Long-term heat acclimation training in mice: Similar metabolic and running performance adaptations despite a lower absolute intensity than training at temperate conditions.

This study investigated the impact of long-term heat acclimation (HA) training on mouse thermoregulation, metabolism, and running performance in temperate (T) and hot (H) environments. Male Swiss mice were divided into 1) Sedentary (SED) mice kept in T (22 °C; SED/T), 2) Endurance Trained mice (ET, 1 h/day, 5 days/week, 8 weeks, 60 % of maximum speed) in T (ET/T), 3) SED kept in H (32 °C; SED/H), and 4) ET in H (ET/H). All groups performed incremental load tests (ILT) in both environments before (pre-ET) and after four and eight weeks of ET. In the pre-ET period, H impaired (∼30 %) performance variables (maximum speed and external work) and increased (1.3 °C) maximum abdominal body temperature compared with T. In T, after four weeks, although ET/H exercised at a lower (∼30 %) absolute intensity than ET/T, performance variables and aerobic power (peak oxygen uptake, VO2peak) were similarly increased in both ET groups compared with SED/T. After eight weeks, the external work was higher in both ET groups compared with SED/T. Only ET/T significantly increased VO2peak (∼11 %) relative to its pre-ET period. In H, only after eight weeks, both ET groups improved (∼19 %) maximum speed and reduced (∼46 %) post-ILT blood lactate concentrations compared with their respective pre-ET values. Liver glycogen content increased (34 %) in both ET groups and SED/H compared with SED/T. Thus, ET/H was performed at a lower absolute intensity but promoted similar effects to ET/T on metabolism, aerobic power, and running performance. Our findings open perspectives for applying HA training as part of a training program or orthopedic and metabolic rehabilitation programs in injured or even obese animals, reducing mechanical load with equivalent or higher physiological demand.

Reduced sympathetic activity is associated with the development of pain and muscle atrophy in a female rat model of fibromyalgia.

Fibromyalgia (FM) is characterized by chronic widespread musculoskeletal pain accompanied by fatigue and muscle atrophy. Although its etiology is not known, studies have shown that FM patients exhibit altered function of the sympathetic nervous system (SNS), which regulates nociception and muscle plasticity. Nevertheless, the precise SNS-mediated mechanisms governing hyperalgesia and skeletal muscle atrophy in FM remain unclear. Thus, we employed two distinct FM-like pain models, involving intramuscular injections of acidic saline (pH 4.0) or carrageenan in prepubertal female rats, and evaluated the catecholamine content, adrenergic signaling and overall muscle proteolysis. Subsequently, we assessed the contribution of the SNS to the development of hyperalgesia and muscle atrophy in acidic saline-injected rats treated with clenbuterol (a selective ß2-adrenergic receptor agonist) and in animals maintained under baseline conditions and subjected to epinephrine depletion through adrenodemedullation (ADM). Seven days after inducing an FM-like model with acidic saline or carrageenan, we observed widespread mechanical hyperalgesia along with loss of strength and/or muscle mass. These changes were associated with reduced catecholamine content, suggesting a common underlying mechanism. Notably, treatment with a ß2-agonist alleviated hyperalgesia and prevented muscle atrophy in acidic saline-induced FM-like pain, while epinephrine depletion induced mechanical hyperalgesia and increased muscle proteolysis in animals under baseline conditions. Together, the results suggest that reduced sympathetic activity is involved in the development of pain and muscle atrophy in the murine model of FM analyzed.

Non-canonical Ca2+- Akt signaling pathway mediates the antiproteolytic effects induced by oxytocin receptor stimulation in skeletal muscle.

Although it has been previously demonstrated that oxytocin (OXT) receptor stimulation can control skeletal muscle mass in vivo, the intracellular mechanisms that mediate this effect are still poorly understood. Thus, rat oxidative skeletal muscles were isolated and incubated with OXT or WAY-267,464, a non-peptide selective OXT receptor (OXTR) agonist, in the presence or absence of atosiban (ATB), an OXTR antagonist, and overall proteolysis was evaluated. The results indicated that both OXT and WAY-267,464 suppressed muscle proteolysis, and this effect was blocked by the addition of ATB. Furthermore, the WAY-induced anti-catabolic action on protein metabolism did not involve the coupling between OXTR and Gαi since it was insensitive to pertussis toxin (PTX). The decrease in overall proteolysis induced by WAY was probably due to the inhibition of the autophagic/lysosomal system, as estimated by the decrease in LC3 (an autophagic/lysosomal marker), and was accompanied by an increase in the content of Ca2+-dependent protein kinase (PKC)-phosphorylated substrates, pSer473-Akt, and pSer256-FoxO1. Most of these effects were blocked by the inhibition of inositol triphosphate receptors (IP3R), which mediate Ca2+ release from the sarcoplasmic reticulum to the cytoplasm, and triciribine, an Akt inhibitor. Taken together, these findings indicate that the stimulation of OXTR directly induces skeletal muscle protein-sparing effects through a Gαq/IP3R/Ca2+-dependent pathway and crosstalk with Akt/FoxO1 signaling, which consequently decreases the expression of genes related to atrophy, such as LC3, as well as muscle proteolysis.

