Hilton Kenji Takahashi

Saturated Fatty Acids Produce an Inflammatory Response Predominantly through the Activation of TLR4 Signaling in Hypothalamus: Implications for the Pathogenesis of Obesity

In animal models of diet-induced obesity, the activation of an inflammatory response in the hypothalamus produces molecular and functional resistance to the anorexigenic hormones insulin and leptin. The primary events triggered by dietary fats that ultimately lead to hypothalamic cytokine expression and inflammatory signaling are unknown. Here, we test the hypothesis that dietary fats act through the activation of toll-like receptors 2/4 and endoplasmic reticulum stress to induce cytokine expression in the hypothalamus of rodents. According to our results, long-chain saturated fatty acids activate predominantly toll-like receptor 4 signaling, which determines not only the induction of local cytokine expression but also promotes endoplasmic reticulum stress. Rats fed on a monounsaturated fat-rich diet do not develop hypothalamic leptin resistance, whereas toll-like receptor 4 loss-of-function mutation and immunopharmacological inhibition of toll-like receptor 4 protects mice from diet-induced obesity. Thus, toll-like receptor 4 acts as a predominant molecular target for saturated fatty acids in the hypothalamus, triggering the intracellular signaling network that induces an inflammatory response, and determines the resistance to anorexigenic signals.

Arachidonic acid actions on functional integrity and attenuation of the negative effects of palmitic acid in a clonal pancreatic β-cell line

Chronic exposure of pancreatic β-cells to saturated non-esterified fatty acids can lead to inhibition of insulin secretion and apoptosis. Several previous studies have demonstrated that saturated fatty acids such as PA (palmitic acid) are detrimental to β-cell function compared with unsaturated fatty acids. In the present study, we describe the effect of the polyunsaturated AA (arachidonic acid) on the function of the clonal pancreatic β-cell line BRIN-BD11 and demonstrate AA-dependent attenuation of PA effects. When added to β-cell incubations at 100 μM, AA can stimulate cell proliferation and chronic (24 h) basal insulin secretion. Microarray analysis and/or real-time PCR indicated significant AA-dependent up-regulation of genes involved in proliferation and fatty acid metabolism [e.g. Angptl (angiopoietin-like protein 4), Ech1 (peroxisomal Δ3,5,Δ2,4-dienoyl-CoA isomerase), Cox-1 (cyclo-oxygenase-1) and Cox-2, P<0.05]. Experiments using specific COX and LOX (lipoxygenase) inhibitors demonstrated the importance of COX-1 activity for acute (20 min) stimulation of insulin secretion, suggesting that AA metabolites may be responsible for the insulinotropic effects. Moreover, concomitant incubation of AA with PA dose-dependently attenuated the detrimental effects of the saturated fatty acid, so reducing apoptosis and decreasing parameters of oxidative stress [ROS (reactive oxygen species) and NO levels] while improving the GSH/GSSG ratio. AA decreased the protein expression of iNOS (inducible NO synthase), the p65 subunit of NF-κB (nuclear factor κB) and the p47 subunit of NADPH oxidase in PA-treated cells. These findings indicate that AA has an important regulatory and protective β-cell action, which may be beneficial to function and survival in the ‘lipotoxic’ environment commonly associated with Type 2 diabetes mellitus.

Activation of survival and apoptotic signaling pathways in lymphocytes exposed to palmitic acid

The toxicity of palmitic acid (PA) towards a human T‐lymphocyte cell line (Jurkat) has been previously investigated but the mechanism(s) of PA action were unknown. In the current study, Jurkat cells were treated with sub‐lethal concentrations of PA (50–150µM) and the activity of various signaling proteins was investigated. PA‐induced apoptosis and mitochondrial dysfunction in a dose‐dependent manner as evaluated by DNA fragmentation assay and depolarization of the mitochondrial membrane, respectively. PA treatment provoked release of cytochrome c from the inner mitochondrial membrane to the cytosol, activated members of the MAPK protein family JNK, p38, ERK, activated caspases 3/9, and increased oxidative/nitrosative stress. Exposure of cells to PA for 12 h increased insulin receptor (IR) and GLUT‐4 levels in the plasma membrane. Insulin treatment (10 mU/ml/30 min) increased the phosphorylation of the IR β‐subunit and Akt. A correlation was found between DNA fragmentation and expression levels of both IR and GLUT‐4. Similar results were obtained for PA‐treated lymphocytes from healthy human donors and from mesenteric lymph nodes of 48‐h starved rats. PA stimulated glucose uptake by Jurkat cells (in the absence of insulin), stimulated accumulation of neutral lipids (triglyceride), and other lipid classes (phospholipids and cholesterol ester) but reduced glucose oxidation. Our results suggest that parameters of insulin signaling and non‐oxidative glucose metabolism are stimulated as part of a coordinated response to prompt survival in lymphocytes exposed to PA but at higher concentrations, apoptosis prevails. These findings may explain aspects of lymphocyte dysfunction associated with diabetes. J. Cell. Physiol. 227: 339–350, 2012.

