Expression Profile and Distribution of Efhc1 Gene Transcript During Rodent Brain Development

One of the putative causative genes for juvenile myoclonic epilepsy (JME) is EFHC1. We report here the expression profile and distribution of Efhc1 messenger RNA (mRNA) during mouse and rat brain development. Real-time polymerase chain reaction revealed that there is no difference in the expression of Efhc1 mRNA between right and left hemispheres in both species. In addition, the highest levels of Efhc1 mRNA were found at intra-uterine stages in mouse and in adulthood in rat. In common, there was a progressive decrease in Efhc1 expression from 1-day-old neonates to 14-day-old animals in both species. In situ hybridization studies showed that rat and mouse Efhc1 mRNAs are expressed in ependymal cells of ventricle walls. Our findings suggest that Efhc1 expression is more important during initial phases of brain development and that at this stage it could be involved in key developmental mechanisms underlying JME.

Expression of alpha-synuclein is increased in the hippocampus of rats with high levels of innate anxiety

A genomic region neighboring the alpha-synuclein gene, on rat chromosome 4, has been associated with anxiety- and alcohol-related behaviors in different rat strains. In this study, we have investigated potential molecular and physiological links between alpha-synuclein and the behavioral differences observed between Lewis (LEW) and Spontaneously Hypertensive (SHR) inbred rats, a genetic model of anxiety. As expected, LEW rats appeared more fearful than SHR rats in three anxiety models: open field, elevated plus maze and light/dark box. Moreover, LEW rats displayed a higher preference for alcohol and consumed higher quantities of alcohol than SHR rats. alpha-Synuclein mRNA and protein concentrations were higher in the hippocampus, but not the hypothalamus of LEW rats. This result inversely correlated with differences in dopamine turnover in the hippocampus of LEW and SHR rats, supporting the hypothesis that alpha-synuclein is important in the downregulation of dopamine neurotransmission. A novel single nucleotide polymorphism was identified in the 30-untranslated region (3`-UTR) of the alpha-synuclein cDNA between these two rat strains. Plasmid constructs based on the LEW 3`-UTR sequence displayed increased expression of a reporter gene in transiently transfected PC12 cells, in accordance with in-vivo findings, suggesting that this nucleotide exchange might participate in the differential expression of alpha-synuclein between LEW and SHR rats. These results are consistent with a novel role for alpha-synuclein in modulating rat anxiety- like behaviors, possibly through dopaminergic mechanisms. Since the behavioral and genetic differences between these two strains are the product of independent evolutionary histories, the possibility that polymorphisms in the alpha-synuclein gene may be associated with vulnerability to anxiety- related disorders in humans requires further investigation. Molecular Psychiatry (2009) 14, 894-905; doi: 10.1038/mp.2008.43; published online 22 April 2008

Chronic resistance training decreases MuRF-1 and Atrogin-1 gene expression but does not modify Akt, GSK-3 beta and p70S6K levels in rats

Long-term adaptation to resistance training is probably due to the cumulative molecular effects of each exercise session. Therefore, we studied in female Wistar rats the molecular effects of a chronic resistance training regimen (3 months) leading to skeletal muscle hypertrophy in the plantaris muscle. Our results demonstrated that muscle proteolytic genes MuRF-1 and Atrogin-1 were significantly decreased in the exercised group measured 24 h after the last resistance exercise session (41.64 and 61.19%, respectively; P < 0.05). Nonetheless, when measured at the same time point, 4EBP-1, GSK-3 beta and eIF2B epsilon mRNA levels and Akt, GSK-3 beta and p70S6K protein levels (regulators of translation initiation) were not modified. Such data suggests that if gene transcription constitutes a control point in the protein synthesis pathway this regulation probably occurs in early adaptation periods or during extreme situations leading to skeletal muscle remodeling. However, proteolytic gene expression is modified even after a prolonged resistance training regimen leading to moderate skeletal muscle hypertrophy.