Glucocorticoids decrease thermogenic capacity and increase triacylglycerol synthesis by glycerokinase activation in the brown adipose tissue of rats.

Although it is well established that glucocorticoids inactivate thermogenesis and promote lipid accumulation in interscapular brown adipose tissue (IBAT), the underlying mechanisms remain unknown. We found that dexamethasone treatment (1 mg/kg) for 7 days in rats decreased the IBAT thermogenic activity, evidenced by its lower responsiveness to noradrenaline injection associated with reduced content of mitochondrial proteins, respiratory chain protein complexes, noradrenaline, and the ß3 -adrenergic receptor. In parallel, to understand better how dexamethasone increases IBAT lipid content, we also investigated the activity of the ATP citrate lyase (ACL), a key enzyme of de novo fatty acid synthesis, glucose-6-phosphate dehydrogenase (G6PD), a rate-limiting enzyme of the pentose phosphate pathway, and the three glycerol-3-P generating pathways: (1) glycolysis, estimated by 2-deoxyglucose uptake, (2) glyceroneogenesis, evaluated by phosphoenolpyruvate carboxykinase activity and pyruvate incorporation into triacylglycerol-glycerol, and (3) direct phosphorylation of glycerol, investigated by the content and activity of glycerokinase. Dexamethasone increased the mass and the lipid content of IBAT as well as plasma levels of glucose, insulin, non-esterified fatty acid, and glycerol. Furthermore, dexamethasone increased ACL and G6PD activities (79% and 48%, respectively). Despite promoting a decrease in the incorporation of U-[14 C]-glycerol into triacylglycerol (~54%), dexamethasone increased the content (~55%) and activity (~41%) of glycerokinase without affecting glucose uptake or glyceroneogenesis. Our data suggest that glucocorticoid administration reduces IBAT thermogenesis through sympathetic inactivation and stimulates glycerokinase activity and content, contributing to increased generation of glycerol-3-P, which is mostly used to esterify fatty acid and increase triacylglycerol content promoting IBAT whitening.

Nuclear PKR in retinal neurons in the early stage of diabetic retinopathy in streptozotocin­induced diabetic rats.

Retinal neuron apoptosis is a key component of diabetic retinopathy (DR), one of the most common complications of diabetes. Stress due to persistent hyperglycaemia and corresponding glucotoxicity represents one of the primary pathogenic mechanisms of diabetes and its complications. Apoptosis of retinal neurons serves a critical role in the pathogenesis of DR observed in patients with diabetes and streptozotocin (STZ)­induced diabetic rats. Retinal neuron apoptosis occurs one month after STZ injection, which is considered the early stage of DR. The molecular mechanism involved in the suppression of retinal neuron apoptosis during the early stage of DR remains unclear. RNA­dependent protein kinase (PKR) is a stress­sensitive pro­apoptotic kinase. Our previous study indicated that PKR­associated protein X, a stress­sensitive activator of PKR, is upregulated in the early stage of STZ­induced diabetes. In order to assess the role of PKR in DR prior to apoptosis of retinal neurons, immunofluorescence and western blotting were performed to investigate the cellular localization and expression of PKR in the retina in the early stage of STZ­induced diabetes in rats. PKR activity was indirectly assessed by expression levels of phosphorylated eukaryotic translation initiation factor 2α (p­eIF2­α) and the presence of apoptotic cells in the retina was investigated by TUNEL assay. The findings revealed that PKR was localized in the nucleus of retinal ganglion and inner nuclear layer cells from normal and diabetic rats. To the best of our knowledge, the present study is the first to demonstrate nuclear localization of PKR in retinal neurons. Immunofluorescence analysis demonstrated that PKR was expressed in the nuclei of retinal neurons at 3 and 6 days and its expression was decreased at 15 days after STZ treatment. In addition, p­eIF2­α expression and cellular localization followed the trend of PKR, suggesting that this pro­apoptotic kinase was active in the nuclei of retinal neurons. These findings are consistent with the hypothesis that nuclear translocation of PKR may be a mechanism to sequester active PKR, thus preventing upregulation of cytosolic signalling pathways that induce apoptosis in retinal neurons. Apoptotic cells were not detected in the retina in the early stage of DR. A model was proposed to explain the mechanism by which apoptosis of retinal neurons by PKR is suppressed in the early stage of DR. The possible role of mitochondrial RNA (mtRNA) and Alu RNA in this phenomenon is also discussed since it was demonstrated that the cellular stress due to prolonged hyperglycaemia induces the release of mtRNA and transcription of Alu RNA. Moreover, it mtRNA activates PKR, whereas Alu RNA inhibits the activation of this protein kinase.

