to leptin action, MCK/SOCS3 mice exhibit suppressed basal and leptin-stimulated activity and phosphorylation of alpha2 AMP-activated protein kinase (α2AMPK) and its downstream target, acetyl-CoA carboxylase (ACC). Muscle SOCS3 overexpression also suppresses leptin-regulated genes involved in fatty acid oxidation and mitochondrial function. These studies demonstrate that SOC3 within skeletal muscle is a critical regulator of leptin and insulin action and that increased SOCS may mediate insulin and leptin resistance in obesity.
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    Resistance exercise training and in vitro skeletal muscle oxidative capacity in older adults
    Flack, Kyle D.; Davy, Brenda M.; DeBerardinis, Martin; Boutagy, Nabil E.; McMillan, Ryan P.; Hulver, Matthew W.; Frisard, Madlyn I.; Anderson, Angela S.; Savla, Jyoti S.; Davy, Kevin P. (2016-07)
    Whether resistance exercise training (RET) improves skeletal muscle substrate oxidative capacity and reduces mitochondrial production of reactive oxygen species in older adults remains unclear. To address this, 19 older males (≥60 years) were randomized to a RET (n = 11) or to a waitlist control group (n = 8) that remained sedentary for 12 weeks. RET was comprised of three upper body and four lower body movements on resistance machines. One set of 8-12 repetitions to failure of each movement was performed on three nonconsecutive days/week. Improvements in chest press and leg press strength were assessed using a three-repetition maximum (3 RM). Body composition was assessed via dual energy X-ray absorptiometry. Muscle biopsies were obtained from the vastus lateralis muscle at baseline and at both 3 weeks and 12 weeks. Palmitate and pyruvate oxidation rates were measured from the (14)CO2 produced from [1-(14)C] palmitic acid and [U-(14)C] pyruvate, respectively, during incubation of muscle homogenates. PGC-1α, TFAM, and PPARδ levels were quantified using qRT-PCR Citrate synthase (CS) and β-HAD activities were determined spectrophotometrically. Mitochondrial production of reactive oxygen species (ROS) were assessed using the Amplex Red Hydrogen Peroxide/Peroxidase assay. There were no significant changes in body weight or body composition following the intervention. Chest press and leg press strength (3RM) increased ~34% (both P < 0.01) with RET There were no significant changes in pyruvate or fatty acid oxidation or in the expression of target genes with the intervention. There was a modest increase (P < 0.05) in βHAD activity with RET at 12 weeks but the change in CS enzyme activity was not significant. In addition, there were no significant changes in ROS production in either group following RET Taken together, the findings of this study suggest that 12 weeks of low volume RET does not increase skeletal muscle oxidative capacity or reduce ROS production in older adults.
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    The Role of Fatty Acids on Toll-like Receptor 4 Regulation of Substrate Metabolism with Obesity
    McMillan, Ryan P. (Virginia Tech, 2009-06-25)
    Growing evidence suggests that obesity and associated metabolic dysregulation occurs in concert with chronic low-grade inflammation. Toll-like receptors (TLR) are transmembrane receptors that play an important role in innate immunity and the induction of inflammatory responses. Our laboratory has observed that TLR4 expression is elevated in the skeletal muscle of obese humans and is associated with reduced fatty acid (FA) oxidation and increased lipid synthesis. Additionally, activation of this pathway by lipopolysaccharide (LPS), ex vivo, results in a shift in substrate metabolism favoring glucose as an energy substrate and preferential storage of FA in intracellular lipid depots. The purpose of this study was to examine the effects of saturated vs. monounsaturated FA on TLR4 transcription and signaling using ex vivo and in vivo models. C2C12 myotubes were incubated in FA-enriched growth medium with varying ratios of palmitate and oleate for 12 hours. Following FA treatment, cells were either collected for measures of mRNA and protein levels of TLR4 or challenged with LPS (500 ng/mL) for 2 hours to assess TLR4 mediated changes in interleukin-6 (IL-6) and glucose and fatty acid metabolism. TLR4 mRNA and protein content were increased in stepwise fashion with higher palmitate concentration (p<0.05). This was associated with an exacerbated LPS effect on IL-6 mRNA and protein levels, and glucose and fatty acid metabolism. To determine if these effects are translated to an in-vivo model, C57BL/6 mice were fed high saturated fat (HSF), high monounsaturated fat (HMF), and control diets for 10 weeks. Following the dietary intervention, animals were challenged with I.P. injections of either saline or LPS (~25μg/mouse), sacrificed 4 hours post-injection, and red and white gastrocnemius muscle were harvested for measures of expression and protein levels of TLR4 and IL-6, and glucose and fatty acid metabolism. TLR4 mRNA and protein levels were not altered with either the HSF or HMF diets. However, there was a heightened LPS response with regards to increases in IL-6 and TNF-α, and enhanced shifts in substrate metabolism following the HSF diet (p<0.05). These effects were not observed in response to the HMF diet. In conclusion, these data demonstrate that a milieu of high saturated fatty acids results in elevated sensitization of the TLR4 pathway in skeletal muscle. These results provide insight into how a westernized diet, one enriched in saturated fat, may link chronic inflammation with obesity-associated metabolic abnormalities.