Effect of lipid infusion on metabolism and force of rat skeletal muscles during intense contractions.

The hypothesis that during intense muscle contraction induced by electrical stimulation, long chain fatty acids (LCFA) might reduce mitochondrial ATP/ADP ratio, raising the contribution of glycolysis for ATP production was examined. The effect of a lipid infusion (Lipovenus emulsion) on UCP-3 mRNA level, lactate, glucose-6-phosphate (G-6P) and glycogen content was investigated in rat. Blood samples for determination of free fatty acids and lactate were collected at 0, 30 and 60 min during rest and at 0, 10 and 20 min during muscle contraction. The content of lactate, glycogen and G-6P was also determined in soleus (SO), red gastrocnemius (RG) and white gastrocnemius (WG) muscles collected immediately after muscle contraction period. In addition, the force level was determined during muscle contractions. The effect of Lipovenus emulsion on respiration of mitochondria isolated from rat skeletal muscle, and content of UCP-3 and lactate in cultured skeletal muscle cells was also determined. The in vivo experiments showed that Lipovenus induced a significant increase of UCP-3 mRNA levels. After Lipovenus infusion, lactate level was increased in RG muscle only, whereas the contents of glycogen and G-6P were decreased in both RG and WG muscles (P < 0.05). Lipovenus infusion failed to exert any effect on muscle force performance (P > 0.05). The in vitro experiments showed that Lipovenus infusion induced a significant increase in mitochondrial respiration, but had no effect on UCP-3 content. Lactate concentration was significantly increased in the culture medium of stimulated cells in the control and Lipovenus groups compared with the respective not-stimulated cells (P< 0.05). We concluded that as mitochondrial function becomes limited by the FFA-uncoupling effect, the ATP demand is mainly supplied by anaerobic glucose metabolism preventing an expected decrease in muscle contraction performance.

Heat shock proteins and heat therapy for type 2 diabetes: pros and cons.

Purpose of reviewHeat therapy, such as sauna and hot tub, has become an increasingly regular therapeutical practice around the world since several studies have shown benefits of heat therapy in metabolic and cardiovascular diseases. The use of heat therapy in people with type 2 diabetes mellitus revealed a striking reduction of 1% unit in the glycated hemoglobin, suggesting this therapy for the treatment of diabetes. Herein, we shall discuss the use of heat therapy and the mechanisms involved, and suggest a provisional guide for the use of heat therapy in obesity and diabetes. Recent findingsHuman studies indicate that heat therapy reduces fasting glycemia, glycated hemoglobin, body weight, and adiposity. Animal studies have indicated that nitric oxide and the increase in heat shock protein 70 expression is involved in the improvements induced by heat therapy on insulin sensitivity, adiposity, inflammation, and vasomotricity. SummaryHeat therapy is a promising and inexpensive tool for the treatment of obesity and diabetes. We proposed that transient increments in nitric oxide and heat shock protein 70 levels may explain the benefits of heat therapy. We suggest that heat therapy (sauna: 80–100°C; hot tub: at 40°C) for 15 min, three times a week, for 3 months, is a safe method to test its efficiency.