The MicroRNA miR-696 is regulated by SNARK and reduces mitochondrial activity in mouse skeletal muscle through Pgc1α inhibition.

OBJECTIVE: MicroRNAs (miRNA) are known to regulate the expression of genes involved in several physiological processes including metabolism, mitochondrial biogenesis, proliferation, differentiation, and cell death. METHODS: Using "in silico" analyses, we identified 219 unique miRNAs that potentially bind to the 3'UTR region of a critical mitochondrial regulator, the peroxisome proliferator-activated receptor gamma coactivator (PGC) 1 alpha (Pgc1α). Of the 219 candidate miRNAs, miR-696 had one of the highest interactions at the 3'UTR of Pgc1α, suggesting that miR-696 may be involved in the regulation of Pgc1α. RESULTS: Consistent with this hypothesis, we found that miR-696 was highly expressed in the skeletal muscle of STZ-induced diabetic mice and chronic high-fat-fed mice. C2C12 muscle cells exposed to palmitic acid also exhibited a higher expression of miR-696. This increased expression corresponded with a reduced expression of oxidative metabolism genes and reduced mitochondrial respiration. Importantly, reducing miR-696 reversed decreases in mitochondrial activity in response to palmitic acid. Using C2C12 cells treated with the AMP-activated protein kinase (AMPK) activator AICAR and skeletal muscle from AMPKα2 dominant-negative (DN) mice, we found that the signaling mechanism regulating miR-696 did not involve AMPK. In contrast, overexpression of SNF1-AMPK-related kinase (SNARK) in C2C12 cells increased miR-696 transcription while knockdown of SNARK significantly decreased miR-696. Moreover, muscle-specific transgenic mice overexpressing SNARK exhibited a lower expression of Pgc1α, elevated levels of miR-696, and reduced amounts of spontaneous activity. CONCLUSIONS: Our findings demonstrate that metabolic stress increases miR-696 expression in skeletal muscle cells, which in turn inhibits Pgc1α, reducing mitochondrial function. SNARK plays a role in this process as a metabolic stress signaling molecule inducing the expression of miR-696.

Obesity-Induced Dysbiosis Exacerbates IFN-γ Production and Pulmonary Inflammation in the Mycobacterium tuberculosis Infection.

The microbiota of the gut-lung axis affects local and far-reaching immune responses and might also trigger chronic and inflammatory diseases. We hypothesized that gut dysbiosis induced by obesity, which coexists in countries with a high tuberculosis burden, aggravates the host susceptibility and the pulmonary damage tolerance. To assess our hypothesis, we used a model of high-fat diet (HFD)-induced obesity, followed by infection of C57BL/6 mice with Mycobacterium tuberculosis. We showed that obesity increased the susceptibility, the pulmonary inflammation and IFN-γ levels in M. tuberculosis-infected mice. During the comorbidity obesity and tuberculosis, there is an increase of Bacteroidetes and Firmicutes in the lungs, and an increase of Firmicutes and butyrate in the feces. Depletion of gut microbiota by antibiotic treatment in the obese infected mice reduced the frequencies of CD4+IFN-γ+IL-17- cells and IFN-γ levels in the lungs, associated with an increase of Lactobacillus. Our findings reinforce the role of the gut-lung axis in chronic infections and suggest that the gut microbiota modulation may be a potential host-directed therapy as an adjuvant to treat TB in the context of IFN-γ-mediated immunopathology.

Oxytocin induces anti-catabolic and anabolic effects on protein metabolism in the female rat oxidative skeletal muscle.

