Transcriptional Regulation Of Metabolic Genes Research Articles

Systems-level transcriptional regulation of Caenorhabditis elegans metabolism.

Metabolism is controlled to ensure organismal development and homeostasis. Several mechanisms regulate metabolism, including allosteric control and transcriptional regulation of metabolic enzymes and transporters. So far, metabolism regulation has mostly been described for individual genes and pathways, and the extent of transcriptional regulation of the entire metabolic network remains largely unknown. Here, we find that three-quarters of all metabolic genes are transcriptionally regulated in the nematode Caenorhabditis elegans. We find that many annotated metabolic pathways are coexpressed, and we use gene expression data and the iCEL1314 metabolic network model to define coregulated subpathways in an unbiased manner. Using a large gene expression compendium, we determine the conditions where subpathways exhibit strong coexpression. Finally, we develop "WormClust," a web application that enables a gene-by-gene query of genes to view their association with metabolic (sub)-pathways. Overall, this study sheds light on the ubiquity of transcriptional regulation of metabolism and provides a blueprint for similar studies in other organisms, including humans.

Megasphaera elsdenii Lactate Degradation Pattern Shifts in Rumen Acidosis Models.

Background: Megasphaera elsdenii is an ecologically important rumen bacterium that metabolizes lactate and relieves rumen acidosis (RA) induced by a high-grain-diet. Understanding the regulatory mechanisms of the lactate metabolism of this species in RA conditions might contribute to developing dietary strategies to alleviate RA.Methods: Megasphaera elsdenii was co-cultured with four lactate producers (Streptococcus bovis, Lactobacilli fermentum, Butyrivibrio fibrisolvens, and Selenomonas ruminantium) and a series of substrate starch doses (1, 3, and 9 g/L) were used to induce one normal and two RA models (subacute rumen acidosis, SARA and acute rumen acidosis, ARA) under batch conditions. The associations between bacterial competition and the shift of organic acids’ (OA) accumulation patterns in both statics and dynamics manners were investigated in RA models. Furthermore, we examined the effects of substrate lactate concentration and pH on Megasphaera elsdenii’s lactate degradation pattern and genes related to the lactate utilizing pathways in the continuous culture.Results and Conclusion: The positive growth of M. elsdenii and B. fibrisolvens caused OA accumulation in the SARA model to shift from lactate to butyrate and resulted in pH recovery. Furthermore, both the quantities of substrate lactate and pH had remarkable effects on M. elsdenii lactate utilization due to the transcriptional regulation of metabolic genes, and the lactate utilization in M. elsdenii was more sensitive to pH changes than to the substrate lactate level. In addition, compared with associations based on statics data, associations discovered from dynamics data showed greater significance and gave additional explanations regarding the relationships between bacterial competition and OA accumulation.

Hyperactivity of the transcription factor Nrf2 causes metabolic reprogramming in mouse esophagus

Mutations in the genes encoding nuclear factor (erythroid-derived 2)-like 2 (NRF2), Kelch-like ECH-associated protein 1 (KEAP1), and cullin 3 (CUL3) are commonly observed in human esophageal squamous cell carcinoma (ESCC) and result in activation of the NRF2 signaling pathway. Moreover, hyperactivity of the transcription factor Nrf2 has been found to cause esophageal hyperproliferation and hyperkeratosis in mice. However, the underlying mechanism is unclear. In this study, we aimed to understand the molecular mechanisms of esophageal hyperproliferation in mice due to hyperactive Nrf2. Esophageal tissues were obtained from genetically modified mice that differed in the status of the Nrf2 gene and genes in the same pathway (Nrf2-/-, Keap1-/-, K5Cre;Pkm2fl/fl;Keap1-/-, and WT) and analyzed for metabolomic profiles, Nrf2 ChIP-seq, and gene expression. We found that hyperactive Nrf2 causes metabolic reprogramming and up-regulation of metabolic genes in the mouse esophagus. One of the glycolysis genes encoding pyruvate kinase M2 (Pkm2) was not only differentially up-regulated, but also glycosylated and oligomerized, resulting in increased ATP biosynthesis. However, constitutive knockout of Pkm2 failed to inhibit this esophageal phenotype in vivo, and this failure may have been due to compensation by Pkm1 up-regulation. Transient inhibition of NRF2 or glycolysis inhibited the growth of human ESCC cells in which NRF2 is hyperactive in vitro In summary, hyperactive Nrf2 causes metabolic reprogramming in the mouse esophagus through its transcriptional regulation of metabolic genes. Blocking glycolysis transiently inhibits cell proliferation and may therefore have therapeutically beneficial effects on NRF2high ESCC in humans.

