PGC-1α mRNA Expression Research Articles

Mitochondrial damage are involved in Aflatoxin B1-induced testicular damage and spermatogenesis disorder in mice

Aflatoxin B1 (AFB1) is an unavoidable environmental pollutants, which seriously endangers human and animal health. AFB1 has male reproductive toxicity, yet the underlying mechanisms remain inconclusive. Mitochondra are a kind of crucial organelle for maintaining spermatogenesis in testis. Thus, we hypothesized that AFB1 can impair mitochondria to aggravate testicular damage and spermatogenesis disorder. To verify this hypothesis, 48 male mice were intragastrically administered with 0, 0.375, 0.75 or 1.5 mg/kg body weight AFB1 for 30 days, respectively. In this study, we found AFB1 caused testicular histopathological lesions and spermatogenesis abnormalities, with the elevation of oxidative stress (increased H2O2, whereas decreased SOD and GSH). Significant mitochondria structure damage of germ cells and Leydig cells, MMP loss, ATP contents reduction, and inhibited activities of mitochondrial complexes I-IV in mice testis were found in AFB1 treatment groups. Besides, AFB1 inhibited mitochondrial biogenesis and mitochondrial dynamics, presenting as the decreased mRNA and protein expressions of PGC-1α, Nrf1, Tfam, Drp1, Fis1, Mfn1 and Opa1. The results revealed that the mitochondrial damage were involved in AFB1-induced testicular damage and spermatogenesis disorder, providing a considerable direction to clarify potential mechanisms of AFB1 reproductive toxicity.

Regulation of metabolism during hibernation in brown bears (Ursus arctos): Involvement of cortisol, PGC-1α and AMPK in adipose tissue and skeletal muscle

The purpose of this study was to investigate changes in expression of known cellular regulators of metabolism during hyperphagia (Sept) and hibernation (Jan) in skeletal muscle and adipose tissue of brown bears and determine whether signaling molecules and transcription factors known to respond to changes in cellular energy state are involved in the regulation of these metabolic adaptations. During hibernation, serum levels of cortisol, glycerol, and triglycerides were elevated, and protein expression and activation of AMPK in skeletal muscle and adipose tissue were reduced. mRNA expression of the co-activator PGC-1α was reduced in all tissues in hibernation whereas mRNA expression of the transcription factor PPAR-α was reduced in the vastus lateralis muscle and adipose tissue only. During hibernation, gene expression of ATGL and CD36 was not altered; however, HSL gene expression was reduced in adipose tissue. During hibernation gene expression of the lipogenic enzyme DGAT in all tissues and the expression of the FA oxidative enzyme LCAD in the vastus lateralis muscle were reduced. Gene and protein expression of the glucose transporter GLUT4 was decreased in adipose tissue in hibernation. Our data suggest that high cortisol levels are a key adaptation during hibernation and link cortisol to a reduced activation of the AMPK/PGC-1α/PPAR-α axis in the regulation of metabolism in skeletal muscle and adipose tissue. Moreover, our results indicate that during this phase of hibernation at a time when metabolic rate is significantly reduced metabolic adaptations in peripheral tissues seek to limit the detrimental effects of unduly large energy dissipation.

Lutein upregulates the PGC-1α, NRF1, and TFAM expression by AMPK activation and downregulates ROS to maintain mtDNA integrity and mitochondrial biogenesis in hyperglycemic ARPE-19 cells and rat retina.

Hyperglycemia (HG) affects cellular organelle including mitochondrion in retina that diminishes mitochondrial biogenesis by downregulation of nuclear transcription factors peroxisome proliferator-activated receptor-γ coactivator-1 (PGC-1α) and mitochondrial transcription factor A (TFAM). Mitochondrial dysfunction has been linked to diabetic retinopathy (DR). Carotenoids reported to modulate mitochondrial biogenesis in HG. Aim of the study was to explore the role of lutein, oxidized lutein (purified upon UV oxidation of lutein) and drug metformin, on mitochondrial biogenesis in HG-induced ARPE-19 cells and rat retina. Results showed higher uptake of lutein and oxidized lutein in ARPE-19 cells and rat retina of HG group than the control groups. Further, lutein and oxidized lutein augmented the AMPK phosphorylation and activation of mitochondrion signaling molecule TFAM (protein expression) and mRNA expression of PGC-1α, TFAM, and nuclear respiratory factor 1 (responsible for mitochondria biogenesis) along with lowered reactive oxygen species in HG compared with control and metformin groups. Higher mRNA expression of nicotinamide adenine dinucleotide dehydrogenase subunits mt-ND1, mt-ND4, mt-ND6, and cytochrome C that aid maintenance of mtDNA integrity was also evidenced. To conclude, lutein and oxidized lutein found to upsurge mitochondrial biogenesis in ARPE-19 cells and rat retina under HG, which may be due to upregulation of AMPK phosphorylation. Finally, lutein and oxidized lutein may provide a therapeutic basis to ameliorate HG-induced DR.

Post-exercise carbohydrate and energy availability induce independent effects on skeletal muscle cell signalling and bone turnover: implications for training adaptation.