AIMS: Although it is well established that skeletal muscle contains oxytocin (OT) receptors and OT-knockout mice show premature development of sarcopenia, the role of OT in controlling skeletal muscle mass is still unknown. Therefore, the present work aimed to determine OT's effects on skeletal muscle protein metabolism. MAIN METHODS: Total proteolysis, proteolytic system activities and protein synthesis were assessed in isolated soleus muscle from prepubertal female rats. Through in vivo experiments, rats received 3-day OT treatment (3UI.kg-1.day-1, i.p.) or saline, and muscles were harvested for mass-gain assessment. KEY FINDINGS: In vitro OT receptor stimulation reduced total proteolysis, specifically through attenuation of the lysosomal and proteasomal proteolytic systems, and in parallel activated the Akt/FoxO1 signaling and suppressed atrogenes (e.g., MuRF-1 and atrogin-1) expression induced by motor denervation. On the other hand, the protein synthesis was not altered by in vitro treatment with the OT receptor-selective agonist. Although short-term OT treatment did not change the atrogene mRNA levels, the protein synthesis was stimulated, resulting in soleus mass gain, probably through an indirect effect. SIGNIFICANCE: Taken together, these data show for the first time that OT directly inhibits the proteolytic activities of the lysosomal and proteasomal systems in rat oxidative skeletal muscle by suppressing atrogene expression via stimulation of Akt/FoxO signaling. Moreover, the data obtained from in vivo experiments suggest OT's ability to control rat oxidative skeletal muscle mass.

Activation of adipose tissue glycerokinase contributes to increased white adipose tissue mass in mice fed a high-fat diet.

PURPOSE: Investigate the pathways of glycerol-3-P (G3P) generation for triacylglycerol (TAG) synthesis in retroperitoneal (RWAT) and epididymal (EWAT) white adipose tissues from high-fat diet (HFD)-fed mice. METHODS: Mice were fed for 8 weeks a HFD and glycolysis, glyceroneogenesis and direct phosphorylation of glycerol were evaluated, respectively, by 2-deoxyglucose uptake, phosphoenolpyruvate carboxykinase (PEPCK-C) activity and pyruvate incorporation into TAG-glycerol, and glycerokinase activity and glycerol incorporation into TAG-glycerol in both tissues. RESULTS: HFD increased body and adipose tissue mass and serum levels of glucose and insulin, which were accompanied by glucose intolerance. RWAT and EWAT from HFD-fed mice had increased rates of de novo fatty acid (FA) synthesis (52% and 255%, respectively). HFD increased lipoprotein lipase (LPL) activity and content in EWAT (107%), but decreased in RWAT (79%). HFD decreased the lipolytic response to norepinephrine (57%, RWAT and 25%, EWAT), ß3-adrenoceptor content (50%), which was accompanied by a decrease in phosphorylated-hormone-sensitive lipase (~80%) and phosphorylated-adipocyte triacylglycerol lipase (~60%) in both tissues. HFD decreased the in vitro rates of glucose uptake (3.5- and 6-fold), as well as in glyceride-glycerol synthesis from pyruvate (~3.5-fold) without changes in PEPCK-C activity and content in RWAT and EWAT, but increased glycerokinase activity(~3-fold) and content (90 and 40%) in both tissues. CONCLUSION: The data suggest that direct phosphorylation of glycerol by glycerokinase may be responsible for maintaining the supply of G3P for the existing rates of FA esterification and TAG synthesis in RWAT and EWAT from HFD-fed mice, contributing, along with a lower lipolytic response to norepinephrine, to higher adiposity.

Increase in liver cytosolic lipases activities and VLDL-TAG secretion rate do not prevent the non-alcoholic fatty liver disease in cafeteria diet-fed rats.

We have previously shown that the cafeteria diet increases body fat mass, plasma triacylglycerol (TAG) and insulin levels, glucose uptake by white and brown adipose tissues, as well as the sympathetic activity to both adipose tissues in Wistar rats. The metabolic pathways responsible for the development of non-alcoholic fatty liver disease (NAFLD) were examined in cafeteria diet-fed rats. After 3 weeks offering cafeteria diet, we evaluated: (i) activity of the sympathetic nervous system by norepinephrine turnover rates; (ii) de novo fatty acid synthesis in vivo using 3H2O; (iii) secretion of very low density lipoprotein (VLDL)-TAG secretion measuring serum TAG levels after administration of lipase lipoprotein inhibitor, (iv) liver cytosolic lipases activities and (v) liver mRNA expression of enzymes involved in lipids secretion and oxidation by RT-PCR. The cafeteria diet induced an increase in TAG (120%) and cholesterol (30%) liver contents. Cafeteria diet did not change the sympathetic nervous system activity to liver, but induced a marked increase in the lipogenesis (approximately four-fold) and significant increase in cytosolic lipases activities (46%) and VLDL-TAG secretion (22%) compared to control diet-fed rats. The cafeteria diet also increased the microsomal triglyceride transfer protein (30%) and carnitine palmitoyltransferase I (130%) mRNA expression but decreased the apolipoprotein B100 (26%) mRNA expression. Our findings demonstrate that the increase in the cytosolic lipases activities and VLDL-TAG secretion rates were not able to compensate for the increased lipogenesis rates induced by the cafeteria diet, resulting in NAFLD.