Chronic supplementation of beta-hydroxy-beta methylbutyrate (HM beta) increases the activity of the GH/IGF-I axis and induces hyperinsulinemia in rats

Objective: Beta-hydroxy-beta-methylbutyrate (HM beta) is a metabolite of leucine widely used for improving sports performance. Although limp is recognized to promote anabolic or anti-catabolic effects on protein metabolism, the impact of its long-term use on skeletal muscle and/or genes that control the skeletal protein balance is not fully known. This study aimed to investigate whether chronic HM beta treatment affects the activity of GH/IGF-I axis and skeletal muscle IGF-I and myostatin mRNA expression. Design: Rats were treated with HK beta (320 mg/kg BW) or vehicle, by gavage, for 4 weeks, and killed by decapitation. Blood was collected for evaluation of serum insulin, glucose and IGF-I concentrations. Samples of pituitary, liver, extensor digitorum longus (EDL) and soleus muscles were collected for total RNA or protein extraction to evaluate the expression of pituitary growth hormone (GH) gene (mRNA and protein), hepatic insulin-like growth factor I (IGF-I) mRNA, skeletal muscle IGF-I and myostatin mRNA by Northern blotting/real time-PCR, or Western blotting. Results: Chronic HM beta treatment increased the content of pituitary GH mRNA and GH, hepatic IGF-I mRNA and serum IGF-I concentration. No changes were detected on skeletal muscle IGF-I and myostatin mRNA expression. However, the HIM-treated rats although normoglycemic, exhibited hyperinsulinemia. Conclusions: The data presented herein extend the body of evidence on the potential role of HM beta-treatment in stimulating GH/IGF-I axis activity. In spite of this effect, HM beta supplementation also induces an apparent insulin resistance state which might limit the beneficial aspects of the former results, at least in rats under normal nutritional status and health conditions. (C) 2010 Growth Hormone Research Society. Published by Elsevier Ltd. All rights reserved.

Absence of Melatonin Induces Night-Time Hepatic Insulin Resistance and Increased Gluconeogenesis Due to Stimulation of Nocturnal Unfolded Protein Response

It is known that the circadian rhythm in hepatic phosphoenolpyruvate carboxykinase expression (a limiting catalytic step of gluconeogenesis) and hepatic glucose production is maintained by both daily oscillation in autonomic inputs to the liver and night feeding behavior. However, increased glycemia and reduced melatonin (Mel) levels have been recently shown to coexist in diabetic patients at the end of the night period. In parallel, pinealectomy (PINX) is known to cause glucose intolerance with increased basal glycemia exclusively at the end of the night. The mechanisms that underlie this metabolic feature are not completely understood. Here, we demonstrate that PINX rats show night-time hepatic insulin resistance characterized by reduced insulin-stimulated RAC-alpha serine/threonine-protein kinase phosphorylation and increased phosphoenolpyruvate carboxykinase expression. In addition, PINX rats display increased conversion of pyruvate into glucose at the end of the night. The regulatory mechanism suggests the participation of unfolded protein response (UPR), because PINX induces night-time increase in activating transcription factor 6 expression and prompts a circadian fashion of immunoglobulin heavy chain-binding protein, activating transcription factor 4, and CCAAT/enhancer-binding protein-homologous protein expression with Zenith values at the dark period. PINX also caused a night-time increase in Tribble 3 and regulatory-associated protein of mammalian target of rapamycin; both were reduced in liver of PINX rats treated with Mel. Treatment of PINX rats with 4-phenyl butyric acid, an inhibitor of UPR, restored night-time hepatic insulin sensitivity and abrogated gluconeogenesis in PINX rats. Altogether, the present data show that a circadian oscillation of UPR occurs in the liver due to the absence of Mel. The nocturnal UPR activation is related with night-time hepatic insulin resistance and increased gluconeogenesis in PINX rats. (Endocrinology 152: 1253-1263, 2011)

Na+-glucose transporter-2 messenger ribonucleic acid expression in kidney of diabetic rats correlates with glycemic levels: Involvement of hepatocyte nuclear factor-1 alpha expression and activity