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    Selective Histone Deacetylase 6 Inhibition Normalizes B Cell Activation and Germinal Center Formation in a Model of Systemic Lupus Erythematosus
    Ren, Jingjing; Catalina, Michelle D.; Eden, Kristin; Liao, Xiaofeng; Read, Kaitlin A.; Luo, Xin M.; McMillan, Ryan P.; Hulver, Matthew W.; Jarpe, Matthew; Bachali, Prathyusha; Grammer, Amrie C.; Lipsky, Peter E.; Reilly, Christopher M. (2019-10-25)
    Autoantibody production by plasma cells (PCs) plays a pivotal role in the pathogenesis of systemic lupus erythematosus (SLE). The molecular pathways by which B cells become pathogenic PC secreting autoantibodies in SLE are incompletely characterized. Histone deactylase 6 (HDAC6) is a unique cytoplasmic HDAC that modifies the interaction of a number of tubulin- associated proteins; inhibition of HDAC6 has been shown to be beneficial in murine models of SLE, but the downstream pathways accounting for the therapeutic benefit have not been clearly delineated. In the current study, we sought to determine whether selective HDAC6 inhibition would abrogate abnormal B cell activation in SLE. We treated NZB/W lupus mice with the selective HDAC6 inhibitor, ACY-738, for 4 weeks beginning at 20 weeks-of age. After only 4 weeks of treatment, manifestation of lupus nephritis (LN) were greatly reduced in these animals. We then used RNAseq to determine the genomic signatures of splenocytes from treated and untreated mice and applied computational cellular and pathway analysis to reveal multiple signaling events associated with B cell activation and differentiation in SLE that were modulated by HDAC6 inhibition. PC development was abrogated and germinal center (GC) formation was greatly reduced. When the HDAC6 inhibitor-treated lupus mouse gene signatures were compared to human lupus patient gene signatures, the results showed numerous immune, and inflammatory pathways increased in active human lupus were significantly decreased in the HDAC6 inhibitor treated animals. Pathway analysis suggested alterations in cellular metabolism might contribute to the normalization of lupus mouse spleen genomic signatures, and this was confirmed by direct measurement of the impact of the HDAC6 inhibitor on metabolic activities of murine spleen cells. Taken together, these studies show HDAC6 inhibition decreases B cell activation signaling pathways and reduces PC differentiation in SLE and suggest that a critical event might be modulation of cellular metabolism.
