Alteration of GSH level, gene expression and cell transformation in NIH3T3 cells by chronic exposure to low dose of arsenic

It is well established that arsenic toxicity is postulated to be primarily due to the binding of As(III) to sulfhydryl-containing enzymes. However, the mechanism of carcinogenesis induced by arsenic is still unclear. The interaction of arsenic with GSH and related enzymes seems a very important issue regarding mechanism of arsenical induced toxicity or carcinogenesis. The purpose of this work is to investigate the effect of chronic exposure to low dose of As(III) on GSH level, gene expression and cell transformation in NIH3T3 cells. The results showed that long-term, low dose arsenic treatment makes 3T3 cell more resistant to acute arsenic treatment. There were morphology changes after long-term arsenic treatment. First, partially immortalized 3T3 cell became immortalized. In addition, the cells were doubling more quickly than the control cells and attained higher density than the control cells at confluence. Second, cells treated with 0.1 µ.M As(III) exhibited anchorage-independent growth. Arsenic could enhance GSH level at 0.5 -10 µM dose of arsenic in 24 h treatment and decrease it at 25 µM and above. In long-term treatment with low dose of arsenic, GSH levels were decreased. As(I1I) can increase both glutathione S-transferase (GST) and glutathione reductase (GR) activities at low dose (0.5-10 M), but decreased GST and GR activities at 25 M and higher dose of arsenic, while in long-term As(III) treatment, GST and GR activities are increased. Both long-term and short-term treatments with As(III) can induce GR gene expression. GPx mRNA levels were decreased both in acute and chronic arsenic treated cells. Chronic treatment with As(III) also decreased the p53 mRNA level. Taken together, our results suggest that As(III) can alter GST, GR enzyme activities as well as GSH level and related gene expression both in long-term and short-term treatment but in a different manner in different doses. Alteration of cellular GSH level by As(III) might play all important role in gene expression and arsenic induced cell transformation.

Differential effects of exercise on insulin-signaling gene expression in human skeletal muscle.

Skeletal muscle insulin sensitivity is enhanced after acute exercise and short-term endurance training. We investigated the impact of exercise on the gene expression of key insulin-signaling proteins in humans. Seven untrained subjects (4 women and 3 men) completed 9 days of cycling at 63 ± 2% of peak O₂ uptake for 60 min/day. Muscle biopsies were taken before, immediately after, and 3 h after the exercise bouts (on days 1 and 9). The gene expression of insulin receptor substrate-2 and the p85α subunit of phosphatidylinositol 3-kinase was significantly higher 3 h after a single exercise bout, although short-term training ameliorated this effect. Gene expression of insulin receptor and insulin receptor substrate-1 was not significantly altered at any time point. These results suggest that exercise may have a transitory impact on the expression of insulin receptor substrate-2 and phosphatidylinositol 3-kinase; however, the predominant actions of exercise on insulin sensitivity appear not to reside in the transcriptional activation of the genes encoding major insulin-signaling proteins.

Creatine transporter protein content, localization, and gene expression in rat skeletal muscle

The present study examined the gene expression and cellular localization of the creatine transporter (CreaT) protein in rat skeletal muscle. Soleus (SOL) and red (RG) and white gastrocnemius (WG) muscles were analyzed for CreaT mRNA, CreaT protein, and total creatine (TCr) content. Cellular location of the CreaT protein was visualized with immunohistochemical analysis of muscle cross sections. TCr was higher (P <= 0.05) in WG than in both RG and SOL, and was higher in RG than in SOL. Total CreaT protein content was greater (P <= 0.05) in SOL and RG than in WG. Two bands (55 and 70 kDa) of the CreaT protein were found in all muscle types. Both the 55-kDa (CreaT-55) and the 70-kDa (CreaT-70) bands were present in greater (P <= 0.05) amounts in SOL and RG than in WG. SOL and RG had a greater amount (P <= 0.05) of CreaT-55 than CreaT-70. Immunohistochemical analysis revealed that the CreaT was mainly associated with the sarcolemmal membrane in all muscle types. CreaT mRNA expression per microgram of total RNA was similar across the three muscle types. These data indicate that rat SOL and RG have an enhanced potential to transport Cr compared with WG, despite a higher TCr in the latter.

