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)

Intensive insulin treatment induces insulin resistance in diabetic rats by impairing glucose metabolism-related mechanisms in muscle and liver

Insulin replacement is the only effective therapy to manage hyperglycemia in type 1 diabetes mellitus (T1DM). Nevertheless, intensive insulin therapy has inadvertently led to insulin resistance. This study investigates mechanisms involved in the insulin resistance induced by hyperinsulinization. Wistar rats were rendered diabetic by alloxan injection, and 2 weeks later received saline or different doses of neutral protamine Hagedorn insulin (1.5, 3, 6, and 9 U/day) over 7 days. Insulinopenic-untreated rats and 6U- and 9U-treated rats developed insulin resistance, whereas 3U-treated rats revealed the highest grade of insulin sensitivity, but did not achieve good glycemic control as 6U- and 9U-treated rats did. This insulin sensitivity profile was in agreement with glucose transporter 4 expression and translocation in skeletal muscle, and insulin signaling, phosphoenolpyruvate carboxykinase/glucose-6-phosphatase expression and glycogen storage in the liver. Under the expectation that insulin resistance develops in hyperinsulinized diabetic patients, we believe insulin sensitizer approaches should be considered in treating T1DM. Journal of Endocrinology (2011) 211, 55-64

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

Nutrient Selection in the Absence of Taste Receptor Signaling

When allowed to choose between different macronutrients, most animals display a strong attraction toward carbohydrates compared with proteins. It remains uncertain, however, whether this food selection pattern depends primarily on the sensory properties intrinsic to each nutrient or, alternatively, metabolic signals can act independently of the hedonic value of sweetness to stimulate elevated sugar intake. Here we show that Trpm5(-/-) mice, which lack the cellular mechanisms required for sweet and several forms of L-amino acid taste transduction, develop a robust preference for D-glucose compared with isocaloric L-serine independently of the perception of sweetness. Moreover, a close relationship was found between glucose oxidation and taste-independent nutrient intake levels, with animals increasing intake as a function of glucose oxidation rates. Furthermore, microdialysis measurements revealed nutrient-specific dopaminergic responses in accumbens and dorsal striatum during intragastric infusions of glucose or serine. Specifically, intragastric infusions of glucose induced significantly higher levels of dopamine release compared with isocaloric serine in both ventral and dorsal striatum. Intragastric stimulation of dopamine release seemed to depend on glucose utilization, because administration of an anti-metabolic glucose analog resulted in lower dopamine levels in striatum, an effect that was reversed by intravenous glucose infusions. Together, our findings suggest that carbohydrate-specific preferences can develop independently of taste quality or caloric load, an effect associated with the ability of a given nutrient to regulate glucose metabolism and stimulate brain dopamine centers.

Smad5 regulates Akt2 expression and insulin-induced glucose uptake in L6 myotubes

Insulin-induced glucose uptake by skeletal muscle results from Akt2 activation and is severely impaired during insulin resistance Recently, we and others have demonstrated that BMP9 improves glucose homeostasis in diabetic and non-diabetic rodents. However, the mechanism by which BMP9 modulates insulin action remains unknown. Here we demonstrate that Smad5. a transcription factor activated by BMP9, and Akt2. are upregulated in differentiated L6 myotubes. Smad5, rather than Smad1/8, is downregulated ""in vivo"" and ""in vitro"" by dexamethasone Smad5 knockdown decreased Akt2 expression and serine phosphorylation and insulin-induced glucose uptake, and increased the expression of the lipid phosphatase Ship2. Additionally, binding of Smad5 to Akt2 gene is decreased in dexamethasone-treated rats and Increased in L6 myotubes compared to myoblasts The present study indicates that Smad5 regulates glucose uptake in skeletal muscle by controlling Akt2 expression and phosphorylation These finding reveals Smad5 as a potential target for the therapeutic of type 2 diabetes. (C) 2010 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.