Abstract 2416: CREB orchestrates metabolic reprogramming in PDGFRA-addicted lung squamous cell cancer

Abstract Aberration of platelet-derived growth factor receptor A (PDGFRA) gene has been increasingly identified in multiple types of human cancers, including hematologic malignancies, gastrointestinal stromal tumor (GIST), medulloblastomas and gliomas. In particular, amplification of 4q12 that contains PDGFRA locus has been reported in 3% to 7% of lung adenocarcinomas and 8% to 10% of lung squamous cell carcinomas. However, it remains unclear how amplified PDGFRA drives malignancy of lung squamous cell cancer. To address this question, we used a lung squamous cell cancer cell line NCI-H1703, which has been previously reported to have 24-fold amplification of the 4q12 region. Either siRNA knockdown or small-molecule inhibition of PDGFRA significantly inhibited cell growth, suggesting that PDGFRA was required for driving cell proliferation. We further showed that both oxidative phosphorylation and aerobic glycolysis were significantly affected followed by interference of PDGFRA, by measuring of oxygen consumption rate (OCR) and extra cellular acidification rate (ECAR). In particular, ATP generation level and maximum respiratory capacity, the key parameters of mitochondrial function, were significantly decreased in mitochondrial pressure testing. These observations suggested that oncogenic PDGFRA drove the metabolic reprogramming and promoted tumor malignancy in lung squamous cell cancer. To further explore the regulatory mechanism of the observed metabolic phenotype, we took the approach of genetic sequencing technology to analyze the transcriptome in PDGFRA-driven NCI-H1703 cells treated with or without PDGFRA inhibitor. It revealed that PDGFRA-driven transcriptional regulation of metabolic genes underpinned the specific metabolic phenotypes of mitochondrial function. In addition, analysis of transcriptome as well as a phosphorylated kinase array revealed that a transcription factor, cAMP response element-binding protein (CREB), regulated the metabolic gene expression in PDGFRA-addicted cells. Knockdown of CREB disrupted metabolic gene expression and the mitochondrial function, consistent with aforementioned findings of PDGFRA, suggesting that transcriptional regulation of metabolic genes was orchestrated by CREB. Together, we herein firstly provide the molecular link between oncogenic PDGFRA and metabolic vulnerability and reveal the important role of CREB in orchestrating metabolic reprogramming in PDGFRA-addicted lung squamous cell cancer. Citation Format: Jun Xu, Nan Jin, Min Huang, Jian Ding, Meiyu Geng. CREB orchestrates metabolic reprogramming in PDGFRA-addicted lung squamous cell cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2416.

Effects of Glucose and Starch on Lactate Production by Newly Isolated Streptococcus bovis S1 from Saanen Goats.