Differential regulation of glyceroneogenesis by glucocorticoids in epididymal and retroperitoneal white adipose tissue from rats.

PURPOSE: Investigate the glycerol-3-phosphate generation pathways in epididymal (EPI) and retroperitoneal (RETRO) adipose tissues from dexamethasone-treated rats. METHODS: Rats were treated with dexamethasone for 7 days. Glycerol-3-phosphate generation pathways via glycolysis, glyceroneogenesis and direct phosphorylation of glycerol were evaluated, respectively, by 2-deoxyglucose uptake, phosphoenolpyruvate carboxykinase (PEPCK-C) activity and pyruvate incorporation into triacylglycerol (TAG)-glycerol, and glycerokinase activity and glycerol incorporation into TAG-glycerol. RESULTS: Dexamethasone treatment markedly decreased the body weight, but increased the weight and lipid content of EPI and RETRO and plasma insulin, glucose, non-esterified fatty acid and TAG levels. EPI and RETRO from dexamethasone-treated rats showed increased rates of de novo fatty acid synthesis (80 and 100%) and basal lipolysis (20%). In EPI, dexamethasone decreased the 2-deoxyglucose uptake (50%), as well as glyceroneogenesis, evidenced by a decrease of PEPCK-C activity (39%) and TAG-glycerol synthesis from pyruvate (66%), but increased the glycerokinase activity (50%) and TAG-glycerol synthesis from glycerol (72%) in this tissue. In spite of a similar reduction in 2-deoxyglucose uptake in RETRO, dexamethasone treatment increased glyceroneogenesis, evidenced by PEPCK activity (96%), and TAG-glycerol synthesis from pyruvate (110%), accompanied by a decrease in glycerokinase activity (50%) and TAG-glycerol synthesis from glycerol (50%). Dexamethasone effects on RETRO were accompanied by a decrease in p-Akt content and by lower insulin effects on the rates of glycerol release in the presence of isoproterenol and on the rates of glucose uptake in isolated adipocytes. CONCLUSION: Our data demonstrated differential regulation of glyceroneogenesis and direct phosphorylation of glycerol by glucocorticoids in EPI and RETRO from rats.

Early dystrophin loss is coincident with the transition of compensated cardiac hypertrophy to heart failure.

Hypertension causes cardiac hypertrophy, one of the most important risk factors for heart failure (HF). Despite the importance of cardiac hypertrophy as a risk factor for the development of HF, not all hypertrophied hearts will ultimately fail. Alterations of cytoskeletal and sarcolemma-associated proteins are considered markers cardiac remodeling during HF. Dystrophin provides mechanical stability to the plasma membrane through its interactions with the actin cytoskeleton and, indirectly, to extracellular matrix proteins. This study was undertaken to evaluate dystrophin and calpain-1 in the transition from compensated cardiac hypertrophy to HF. Wistar rats were subjected to abdominal aorta constriction and killed at 30, 60 and 90 days post surgery (dps). Cardiac function and blood pressure were evaluated. The hearts were collected and Western blotting and immunofluorescence performed for dystrophin, calpain-1, alpha-fodrin and calpastatin. Statistical analyses were performed and considered significant when p<0.05. After 90 dps, 70% of the animals showed hypertrophic hearts (HH) and 30% hypertrophic+dilated hearts (HD). Systolic and diastolic functions were preserved at 30 and 60 dps, however, decreased in the HD group. Blood pressure, cardiomyocyte diameter and collagen content were increased at all time points. Dystrophin expression was lightly increased at 30 and 60 dps and HH group. HD group showed decreased expression of dystrophin and calpastatin and increased expression of calpain-1 and alpha-fodrin fragments. The first signals of dystrophin reduction were observed as early as 60 dps. In conclusion, some hearts present a distinct molecular pattern at an early stage of the disease; this pattern could provide an opportunity to identify these failure-prone hearts during the development of the cardiac disease. We showed that decreased expression of dystrophin and increased expression of calpains are coincident and could work as possible therapeutic targets to prevent heart failure as a consequence of cardiac hypertrophy.