Reduced carbohydrate (CHO) availability before and after exercise may augment endurance training-induced adaptations of human skeletal muscle, as mediated via modulation of cell signalling pathways. However, it is not known whether such responses are mediated by CHO restriction, energy restriction or a combination of both. In recovery from a twice per day training protocol where muscle glycogen concentration is maintained within 200-350mmolkg-1 dry weight (dw), we demonstrate that acute post-exercise CHO and energy restriction (i.e. <24h) does not potentiate potent cell signalling pathways that regulate hallmark adaptations associated with endurance training. In contrast, consuming CHO before, during and after an acute training session attenuated markers of bone resorption, effects that are independent of energy availability. Whilst the enhanced muscle adaptations associated with CHO restriction may be regulated by absolute muscle glycogen concentration, the acute within-day fluctuations in CHO availability inherent to twice per day training may have chronic implications for bone turnover. We examined the effects of post-exercise carbohydrate (CHO) and energy availability (EA) on potent skeletal muscle cell signalling pathways (regulating mitochondrial biogenesis and lipid metabolism) and indicators of bone metabolism. In a repeated measures design, nine males completed a morning (AM) and afternoon (PM) high-intensity interval (HIT) (8×5min at 85% ) running protocol (interspersed by 3.5h) under dietary conditions of (1) high CHO availability (HCHO: CHO ∼12gkg-1 , EA∼ 60kcalkg-1 fat free mass (FFM)), (2) reduced CHO but high fat availability (LCHF: CHO ∼3(-1 , EA∼ 60kcalkg-1 FFM) or (3), reduced CHO and reduced energy availability (LCAL: CHO ∼3gkg-1 , EA∼ 20kcalkg-1 FFM). Muscle glycogen was reduced to ∼200mmolkg-1 dw in all trials immediately post PM HIT (P<0.01) and remained lower at 17h (171, 194 and 316mmolkg-1 dw) post PM HIT in LCHF and LCAL (P<0.001) compared to HCHO. Exercise induced comparable p38MAPK phosphorylation (P<0.05) immediately post PM HIT and similar mRNA expression (all P<0.05) of PGC-1α, p53 and CPT1 mRNA in HCHO, LCHF and LCAL. Post-exercise circulating βCTX was lower in HCHO (P<0.05) compared to LCHF and LCAL whereas exercise-induced increases in IL-6 were larger in LCAL (P<0.05) compared to LCHF and HCHO. In conditions where glycogen concentration is maintained within 200-350mmolkg-1 dw, we conclude post-exercise CHO and energy restriction (i.e. <24h) does not potentiate cell signalling pathways that regulate hallmark adaptations associated with endurance training. In contrast, consuming CHO before, during and after HIT running attenuates bone resorption, effects that are independent of energy availability and circulating IL-6.

An anti-inflammatory phenotype in visceral adipose tissue of old lean mice, augmented by exercise

Visceral adipose tissue is an immunogenic tissue, which turns detrimental during obesity by activation of proinflammatory macrophages. During aging, chronic inflammation increases proportional to visceral adipose tissue (VAT) mass and associates with escalating morbidity and mortality. Here, we utilize a mouse model to investigate the inflammatory status of visceral adipose tissue in lean aging mice and assess the effects of exercise training interventions. We randomized adult (11 months; n = 21) and old (23 months; n = 27) mice to resistance training (RT) or endurance training (ET), or to a sedentary control group (S). Strikingly, we observed an anti-inflammatory phenotype in the old mice, consisting of higher accumulation of M2 macrophages and IL-10 expression, compared to the adult mice. In concordance, old mice also had less VAT mass and smaller adipocytes compared to adult mice. In both age groups, exercise training enhanced the anti-inflammatory phenotype and increased PGC1-α mRNA expression. Intriguingly, the brown adipose tissue marker UCP1 was modestly higher in old mice, while remained unchanged by the intervention. In conclusion, in the absence of obesity, visceral adipose tissue possesses a pronounced anti-inflammatory phenotype during aging which is further enhanced by exercise.

Regulatory effects of the fruit extract of Lycium chinense and its active compound, betaine, on muscle differentiation and mitochondrial biogenesis in C2C12 cells

Our study was conducted to investigate the effects of the fruits of Lycium chinense Mill. (Lycii Fructus, LF) and its bioactive compound, betaine, on muscle differentiation and mitochondrial biogenesis in C2C12 cells. LF extract and betaine was analyzed by high-performance liquid chromatography (HPLC). The expression of myosin heavy chain (MyHC) and peroxisome proliferator-activated receptor gamma coactivator1-alpha (PGC-1α), sirtuin-1(Sirt-1), nuclear respiratory factor-1 (NRF-1), transcription factor A, mitochondrial (TFAM) and the phosphorylation of AMP-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC), were determined in cellular or mitochondrial levels by quantitative polymerase chain reaction (qPCR) or Western blot, respectively. The glucose levels and total ATP contents were measured by the glucose consumption in a culture medium, cellular glucose uptake and ATP assays. LF extract at 4 mg/ml and betaine at 2 and 5 mM significantly increased the expression of MyHC in C2C12 myotubes, compared with non-treated cells. LF extract and betaine significantly increased the expression of PGC-1α, Sirt-1, NRF-1 and TFAM mRNA and protein in the myotubes, as well as phosphorylation of AMPK and ACC. Furthermore, LF extract and betaine significantly increased the mitochondrial protein contents, as the TFAM and NRF-1 expressions were increased. LF extract and betaine also significantly increased the glucose uptake and ATP contents in the myotubes. The LF extract contained 3.18% betaine was quantitated by HPLC. LF extract and betaine enhanced muscle differentiation and energy metabolism through the up-regulation of mitochondrial biogenesis-regulating factors, suggesting that LF extract and betaine can help to prevent the dysfunction of skeletal muscle.