When ruminants are fed high-concentrate diets, Streptococcus bovis proliferates rapidly and produces lactate, potentially causing rumen acidosis. Understanding the regulatory mechanisms of the metabolism of this species might help in developing dietary strategies to alleviate rumen acidosis. S. bovis strain S1 was newly isolated from the ruminal fluid of Saanen dairy goats and then used to examine the effects of glucose and starch on bacterial metabolism and gene regulation of the organic acid-producing pathway in cultures at a pH of 6.5. Glucose or starch was added to the culture medium at 1 g/liter, 3 g/liter (close to a normal range in the rumen fluid), or 9 g/liter (excessive level). Lactate was the dominant acid produced during the fermentation, and levels increased with the amount of glucose or starch in a dose-dependent manner (P < 0.001). The production of formate and acetate in the fermentation media fluctuated slightly with the dose but accounted for small fractions of the total acids. The activities of lactate dehydrogenase (LDH) and α-amylase (α-AMY) increased with the starch dose (P < 0.05), but the α-AMY activity did not change with the glucose dose. The relative expression levels of the genes ldh, pfl (encoding pyruvate formate lyase), ccpA (encoding catabolite control protein A), and α-amy were higher at a dose of 9 g/liter than at 1 g/liter (P < 0.05). Expression levels of pfl and α-amy genes were higher at 3 g/liter than at 1 g/liter (P < 0.05). The fructose 1,6-diphosphate (FDP) concentration tended to increase with the glucose and starch concentrations. In addition, the S. bovis S1 isolate fermented glucose much faster than starch. We conclude that the quantities of glucose and soluble starch had a major effect on lactate production due to the transcriptional regulation of metabolic genes. This work used a newly isolated S. bovis strain S1 from the rumen fluid of Saanen goats and examined the effects of glucose and soluble starch on organic acid patterns, enzyme activity, and expression of genes for in vitro fermentation. It was found that lactate was the dominant product from S. bovis strain S1, and the quantities of both glucose and starch in the medium were highly correlated with lactate production and with the corresponding changes in associated enzymes and genes. Therefore, manipulating the metabolic pathway of S. bovis to alter the dietary level of readily fermentable sugar and carbohydrates may be a strategy to alleviate rumen acidosis.

Metabolic reprogramming during neuronal differentiation from aerobic glycolysis to neuronal oxidative phosphorylation.

How metabolism is reprogrammed during neuronal differentiation is unknown. We found that the loss of hexokinase (HK2) and lactate dehydrogenase (LDHA) expression, together with a switch in pyruvate kinase gene splicing from PKM2 to PKM1, marks the transition from aerobic glycolysis in neural progenitor cells (NPC) to neuronal oxidative phosphorylation. The protein levels of c-MYC and N-MYC, transcriptional activators of the HK2 and LDHA genes, decrease dramatically. Constitutive expression of HK2 and LDHA during differentiation leads to neuronal cell death, indicating that the shut-off aerobic glycolysis is essential for neuronal survival. The metabolic regulators PGC-1α and ERRγ increase significantly upon neuronal differentiation to sustain the transcription of metabolic and mitochondrial genes, whose levels are unchanged compared to NPCs, revealing distinct transcriptional regulation of metabolic genes in the proliferation and post-mitotic differentiation states. Mitochondrial mass increases proportionally with neuronal mass growth, indicating an unknown mechanism linking mitochondrial biogenesis to cell size.

Hepatic CREB3L3 controls whole-body energy homeostasis and improves obesity and diabetes.

Transcriptional regulation of metabolic genes in the liver is the key to maintaining systemic energy homeostasis during starvation. The membrane-bound transcription factor cAMP-responsive element-binding protein 3-like 3 (CREB3L3) has been reported to be activated during fasting and to regulate triglyceride metabolism. Here, we show that CREB3L3 confers a wide spectrum of metabolic responses to starvation in vivo. Adenoviral and transgenic overexpression of nuclear CREB3L3 induced systemic lipolysis, hepatic ketogenesis, and insulin sensitivity with increased energy expenditure, leading to marked reduction in body weight, plasma lipid levels, and glucose levels. CREB3L3 overexpression activated gene expression levels and plasma levels of antidiabetic hormones, including fibroblast growth factor 21 and IGF-binding protein 2. Amelioration of diabetes by hepatic activation of CREB3L3 was also observed in several types of diabetic obese mice. Nuclear CREB3L3 mutually activates the peroxisome proliferator-activated receptor (PPAR) α promoter in an autoloop fashion and is crucial for the ligand transactivation of PPARα by interacting with its transcriptional regulator, peroxisome proliferator-activated receptor gamma coactivator-1α. CREB3L3 directly and indirectly controls fibroblast growth factor 21 expression and its plasma level, which contributes at least partially to the catabolic effects of CREB3L3 on systemic energy homeostasis in the entire body. Therefore, CREB3L3 is a therapeutic target for obesity and diabetes.