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    Serum endotoxin, gut permeability and skeletal muscle metabolic adaptations following a short term high fat diet in humans
    Bowser, Suzanne M.; McMillan, Ryan P.; Boutagy, Nabil E.; Tarpey, Michael D.; Smithson, Andrew T.; Osterberg, Kristin L.; Neilson, Andrew P.; Davy, Brenda M.; Davy, Kevin P.; Hulver, Matthew W. (2019-11-27)
    Background: Our previous work demonstrated that a short-term high fat diet (HFD) increased fasting serum endotoxin, altered postprandial excursions of serum endotoxin, and led to metabolic and transcriptional responses in skeletal muscle in young, healthy male humans. Purpose: The purpose of the present study was to determine if a short-term high fat diet: 1) increases intestinal permeability and, in turn, fasting endotoxin concentrations and 2) decreases postprandial skeletal muscle fat oxidation. Methods: Thirteen normal weight young adult males (BMI 23.1 +/- 0.8 kg/m(2), age 22.2 +/- 0.4 years) were fed a control diet (55% carbohydrate, 30% fat, 9% of which was saturated, 15% protein) for two weeks, followed by 5 days of an isocaloric HFD (30% carbohydrate, 55% fat, 25% of which was saturated, 15% protein, isocaloric to the control diet). Intestinal permeability (via four sugar probe test) was assessed in the fasting state. Both before and after the HFD, a high fat meal challenge (HFM, 820 kcal, 25% carbohydrate, 63% fat, 26% of which was saturated, and 12% protein) was administered. After an overnight fast, blood samples were collected before and every hour for 4 h after the HFM to assess endotoxin, and other serum blood measures. Muscle biopsies were obtained from the vastus lateralis before and 4 h after the HFM in order to assess substrate oxidation (glucose, fatty acid and pyruvate) using radiolabeled techniques. Insulin sensitivity was assessed via intravenous glucose tolerance test. Intestinal permeability, blood samples and muscle biopsies were assessed in the same manner before and following the HFD. Main findings: Intestinal permeability was not affected by HFD (p > 0.05), but fasting endotoxin increased two fold following the HFD (p = 0.04). Glucose oxidation and fatty acid oxidation in skeletal muscle homogenates significantly increased after the HFM before the HFD (+97%, and +106% respectively) but declined after the HFM following 5 days of the HFD (-24% and +16% respectively). Fatty acid suppressibility of pyruvate oxidation increased significantly after the HFM (+32%) but this physiological effect was abolished following 5 days of the HFD (+7%). Insulin sensitivity did not change following the HFD. Conclusion: These findings demonstrate that in healthy young men, consuming an isocaloric HFD for 5 days increases fasting endotoxin, independent of changes in gut permeability. These changes in endotoxin are accompanied by a broad effect on skeletal muscle substrate metabolism including increases in postprandial fat oxidation. Importantly, the latter occurs independent of changes in body weight and whole-body insulin sensitivity.
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    Skeletal muscle autophagy and mitophagy in endurance-trained runners before and after a high-fat meal
    Tarpey, Michael D.; Davy, Kevin P.; McMillan, Ryan P.; Bowser, Suzanne M.; Halliday, Tanya M.; Boutagy, Nabil E.; Davy, Brenda M.; Frisard, Madlyn I.; Hulver, Matthew W. (Elsevier, 2017-10-24)
    Objective: We tested the hypothesis that skeletal muscle of endurance-trained male runners would exhibit elevated autophagy and mitophagy markers, which would be associated with greater metabolic flexibility following a high-fat meal (HFM). Methods: Muscle biopsies were collected to determine differences in autophagy and mitophagy protein markers and metabolic flexibility under fasting conditions and 4 h following a HFM between endurance-trained male runners (n =10) and sedentary, non-obese controls (n = 9). Results: Maximal oxygen consumption (ml・kg・min⁻¹) was approximately 50% higher (p < 0.05) in endurance-trained runners compared with sedentary controls (65.8 ±2.3 and 43.1 ±3.4, respectively). Autophagy markers were similar between groups. Mitophagy and mitochondrial dynamics protein markers were significantly higher in skeletal muscle of endurance-trained runners compared with sedentary controls in the fasted state, although unaffected by the HFM. Skeletal muscle metabolic flexibility was similar between groups when fasted (p > 0.05), but increased in response to the HFM in endurance-trained athletes only (p < 0.005). Key mitophagy markers, phospho-Pink1Thr257 and phospho- ParkinS⁶⁵(r = 0.64, p < 0.005), and phospo-ParkinSer⁶⁵ and phospho-Drp1Ser⁶¹⁶ (r = 0.70, p < 0.05) were correlated only within the endurance-trained group. Autophagy and mitophagy markers were not correlated with metabolic flexibility. Conclusion: In summary, mitophagy may be enhanced in endurance-trained runners based on elevated markers of mitophagy and mitochondrial dynamics. The HFM did not alter autophagy or mitophagy in either group. The absence of a relationship between mitophagy markers and metabolic flexibility suggests that mitophagy is not a key determinant of metabolic flexibility in a healthy population, but further investigation is warranted.