Effect of metformin on myotube BCAA catabolism.

Metformin has antihyperglycemic properties and is a commonly prescribed drug for type II diabetes mellitus. Metformin functions in part by activating 5'-AMP-activated protein kinase, reducing hepatic gluconeogenesis and blood glucose. Metformin also upregulates peroxisome proliferator-activated receptor-gamma coactivator-1α (PGC-1α). Several population studies have shown levels of circulating branched-chain amino acids (BCAA) positively correlate with insulin resistance. Because BCAA catabolic enzyme content is regulated by PGC-1α, we hypothesized metformin may alter BCAA catabolism. Therefore, the purpose of this work was to investigate the effect of metformin at varying concentrations on myotube metabolism and related gene and protein expression. C2C12 myotubes were treated with metformin at 30 uM (physiological) or 2 mM (supraphysiological) for up to 24 hours. Metabolic gene expression was measured via quantitative real time polymerase chain reaction, protein expression was measured using Western blot, and mitochondrial and glycolytic metabolism were measured via oxygen consumption and extracellular acidification rate, respectively. Supraphysiological metformin upregulated PGC-1α mRNA expression along withrelated downstream targets, yet the reduced expression of electron transport chain components as well as basal and peak cell metabolism. Supraphysiological metformin also suppressed branched-chain aminotransferase 2 (BCAT2) and branched-chain-alpha-keto acid dehydrogenase E1a (BCKDHa) mRNA expression as well as BCAT2 protein expression and BCKDHa activity, which was accompanied by decreased Kruppel-like factor 15 protein expression. Physiological levels of metformin suppressed BCKDHa and cytochrome c oxidase mRNA expression at early time points (4-12 hours) but had no effect on any other outcomes. Together these data suggest metformin may suppress BCAA catabolic enzyme expression or activity, possibly reducing levels of circulating gluconeogenic substrates.

Electroacupuncture Reduces Body Weight by Regulating Fat Browning-Related Proteins of Adipose Tissue in HFD-Induced Obese Mice.

Objective: This study investigated the influence of electroacupuncture (EA) and its potential underlying mechanisms on adipose tissue in obese mice. Methods: Three-week-old male C56BL/6 mice were randomly divided to feed or not to feed high-fat diet (HFD), named HFD group and chow diet (CD) group, respectively. After 12 weeks, CD and HFD mice were randomly divided into two groups, respectively, to receive or not receive EA for 4 weeks. Body weight (BW) was monitored. Intraperitoneal glucose tolerance test and metabolic chamber recordings were performed. Blood samples and adipose tissue were collected for the analysis of leptin, triglyceride levels, and fat browning-related proteins. Results: EA significantly reduced food intake, BW, and white adipose tissue (WAT)/BW ratio; decreased the adipocyte size and serum concentrations of triglyceride (TG) and cholesterol; and increased oxygen consumption in HFD mice. Compared with the CD mice, the HFD mice had elevated fasting serum glucose level and impaired glucose tolerance; however, these parameters were decreased by EA treatment. Meanwhile, EA promoted the protein and mRNA expressions of UCP1, PRDM16, and PGC-1α in adipose tissue, and activated sympathetic nerves via p-TH, A2AR, and β3AR in white adipose tissue. Conclusions: EA reduced food intake, BW, TG, and cholesterol, and improved glucose tolerance in HFD mice. This ameliorative effect of EA on obesity-related symptoms associated with its promoted adipose tissue plasticity via activating sympathetic nerves.

1885-P: Caveolin1 Attenuates Palmitic Acid-Induced Hepatic Steatosis through Upregulating PGC1α-sirt3 Axis

Nonalcoholic fatty liver disease (NAFLD) is the most common liver disease worldwide. Caveolae are flask-shaped invaginations of the plasma membrane enriched with cholesterol and phospholipid. Caveolin1 is the resident structural protein of caveolae and has previously been reported to involve in hepatic steatosis and NAFLD, while the mechanism remains elusive. Here we examined the effects of caveolin1 on palmitic acid (PA)-induced hepatic steatosis. Stable caveolin1 overexpressing HepG2 cells were developed by transfection with lentiviral particles containing human CAV1 protein (Lenti-CAV1). Lenti-Control served as a negative control. Compared with BSA treatment, PA treatment of HepG2 cells for 24 hours increased the lipid droplets and TG level (P&amp;lt;0.01). Compared with negative control, overexpression of caveolin1 attenuated the accumulation of lipid droplets and triglyceride content in HepG2 cells (P&amp;lt;0.05). At the same time, quantitative real-time PCR revealed that the mRNA expression of PGC1α and sirt3 were increased in caveolin1 overexpressing cells (P&amp;lt;0.05). In conclusion, caveolin1 may improve PA-induced hepatic steatosis through up-regulating PGC1α-sirt3 axis. Disclosure C. Lin: None. W. Zeng: None. S. Lin: None. K. Liu: None. L. Zeng: None.