Soybean and sunflower oil‐induced insulin resistance correlates with impaired GLUT4 protein expression and translocation specifically in white adipose tissue

Free fatty acids are known for playing a crucial role in the development of insulin resistance. High fat intake is known for impairing insulin sensitivity; however, the effect of vegetable‐oil injections have never been investigated. The present study investigated the effects of daily subcutaneous injections (100 µL) of soybean (SB) and sunflower (SF) oils, during 7 days. Both treated groups developed insulin resistance as assessed by insulin tolerance test. The mechanism underlying the SB‐ and SF‐induced insulin resistance was shown to involve GLUT4. In SB‐ and SF‐treated animals, the GLUT4 protein expression was reduced ∼20% and 10 min after an acute in vivo stimulus with insulin, the plasma membrane GLUT4 content was ∼60% lower in white adipose tissue (WAT). No effects were observed in skeletal muscle. Additionally, both oil treatments increased mainly the content of palmitic acid (∼150%) in WAT, which can contribute to explain the GLUT4 regulations. Altogether, the present study collects evidence that those oil treatments might generate insulin resistance by targeting GLUT4 expression and translocation specifically in WAT. These alterations are likely to be caused due to the specific local increase in saturated fatty acids that occurred as a consequence of oil daily injections. Copyright

Lonomia obliqua (Lepidoptera, Saturniidae) caterpillar bristle extract induces direct lysis by cleaving erythrocyte membrane glycoproteins.

Lonomia obliqua caterpillar bristle extract induces hemolysis in vitro on washed human and rat erythrocytes, in either the absence or presence of exogenous lecithin. In the former condition, phospholipases A(2) are key enzymes involved in hemolysis. However, the mechanism whereby this extract causes direct hemolysis is not known. Thus, the aim of this study was to investigate the hemolytic mechanism of the crude extract of the caterpillar L. obliqua on human erythrocytes in the absence of lecithin. The extract significantly increased the erythrocyte osmotic fragility and promoted the removal of glycophorins A and C, and band 3 from the erythrocyte membrane. The use of Ca2+ and Mg2+ ions significantly potentiated glycoprotein removal, remarkably of erythrocyte band 3. The composition of fatty acids was analyzed by HPLC in both L. obliqua caterpillar bristle extract and human erythrocyte membranes incubated with the extract. The levels of unsaturated fatty acids were remarkably augmented in erythrocytes incubated with the extract than in control erythrocytes, modifying thereby the saturated/unsaturated fatty acid ratio. Altogether, evidence is provided here that the interplay of at least three mechanisms of action accounts for the direct activity of the bristle extract on erythrocyte membrane, leading to hemolysis: the removal of glycoproteins and band 3; the insertion of fatty acids; and the action of phospholipases. Such mechanisms might affect erythrocyte flexibility and deformability, which may induce hemolysis by increasing erythrocyte fragility. However, whether the direct hemolytic activity of L. obliqua caterpillar is the major cause of intravascular hemolysis during envenomation still needs further investigation.

Regulatory principles in metabolism–then and now

The importance of metabolic pathways for life and the nature of participating reactions have challenged physiologists and biochemists for over a hundred years. Eric Arthur Newsholme contributed many original hypotheses and concepts to the field of metabolic regulation, demonstrating that metabolic pathways have a fundamental thermodynamic structure and that near identical regulatory mechanisms exist in multiple species across the animal kingdom. His work at Oxford University from the 1970s to 1990s was groundbreaking and led to better understanding of development and demise across the lifespan as well as the basis of metabolic disruption responsible for the development of obesity, diabetes and many other conditions. In the present review we describe some of the original work of Eric Newsholme, its relevance to metabolic homoeostasis and disease and application to present state-of-the-art studies, which generate substantial amounts of data that are extremely difficult to interpret without a fundamental understanding of regulatory principles. Erics work is a classical example of how one can unravel very complex problems by considering regulation from a cell, tissue and whole body perspective, thus bringing together metabolic biochemistry, physiology and pathophysiology, opening new avenues that now drive discovery decades thereafter.

