Maria Tereza Nunes

Changes in sodium or glucose filtration rate modulate expression of glucose transporters in renal proximal tubular cells of rat.

Abstract. Renal glucose reabsorption is mediated by luminal sodium-glucose cotransporters (SGLTs) and basolateral facilitative glucose transporters (GLUTs). The modulators of these transporters are not known, and their substrates glucose and Na+ are potential candidates. In this study we examined the role of glucose and Na+ filtration rate on gene expression of glucose transporters in renal proximal tubule. SGLT1, SGLT2, GLUT1 and GLUT2 mRNAs were assessed by Northern blotting; and GLUT1 and GLUT2 proteins were assessed by Western blotting. Renal cortex and medulla samples from control rats (C), diabetic rats (D) with glycosuria, and insulin-resistant 15-month old rats (I) without glycosuria; and from normal (NS), low (LS), and high (HS) Na+-diet fed rats were studied. Compared to C and I rats, D rats increased (P < 0.05) gene expression of SGLT2 by ∼36%, SGLT1 by ∼20%, and GLUT2 by ∼100%, and reduced (P < 0.05) gene expression of GLUT1 by more than 50%. Compared to NS rats, HS rats increased (P < 0.05) SGLT2, GLUT2, and GLUT1 expression by ∼100%, with no change in SGLT1 mRNA expression, and LS rats increased (P < 0.05) GLUT1 gene expression by ∼150%, with no changes in other transporters. In summary, the results showed that changes in glucose or Na+ filtrated rate modulate the glucose transporters gene expression in epithelial cells of the renal proximal tubule.

Comparative toxicity of fatty acids on a macrophage cell line (J774)

In the present study, the cytotoxicity of palmitic, stearic, oleic, linoleic, arachidonic, docosahexaenoic and eicosapentaenoic acids on a macrophage cell line (J774) was investigated. The induction of toxicity was investigated by changes in cell size, granularity, membrane integrity, DNA fragmentation and phosphatidylserine externalization by using flow cytometry. Fluorescence microscopy was used to determine the type of cell death (Acridine Orange/ethidium bromide assay). The possible mechanisms involved were examined by measuring mitochondrial depolarization, lipid accumulation and PPARgamma (peroxisome-proliferator-activated receptor gamma) activation. The results demonstrate that fatty acids induce apoptosis and necrosis of J774 cells. At high concentrations, fatty acids cause macrophage death mainly by necrosis. The cytotoxicity of the fatty acids was not strictly related to the number of double bonds in the molecules: palmitic acid>docosahexaenoic acid>stearic acid=eicosapentaenoic acid=arachidonic acid>oleic acid>linoleic acid. The induction of cell death did not involve PPARgamma activation. The mechanisms of fatty acids to induce cell death involved changes in mitochondrial transmembrane potential and intracellular neutral lipid accumulation. Fatty acids poorly incorporated into triacylglycerol had the highest toxicity.

NF-κB, MEF2A, MEF2D and HIF1-a involvement on insulin- and contraction-induced regulation of GLUT4 gene expression in soleus muscle

The GLUT4 gene transcriptional activity has a profound impact on the insulin-mediated glucose disposal and it is, therefore, important to understand the mechanisms underlying it. Insulin and exercise modulate GLUT4 expression in vivo, but the net control and involved mechanisms of each one have not been established yet. This paper sought to discriminate, in soleus muscle, the effects of insulin and muscle contraction on GLUT4 gene expression, and the involvement of transcriptional factors: myocite enhancer factor 2 (MEF2 A/C/D), hypoxia inducible factor 1-a (HIF1-a) and nuclear factor-kappa B (NF-kappaB). The GLUT4 mRNA was reduced by fasting (40%), and increased by in vitro incubation with insulin (25%) or insulin plus glucose (40%), which was accompanied by opposite regulations of NF-kappaB mRNA. Differently, in vitro, muscle contraction led to a rapid increase (35-80%) in GLUT4, MEF2A, MEF2D and HIF1-a mRNAs. Additionally, electrophoretic mobility shift assay confirmed changes in the binding activity of nuclear proteins to consensus NF-kappaB, GLUT4-Ebox and GLUT4-AT-rich element probes, parallel to the mRNA changes of their respective transcriptional factors NF-kappaB, HIF1-a and MEF2s. Concluding, insulin- and contraction-induced regulation of GLUT4 expression involves distinct transcriptional factors.

GLUT4 protein is differently modulated during development of obesity in monosodium glutamate-treated mice.