Acute Exercise Intervention Combined with Metformin’s Influences on Glucose Homeostasis in T2D Mice

PURPOSE: The aim of our investigation was to determine the effects of different ways of acute exercise intervention combined with high dose of metformin on glucose homeostasis and its relative molecular mechanisms in type 2 diabetic mice. METHODS: 4-week high fat diet (HFD) and one-time Streptozocin (100mg/kg) intraperitoneal injection were used for building T2D mice. 24 mice were divided into normal control (NC), normal acute resistance training (NCR) and normal acute endurance training (NCE) group, all n=8, fed in normal chow. Finally 48 mice were developing T2D and divided into diabetic control (DC), diabetic acute resistance training (DCR), diabetic acute endurance training (DCE), high dose of metformin (200mg/kg) control (HMC), metformin combined with acute resistance training (HMR) and metformin combined with acute endurance continuous training (HME) group, all n=8. RESULTS: The two ways also enhanced blood glucose and lipid metabolism in T2D mice. Compared to HMC group, hepatic G6Pase mRNA expression in HMR and HME group was significantly escalated and hepatic FBP1 mRNA expression of both groups were significantly declined. Compared to HMC group, hepatic GLUT2 and Gck mRNA expression in HMR and HME group showed opposite trends, one was down and the other was up. Compared to HMC group, hepatic PEPCK mRNA expression in HMR group mice was notably raised and hepatic AMPKα2, PGC-1α and CREB mRNA expression in HMR and HME group mice were notably increased and only hepatic AMPKα1 mRNA expression in HMR group was significantly increased. CONCLUSIONS: Acute resistance training (ART) and acute endurance training (AET) combined with metformin can effectively improve glucose homeostasis in T2D mice. And the two ways can improve blood glucose and lipid metabolism in T2D mice. ART combined with metformin was better to improve glucose homeostasis and inhibit hepatic gluconeogenesis relative mRNA expression in T2D mice probably via the signaling pathway of AMPKα-PGC-1α-CREB.

The Effect of 10%O2 Microenvironment and Electric Pulse Stimulation on Glucose and Lipid Metabolism

PURPOSE: There is currently no successful cell contraction model in vitro. Electrical pulse stimulation (EPS) can induce muscle cell contraction. 10%O2 microenvironment is closer to the human skeletal muscle environment. In this study, EPS was applied to myotubes to test the changes of the metabolism of glucose and lipid, and to establish successful cell contraction model in a 10%O2 microenvironment in vitro. METHODS: After seven days of differentiation in 5% CO2(20%O2) incubator, the mouse myoblast cell line C2C12 were transferred into the 10%O2 incubator. After adaptation to the hypoxia for 12h, the myotubes were subjected to EPS. The EPS stimulation was performed for 120min each, total 4 consecutive days. And cell extracts obtained from each were prepared 3h after EPS. The content of PGC-1α,MyHCI, MyHCIIa, MyHCIIb and MyHCIIx were determined by ELISA. And RT-PCR analysis was applied to determine mRNA expression of PGC-1α, MCAD, Cpt1B, GLUT1, GLUT4, PDH, LDH and GAPDH. RESULTS: Both 10%O2 microenvironment and EPS significantly increased the protein level of PGC1-α, and the synergistic effect of hypoxia and EPS was more significantly. 10%O2 microenvironment significantly decreased the content of MyHCI, MyHCIIx and MyHCIIa in myotubes. EPS significantly decreased the content of MyHCI and MyHCIIx but significantly increased the content of MyHCIIa in myotubes. 10%O2 microenvironment significant decreased the mRNA level of GLTT1 and GLTU4.However, in stimulated myotubes, the mRNA level of GLUT1 was significantly elevated, but the RNA level of GLUT4 was decreased. Meanwhile, 10%O2 microenvironment significantly decreases the mRNA level of MCAD, CPT1B and PDH, and EPS increased the mRNA level of MCAD. CONCLUSIONS: 4 days of 10%O2 microenvironment combined with EPS successfully established myotubes cell contraction model. 10%O2 microenvironment inhibited the proliferation and glycolipid metabolism in myotubes. The usage energy of EPS-induced contraction is based on aerobic oxidation of glucose and fatty. The pulse frequency increases from 1 Hz to 2 Hz in the EPS protocol, were association with substrate from fat to glucose.

Peroxisome proliferator-activated receptor-γ coactivator 1α-mediated pathway as a possible therapeutic target in endometriosis.