Morphological and molecular aspects of immobilization-induced muscle atrophy in rats at different stages of postnatal development: the role of autophagy.

Muscle loss occurs following injury and immobilization in adulthood and childhood, which impairs the rehabilitation process; however, far fewer studies have been conducted analyzing atrophic response in infants. This work investigated first the morphological and molecular mechanisms involved in immobilization-induced atrophy in soleus muscles from rats at different stages of postnatal development [i.e., weanling (WR) and adult (AR) rats] and, second, the role of autophagy in regulating muscle plasticity during immobilization. Hindlimb immobilization for 10 days reduced muscle mass and fiber cross-sectional area, with more pronounced atrophy in WR, and induced slow-to-fast fiber switching. These effects were accompanied by a decrease in markers of protein synthesis and an increase in autophagy. The ubiquitin (Ub)-ligase MuRF1 and the ubiquitinated proteins were upregulated by immobilization in AR while the autolyzed form of µ-calpain was increased in WR. To further explore the role of autophagy in muscle abnormalities, AR were concomitantly immobilized and treated with colchicine, which blocks autophagosome-lysosome fusion. Colchicine-treated immobilized muscles had exacerbated atrophy and presented degenerative features. Despite Igf1/Akt signaling was downregulated in immobilized muscles from both age groups, Foxo1 and 4 phosphorylation was increased in WR. In the same group of animals, Foxo1 acetylation and Foxo1 and 4 content was increased and decreased, respectively. Our data show that muscle disorders induced by 10-day-immobilization occur in both age-dependent and -independent manners, an understanding that may optimize treatment outcomes in infants. We also provide further evidence that the strong inhibition of autophagy may be ineffective for treating muscle atrophy.

Higher insulin sensitivity in EDL muscle of rats fed a low-protein, high-carbohydrate diet inhibits the caspase-3 and ubiquitin-proteasome proteolytic systems but does not increase protein synthesis.

Compared with the extensor digitorum longus (EDL) muscle of control rats (C), the EDL muscle of rats fed a low-protein, high-carbohydrate diet (LPHC) showed a 36% reduction in mass. Muscle mass is determined by the balance between protein synthesis and proteolysis; thus, the aim of this work was to evaluate the components involved in these processes. Compared with the muscle from C rats, the EDL muscle from LPHC diet-fed rats showed a reduction (34%) in the in vitro basal protein synthesis and a 22% reduction in the in vitro basal proteolysis suggesting that the reduction in the mass can be associated with a change in the rate of the two processes. Soon after euthanasia, in the EDL muscles of the rats fed the LPHC diet for 15days, the activity of caspase-3 and that of components of the ubiquitin-proteasome system (atrogin-1 content and chymotrypsin-like activity) were decreased. The phosphorylation of p70(S6K) and 4E-BP1, proteins involved in protein synthesis, was also decreased. We observed an increase in the insulin-stimulated protein content of p-Akt. Thus, the higher insulin sensitivity in the EDL muscle of LPHC rats seemed to contribute to the lower proteolysis in LPHC rats. However, even with the higher insulin sensitivity, the reduction in p-E4-BP1 and p70(S6K) indicates a reduction in protein synthesis, showing that factors other than insulin can have a greater effect on the control of protein synthesis.

A low-protein, high-carbohydrate diet increases de novo fatty acid synthesis from glycerol and glycerokinase content in the liver of growing rats.

We had previously shown that adipose tissue increased in rats fed a low-protein, high-carbohydrate (LPHC) diet (6% protein, 74% carbohydrate) without a simultaneous increase in the de novo fatty acids (FA) synthesis. In addition, impairment in insulin signaling in adipose tissues was observed in these rats. For this study, we hypothesized that the insulin signaling pathway is preserved in the livers from these rats, which contributes to an increase in liver lipogenesis and, consequently, an increase in the weight of the adipose tissue. We also hypothesized that glycerol from triacylglycerol is an important substrate for FA synthesis. Our results showed that administration of the LPHC diet induced an increase in the in vivo rate of total FA synthesis (150%) as well as FA synthesis from glucose (270%) in the liver. There were also increased rates of [U-¹4C]glycerol incorporation into glyceride-FA (15-fold), accompanied by increased glycerokinase content (30%) compared with livers of rats fed the control diet. The LPHC diet did not change the glycerol-3-phosphate generation from either glucose or glyceroneogenesis. There was an increase in the insulin sensitivity in liver from LPHC-fed rats, as evidenced by increases in IR(ß) (35%) levels and serine/threonine protein kinase (AKT) levels (75%), and basal (95%) and insulin-stimulated AKT phosphorylation (105%) levels. The LPHC diet also induced an increase in the liver sterol regulatory element-binding protein-1c content (50%). In summary, these data confirmed the hypothesis that lipogenesis and insulin signaling are increased in the livers of LPHC-fed rats and that glycerol is important not only for FA esterification but also for FA synthesis.