The aim of the present study was to investigate the GLUT4 protein expression during the development of obesity in monosodium glutamate- (MSG) treated mice. Control (C) and neonatally MSG-treated 2-month-old (2-mo), 4-month-old (4-mo) and 7-month-old (7-mo) mice were analyzed. Anthropometric data, basal glycemia and insulinemia were measured; and the GLUT4 protein was assessed by Western blotting in white adipose tissue (WAT), skeletal muscle gastrocnemius (SM) and heart (H). Compared to age-matched C mice, the 2-mo and 4-mo MSG mice were already obese, but metabolically they showed increased or preserved whole-body insulin sensitivity, respectively. At these ages they showed unchanged total GLUT4 content in SM and H. However, in plasma membrane fraction from WAT, the MSG showed increased GLUT4 content at both 2- (by 60%) and 4-month (by 45%) of age. When the GLUT4 protein was expressed by unit of adipocyte surface area the protein amount was increased by 36 and 220% in 2-mo and 4-mo MSG mice, respectively. At 7 months of age, obesity was fully established in MSG mice, showing a strongly insulin resistant condition. Additionally, in the 7-mo MSG-mice the GLUT4 protein was reduced in SM (by 40%), H (by 28%), PM and M fractions of WAT (by approximately 70%), and PM expressed by unit of adipocyte surface area (by 92%). The data demonstrate that early, during the accelerated development of obesity in MSG-treated mice, the GLUT4 content was increased in WAT, and that may play a key role in the development of obesity. Later on, when obesity is fully established, the GLUT4 protein was reduced in SM, heart and WAT, and that may be involved in the insulin resistance present in this condition.

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

OBJECTIVE Beta-hydroxy-beta-methylbutyrate (HMβ) is a metabolite of leucine widely used for improving sports performance. Although HMβ 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 HMβ treatment affects the activity of GH/IGF-I axis and skeletal muscle IGF-I and myostatin mRNA expression. DESIGN Rats were treated with HMβ (320mg/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 HMβ 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 HMβ-treated rats although normoglycemic, exhibited hyperinsulinemia. CONCLUSIONS The data presented herein extend the body of evidence on the potential role of HMβ-treatment in stimulating GH/IGF-I axis activity. In spite of this effect, HMβ 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.

Local Injections of Adipose-Derived Mesenchymal Stem Cells Modulate Inflammation and Increase Angiogenesis Ameliorating the Dystrophic Phenotype in Dystrophin-Deficient Skeletal Muscle

The effects of adipose-derived mesenchymal stem cells (ADMSC) transplantation on degeneration, regeneration and skeletal muscle function were investigated in dystrophin-deficient mice (24-week-old). ADMSC transplantation improved muscle strength and, resistance to fatigue. An increase in fiber cross-sectional area and in the number of fibers with centralized nuclei and augment of myogenin content were observed. In ADMSC-treated muscles a decrease in muscle content of TNF-α, IL-6 and oxidative stress measured by Amplex® reagent were observed. The level of TGF-β1 was lowered whereas that of VEGF, IL-10 and IL-4 were increased by ADMSC treatment. An increase in markers of macrophage M1 (CD11 and F4-80) and a decrease in T lymphocyte marker (CD3) and arginase-1 were also observed in ADMSCs-treated dystrophic muscle. No change was observed in iNOS expression. Increased phosphorylation of Akt, p70S6k and 4E-BP1 was found in dystrophic muscles treated with ADMSC. These results suggest that ADMSC transplantation modulates inflammation and improves muscle tissue regeneration, ameliorating the dystrophic phenotype in dystrophin-deficient mice.

The Acute Inhibitory Effect of Iodide Excess on Sodium/Iodide Symporter Expression and Activity Involves the PI3K/Akt Signaling Pathway

Iodide (I(-)) is an irreplaceable constituent of thyroid hormones and an important regulator of thyroid function, because high concentrations of I(-) down-regulate sodium/iodide symporter (NIS) expression and function. In thyrocytes, activation of phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) cascade also inhibits NIS expression and function. Because I(-) excess and PI3K/Akt signaling pathway induce similar inhibitory effects on NIS expression, we aimed to study whether the PI3K/Akt cascade mediates the acute and rapid inhibitory effect of I(-) excess on NIS expression/activity. Here, we reported that the treatment of PCCl3 cells with I(-) excess increased Akt phosphorylation under normal or TSH/insulin-starving conditions. I(-) stimulated Akt phosphorylation in a PI3K-dependent manner, because the use of PI3K inhibitors (wortmannin or 2-(4-Morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) abrogated the induction of I(-) effect. Moreover, I(-) inhibitory effect on NIS expression and function were abolished when the cells were previously treated with specific inhibitors of PI3K or Akt (Akt1/2 kinase inhibitor). Importantly, we also found that the effect of I(-) on NIS expression involved the generation of reactive oxygen species (ROS). Using the fluorogenic probes dihydroethidium and mitochondrial superoxide indicator (MitoSOX Red), we observed that I(-) excess increased ROS production in thyrocytes and determined that mitochondria were the source of anion superoxide. Furthermore, the ROS scavengers N-acetyl cysteine and 2-phenyl-1,2-benzisoselenazol-3-(2H)-one blocked the effect of I(-) on Akt phosphorylation. Overall, our data demonstrated the involvement of the PI3K/Akt signaling pathway as a novel mediator of the I(-)-induced thyroid autoregulation, linking the role of thyroid oxidative state to the Wolff-Chaikoff effect.