Is a peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α)-mediated pathway involved in the development of endometriosis? PGC-1α plays critical roles in inflammation and cell proliferation of endometriotic tissues and may be involved in the development of endometriosis. Expression levels of PGC-1α are higher in ovarian endometrioma (OE) than normal endometrium (NE). PGC-1α also stimulates aromatase activity and promotes local estrogen biosynthesis in OE. This is a case-controlled biological study using endometrial cells and tissues derived from 23 women with, and 10 women without, OE. Ectopic endometriotic and eutopic endometrial stromal cells (SCs) were isolated and maintained in culture. PGC-1α was either overexpressed in the cells or knocked down using siRNA. The expression of PGC-1α and other factors during endometriosis was examined using real-time PCR and western blotting, cell proliferation was measured using Cell Counting Kit-8 (WST-8) assays and transcriptional activity was assessed using luciferase reporter assays. PGC-1α overexpression promoted the proliferation of OESCs in a time-dependent manner (P < 0.01 versus control) but not NESCs. PGC-1α stimulated aromatase (P < 0.01 versus control) and interleukin (IL)-6/IL-8 mRNA expression levels (P < 0.05 versus control for each) and led to inhibitor kappa B phosphorylation protein expression and upregulation of the apoptosis inhibitors X-linked inhibitor of apoptosis protein and survivin at mRNA level (P < 0.05 versus control for each). HX531, a selective retinoid-X receptor-α (RXRα) antagonist, suppressed the PGC-1α-induced cell proliferation (P < 0.05 versus control), aromatase/IL-6/IL-8/survivin mRNA expression (P < 0.05 versus control for each) and transcription reporter activity of PGC-1α in a dose-dependent manner (P < 0.01 versus control). Moreover, HX531 downregulated PGC-1α-induced aromatase-promoter PI.3-II transcripts in OESCs, and PGC-1α knockdown reduced aromatase, IL-6/IL-8 and antiapoptotic factors mRNA expression (P < 0.05 versus control for each). Notably, the Histogram score, which was used for quantifying RXRα status, was markedly higher in OE than in NE tissue (P < 0.01). N/A. Only OE tissues were included in this study, while peritoneal and deep infiltrating endometriotic tissues were not. Therefore, these findings might not be generalized to other types of endometriosis. In OESC, PGC-1α stimulated cell proliferation and was involved in local estrogen biosynthesis, inflammation and apoptosis, and these effects of PGC-1α were inhibited by HX531. The suppression of PGC-1α-induced proliferation by HX531 in OESCs but not NESCs suggests that the PGC-1α-RXRα axis could play critical roles in promoting endometriosis. This is the first report of a relationship between PGC-1α and inhibitor of apoptosis proteins in endometriosis. Based on these findings, the PGC-1α-mediated pathway could represent a potential target in molecular therapy of endometriosis. The study is supported in part by Grants-in-Aid for Scientific Research (15 K10681 and 15 K10726) from the Ministry of Education, Culture, Sports, Science, and Technology (Japan). The authors have no conflicts of interest to disclose.

Role of epicardial adipose tissue NPR-C in acute coronary syndrome

Background and aimsIt has been suggested that epicardial adipose tissue (EAT) thermogenesis plays a role in coronary artery disease (CAD). Recent evidence indicates that natriuretic peptide receptors (NPRs) are critical for thermogenesis. We determined the expression and signaling of NPRs in EAT in the context of CAD progression and their association with brown fat-related genes, such as uncoupling protein 1 (UCP1) and peroxisome proliferator-activated receptor gamma coactivator alpha (PGC1α). MethodsNPR-A, NPR-B and NPR-C mRNA and protein expression levels were analyzed in EAT and thoracic subcutaneous adipose tissue (SAT) from non-CAD (NCAD), stable CAD and acute coronary syndrome (ACS) patients. The associations of NPRs with thermogenic genes were also evaluated. ResultsThe EAT of ACS patients showed lower NPR-C gene and protein expression levels compared with that of stable CAD or NCAD patients. NPR-C mRNA expression in EAT also decreased as the number of injured arteries rose, and correlated positively with left ventricular ejection fraction and EAT PGC1α mRNA expression. EAT PGC1α and UCP1 gene expression levels also decreased in the ACS group. Linear and logistic regression models showed associations of EAT NPR-C mRNA levels with EAT PGC1α mRNA levels and the presence of ACS. Furthermore, the EAT of ACS patients showed reduced p38 mitogen-activated protein kinase (p38 MAPK) phosphorylation levels, which correlated positively with NPR-C protein levels. ConclusionsThe EAT of patients with ACS is characterized by decreased NPR-C, reduced UCP1 and PGC1α mRNA expression levels and reduced activation of the p38 MAPK pathway. The associations among the expression of EAT NPR-C and ACS, and brown fat markers suggest that NPR-C may play a role in ACS and in the regulation of EAT brown-like fat features in humans.