Estimating biological elementary flux modes that decompose a flux distribution by the minimal branching property.

Elementary flux mode (EFM) is a useful tool in constraint-based modeling of metabolic networks. The property that every flux distribution can be decomposed as a weighted sum of EFMs allows certain applications of EFMs to studying flux distributions. The existence of biologically infeasible EFMs and the non-uniqueness of the decomposition, however, undermine the applicability of such methods. Efforts have been made to find biologically feasible EFMs by incorporating information from transcriptional regulation and thermodynamics. Yet, no attempt has been made to distinguish biologically feasible EFMs by considering their graphical properties. A previous study on the transcriptional regulation of metabolic genes found that distinct branches at a branch point metabolite usually belong to distinct metabolic pathways. This suggests an intuitive property of biologically feasible EFMs, i.e. minimal branching. We developed the concept of minimal branching EFM and derived the minimal branching decomposition (MBD) to decompose flux distributions. Testing in the core Escherichia coli metabolic network indicated that MBD can distinguish branches at branch points and greatly reduced the solution space in which the decomposition is often unique. An experimental flux distribution from a previous study on mouse cardiomyocyte was decomposed using MBD. Comparison with decomposition by a minimum number of EFMs showed that MBD found EFMs more consistent with established biological knowledge, which facilitates interpretation. Comparison of the methods applied to a complex flux distribution in Lactococcus lactis similarly showed the advantages of MBD. The minimal branching EFM concept underlying MBD should be useful in other applications. sinhu@bio.dtu.dk or p.ji@polyu.edu.hk Supplementary data are available at Bioinformatics online.

Abstract 18251: Sirt1 Mediates Pressure Overload-Induced Cardiac Hypertrophy and Failure in vivo

Mitochondrial dysfunction is a hallmark of the failing heart. Sirt1, a class III histone deacetylase, plays an important role in mediating transcriptional regulation of metabolic genes in response to stress. The function of endogenous Sirt1 during heart failure is poorly understood. Here we investigated the role of Sirt1 in pressure overload-induced cardiac hypertrophy and failure using genetically altered mouse models, namely cardiac specific Sirt1 heterozygous (Sirt1 +/- ) and homozygous (Sirt1 -/- ) knockout (KO) (C57BL/6) and Sirt1 overexpression (Tg-Sirt1) mice (FVB). These mice were subjected to pressure overload with transverse aortic constriction (TAC). Notably, C57BL/6 mice are more susceptible to TAC-induced cardiac hypertrophy and failure than FVB mice. Cardiac hypertrophy induced by 4 weeks of TAC was exacerbated in Tg-Sirt1 (heart weight/body weight (HW/BW, mg/g), Wild type (WT) 6.0, Tg-Sirt1 8.4*, p&lt;0.05 vs WT), whereas it was ameliorated in Sirt1 KO mice (HW/BW, (mg/g), WT 7.9, Sirt1 +/- 6.9*, Sirt1 -/- 6.0*, * p&lt;0.05 vs WT). TAC-induced systolic dysfunction was also exacerbated in Tg-Sirt1 (ejection fraction (EF) (%), WT 71, Tg-Sirt1 48*, p&lt;0.05 vs WT), whereas it was ameliorated in Sirt1 KO mice (EF (%), WT 55, Sirt1 +/- 65*, Sirt1 -/- 63*, * p&lt;0.05 vs WT). The expression of nuclear encoded mitochondrial genes, such as Cpt2, Sdha, Fh1 and Ndufv1, was downregulated by TAC in WT mice, an effect that was normalized in Sirt1 -/- mice (relative mRNA levels vs WT sham, Sdha: WT TAC 0.56, Sirt1 -/- sham 1.2, Sirt1 -/- TAC 0.81*, Ndufv1: WT TAC 0.59, Sirt1 -/- sham 0.84, Sirt1 -/- TAC 1.1*, * p&lt;0.05 vs WT TAC). On the other hand, ATP content was decreased in Tg-Sirt1 mice after TAC (45% reduction vs WT). Chromatin immunoprecipitation assays revealed that pressure overload induces recruitment of Sirt1 to the promoter region of these mitochondrial genes (relative promoter occupancy after 4 weeks of TAC, Cpt2 2.6*, Sdha 7.4*, Fh1 5.4*, Ndufv1 5.6*, * p&lt;0.05 vs sham), indicating that endogenous Sirt1 interacts with mitochondrial genes, thereby negatively regulating their expression during TAC. These results suggest that Sirt1 mediates pressure overload-induced cardiac hypertrophy and failure, possibly through attenuation of mitochondrial function.