Elevated hypothalamic beacon gene expression in Psammomys obesus prone to develop obesity and type 2 diabetes

Objective: To investigate hypothalamic beacon gene expression at various developmental stages in genetically selected diabetes-resistant and diabetes-prone Psammomys obesus. In addition, effects of dietary energy composition on beacon gene expression were investigated in diabetes-prone P. obesus. Methods: Hypothalamic beacon gene expression was measured using TaqmanÔ fluorogenic PCR in 4-, 8- and 16-week-old animals from each genetically selected line. Results: Expression of beacon was elevated in the diabetes-prone compared with diabetes-resistant P. obesus at 4 weeks of age despite no difference in body weight between the groups. At 8 weeks of age, hypothalamic beacon gene expression was elevated in diabetes-prone animals fed a high-energy diet, and was correlated with serum insulin concentration. Conclusion: P. obesus with a genetic predisposition for the development of obesity and type 2 diabetes have elevated hypothalamic beacon gene expression at an early age. Overexpression of beacon may contribute to the development of obesity and insulin resistance in these animals.

Calpain 3 gene expression in skeletal muscle is associated with body fat content and measures of insulin resistance

OBJECTIVE: To investigate whether skeletal muscle gene expression of calpain 3 is related to obesity and insulin resistance.

DESIGN: Cross-sectional studies in 27 non-diabetic human subjects and in Psammomys obesus, a polygenic animal model of obesity and type 2 diabetes.

MEASUREMENTS: Expression of CAPN3 in skeletal muscle was measured using Taqman fluorogenic PCR. In the human subjects, body composition was assessed by DEXA and insulin sensitivity was measured by euglycemic-hyperinsulinemic clamp. In Psammomys obesus, body composition was determined by carcass analysis, and substrate oxidation rates, physical activity and energy expenditure were measured by whole-body indirect calorimetry.

RESULTS: In human subjects, calpain 3 gene expression was negatively correlated with total (P=0.022) and central abdominal fat mass (P=0.034), and with blood glucose concentration in non-obese subjects (P=0.017). In Psammomys obesus, calpain 3 gene expression was negatively correlated with circulating glucose (P=0.013) and insulin (P=0.034), and with body fat mass (P=0.049). Indirect calorimetry revealed associations between calpain 3 gene expression and carbohydrate oxidation (P=0.009) and energy expenditure (P=0.013).

CONCLUSION/INTERPRETATION: Lower levels of expression of calpain 3 in skeletal muscle were associated with reduced carbohydrate oxidation and elevated circulating glucose and insulin concentrations, and also with increased body fat and in particular abdominal fat. Therefore, reduced expression of calpain 3 in both humans and Psammomys obesus was associated with phenotypes related to obesity and insulin resistance.

Fasting activates the gene expression of UCP3 independent of genes necessary for lipid transport and oxidation in skeletal muscle

Fasting triggers a complex array of adaptive metabolic and hormonal responses including an augmentation in the capacity for mitochondrial fatty acid (FA) oxidation in skeletal muscle. This study hypothesized that this adaptive response is mediated by increased mRNA of key genes central to the regulation of fat oxidation in human skeletal muscle. Fasting dramatically increased UCP3 gene expression, by 5-fold at 15 h and 10-fold at 40 h. However the expression of key genes responsible for the uptake, transport, oxidation, and re-esterification of FA remained unchanged following 15 and 40 h of fasting. Likewise there was no change in the mRNA abundance of transcription factors. This suggests a unique role for UCP3 in the regulation of FA homeostasis during fasting as adaptation to 40 h of fasting does not require alterations in the expression of other genes necessary for lipid metabolism.