Mutations in Na+-glucose transporters (SGLT)-2 and hepatocyte nuclear factor (HNF)-1 alpha genes have been related to renal glycosuria and maturity-onset diabetes of the young 3, respectively. However, the expression of these genes have not been investigated in type 1 and type 2 diabetes. Here in kidney of diabetic rats, we tested the hypotheses that SGLT2 mRNA expression is altered; HNF-1 alpha is involved in this regulation; and glycemic homeostasis is a related mechanism. The in vivo binding of HNF-1 alpha into the SGLT2 promoter region in renal cortex was confirmed by chromatin immunoprecipitation assay. SGLT2 and HNF-1 alpha mRNA expression (by Northern and RT-PCR analysis) and HNF-1 binding activity of nuclear proteins (by EMSA) were investigated in diabetic rats and treated or not with insulin or phlorizin (an inhibitor of SGLT2). Results showed that diabetes increases SGLT2 and HNF-1 alpha mRNA expression (similar to 50%) and binding of nuclear proteins to a HNF-1 consensus motif (similar to 100%). Six days of insulin or phlorizin treatment restores these parameters to nondiabetic-rat levels. Moreover, both treatments similarly reduced glycemia, despite the differences in plasma insulin and urinary glucose concentrations, highlighting the plasma glucose levels as involved in the observed modulations. This study shows that SGLT2 mRNA expression and HNF-1 alpha expression and activity correlate positively in kidney of diabetic rats. It also shows that diabetes-induced changes are reversed by lowering glycemia, independently of insulinemia. Our demonstration that HNF-1 alpha binds DNA that encodes SGLT2 supports the hypothesis that HNF-1 alpha, as a modulator of SGLT2 expression, may be involved in diabetic kidney disease.

Akt Expression is Diminished by Physical Inactivity in Early Stages of Development in Soleus Muscle of Rats

Metabolic Syndrome is a group of conditions related to obesity and physical inactivity. Little is known about the role of physical inactivity, in early stages of development, in the susceptibility to insulin resistant phenotype induced by high fat diet. Akt plays a key role in protein synthesis and glucose transport in skeletal muscle and has been regulated by muscle activity. The objective of present study was to determine the effect of early physical inactivity on muscle growth and susceptibility to acquire a diabetic phenotype and to assess its relationship with Akt expression. Forty Wistar male rats were distributed in two groups (standard group, Std) and movement restriction (RM). Between days 23 and 70 after birth, RM group was kept in small cages that did not allow them to perform relevant motor activity. From day 71 to 102 after birth, 10 rats of each group were fed with hyperlipidic diet (groups Std-DAG and RM-DAG). No differences were observed in total body weight although DAG increased epididymal fat pad weight. RM decreased significantly the soleus weight. Insulin-mediated glucose uptake was lower in RM-DAG group. Akt protein levels were lower in RM groups. Real time RT-PCR analysis showed that movement restriction decreased mRNA levels of AKT1 in soleus muscle, regardless of supplied diet. These findings suggest that early physical inactivity limits muscle`s growth and contributes to instauration of insulin resistant phenotype, which can be partly explained by dysregulation of Akt expression.

Molecular Mechanisms by Which Saturated Fatty Acids Modulate TNF-alpha Expression in Mouse Macrophage Lineage

Many macrophage functions are modulated by fatty acids (FAs), including cytokine release, such as tumor necrosis factor-alpha (TNF-alpha). TNF-alpha is of great interest due to its role in the inflammation process observed in several diseases such as rheumatoid arthritis, atherosclerosis, and obesity. However, the mechanisms by which FA effects occur have not been completely elucidated yet. In this study, we used a mouse monocyte lineage (J774 cells) to evaluate the effect of 50 and 100 mu M of saturated (palmitic and stearic acids), monounsaturated (oleic acid) and polyunsaturated (linoleic acid) FAs on TNF-alpha production. Alterations in gene expression, poly(A) tail length and activation of transcription factors were evaluated. Oleic and linoleic acids, usually known as neutral or pro-inflammatory FA, inhibited LPS-induced TNF-alpha secretion by the cells. Saturated FAs were potent inducers of TNF-alpha expression and secretion under basal and inflammatory conditions (in the presence of LPS). Although the effect of the saturated FA was similar, the mechanism involved in each case seem to be distinct, as palmitic acid increased EGR-1 and CREB binding activity and stearic acid increased mRNA poly(A) tail. These results may contribute to the understanding of the molecular mechanisms by which saturated FAs modulate the inflammatory response and may lead to design of associations of dietary and pharmacological strategies to counteract the pathological effects of TNF-alpha.