Transcriptional regulation of metabolic genes in the postprandial python heart: an extreme example of physiologic adaptation

In order to cope with the physiologic stresses of digesting large meals, the heart of the Burmese python undergoes significant hypertrophy in as little as 24 hours, returning to the fasted state a few days afterwards. This remarkable response has led us to investigate the molecular mechanisms utilized by python ventricular myocytes during the onset and regression of hypertrophy. We have initially focused on using quantitative PCR to examine changes in the transcripts of genes involved in metabolic processes and cardiac function between fasted and post‐fed states in the heart. We have demonstrated that genes involved in glycolysis and lipid import are downregulated during hypertrophy, while the expression of genes that control lipolytic processes are maintained in response to elevated serum lipid concentrations. Moreover, we have shown that the hypertrophic python heart favors the expression of genes that maintain a highly contractile state, and also exhibits a striking pattern of gene markers that would indicate both physiology and pathology in a mammalian system. These results implicate a novel mechanism of molecular regulation utilized by the python in order to facilitate the dynamic changes inherent to cardiac hypertrophy. This research was supported by a grant from Hiberna, Inc.

Resistance Exercise Produces A Metabolic Gene Expression Signature Similar To Endurance Exercise

Resistance training (RT) has been proposed as a beneficial intervention modality for patients with metabolic syndrome and type II diabetes because of increasing evidence that similar metabolic benefits may be achieved with RT as with endurance training. Much of the improvements resulted from exercise training are associated with transient modifications in skeletal muscle induced by an acute bout of exercise. PURPOSE: to examine the transcriptional regulation of metabolic genes in skeletal muscle induced by an acute bout of resistance exercise (RE). METHODS: Subjects (11 female and 12 male, 18 to 40 y) trained the non-dominant elbow flexors for 12 weeks resulting in a 14.2 +/- 6.2 % increase in muscle volume and a 59.7 +/- 20.4% increase in 1RM strength. Six days after the last exercise session, an acute bout of RE was performed and bilateral biopsies were taken 4 hr post-exercise from both the trained and the untrained arms. RNA was extracted and prepared for expression profiling using the Affymetrix Human Genome U133 Plus 2 chips. Absolute expression values were calculated using MAS.5 and dChip and analysis was carried out using Gene Spring. RESULTS: RE induced augmented mRNA levels of regulators implicated in 1) mitochondrial biogenesis including PGC1-a (↑2.88, p=0.0015), SIRT1(↑1.25, p=0.0152) and PRKAG2 (↑4.80, p=0.0009); 2) insulin signaling including IRS2 (↑3.42, p=0.0010), RRAD (↑5.01, p=0.0007) and NR4A1 (↑4.48, p=0.0020); and 3) capillary biogenesis including VEGF (↑1.85, p=0.0027), HIF1A (↑1.67, p=0.0004) and CYR61 (↑3.02, p=0.0045). Transcriptional modification of key lipid metabolism genes favored fatty acid repartitioning towards esterification with mRNA levels for LPL and CPT1B being decreased (LPL, ↓1.71 fold, p=0.0001; CPT1B, ↓1.42 fold, p=0.0246), and for FABPs being increased (FABP4, ↑1.56 fold, p=0.0003; FABP5, ↑1.50, p=0.0065). CONCLUSION: Similar to endurance exercise, acute RE exhibits favorable regulation of metabolic gene transcription in skeletal muscle, implying, in part, an explanation for the beneficial role of resistance training in treating metabolic disturbances.