Exercise, diet and skeletal muscle gene expression

Skeletal muscle, as a consequence of its mass and great capacity for altered metabolism, has a major impact on whole-body metabolic homeostasis and is capable of remarkable adaptation in response to various physiological stimuli, including exercise and dietary intervention. Exercise-induced increases in skeletal muscle mRNA levels of a number of genes have been reported, due to transcriptional activation and/or increased mRNA stability. The cellular adaptations to exercise training appear to be due to the cumulative effects of transient increases in gene transcription after repeated exercise bouts. The relative importance of transcriptional (mRNA synthesis) and translational (mRNA stability or translational efficiency) mechanisms for the training-induced increases in skeletal muscle protein abundance remains to be fully elucidated. Dietary manipulation, and the associated alterations in nutrient availability and hormone levels, can also modify skeletal muscle gene expression, although fewer studies have been reported. A major challenge is to understand how exercise and diet exert their effects on gene and protein expression in skeletal muscle. In relation to exercise, potential stimuli include stretch and muscle tension, the pattern of motor nerve activity and the resultant calcium transients, the energy charge of the cell and substrate availability, oxygen tension and circulating hormones. These are detected by various cellular signaling mechanisms, acting on a range of downstream targets and a wide range of putative transcription factors. A key goal in the years ahead is to identify how alterations at the level of gene expression are coupled to the changes in skeletal muscle phenotype. It is clear that gene expression, although representing a specific site of regulation, is only one step in a complex cascade from the initial stimulus to the final phenotypic adaptation and integrated physiological response.

Exercise training increases lipid metabolism gene expression in human skeletal muscle

The effects of a single bout of exercise and exercise training on the expression of genes necessary for the transport and beta -oxidation of fatty acids (FA), together with the gene expression of transcription factors implicated in the regulation of FA homeostasis were investigated. Seven human subjects (3 male, 4 female, 28.9 ± 3.1 yr of age, range 20-42 yr, body mass index 22.6 kg/m², range 17-26 kg/m²) underwent a 9-day exercise training program of 60 min cycling per day at 63% peak oxygen uptake (V_{O2 peak}; 104 ± 14 W). On days 1 and 9 of the program, muscle biopsies were sampled from the vastus lateralis muscle at rest, at the completion of exercise, and again 3 h postexercise. Gene expression of key components of FA transport [FA translocase (FAT/CD36), plasma membrane-associated FA-binding protein], beta -oxidation [carntine palmitoyltransferase(CPT) I, beta -hydroxyacyl-CoA dehydrogenase] and transcriptional control [peroxisome proliferator-activated receptor (PPAR)alpha , PPARgamma , PPARgamma coactivator 1, sterol regulatory element-binding protein-1c] were unaltered by exercise when measured at the completion and at 3 h postexercise. Training increased total lipid oxidation by 24% (P < 0.05) for the 1-h cycling bout. This increased capacity for lipid oxidation was accompanied by an increased expression of FAT/CD36 and CPT I mRNA. Similarly, FAT/CD36 protein abundance was also upregulated by exercise training. We conclude that enhanced fat oxidation after exercise training is most closely associated with the genes involved in regulating FA uptake across the plasma membrane (FAT/CD36) and across the mitochondrial membrane (CPT I).

Exercise and skeletal muscle gene expression

1. Skeletal muscle is a complex and heterogenous tissue capable of remarkable adaptation in response to exercise training. The role of gene transcription, as an initial target to control protein synthesis, is poorly understood.
2. Mature myofibres contain several hundred nuclei, all of which maintain transcriptional competency, although the localized responsiveness of nuclei is not well known. Myofibres are capable of hypertrophy. These processes require the activation and myogenic differentiation of mononuclear satellite cells that fuse with the enlarging or repairing myofibre.
3. A single bout of exercise in human subjects is capable of activating the expression of many diverse groups of genes.
4. The impact of repeated exercise bouts, typical of exercise training, on gene expression has yet to receive systematic investigation.
5. The molecular programme elicited by resistance exercise and endurance exercise differs markedly. Muscular hypertrophy following resistance exercise is dependent on the activation of satellite cells and their subsequent myogenic maturation. Endurance exercise requires the simultaneous activation of mitochondrial and nuclear genes to enable mitochondrial biogenesis.
6. Future analysis of the regulation of genes by exercise may combine high-throughput technologies, such as gene-chips, enabling the rapid detection and analysis of changes in the expression of many thousands of genes.