Transepithelial transport of glucose and mRNA of glucose transporters in the small intestine of rats with iron-deficiency anemia

Objective: To evaluate the transepithelial transport of sodium, glucose, potassium, and water and the mRNA level of the sodium-glucose cotransporter (SGLT1) and the facilitated sugar transporter (GLUT2) in the small intestine of iron-deficient rats. Methods: After 6 wk of receiving diets with low or normal iron content, rats (Wistar-EPM) were subjected to two experiments: 1) evaluation of the transepithelial transport of sodium, glucose, potassium, and water by an ""in vivo"" experimental model of intestinal perfusion and 2) determination of relative SGLT1 and GLUT2 mRNA levels in the proximal, intermediate, and distal portions of the small intestine by the northern blotting technique. Results: Hemoglobin and hepatic iron levels were statistically lower in the anemic rats. The mean transepithelial transports of sodium (-33.0 mu Eq . min(-1) . cm(-1)), glucose (426.0 mu M . min(-1) . cm(-1)), and water (0.4 mu L . min(-1) . cm(-1)) in the small intestine of the anemic rats were significantly lower than in the control group (349.1 mu Eq . min(-1) cm(-1), 842.6 mu M . min(-1) . cm(-1), and 4.3 mu l . min(-1) cm(-1), respectively, P < 0.05). The transepithelial transport of potassium was similar for both groups. The relative SGLT1 mRNA levels of the anemic rats in the intermediate (1.796 +/- 0.659 AU) and distal (1.901 +/- 0.766 AU) segments were significantly higher than the values for the control rats (intermediate 1.262 +/- 0.450 AU, distal 1.244 +/- 0.407 AU). No significant difference was observed for the relative SLGT1 mRNA levels in the proximal segment or for the GLUT2 mRNA levels in all segments. Conclusion: Iron deficiency decreases the absorption of glucose, sodium, and water and increases SGLT1 mRNA in the intermediate and distal segments of the small intestine of rats. (C) 2011 Elsevier Inc. All rights reserved.

UPR-mediated TRIB3 expression correlates with reduced AKT phosphorylation and inability of interleukin 6 to overcome palmitate-induced apoptosis in RINm5F cells

Unfolded protein response (UPR)-mediated pancreatic beta-cell death has been described as a common mechanism by which palmitate (PA) and pro-inflammatory cytokines contribute to the development of diabetes. There are evidences that interleukin 6 (IL6) has a protective action against beta-cell death induced by proinflammatory cytokines; the effects of IL6 on PA-induced apoptosis have not been investigated yet. In the present study, we have demonstrated that PA selectively disrupts IL6-induced RAC-alpha serine/threonine-protein kinase (AKT) activation without interfering with signal transducer and activator of transcription 3 phosphorylation in RINm5F cells. The inability of IL6 to activate AKT in the presence of PA correlated with an inefficient protection against PA-induced apoptosis. In contrast to PA, IL6 efficiently reduced apoptosis induced by pro-inflammatory cytokines. In addition, we have demonstrated that IL6 is unable to overcome PA-stimulated UPR, as assessed by activating transcription factor 4 (ATF4) andC/EBP homologous protein (CHOP) expression, X-box binding protein-1 gene mRNA splicing, and pancreatic eukaryotic initiation factor-2 alpha kinase phosphorylation, whereas no significant induction of UPR by pro-inflammatory cytokines was detected. This unconditional stimulation of UPR and apoptosis by PA was accompanied by the stimulation of CHOP and tribble3 (TRIB3) expression, irrespective of the presence of IL6. These findings suggest that IL6 is unable to protect pancreatic beta-cells from PA-induced apoptosis because it does not repress UPR activation. In this way, CHOP and ATF4 might mediate PA-induced TRIB3 expression and, by extension, the suppression of IL6 activation of pro-survival kinase AKT. Journal of Endocrinology (2010) 206, 183-193