Effect of short-term and long-term fasting on transcriptional regulation of metabolic genes in rat tissues

Ninety-eight genes/ESTs with differential expressions in epididymal adipose tissue of fed and 3-day fasting ( F3) rats were identified by microarray analysis. Genes for lipogenesis, glycolysis, and glucose aerobic oxidation were decreased in response to starvation. Further study was performed to investigate the expression patterns of these genes in rat tissues after short- and long-term starvations. The results of the increased expression of the pyruvate dehydrogenase kinase 4 (PDK4) gene and decreased pyruvate dehydrogenase (PDH) in rat muscle together with decreased fatty acid synthase (FAS) in rat adipose tissue after 1 day of fasting ( F1) suggested from transcriptional level that glucose aerobic oxidation was down-regulated in rat muscle and synthesis of saturated fatty acids was inhibited in rat adipose tissue after short-term fasting. It was noted that the transcriptions of genes involved in the fatty acid oxidation, such as very-long-chain Acyl-CoA dehydrogenase (LCAH), Acyl-CoA oxidase (ACO), carnitine palmitoyltransferase-I (CPT-I), and carntine-acylcarnitine translocase(CAT)L, were greatly increased in F1 rat liver, then began to decrease in F3 and 5-day fasting ( F5) rat liver, combined with significantly increased serum non-esterified fatty acids (NEFA) in F1 rats and increased urea in F5 rats, suggesting that inhibition of the oxidation of lipid and not the decreased availability of these fuels may play an important role in the phase II–phase III of fasting transition in the long-term fasting rats.

Nutritional Stress Activates C/EBPb and Induces Hepatic Steatosis In Vivo: Attenuation by AMP Kinase

CCAAT/Enhancer Binding Protein β plays an important role in transcriptional regulation of metabolic genes in the liver and is induced by ER stress agents, suggesting it may be important for coordinating common metabolic and stress remediation strategies. Here we show that mice deficient in C/EBPβ are protected from hepatic steatosis, while C/EBPβ over-expression in the liver using adenovirus-mediated gene delivery triggers ER stress response marker proteins (pPERK, p-eIF2α, pJNK), down-regulates pAMPK, and triggers triglyceride accumulation in the liver of normal mice. To investigate the role of AMP Kinase further on these events, FAO hepatoma cells were treated with AICAR, a pharmacological activator of AMPK, or adenovirus expressing a dominant negative subunit of AMPKα1 (adv-DN-AMPK). Pretreatment with 0.5 mM AICAR suppressed ER stress-induced C/EBPβ expression (using either thapsigargin or 0.5 mM glucose) and prevented ER stress protein activation (pPERK, p-eIF2α), while DN-AMPKα1 blocked AICAR-mediated C/EBPβ suppression, suggesting that regulation of C/EBPβ in response to ER stress is AMPK dependent. Finally, a mouse model of non-alcoholic steatosis (NASH) fed a methionine-choline deficient diet for 3 weeks induced a 3-fold increase in liver TG content along with significantly increasing C/EBPβ, PPARγ, p-PERK and p-eIF2, while significantly depleting p-AMPK and levels of the adiponectin receptor. These findings suggest that C/EBPβ plays an important role in ER stress mediated hepatic steatosis, which may be ameliorated by AMPK. Supported by NIH-DK05967

Substrate availability and transcriptional regulation of metabolic genes in human skeletal muscle during recovery from exercise