Low doses of hydrogen peroxide impair glucose-stimulated insulin secretion via inhibition of glucose metabolism and intracellular calcium oscillations

The inhibitory effect of hydrogen peroxide (H(2)O(2)) on glucose-stimulated insulin secretion was previously reported. However, the precise mechanism involved was not systematically investigated. In this study, the effects of low concentrations of H(2)O(2) (5-10 mu mol/L) on glucose metabolism, intracellular calcium ([Ca(2+)](i)) oscillations, and dynamic insulin secretion in rat pancreatic islets were investigated. Low concentrations of H(2)O(2) impaired insulin secretion in the presence of high glucose levels (16.7 mmol/L). This phenomenon was observed already after 2 minutes of exposure to H(2)O(2). Glucose oxidation and the amplitude of [Ca(2+)](i); oscillations were dose-dependently suppressed by H(2)O(2). These findings indicate that low concentrations of H(2)O(2) reduce insulin secretion in the presence of high glucose levels via inhibition of glucose metabolism and consequent impairment in [Ca(2+)](i); handling. (C) 2010 Elsevier Inc. All rights reserved.

Effect of exercise on glutamine metabolism in macrophages of trained rats

This study investigated the effect of exercise on glutamine metabolism in macrophages of trained rats. Rats were divided into three groups: sedentary (SED); moderately trained (MOD) rats that were swim trained 1 h/day, 5 days/week for 6 weeks; and exhaustively trained (EXT) rats that were similarly trained as MOD for 5 weeks and, in the 6th week, trained in three 1-h sessions/day with 150 min of rest between sessions. The animals swam with a load equivalent to 5.5% of their body weight and were killed 1 h after the last exercise session. Cells were collected, and glutamine metabolism in macrophage and function were assayed. Exercise increased phagocytosis in MOD when compared to SED (34.48 +/- 1.79 vs 15.21 +/- 2.91%, P < 0.05); however, H(2)O(2) production was higher in MOD (75.40 +/- 3.48 nmol h x 10(5) cell(-1)) and EXT (79.20 +/- 1.18 nmol h x 10(5) cell(-1)) in relation to SED (32.60 +/- 2.51 nmol h x 10(5) cell(-1), P < 0.05). Glutamine consumption increased in MOD and EXT (26.53 +/- 3.62 and 19.82 +/- 2.62 nmol h x 10(5) cell(-1), respectively) relative to SED (6.72 +/- 0.57 nmol h x 10(5) cell(-1), P < 0.05). Aspartate increased in EXT (9.72 +/- 1.14 nmol h x 10(5) cell(-1)) as compared to SED (1.10 +/- 0.19 nmol h x 10(5) cell(-1), P < 0.05). Glutamine decarboxylation was increased in MOD (12.10 +/- 0.27 nmol h x 10(5) cell(-1)) and EXT (16.40 +/-\ 2.17 nmol h x 10(5) cell(-1)) relative to SED (1.10 +/- 0.06 nmol h x 10(5) cell(-1), P < 0.05). This study suggests an increase in macrophage function post-exercise, which was supported by enhanced glutamine consumption and metabolism, and highlights the importance for glutamine after exercise.

Increased aerobic metabolism is essential for the beneficial effects of caloric restriction on yeast life span

Calorie restriction is a dietary regimen capable of extending life span in a variety of multicellular organisms. A yeast model of calorie restriction has been developed in which limiting the concentration of glucose in the growth media of Saccharomyces cerevisiae leads to enhanced replicative and chronological longevity. Since S. cerevisiae are Crabtree-positive cells that present repression of aerobic catabolism when grown in high glucose concentrations, we investigated if this phenomenon participates in life span regulation in yeast. S. cerevisiae only exhibited an increase in chronological life span when incubated in limited concentrations of glucose. Limitation of galactose, raffinose or glycerol plus ethanol as substrates did not enhance life span. Furthermore, in Kluyveromyces lactis, a Crabtree-negative yeast, glucose limitation did not promote an enhancement of respiratory capacity nor a decrease in reactive oxygen species formation, as is characteristic of conditions of caloric restriction in S. cerevisiae. In addition, K. lactis did not present an increase in longevity when incubated in lower glucose concentrations. Altogether, our results indicate that release from repression of aerobic catabolism is essential for the beneficial effects of glucose limitation in the yeast calorie restriction model. Potential parallels between these changes in yeast and hormonal regulation of respiratory rates in animals are discussed.