Chronic low frequency/low volume resistance training reduces pro-inflammatory cytokine protein levels and TLR4 mRNA in rat skeletal muscle

Skeletal muscle is the source of pro- and anti-inflammatory cytokines, and recently, it has been recognized as an important source of interleukin 6 (IL-6), a cytokine that exerts inhibitory effects on several pro-inflammatory cytokines. Although dynamic chronic resistance training has been shown to produce the known ""repeated bout effect"", which abolishes the acute muscle damage, performing of high-intensity resistance training has been regarded highly advisable, at least from the hypertrophy perspective. On the other hand, a more therapeutic, ""non-damaging"" resistance training program, mainly composed of concentric forces, low frequency/low volume of training, and the same exercise, could theoretically benefit the muscle when the main issue is to avoid muscle inflammation (as in the treatment of several ""low-grade"" inflammatory diseases) because the acute effect of each resistance exercise session could be diminished/avoided, at the same time that the muscle is still being overloaded in a concentric manner. However, the benefits of such ""less demanding"" resistance training schedule on the muscle inflammatory profile have never been investigated. Therefore, we assessed the protein expression of IL-6, TNF-alpha, IL-10, IL-10/TNF-alpha ratio, and HSP70 levels and mRNA expression of SCF(beta-TrCP), IL-15, and TLR-4 in the skeletal muscle of rats submitted to resistance training. Briefly, animals were randomly assigned to either a control group (S, n = 8) or a resistance-trained group (T, n = 7). Trained rats were exercised over a duration of 12 weeks (two times per day, two times per week). Detection of IL-6, TNF-alpha, IL-10, and HSP70 protein expression was carried out by western blotting and SCF(beta-TrCP) (SKP Cullin F-Box Protein Ligases), a class of enzymes involved in the ubiquitination of protein substrates to proteasomal degradation, IL-15, and TLR-4 by RT-PCR. Our results show a decreased expression of TNF-alpha and TLR4 mRNA (40 and 60%, respectively; p < 0.05) in the plantar muscle from trained, when compared with control rats. In conclusion, exercise training induced decreased TNF-alpha and TLR-4 expressions, resulting in a modified IL-10/TNF-alpha ratio in the skeletal muscle. These data show that, in healthy rats, 12-week resistance training, predominantly composed of concentric stimuli and low frequency/low volume schedule, down regulates skeletal muscle production of cytokines involved in the onset, maintenance, and regulation of inXammation.

T3 acutely increases GH mRNA translation rate and GH secretion in hypothyroid rats

Cytoskeleton controls the stability of transcripts, by mechanisms that involve mRNAs and eEF1A attachment to it. Besides, it plays a key role in protein synthesis and secretion, which seems to be impaired in somatotrophs of hypothyroid rats, whose cytoskeleton is disarranged. This study investigated the: eEF1A and GH mRNA binding to cytoskeleton plus GH mRNA translation rate and GH secretion, in sham-operated and thyroidectomized rats treated with T3 or saline, and killed 30 min thereafter. Thyroidectomy reduced: (a) pituitary F-actin content, and eEF1A plus GH mRNA binding to it; (b) GH mRNA recruitment to polysome; and (c) liver IGF-1 mRNA expression, indicating that GH mRNA stability and translation rate, as well as GH secretion were impaired. T3 acutely reversed all these changes, which points toward a nongenomic action of T3 on cytoskeleton rearrangement, which might contribute to the increase on GH mRNA translation rate and GH secretion. (C) 2009 Elsevier Ireland Ltd. All rights reserved.