Graded reductions in preexercise muscle glycogen impair exercise capacity but do not augment skeletal muscle cell signaling: implications for CHO periodization

We examined the effects of graded muscle glycogen on exercise capacity and modulation of skeletal muscle signaling pathways associated with the regulation of mitochondrial biogenesis. In a repeated-measures design, eight men completed a sleep-low, train-low model comprising an evening glycogen-depleting cycling protocol followed by an exhaustive exercise capacity test [8 × 3 min at 80% peak power output (PPO), followed by 1-min efforts at 80% PPO until exhaustion] the subsequent morning. After glycogen-depleting exercise, subjects ingested a total of 0 g/kg (L-CHO), 3.6 g/kg (M-CHO), or 7.6 g/kg (H-CHO) of carbohydrate (CHO) during a 6-h period before sleeping, such that exercise was commenced the next morning with graded (P < 0.05) muscle glycogen concentrations (means ± SD: L-CHO: 88 ± 43, M-CHO: 185 ± 62, H-CHO: 278 ± 47 mmol/kg dry wt). Despite differences (P < 0.05) in exercise capacity at 80% PPO between trials (L-CHO: 18 ± 7, M-CHO: 36 ± 3, H-CHO: 44 ± 9 min), exercise induced comparable AMPKThr172 phosphorylation (~4-fold) and PGC-1α mRNA expression (~5-fold) after exercise and 3 h after exercise, respectively. In contrast, neither exercise nor CHO availability affected the phosphorylation of p38MAPKThr180/Tyr182 or CaMKIIThr268 or mRNA expression of p53, Tfam, CPT-1, CD36, or PDK4. Data demonstrate that when exercise is commenced with muscle glycogen < 300 mmol/kg dry wt, further graded reductions of 100 mmol/kg dry weight impair exercise capacity but do not augment skeletal muscle cell signaling. NEW & NOTEWORTHY We provide novel data demonstrating that when exercise is commenced with muscle glycogen below 300 mmol/kg dry wt (as achieved with the sleep-low, train-low model) further graded reductions in preexercise muscle glycogen of 100 mmol/kg dry wt reduce exercise capacity at 80% peak power output by 20-50% but do not augment skeletal muscle cell signaling.

Exercise Induced Irisin Alleviates Type 1 Diabetic Nephropathy by Promoting Mitochondria Biogenesis and Function

Mitochondrial dysfunction is known to play an important role in the development and progression of chronic kidney disease. Improving mitochondrial homeostasis therefore has the potential to restore renal function in diabetic kidney disease. Exercise training was shown to be beneficial in improving skeletal muscle and cardiovascular system pathology by improving mitochondrial function, reduction of oxidative stress and metabolic reprogramming. Recent studies have shown that Irisin, a molecule secreted by the skeletal muscles during exercise protects the mitochondria from ischemia reperfusion injury in the lung and heart. In clinical studies, involving type 1 diabetes, high and low levels of Irisin has been reported however, its functional significance remains unclear. Exercise induces the expression of PGC‐1α, leading to the expression of the fibronectin type III domain containing 5 (FNDC5). Irisin derives from the proteolytic cleavage of the peptide FNDC5 by yet unknown enzymes. The purpose of our study was to investigate whether exercise induced Irisin improves mitochondrial biogenesis and function in type 1 diabetic nephropathy. C57BL/6‐Ins2Akita/J (Type 1 diabetes, Akita) and C57BL/6J (Wild type, WT) mice were subjected to 16‐week exercise regimen of 280m run on treadmill at speed of 7m/min for 5 days/week for 2 weeks and increased to 12m/min for the remaining 14 weeks. In vitro experiments were done in mouse glomerular endothelial cells (MGECs) treated with recombinant Irisin. In Akita and WT mice, exercise training improved renal vascularity, cortical blood flow and glomerular filtration rate compared to sedentary groups. Exercise upregulated Irisin expression in Akita and WT mice compared to sedentary mice but to a greater extent in WT mice. The protein and mRNA expression of PGC1α, PPARα and PPARγ were decreased in sedentary Akita mice and increased following exercise training suggesting induction of mitochondrial biogenesis. Sedentary Akita mice showed decreased expression of regulatory markers of mitochondrial DNA transcription, Nrf1, Nrf2, and Tfam and exercise normalized their expression. The expression of MnSOD and catalase were decreased in Akita mice suggesting impaired antioxidant defense and exercise restored their levels to normal. In conclusion, our results suggest that exercise induced Irisin protects the kidney in type 1 diabetes by restoring mitochondrial homeostasis.Support or Funding InformationThis study was supported, in part, by NIH grant HL‐104103 and DK 104653 to US and AHA grant: 15SDG25840013 to SP.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Effects of phlegm-resolving and stasis-removing herbal drugs on PGC-1α mRNA expressions and insulin resistance in NAFLD rats