Laterally transferred genomic islands in Xanthomonadales related to pathogenicity and primary metabolism

Lateral gene transfer (LGT) is considered as one of the drivers in bacterial genome evolution, usually associated with increased fitness and/or changes in behavior, especially if one considers pathogenic vs. non-pathogenic bacterial groups. The genomes of two phytopathogens, Xanthomonas campestris pv. campestris and Xanthomonas axonopodis pv. citri, were previously inspected for genome islands originating from LGT events, and, in this work, potentially early and late LGT events were identified according to their altered nucleotide composition. The biological role of the islands was also assessed, and pathogenicity, virulence and secondary metabolism pathways were functions highly represented, especially in islands that were found to be recently transferred. However, old islands are composed of a high proportion of genes related to cell primary metabolic functions. These old islands, normally undetected by traditional atypical composition analysis, but confirmed as product of LGT by atypical phylogenetic reconstruction, reveal the role of LGT events by replacing core metabolic genes normally inherited by vertical processes.

Serine-like proteolytic enzymes correlated with differential pathogenicity in patients with acute Acanthamoeba keratitis

P>Acute ocular infection due to free-living amoebae of the genus Acanthamoeba is characterized by severe pain, loss of corneal transparency and, eventually, blindness. Proteolytic enzymes secreted by trophozoites of virulent Acanthamoeba strains have an essential role in the mechanisms of pathogenesis, including adhesion, invasion and destruction of the corneal stroma. In this study, we analysed the relationship between the extracellular proteases secreted by clinical isolates of Acanthamoeba and the clinical manifestations and severity of disease that they caused. Clinical isolates were obtained from patients who showed typical symptoms of Acanthamoeba keratitis. Trophozoites were cultivated axenically, and extracellular proteins were collected from cell culture supernatants. Secreted enzymes were partially characterized by gelatin and collagen zymography. Acanthamoeba trophozoites secreted proteases with different molecular masses, proteolysis rates and substrate specificities, mostly serine-like proteases. Different enzymatic patterns of collagenases were observed, varying between single and multiple collagenolytic activities. Low molecular weight serine proteases were secreted by trophozoites associated with worse clinical manifestations. Consequently, proteolytic enzymes of some Acanthamoeba trophozoites could be related to the degree of their virulence and clinical manifestations of disease in the human cornea.

Effect of endurance training upon lipid metabolism in the liver of cachectic tumour-bearing rats

The syndrome of cancer cachexia is accompanied by several alterations in lipid metabolism, and the liver is markedly affected. Previous Studies showed that moderate exercise training may prevent liver fill accumulation through diminished delivery of lipids to the liver, increased hepatic oxidation and increased incorporation of triacylglycerol (TAG) into very low density lipoprotein (VLDL). Our aim was to examine the influence of moderate intensity training (8 weeks) upon TAG content, VLDL assembly and secretion, apolipoprotein B (apoB) and microsomal transfer protein (MTP) gene expression in the liver of cachectic tumour-bearing rats. Animals were randomly assigned to a sedentary control (SC), sedentary tumour-bearing (ST) or exercise-trained control (EC) or to all exercise trained tumour-bearing (ET) group. Trained rats ran on a treadmill (60% VO2max) for 60 min day(-1), 5 day week(-1), for 8 weeks. TAG content and the rate of VLDL secretion (followed for 3 h), its well its mRNA expression of apoB and MTP, and total cholesterol, VLDL-TAG, VLDL-cholesterol, high density lipoprotein cholesterol (HDL-cholesterol) and tumor weight were evaluated. VLDL-cholesterol showed a decrease in ST (p < 0.05) in relation to SC. Serum TAG, VLDL-TAG and tissue TAG content were all increased in ST (p < 0.01), when compared with SC. ST showed a lower rate of VLDL secretion (p < 0.05) and reduced expression of apoB (p < 0.001) and MTP (p < 0.001), when compared with SC. These parameters were restored to control values (p < 0.05) when the animals were submitted to the exercise training protocol. Tumour weight decreased 10-fold after training (p < 0.001). It is possible to affirm, therefore, that endurance training promoted the re-establishment of lipid metabolism in cachectic tumour-bearing animals, especially in relation to VLDL secretion and assembly. Copyright (C) 2008 John Wiley & Sons, Ltd.