Effect of arsenic on transcription factor AP-1 and NF-kB DNA binding activity and related gene expression

Both acute (24 h) and chronic (10–20 week) exposure of human fibroblast cells to low dose sodium arsenite (As(III)) significantly affects activating protein-1 (AP-1) and nuclear factor kappa B (NF-κB) DNA binding activity. Short-term treatment with 0.1–5 μM As(III) up-regulates expression of c-Fos and c-Jun and the redox regulators, thioredoxin (Trx) and Redox factor-1 (Ref-1) and activates both AP-1 and NF-κB binding. Chronic exposure to 0.1 or 0.5 μM As(III) decreased c-Jun, c-Fos and Ref-1 protein levels and AP-1 and NF-κB binding activity, but increased Trx expression. Short term exposure to phorbol 12-myristate 13-acetate (TPA), a phorbol ester tumour promoter, or hydrogen peroxide (H₂O₂) also activates AP-1 and NF-κB binding. However, pre-treatment with As(III) prevents this increase. These results suggest that As(III) may alter AP-1 and NF-κB activity, in part, by up-regulating Trx and Ref-1. The different effects of short- versus long-term As(III) treatment on acute-phase response to oxidative stress reflect changes in the expression of Ref-1, c-Fos and c-Jun, but not Trx.

Dietary regulation of fat oxidative gene expression in different skeletal musle fiber types

Objective: To determine the effect of a high-fat diet on the expression of genes important for fat oxidation, the protein abundance of the transcription factors peroxisome proliferator-activated receptor (PPAR) isoforms α and γ, and selected enzyme activities in type I and II skeletal muscle. Research Methods and Procedures: Sprague-Dawley rats consumed either a high-fat (HF: 78% energy, n = 8) or high-carbohydrate (64% energy, n = 8) diet for 8 weeks while remaining sedentary. Results: The expression of genes important for fat oxidation tended to increase in both type I (soleus) and type II (extensor digitorum longus) fiber types after an HF dietary intervention. However, the expression of muscle type carnitine palmitoyltransferase I was not increased in extensor digitorum longus. Analysis of the gene expression of both peroxisome proliferator-activated receptor-γ coactivator and forkhead transcription factor O1 demonstrated no alteration in response to the HF diet. Similarly, PPARα and PPARγ protein levels were also not altered by the HF diet. Discussion: An HF diet increased the expression of an array of genes involved in lipid metabolism, with only subtle differences evident in the response within differing skeletal muscle fiber types. Despite changes in gene expression, there were no effects of diet on peroxisome proliferator-activated receptor-gamma coactivator and forkhead transcription factor O1 mRNA and the protein abundance of PPARα and PPARγ.

A short-term, high-fat diet up-regulates lipid metabolism and gene expression in human skeletal muscle

Background: Dietary fatty acids may be important in regulating gene expression. However, little is known about the effect of changes in dietary fatty acids on gene regulation in human skeletal muscle.
Objective: The objective was to determine the effect of altered dietary fat intake on the expression of genes encoding proteins necessary for fatty acid transport and &szlig;-oxidation in skeletal muscle.
Design: Fourteen well-trained male cyclists and triathletes with a mean (&plusmn; SE) age of 26.9 &plusmn; 1.7 y, weight of 73.7 &plusmn; 1.7 kg, and peak oxygen uptake of 67.0 &plusmn; 1.3 mL &dot; kg-1 &dot; min-1 consumed either a high-fat diet (HFat: > 65% of energy as lipids) or an isoenergetic high-carbohydrate diet (HCho: 70–75% of energy as carbohydrate) for 5 d in a crossover design. On day 1 (baseline) and again after 5 d of dietary intervention, resting muscle and blood samples were taken. Muscle samples were analyzed for gene expression [fatty acid translocase (FAT/CD36), plasma membrane fatty acid binding protein (FABPpm), carnitine palmitoyltransferase I (CPT I), &szlig;-hydroxyacyl-CoA dehydrogenase (&szlig;-HAD), and uncoupling protein 3 (UCP3)] and concentrations of the proteins FAT/CD36 and FABPpm.
Results: The gene expression of FAT/CD36 and &szlig; -HAD and the gene abundance of FAT/CD36 were greater after the HFat than after the HCho diet (P < 0.05). Messenger RNA expression of FABPpm, CPT I, and UCP-3 did not change significantly with either diet.
Conclusions: A rapid and marked capacity for changes in dietary fatty acid availability to modulate the expression of mRNA-encoding proteins is necessary for fatty acid transport and oxidative metabolism. This finding is evidence of nutrient-gene interactions in human skeletal muscle.