Objective To explore the mechanism of phlegm-resolving and stasis- removing herbals on NAFLD by observing expressions of PGC1α mRNA and insulin resistance. Methods A total of 60 male SD rats were randomly divided into the normal group, model group, positive medication control group, high-dose, middle-dose and low-dose group. The rats were fed with high-fat forage for 8 weeks. The positive medication control group were gavaged with Dongbao-Gantai liquid (0.9 g/kg/day), the high-dose, middle-dose and low-dose group were gavaged with Xiaotan-Huayu liquid (43.34、32.50、21.67 g/kg/day), and normal group, model group were gavaged with equal volume of distilled water. The drugs were given by 1 ml/100 g and last for 8 weeks. The levels of TC, TG, FFA, ALT, AST, FBG, FINS, and HOMA-IR in serum, and levels of TC, TG, and PGC-1α mRNA and pathological morphological changes in hepatic tissue were observed after 8 weeks. Results The levels of TG (0.55 ± 0.10 mmol/L, 0.58 ± 0.09 mmol/L, 0.67 ± 0.11 mmol/L vs. 1.18 ± 0.15 mmol/L), TC (1.48 ± 0.24 mmol/L, 1.69 ± 0.27 mmol/L, 1.74 ± 0.27 mmol/L vs. 3.29 ± 0.26 mmol/L), FFA (251.08 ± 48.18 μmol/L, 277.53 ± 56.73 μmol/L, 291.82 ± 48.67 μmol/L vs. 432.19 ± 67.83 μmol/L), ALT (29.32 ± 4.17 U/L, 31.26 ± 4.74 U/L, 33.56 ± 5.18 U/L vs. 47.21 ± 8.67 U/L), AST (11.05 ± 2.18 U/L, 12.15 ± 2.67 U/L, 12.96 ± 2.93 U/L vs. 19.43 ± 3.68 U/L), FBG (5.68 ± 1.22 mmol/L, 6.86 ± 1.36 mmol/L, 7.94 ± 1.82 mmol/L vs. 11.88 ± 2.54 mmol/L), FINS (8.48 ± 1.22 mmol/L, 9.55 ± 1.95 mmol/L, 9.96 ± 1.74 mmol/L vs. 12.96 ± 2.67 mmol/L), HOMA-IR (1.91 ± 0.26, 2.91 ± 0.65, 3.52 ± 0.58 vs. 6.89 ± 1.21) in serum of high-dose, middle-dose and low-dose groups were decreased than model group. Levels of FFA (242.19 ± 35.13 μmol/L, 259.78 ± 29.33 μmol/L, 277.62 ± 34.29 μmol/L vs. 436.48 ± 52.15 μmol/L), TG (23.65 ± 3.28 mmol/L, 24.41 ± 3.15 mmol/L, 25.37 ± 3.59 mmol/L vs. 15.98 ± 2.37 mmol/L), TC (7.15 ± 0.82 mmol/L, 8.60 ± 0.95 mmol/L, 8.86 ± 1.04 mmol/L vs. 36.98 ± 4.28 mmol/L) were in hepatic tissue of high-dose, middle-dose and low-dose groups were significantly lower than the model group. The levels of PGC-1α mRNA (1.24 ± 0.06, 1.02 ± 0.07, 0.99 ± 0.08 vs. 0.43 ± 0.06) in hepatic tissue of high-dose, middle-dose and low-dose groups were significantly higher than model group. Conclusions The phlegm-resolving and stasis-removing herbals may improve lipid metabolism by regulating the expression of PGC-1α mRNA, inhibiting gluconeogenesis and liver sugar output, correcting disturbance of lipid metabolism and improving insulin resistance. Key words: Non-alcoholic fatty liver disease; Phlegm-resolving and stasis-removing; PGC-1α; Rats

Repeatability of exercise‐induced changes in mRNA expression and technical considerations for qPCR analysis in human skeletal muscle

What is the central question of this study? Are individual changes in exercise-induced mRNA expression repeatable (i.e. representative of the true response to exercise rather than random error)? What is the main finding and its importance? Exercise-induced changes in mRNA expression are not repeatable even under identical experimental conditions, thereby challenging the use of mRNA expression as a biomarker of adaptive potential and/or individual responsiveness to exercise. It remains unknown if (1) the observed change in mRNA expression reflects an individual's true response to exercise or random (technical and/or biological) error, and (2) the individual responsiveness to exercise is protocol-specific. We examined the repeatability of skeletal muscle PGC-1α, PDK4, NRF-1, VEGF-A, HSP72 and p53 mRNA expression following two identical endurance exercise (END) bouts (END-1, END-2; 30min of cycling at 65% of peak work rate (WRpeak ), n=11) and inter-individual variability in PGC-1α and PDK4 mRNA expression following END and sprint interval training (SIT; 8×20s cycling intervals at ∼170% WRpeak , n=10) in active young males. The repeatability of key gene analysis steps (RNA extraction, reverse transcription, qPCR) and within-sample fibre-type distribution (n=8) was also determined to examine potential sources of technical error in our analyses. Despite highly repeatable exercise bout characteristics (work rate, heart rate, blood lactate; ICC>0.71; CV<10%; r>0.85, P<0.01), gene analysis steps (ICC>0.73; CV<24%; r>0.75, P<0.01), and similar group-level changes in mRNA expression, individual changes in PGC-1α, PDK4, VEGF-A and p53 mRNA expression were not repeatable (ICC<0.22; CV>20%; r<0.21). Fibre-type distribution in two portions of the same muscle biopsy was highly variable and not significantly related (ICC=0.39; CV=26%; r=0.37, P=0.37). Since individual changes in mRNA expression following identical exercise bouts were not repeatable, inferences regarding individual responsiveness to END or SIT were not made. Substantial random error exists in changes in mRNA expression following acute exercise, thereby challenging the use of mRNA expression for analysing individual responsiveness to exercise.

The decline of whole-body glucose metabolism in ovariectomized rats.