Disruption of the circadian clock within the cardiomyocyte influences myocardial contractile function, metabolism, and gene expression

Virtually every mammalian cell, including cardiomyocytes, possesses an intrinsic circadian clock. The role of this transcriptionally based molecular mechanism in cardiovascular biology is poorly understood. We hypothesized that the circadian clock within the cardiomyocyte influences diurnal variations in myocardial biology. We, therefore, generated a cardiomyocyte-specific circadian clock mutant (CCM) mouse to test this hypothesis. At 12 wk of age, CCM mice exhibit normal myocardial contractile function in vivo, as assessed by echocardiography. Radiotelemetry studies reveal attenuation of heart rate diurnal variations and bradycardia in CCM mice (in the absence of conduction system abnormalities). Reduced heart rate persisted in CCM hearts perfused ex vivo in the working mode, highlighting the intrinsic nature of this phenotype. Wild-type, but not CCM, hearts exhibited a marked diurnal variation in responsiveness to an elevation in workload (80 mmHg plus 1 mu M epinephrine) ex vivo, with a greater increase in cardiac power and efficiency during the dark (active) phase vs. the light (inactive) phase. Moreover, myocardial oxygen consumption and fatty acid oxidation rates were increased, whereas cardiac efficiency was decreased, in CCM hearts. These observations were associated with no alterations in mitochondrial content or structure and modest mitochondrial dysfunction in CCM hearts. Gene expression microarray analysis identified 548 and 176 genes in atria and ventricles, respectively, whose normal diurnal expression patterns were altered in CCM mice. These studies suggest that the cardiomyocyte circadian clock influences myocardial contractile function, metabolism, and gene expression.

Biomimetic biosensor based on lipidic layers containing tyrosinase and lutetium bisphthalocyanine for the detection of antioxidants

This paper describes the preparation of a biomimetic Langmuir-Blodgett film of tyrosinase incorporated in a lipidic layer and the use of lutetium bisphthalocyanine as an electron mediator for the voltammetric detection of phenol derivatives, which include one monophenol (vanillic acid), two diphenols (catechol and caffeic acid) and two triphenols (gallic acid and pyrogallol). The first redox process of the voltammetric responses is associated with the reduction of the enzymatically formed o-quinone and is favoured by the lutetium bisphthalocyanine because significant signal amplification is observed, while the second is associated with the electrochemical oxidation of the antioxidant and occurs at lower potentials in the presence of an electron mediator. The biosensor shows low detection limit (1.98 x 10(-6)-27.49 x 10(-6) M), good reproducibility, and high affinity to antioxidants (Km in the range of 62.31-144.87 mu M). The excellent functionality of the enzyme obtained using a biomimetic immobilisation method, the selectivity afforded by enzyme catalysis, the signal enhancement caused by the lutetium bisphthalocyanine mediator and the increased selectivity of the curves due to the occurrence of two redox processes make these sensors exceptionally suitable for the detection of phenolic compounds. (C) 2010 Elsevier B.V. All rights reserved.

Mitochondrial energy metabolism in neurodegeneration associated with methylmalonic acidemia

Methylmalonic acidemia is one of the most prevalent inherited metabolic disorders involving neurological deficits. In vitro experiments, animal model studies and tissue analyses from human patients suggest extensive impairment of mitochondrial energy metabolism in this disease. This review summarizes changes in mitochondrial energy metabolism occurring in methylmalonic acidemia, focusing mainly on the effects of accumulated methylmalonic acid, and gives an overview of the results found in different experimental models. Overall, experiments to date suggest that mitochondrial impairment in this disease occurs through a combination of the inhibition of specific enzymes and transporters, limitation in the availability of substrates for mitochondrial metabolic pathways and oxidative damage.