Regulation of glycolysis and expression of glucose metabolism-related genes by reactive oxygen species in contracting skeletal muscle cells

Contractile activity induces a marked increase in glycolytic activity and gene expression of enzymes and transporters involved in glucose metabolism in skeletal muscle. Muscle contraction also increases the production of reactive oxygen species (ROS). In this study, the effects of treatment with N-acetylcysteine (NAC), a potent antioxidant compound, on contraction-stimulated glycolysis were investigated in electrically stimulated primary rat skeletal muscle cells. The following parameters were measured: 2-[(3)H]deoxyglucose (2-DG) uptake; activities of hexokinase, phosphofructokinase (PFK), and glucose-6-phosphate dehydrogenase (G6PDH); lactate production; and expression of the glucose transporter 4 (GLUT4), hexokinase II (HKII), and PFK genes after one bout of electrical stimulation in primary rat myotubes. NAC treatment decreased ROS signal by 49% in resting muscle cells and abolished the muscle contraction-induced increase in ROS levels. In resting cells, NAC decreased mRNA and protein contents of GLUT4, mRNA content and activity of PFK, and lactate production. NAC treatment suppressed the contraction-mediated increase in 2-DG uptake; lactate production; hexokinase, PFK, and G6PDH activities; and gene expression of GLUT4. HKII, and PFK. Similar to muscle contraction, exogenous H(2)O(2) (500 nM) administration increased 2-DG uptake; lactate production; hexokinase, PFK, and G6PDH activities; and gene expression of GLUT4. HKII, and PFK. These findings support the proposition that ROS endogenously produced play an important role in the changes in glycolytic activity and gene expression of GLUT4, HKII, and PFK induced by contraction in skeletal muscle cells. (C) 2010 Elsevier Inc. All rights reserved.

The role of proliferator-activated receptor gamma coactivator-1 alpha in the fatty-acid -dependent transcriptional control of interleukin-10 in hepatic cells of rodents

Interleukin-10 (IL-10) is an endogenous factor that restrains hepatic insulin resistance in diet-induced steatosis Reducing IL-10 expression increases proinflammatory activity in the steatotic liver and worsens insulin resistance As the transcriptional coactivator proliferator-activated receptor gamma coactivator-1 alpha (PGC-1 alpha) plays a central role in dysfunctional hepatocytic activity in diet-induced steatosis, we hypothesized that at least part of the action of PGC-1 alpha could be mediated by reducing the transcription of the IL-10 gene Here, we used immunoblotting, real-time polymerase chain reaction, immunocytochemistry, and chromatin immunoprecipitation assay to investigate the role of PGC-1 alpha in the control of IL-10 expression in hepatic cells First, we show that, in the intact steatotic liver, the expressions of IL-10 and PGC-1 alpha are increased Inhibiting PGC-1 alpha expression by antisense oligonucleotide increases IL-10 expression and reduces the steatotic phenotype. In cultured hepatocytes, the treatment with saturated and unsaturated fatty acids increased IL-10 expression. This was accompanied by increased association of PGC-1 alpha with c-Maf and p50-nuclear factor (NF) kappa B, 2 transcription factors known to modulate IL-10 expression In addition, after fatty acid treatment. PGC-1 alpha, c-Maf, and p50-NF kappa B migrate from the cytosol to the nuclei of hepatocytes and bind to the IL-10 promoter region Inhibiting NF kappa B activation with salicylate reduces IL-10 expression and the association of PGC-1 alpha with p50-NF kappa B Thus, PGC-1 alpha emerges as a potential transcriptional regulator of the inflammatory phenomenon taking place in the steatotic liver (C) 2010 Elsevier Inc All rights reserved

Proinsulin C-peptide induces c-Jun N-terminal kinase 1 expression in LEII mouse lung capillary endothelial cells

To characterize the roles of C-peptide in vascular homeostatic processes, we examined the genes regulated by C-peptide in LEII mouse lung microvascular endothelial cells. Treatment of the cells with C-peptide increased the expression of c-Jun N-terminal kinase 1 (JNK1) mRNA dose-dependently, accompanied by an increase in JNK1 protein content. Prior treatment of the cells with PD98059, an ERK kinase inhibitor or SB203580, a p38MAPK inhibitor, abrogated the C-peptide-elicited JNK1 mRNA expression. These results indicate that C-peptide increases JNK1 protein levels, possibly through ERK- and p38MAPK-dependent activation of JNK. gene transcription.