Age is a major risk factor for developing chronic diseases, including type 2 diabetes and osteoporosis. Emerging evidences suggest that the disorder of bone metabolism in osteoporosis is involved in the pathogenesis of glucose intolerance, insulin resistance and type 2 diabetes. However, their etiology and relative regulatory factors still remain elusive to clinicians and researchers. In this study, rats were divided into two groups: normal sham surgery control and ovariectomized (OVX) groups. We evaluated the global bone parameters, glucose metabolism, protein and gene expressions in both skeletal muscle and adipocytes. The present findings showed that the bone mineral density (BMD) and compression load of bone were markedly reduced in OVX rats as revealed by micro-CT, dual energy X-ray absorptiometry and bone biomechanics analysis. Besides, plasma estrogen, total alkaline phosphatase (TALP) and osteocalcin levels were significantly decreased in the OVX rats, but body weight, fat mass and plasma tartrate-resistant acid phosphatase (TRAP) and chemerin levels were significantly increased in the OVX rats. More interestingly, we found that p-AKT, p-P38MAPK, glucose transporter 4 (GLUT4) and peroxisome proliferator-activated receptor gamma coactivator 1 alpha (PGC-1α) contents as well as GLUT4 and PGC-1α mRNA expression were significantly decreased in skeletal muscle and adipocytes of OVX rats. In conclusion, our results indicated that whole-body glucose metabolism and glucose intolerance in OVX rats was degressive, suggesting there was a novel link between osteoporosis and whole body glucose homeostasis, which are controlled by the P38MAPK/PGC-1α/GLUT4 signaling pathway.

PO-062 Acute exercise intervention combined with metformin’s influences on glucose homeostasis in T2D mice

PO-062 Acute exercise intervention combined with metformin’s influences on glucose homeostasis in T2D mice

PO-111 Effects of aerobic exercise on fatty acid metabolism in liver of NAFLD Rats

Objective Aerobic exercise can improve liver metabolic diseases such as non-alcoholic fatty liverdisease (NAFLD), but the molecular mechanism is not completely clear, which limits the applicationof aerobic exercise in the treatment and improvement of NAFLD. In this paper, the effects of aerobicexercise on the expression levels of important genes regulating fatty acid metabolism in the liver ofNAFLD rats were studied in order to explore the possible molecular mechanism of aerobic exerciseregulating hepatic fatty acid metabolism.Methods 1. Animal grouping. 36 male Sprague Dawley (SD) rats aged 21 days old were randomlydivided into three groups after 1 week of adaptive feeding: 10 in the normal diet quiet group (NCgroup), 10 in the high-fat diet quiet group (HC group) and 16 in the high-fat diet exercise group (HTgroup). 2. Training arrangements. After 12 weeks, the rats in the HT group underwent 8 weeks ofaerobic exercise with a training intensity of 25m/min, and continued exercise for 1h/d, 6d/w for atotal of 8 weeks. 3. Sample collection. After an overnight fast, rats in the three groups were weighedand anesthetized with an intraperitoneal injection of 10% chloral hydrate (0.3 ml/100 g bodyweight). Blood samples were collected from the aortaventralis, centrifuged, and kept frozen at -80 °Cfor chemical assays. The rat liver was removed, frozen in liquid nitrogen, and stored in a -80 °Cfreezer. 4. Serum lipid concentration and liver function detection. Serum levels of total cholesterol(TC), triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoproteincholesterol (LDL-C), free fatty acid (FFA), aspartate aminotransferase (AST) and alanineaminotransferase (ALT) were detected in three groups of rats. 5. Detection of expression levels offatty acid metabolism related genes in liver. The expression levels of AMP-activated protein kinase(AMPK), silence information regulator 1 (SIRT1), peroxisome proliferator-activated receptor γcoactivator 1α (PGC-1α) in rat liver were detected by SYBR Green real-time PCR. 6. Data statistics.Results were expressed in the form of mean±standard deviation. Spss17.0 statistical software wasused to conduct one-way ANOVA to compare the differences between the groups, p&lt;0.05 indicatessignificant difference between groups.Results 1. Effects of aerobic exercise on Blood lipids and liver function. The serum levels of TC, TG,LDL-C, FFA, AST and ALT in HC group were significantly higher than those in NC group (p &lt; 0.05),while HDL-C level was significantly lower (p &lt; 0.05). The serum concentrations of TC, TG, LDL-C, FFA,AST and ALT in HT group were significantly lower than those in HC group (p &lt; 0.05), while HDL-Clevel was significantly higher (p &lt; 0.05). 2.Effects of aerobic exercise on the expression of fatty acidmetabolism regulatory genes in liver of rats. The mRNA expression levels of AMPK, SIRT1 and PGC-1α in liver of HC group were significantly lower than those of NC group (p &lt; 0. 05). While the mRNAexpression levels of AMPK, SIRT1 and PGC-1 α in liver of HT group were significantly higher thanthose of HC group (p &lt; 0. 05).Conclusions Aerobic exercise can improve serum lipid concentration and liver function in NAFLDrats. It increased the consumption of ATP, led to an increase in AMP/ATP ratio, enhanced the mRNAexpression of AMPK, SIRT1 and PGC-1α, activated AMPK - SIRT1 - PGC-1α signaling pathway,reduced hepatic fat synthesis and accelerated fatty acid oxidation in NAFLD rats liver.&#x0D;