The sympathetic nervous system regulates the three glycerol-3P generation pathways in white adipose tissue of fasted, diabetic and high-protein diet-fed rats.

The aim of the present study was to investigate the participation of the sympathetic nervous system (SNS) in the control of glycerol-3-P (G3P) generating pathways in white adipose tissue (WAT) of rats in three situations in which the plasma insulin levels are low. WAT from 48 h fasted animals, 3 day-streptozotocin diabetic animals and high-protein, carbohydrate-free (HP) diet-fed rats was surgical denervated and the G3P generation pathways were evaluated. Food deprivation, diabetes and the HP diet provoke a marked decrease in the rate of glucose uptake and glycerokinase (GyK) activity, but a significant increase in the glyceroneogenesis, estimated by the phosphoenolpyruvate carboxykinase (PEPCK) activity and the incorporation of 1-[(14)C]-pyruvate into glycerol-TAG. The denervation provokes a reduction (~70%) in the NE content of WAT in fasted, diabetic and HP diet-fed rats. The denervation induced an increase in WAT glucose uptake of fed, fasted, diabetic and HP diet-fed rats (40%, 60%, 3.2 fold and 35%, respectively). TAG-glycerol synthesis from pyruvate was reduced by denervation in adipocytes of fed (58%) and fasted (36%), saline-treated (58%) and diabetic (23%), and HP diet-fed rats (11%). In these same groups the denervation reduced the PEPCK mRNA expression (75%-95%) and the PEPCK activity (35%-60%). The denervation caused a ~35% decrease in GyK activity of control rats and a further ~35% reduction in the already low enzyme activity of fasted, diabetic and HP diet-fed rats. These data suggest that the SNS plays an important role in modulating G3P generating pathways in WAT, in situations where insulin levels are low.

Low protein diet changes the energetic balance and sympathetic activity in brown adipose tissue of growing rats.

OBJECTIVE: The aim of this study was to assess the effects of protein restriction in growing rats. METHODS: Rats (approximate weight, 100g) were maintained with low-protein (LP; 6%) or normoproteic (control; 17%) diets, and at the end of the 15th day, hormonal and biochemistry parameters and energetic balance were evaluated. Data were analyzed using Student's t test (with statistical significance set at P < or = .05). RESULTS: LP animals were hyperphagic and showed increased energetic gain (24%) and energy expenditure (EE) compared with controls. The increase in EE was followed by increased sympathetic activity in brown adipose tissue, evidenced by increased norepinephrine turnover, suggesting increased thermogenesis. In spite of hyperphagia, protein ingestion in LP animals was lower than that of controls (P<0.01). The LP diet impaired body growth and caused deep alterations in body chemical composition, with an increase in carcass lipid content (64%) and reductions of protein and water. In LP animals, postprandial glycemia was unchanged, and insulinemia was lower than in controls (P < or = .01). Reduction in fasting glycemia without changes in insulinemia also was detected (P < .01), suggesting increased insulin sensitivity. The LP diet caused a 100% increase in serum leptin (P < .01). CONCLUSIONS: Protein restriction led to an increase in EE, with probable activation of thermogenesis in brown adipose tissue, evidenced by an increase in catecholamines levels. Despite the higher EE, energetic gain and lipids increased. The high level of leptin associated with hyperphagia led to the supposition that these animals are leptin resistant, and the increase in insulin sensitivity, suggested by the relation between insulin and glycemia in fasting and fed animals, might contribute to lipid accumulation.

Glyceroneogenesis and the supply of glycerol-3-phosphate for glyceride-glycerol synthesis in liver slices of fasted and diabetic rats.