In skeletal muscle of humans, transcription of several metabolic genes is transiently induced during recovery from exercise when no food is consumed. To determine the potential influence of substrate availability on the transcriptional regulation of metabolic genes during recovery from exercise, 9 male subjects (aged 22-27) completed 75 minutes of cycling exercise at 75% V˙ o 2max on 2 occasions, consuming either a high-carbohydrate (HC) or low-carbohydrate (LC) diet during the subsequent 24 hours of recovery. Nuclei were isolated and tissue frozen from vastus lateralis muscle biopsies obtained before exercise and 2, 5, 8, and 24 hours after exercise. Muscle glycogen was restored to near resting levels within 5 hours in the HC trial, but remained depressed through 24 hours in the LC trial. During the 2- to 8-hour recovery period, leg glucose uptake was 5- to 15-fold higher with HC ingestion, whereas arterial plasma free fatty acid levels were ∼3- to 7-fold higher with LC ingestion. Exercise increased ( P < .05) transcription and/or mRNA content of the pyruvate dehydrogenase kinase 4, uncoupling protein 3, lipoprotein lipase, carnitine palmitoyltransferase I, hexokinase II, peroxisome proliferator activated receptor γ coactivator-1 α, and peroxisome proliferator activated receptor α. Providing HC during recovery reversed the activation of pyruvate dehydrogenase kinase 4, uncoupling protein 3, lipoprotein lipase, and carnitine palmitoyltransferase I within 5 to 8 hours after exercise, whereas providing LC during recovery elicited a sustained/enhanced increase in activation of these genes through 8 to 24 hours of recovery. These findings provide evidence that factors associated with substrate availability and/or cellular metabolic recovery (eg, muscle glycogen restoration) influence the transcriptional regulation of metabolic genes in skeletal muscle of humans during recovery from exercise.

Suppression of plasma free fatty acids upregulates peroxisome proliferator-activated receptor (PPAR) α and δ and PPAR coactivator 1α in human skeletal muscle, but not lipid regulatory genes

Fatty acids are an important ligand for peroxisome proliferator-activated receptor (PPAR) activation and transcriptional regulation of metabolic genes. To examine whether reduced plasma free fatty acid (FFA) availability affects the mRNA content of proteins involved in fuel metabolism in vivo, the skeletal muscle mRNA content of various transcription factors, transcriptional coactivators and genes encoding for lipid regulatory proteins were examined before and after 3 h of cycle exercise with (NA) and without (CON) pre-exercise ingestion of nicotinic acid (NA). NA resulted in a marked (3- to 6-fold) increase (P<0.05) in PPARalpha, PPARdelta and PPAR coactivator 1alpha (PGC1alpha) mRNA, but was without effect on nuclear respiratory factor-1 and Forkhead transcription factor, fatty acid transcolase/CD36, carnitine palmitoyl transferase 1, hormone sensitive lipase (HSL) and pyruvate dehydrogenase kinase 4. Exercise in CON was associated with increased (P<0.05) PPARalpha, PPARdelta and PGC1alpha mRNA, which was similar in magnitude to levels observed with NA at rest. Exercise was generally without effect on the mRNA content of lipid regulatory proteins in CON and did not affect the mRNA content of the measured subset of transcription factors, transcriptional co-activators and lipid regulatory proteins during NA. To determine the possible mechanisms by which NA might affect PGC1alpha expression, we measured p38 MAP kinase (MAPK) and plasma epinephrine. Phosphorylation of p38 MAPK was increased (P<0.05) by NA treatment at rest, and this correlated (r2=0.84, P<0.01) with increased PGC1alpha. Despite this close relationship, increasing p38 MAPK in human primary myotubes was without effect on PGC1alpha mRNA content. Plasma epinephrine was elevated (P<0.05) by NA at rest (CON: 0.27+/-0.06, NA: 0.72+/-0.11 nM) and throughout exercise. Incubating human primary myotubes with epinephrine increased PGC1alpha independently of changes in p38 MAPK phosphorylation. Hence, despite the fact that NA ingestion decreased FFA availability, it promoted the induction of PPARalpha/delta and PGC1alpha gene expression to a similar degree as prolonged exercise. We suggest that the increase in PGC1alpha may be due to the elevated plasma epinephrine levels. Despite these changes in transcription factors/coactivators, the mRNA content of lipid regulatory proteins was generally unaffected by plasma FFA availability.

Differential transcriptional activation of select metabolic genes in response to variations in exercise intensity and duration.