Analysis of zinc transporter, hZnT4(Slc30A4), gene expression in a mammary gland disorder leading to reduced zinc secretion into milk

Zinc deficiency, causing impaired growth and development, may have a nutritional or genetic basis. We investigated two cases of inherited zinc deficiency found in breast-fed neonates, caused by low levels of zinc in the maternal milk. This condition is different from acrodermatitis enteropathica but has similarities to the "lethal milk" mouse, where low levels of zinc in the milk of lactating dams leads to zinc deficiency in pups. The mouse disorder has been attributed to a defect in the ZnT4 gene. Little is known about the expression of the human orthologue, hZnT4 (Slc30A4). Sequence analysis of cDNA, real-time PCR and Western blot analysis of hZnT4, carried out on control cells and cells from unrelated mothers of two infants with zinc deficiency, showed no differences. The hZnT4 gene was highly expressed in mouthwash buccal cells compared with lymphoblasts and fibroblasts. The hZnT4 protein did not co-localise with intracellular free zinc pools, suggesting that hZnT4 is not involved in transport of zinc into vesicles destined for secretion into milk. This observation, combined with phenotypic differences between the "lethal milk" mouse and the human disorder, suggests that the "lethal milk" mouse is not the corresponding model for the human zinc deficiency condition.

Skeletal muscle total creatine content and creatine transporter gene expression in vegetarians prior to and following creatine supplementation

This study examined the effect of vegetarianism on skeletal muscle total creatine (TCr) content and creatine transporter (CreaT) gene expression, prior to and during 5 d of Cr supplementation (CrS). In a double-blind, crossover design, 7 vegetarians (VEG) and nonvegetarians (NVEG) were assigned Cr or placebo supplements for 5 d and after 5 wk, received the alternative treatment. Muscle sampling occurred before, and after 1 and 5 d of treatment ingestion. Basal muscle TCr content was lower (P < 0.05) in VEG compared with NVEG. Muscle TCr increased (P < 0.05) throughout the Cr trial in both groups but was greater (P < 0.05) in VEG compared with NVEG, at days 1 and 5. CreaT gene expression was not different between VEG and NVEG. The results indicate that VEG have a lower muscle TCr content and an increased capacity to load Cr into muscle following CrS. Muscle CreaT gene expression does not appear to be affected by vegetarianism.

Effect of elevated lipid concentrations on human skeletal muscle gene expression

Dietary fatty acids regulate the abundance and activity of various proteins involved in the regulation of fat oxidation by functioning as regulators of gene transcription. To determine whether the transcription of key lipid metabolic proteins necessary for fat metabolism within human skeletal muscle are regulated by acute elevations in circulating free fatty acid (FFA) concentrations, 7 healthy men underwent 3 randomized resting infusions of Intralipid (20%) with heparin sodium, saline and heparin sodium, or saline only for 5 hours. These infusions significantly elevated plasma FFA concentrations by 15-fold (to 1.67 ± 0.13 mmol/L) in the Intralipid infusion trial, with modest elevations observed in the saline and heparin sodium and saline alone infusion groups (0.67 ± 0.09 and 0.49 ± 0.087 mmol/L, P < .01 both vs Intralipid infusion). Analysis of messenger RNA (mRNA) concentration demonstrated that pyruvate dehydrogenase kinase isoform 4 (PDK4) mRNA, a key negative regulator of glucose oxidation, was increased in all trials with a 24-fold response after Intralipid infusion, 15-fold after saline and heparin infusion, and 9-fold after saline alone. The PDK4 increases were not significantly different between the 3 trials. The mRNA concentration of the major uncoupling protein within skeletal muscle, uncoupling protein 3, was not elevated in parallel to the increased plasma FFA as similar (not, vert, similar2-fold) increases were evident in all trials. Additional genes involved in lipid transport (fatty acid translocase/CD36), oxidation (carnitine palmitoyltransferase I), and metabolism (1-acylglycerol-3-phosphate O-acyltransferase 1, hormone-sensitive lipase, and peroxisomal proliferator-activated receptor-γ coactivator-1α) were not altered by increased circulating FFA concentrations. The present data demonstrate that of the genes analyzed that encode proteins that are key regulators of lipid homeostasis within skeletal muscle, only the PDK4 gene is uniquely sensitive to increasing FFA concentrations after increased plasma FFA achieved by intravenous lipid infusion.