The pathways of glycerol-3-phosphate (G3P) generation for glyceride synthesis were examined in precision-cut liver slices of fasted and diabetic rats. The incorporation of 5 mM [U-(14)C]glucose into glyceride-glycerol, used to evaluate G3P generation via glycolysis, was reduced by approximately 26-36% in liver slices of fasted and diabetic rats. The glycolytic flux was reduced by approximately 60% in both groups. The incorporation of 1.0 mM [2-(14)C]pyruvate into glyceride-glycerol (glyceroneogenesis) increased approximately 50% and approximately 36% in slices of fasted and diabetic rats, respectively, which also showed a two-fold increase in the activity phosphoenolpyruvate carboxykinase. The increased incorporation of 1.0 mM [2-(14)C]pyruvate into glyceride-glycerol by slices of fasted rats was not affected by the addition of 5 mM glucose to the incubation medium. The activity of glycerokinase and the incorporation of 1 mM [U-(14)C]glycerol into glyceride-glycerol, evaluators of G3P formation by direct glycerol phosphorylation, did not differ significantly from controls in slices of the two experimental groups. Rates of incorporation of 1 mM [2-(14)C]pyruvate and [U-(14)C]glycerol into glucose of incubation medium (gluconeogenesis) were approximately 140 and approximately 20% higher in fasted and diabetic slices than in control slices. It could be estimated that glyceroneogenesis by liver slices of fasted rats contributed with approximately 20% of G3P generated for glyceride-glycerol synthesis, the glycolytic pathway with approximately 5%, and direct phosphorylation of glycerol by glycerokinase with approximately 75%. Pyruvate contributed with 54% and glycerol with 46% of gluconeogenesis. The present data indicate that glyceroneogenesis has a significant participation in the generation of G3P needed for the increased glyceride-glycerol synthesis in liver during fasting and diabetes.

Effect of cyclosporine a on glucose interstitial concentration in renal cortex and medulla from rats.

AIM: To standardize microdialysis in rat kidneys and address cyclosporine A (CsA) effects on renal cortex and medulla interstitial glucose. METHODS: Munich-Wistar rats were treated with vehicle or CsA (15 mg/kg/day) for 3 weeks. Glucose was assessed by spectrophotometry in dialysate samples from cortex, medulla and arterial plasma. Plasma insulin was measured by radioimmunoassay. Renal blood flow (RBF) was measured by Doppler ultrasound. Creatinine and urea were measured by spectrophotometry. RESULTS: CsA significantly increased the plasma levels of urea and creatinine (1.5 +/- 0.20 vs. 0.73 +/- 0.03 mg/dl in controls, p < 0.05). Medullary glucose in control was 44% lower than arterial glucose (56 +/- 6 vs. 101 +/- 8 mg/dl, p < 0.05). At the same time, CsA increased arterial (163 +/- 35 vs. 101 +/- 8 mg/dl in controls, p < 0.05) and medullary interstitial glucose (100 +/- 18 vs. 56 +/- 6 mg/dl in controls, p < 0.05), but did not affect cortical glucose (114 +/- 21 vs. 90 +/- 11 mg/dl in controls). These changes occurred in the presence of a decreased plasma insulin level (2.7 +/- 0.2 vs. 9.3 +/- 0.4 microU/ml in controls, p < 0.05). The increment in medullary glucose in CsA group occurred despite a reduction in RBF (4.6 +/- 0.8 vs. 6.5 +/- 1.0 ml/min/kidney in controls, p < 0.05). CONCLUSIONS: Microdialysis was an adequate tool to investigate in vivo regulation of renal glucose metabolism. Renal glucose uptake was dependent on medullary cells and CsA treatment induced diabetogenic effects on renal medulla in situ.

Glyconeogenic pathway in isolated skeletal muscles of rats.

Although the conversion of lactate to glycogen (glyconeogenesis) in muscle was demonstrated a long time ago, the biochemical reactions responsible for this process are still a controversial matter. In the present study, advantage was taken from the specific inhibition induced by phenylalanine on muscle pyruvate kinase (PK) to investigate the role of reverse PK activity in muscle glyconeogenesis. Addition of phenylalanine to the incubation medium of a preparation of isolated, intact skeletal muscles that maintain metabolic activity for several hours reduced by 50% the rate of incorporation of [14C]lactate or [14C]bicarbonate into muscle glycogen. Muscle extracts presented high levels of maximal activity of PK in the reverse direction, which was completely blocked in the presence of phenylalanine. In contrast, mercaptopicolinic acid, an inhibitor of phosphoenolpyruvate carboxykinase (PEPCK), did not affect the incorporation of 14C from either lactate or bicarbonate into muscle glycogen. Maximal PEPCK activity was much lower in muscle extracts than in gluconeogenic or glyceroneogenic tissues and was suppressed in the presence of mercaptopicolinic acid. The data suggest that a reversal of the metabolic flux through the reaction catalyzed by PK contributes to the accumulation of lactate-derived glycogen that occurs in skeletal muscle under certain physiological conditions.