Cellular adaptations to endurance training are influenced by the intensity and duration of exercise. To examine the impact of exercise intensity and duration on the acute transcriptional regulation of metabolic genes in red (RG) and white (WG) gastrocnemius muscle, rats completed either low-intensity [ approximately 50% maximal O2 uptake (VO2 max)] treadmill exercise (LIE) for 45 min, LIE for 180 min, or high-intensity ( approximately 75% VO2 max) exercise (HIE) for 45 min. LIE for 45 min activated (P < 0.05) transcription of the pyruvate dehydrogenase kinase-4 (PDK4), uncoupling protein-3 (UCP3), heme oxygenase-1 (HO-1), and hexokinase II (HK II) genes in RG within 1 h after exercise. In WG, transcription of PDK4, UCP3, HKII, and lipoprotein lipase (LPL) was also induced, whereas transcription of the HO-1 gene did not change. In RG, extending LIE duration from 45 to 180 min elicited a similar activation of PDK4 and UCP3 ( approximately 15-fold) but a far greater increase in HO-1 (>30-fold) and HKII transcription (>25-fold). In WG, extending LIE for 180 min induced a much greater and prolonged (through 2- to 4-h recovery) activation of PDK4, UCP3 (both >200-fold), and HO-1 (>10-fold). HIE elicited a similar pattern of gene activation to LIE in both RG and WG, with the exception that HIE triggered >10-fold activation of HO-1 in WG. These data provide evidence that both the intensity and the duration of exercise affect the transcriptional regulation of metabolic genes in muscle in a fiber type-specific manner, possibly reflecting the relative stress imposed by the exercise bout.

Effect of short-term fasting and refeeding on transcriptional regulation of metabolic genes in human skeletal muscle.

During short-term fasting, substrate utilization in skeletal muscle shifts from predominantly carbohydrate to fat as a means of conserving glucose. To examine the potential influence of short-term fasting and refeeding on transcriptional regulation in skeletal muscle, muscle biopsies were obtained from nine male subjects at rest, after 20 h of fasting, and 1 h after consuming either a high-carbohydrate (CHO trial) or a low-carbohydrate (FAT trial) meal. Fasting induced an increase in transcription of the pyruvate dehydrogenase kinase 4 (PDK4) (10-fold), lipoprotein lipase (LPL) ( approximately 2-fold), uncoupling protein 3 (UCP3) ( approximately 5-fold), and carnitine palmitoyltransferase I (CPT I) ( approximately 2.5-fold) genes. Surprisingly, transcription of PDK4 and LPL increased further in response to refeeding (both trials) to more than 50-fold and 6- to 10-fold, respectively, over prefasting levels. However, responses varied among subjects with two subjects in particular displaying far greater activation of PDK4 (>100-fold) and LPL (>20-fold) than the other subjects (mean approximately 8-fold and approximately 2-fold, respectively). Transcription of UCP3 decreased to basal levels after the CHO meal but remained elevated after the FAT meal, whereas CPT I remained elevated after both refeeding meals. The present findings demonstrate that short-term fasting/refeeding in humans alters the transcription of several genes in skeletal muscle related to lipid metabolism. Marked heterogeneity in the transcriptional response to the fasting/refeeding protocol suggests that individual differences in genetic profile may play an important role in adaptive molecular responses to metabolic challenges.

Hypoxia Inhibits G1/S Transition through Regulation of p27 Expression

Mammalian cellular responses to hypoxia include adaptive metabolic changes and a G1 cell cycle arrest. Although transcriptional regulation of metabolic genes by the hypoxia-induced transcription factor (HIF-1) has been established, the mechanism for the hypoxia-induced G1 arrest is not known. By using genetically defined primary wild-type murine embryo fibroblasts and those nullizygous for regulators of the G1/S checkpoint, we observed that the retinoblastoma protein is essential for the G1/S hypoxia-induced checkpoint, whereas p53 and p21 are not required. In addition, we found that the cyclin-dependent kinase inhibitor p27 is induced by hypoxia, thereby inhibiting CDK2 activity and forestalling S phase entry through retinoblastoma protein hypophosphorylation. Reduction or absence of p27 abrogated the hypoxia-induced G1 checkpoint, suggesting that it is a key regulator of G1/S transition in hypoxic cells. Intriguingly, hypoxic induction of p27 appears to be transcriptional and through an HIF-1-independent region of its proximal promoter. This demonstration of the molecular mechanism of hypoxia-induced G1/S regulation provides insight into a fundamental response of mammalian cells to low oxygen tension.