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    Small Molecule Kaempferol Promotes Insulin Sensitivity and Preserved Pancreatic β-Cell Mass in Middle-Aged Obese Diabetic Mice
    Alkhalidy, Hana; Moore, William B.; Zhang, Yanling; McMillan, Ryan P.; Wang, Aihua; Ali, Mostafa; Suh, Kyung-Shin; Zhen, Wei; Cheng, Zhiyong; Jia, Zhenquan; Hulver, Matthew W.; Liu, Dongmin (Hindawi, 2015-05-07)
    Insulin resistance and a progressive decline in functional β-cell mass are hallmarks of developing type 2 diabetes (T2D). Thus, searching for natural, low-cost compounds to target these two defects could be a promising strategy to prevent the pathogenesis of T2D. Here, we show that dietary intake of flavonol kaempferol (0.05% in the diet) significantly ameliorated hyperglycemia, hyperinsulinemia, and circulating lipid profile, which were associated with the improved peripheral insulin sensitivity in middle-aged obese mice fed a high-fat (HF) diet. Kaempferol treatment reversed HF diet impaired glucose transport-4 (Glut4) and AMP-dependent protein kinase (AMPK) expression in both muscle and adipose tissues from obese mice. In vitro, kaempferol increased lipolysis and prevented high fatty acid-impaired glucose uptake, glycogen synthesis, AMPK activity, and Glut4 expression in skeletal muscle cells. Using another mouse model of T2D generated by HF diet feeding and low doses of streptozotocin injection, we found that kaempferol treatment significantly improved hyperglycemia, glucose tolerance, and blood insulin levels in obese diabetic mice, which are associated with the improved islet β-cell mass. These results demonstrate that kaempferol may be a naturally occurring anti-diabetic agent by improving peripheral insulin sensitivity and protecting against pancreatic β-cell dysfunction.
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    Temporal examination of DNA methylation profile reprogramming in the promoter region of PGC-1α during the progression of insulin resistance and type 2 diabetes mellitus in rodent models
    Donnelly, Sarah Rebecca (Virginia Tech, 2019-07-31)
    Type 2 Diabetes Mellitus (T2DM), a metabolic disorder denoted by elevated blood glucose levels and insufficient insulin action, is growing in prevalence worldwide . Barriers to improving disease outcome resolve primarily around identifying and intervening during the preliminary stages of insulin resistance, a state clinically referred to as pre-diabetes. Emerging evidence suggests that mitochondrial dysfunction may underlie , and potentially precede, progressive insulin resistance, suggesting that biomarkers indicative of mitochondrial dysfunction could predict disease risk and status. In this study, we examined epigenetic modifications, in the form of DNA methylation, in the promoter region of peroxisome proliferator activated receptor gamma coactivator 1 alpha (PGC-1α), a known regulator of mitochondrial biogenesis. Following the initiation of a high fat diet, we observed significant genotypic (DNA methylation) and phenotypic (mitochondrial copy number) alterations in C57/BL6 rodent models. These changes preceded overt disease onset, as classified by clinically utilized indices, which included the homeostatic model assessment for insulin resistance (HOMA-IR), the homeostatic model assessment for β-cell dysfunction (HOMA- β), and the quantitative insulin-sensitivity check index (QUICKI). Our data indicate that methylation analysis may serve as an effective clinical parameter to use in conjunction with physiological criterion for the diagnosis of pre-diabetes and the assessment of T2DM disease risk, and adds to the growing body of work seeking to elucidate the role.