Posttranscriptional regulation of sodium-iodide symporter mRNA expression in the rat thyroid gland by acute iodide administration

Iodide is an important regulator of thyroid activity. Its excess elicits the Wolff-Chaikoff effect, characterized by an acute suppression of thyroid hormone synthesis, which has been ascribed to serum TSH reduction or TGF-beta increase and production of iodolipids in the thyroid. These alterations take hours/days to occur, contrasting with the promptness of Wolff-Chaikoff effect. We investigated whether acute iodide administration could trigger events that precede those changes, such as reduction of sodium-iodide symporter (NIS) mRNA abundance and adenylation, and if perchlorate treatment could counteract them. Rats subjected or not to methylmercaptoimidazole treatment (0.03%) received NaI (2,000 microg/0.5 ml saline) or saline intraperitoneally and were killed 30 min up to 24 h later. Another set of animals was treated with iodide and perchlorate, in equimolar doses. NIS mRNA content was evaluated by Northern blotting and real-time PCR, and NIS mRNA poly(A) tail length by rapid amplification of cDNA ends-poly(A) test (RACE-PAT). We observed that NIS mRNA abundance and poly(A) tail length were significantly reduced in all periods of iodide treatment. Perchlorate reversed these effects, indicating that iodide was the agent that triggered the modifications observed. Since the poly(A) tail length of mRNAs is directly associated with their stability and translation efficiency, we can assume that the rapid decay of NIS mRNA abundance observed was due to a reduction of its stability, a condition in which its translation could be impaired. Our data show for the first time that iodide regulates NIS mRNA expression at posttranscriptional level, providing a new mechanism by which iodide exerts its autoregulatory effect on thyroid.

Acute and short-term insulin-induced molecular adaptations of GLUT2 gene expression in the renal cortex of diabetic rats.

Increased GLUT2 gene expression in the renal proximal tubule of diabetic rats is an adaptive condition, which may be important in the diabetic nephropathy development. We investigated the effects of insulin treatment upon the renal GLUT2 overexpression of diabetic rats. Acute treatment, surprisingly, induced a rapid further increase in GLUT2 mRNA content. Twelve hours after insulin injection, GLUT2 mRNA was twice the value of saline-injected rats (P<0.001), when GLUT2 protein remained unchanged. In response to short-term treatment, both GLUT2 mRNA and protein were increased in 1-day treated rats (P<0.05 versus saline-injected), decreasing after that, and reaching, within 6 days, values close to those of non-diabetic rats. Concluding, insulin treatment induced: initially, an additional upregulation of GLUT2 gene expression, involving posttranscriptional modulation; thereafter, downregulation of GLUT2 expression, which returns to non-diabetic levels. The former may be related to increased insulin concentration, the latter may be due to glycemic control.

Thyroid Hormone Stimulates Myoglobin Expression in Soleus and Extensorum Digitalis Longus Muscles of Rats: Concomitant Alterations in the Activities of Krebs Cycle Oxidative Enzymes

Myoglobin (Mb) gene expression, Citrate Synthase (CS) and Succinate Dehydrogenase (SDH) activities of Soleus (S) and Extensorum Digitalis Longus (EDL) muscles were studied in intact, thyroidectomized and T3-treated (25 microg/100g, BW, ip, 15 days) rats. The fiber type composition of S muscle was also evaluated and used as control of the T3-induced effects. In the S muscle, the T3 treatment increased the Mb mRNA and protein expression, as well as the CS and SDH activity. These changes occurred parallel to the expected increase in type II (fast) and decrease in type I (slow)-fibers in S muscle. In the hypothyroid state, the Mb mRNA was decreased, while the Mb expression and CS activity tended to decrease. In contrast the SDH activity was increased, probably due to the enhanced motor activity that occurs as a short-term response to the hypothermia induced by hypothyroidism. In the EDL, the alterations were milder than those in S muscle in both thyroid states. These findings show that Mb gene expression is induced by T3. This is concomitant with the enhancement of Krebs Cycle enzyme activities and provides additional evidence that thyroid hormone increases the aerobic potential of skeletal muscles, as well as the speed of muscle contraction.