Molecular Mechanisms Involved in Inflammation and Insulin Resistance in Chronic Diseases and Possible Interventions

Inflammation and insulin resistance are present in several chronic diseases, including obesity, type 2 diabetes mellitus, metabolic syndrome, cancer, and cardiovascular diseases. Recent studies showed a close relationship between these two conditions, although the precise mechanisms are not completely understood. This special issue aimed to join original research and review articles related to the involvement of inflammation and/or insulin resistance with the development of chronic diseases as well as the interaction between these two factors and possible interventions based on important molecular targets. This issue was interested in articles that explore molecular aspects of inflammatory pathways, insulin resistance, and the crosstalk between them, in humans and in cell and animal models. Moreover, we accepted studies that explored interventions based on molecular targets for preventing or treating correlated disorders and advances for a better characterization and understanding of the mechanisms and mediators involved with the different inflammatory and insulin resistance conditions, addressing biotechnological studies for the development of new potential therapies and interventions. The potential topics for this special issue included (a) molecular basis of inflammation and insulin resistance; (b) crosstalk between inflammatory pathways and insulin signaling; (c) cell and animal models to test and understand the role of inflammation and insulin resistance as well as the interaction of these two factors on the development of chronic diseases; (d) interaction of inflammation and/or insulin resistance in human pathology conditions; (e) factors leading to inflammation and/or insulin resistance in models of chronic diseases; (f) identification of new biomarkers of chronic inflammation and insulin resistance in different models; (g) identification of new molecular targets for reducing inflammation and insulin resistance in chronic disease models; (h) new interventions for preventing or reducing insulin resistance and inflammation based on molecular targets in signaling pathways. In the review article from S. Hirabara et al., the authors discussed the recent findings concerning insulin resistance and inflammation, the relationship between these two factors, possible mechanisms involved, and potential interventions for the disorders related. Authors pointed the advances in the field and new biotechnological tools and methodologies that have aided to understand these processes. Discovery and identification of new biomarkers involved with the development of chronic diseases characterized by increased inflammation and insulin resistance, as well as their relevance in the comprehension of interaction of these processes, were also discussed and will allow the study of treatment or prevention for related disorders. L. Masi et al. characterized the effects of sunflower oil supplementation on insulin resistance and inflammation in mice submitted to high-fat diet (HFD). Briefly, the authors observed that the sunflower oil supplementation induces proinflammatory responses in macrophages and insulin-sensitive peripheral tissues, as well as insulin resistance. These responses were observed in control mice and pronounced in mice submitted to HFD. Interestingly, although sunflower oil supplementation was able to improve dislipidemia in mice fed with HFD, it increased inflammatory condition and insulin resistance state induced by the diet. P. Li et al. studied gene variants of mitofusin-2 (MFN-2) in subjects with type 2 diabetes mellitus. This gene is involved in the mitochondrial fusion, regulating the morphology and distribution of this organelle, especially in cells and tissues with high demand of energy, including skeletal muscle and heart. Authors found a relationship between MFN-2 gene polymorphisms and type 2 diabetes mellitus, as well as between some MFN-2 gene variants and two other gene polymorphisms: peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) and estrogen-related receptor-α (ESRRA) genes, which are important transcriptional factors that also regulate mitochondrial biogenesis and oxidative capacity. These findings point MFN-2 gene as a new potential target for further studies aiming at preventive or therapeutic interventions. In the study of G. Rodrigues et al., authors investigated the effects of the treatment with aqueous extract from Croton cajucara plant on hepatic oxidative stress in streptozotocin-induced diabetic rats. Croton cajucara is a Brazilian plant rich in several compounds, including flavonoids, coumarins, and alkaloids, that has been suggested to have important antioxidant effects, being used in various diseases, including diabetes mellitus, hypercholesterolemia, diarrhea, malaria, fever, gastrointestinal, renal, and hepatic disorders. The results found in this study suggest that the treatment with Croton cajucara aqueous extract is effective in decreasing hepatic oxidative stress and cell damage in streptozotocin-induced diabetic rats, indicating for the potential therapeutic use of this plant in related diseases. M.-Liao et al. reviewed the role of insulin resistance in the development of chronic kidney disease and the potential mechanisms involved. They discuss how different factors, including chronic inflammation, cellular and endoplasmic reticulum oxidative stress, decreased serum erythropoietin, elevated plasma adipokine and fetuin-A, and vitamin D deficiency, can interfere in insulin sensitivity, contributing to the insulin resistance establishment and consequently to the development of chronic kidney disease. C.-Sung et al. discussed the role of vitamin D on insulin resistance in several pathological conditions, including diabetes mellitus, hypertension, and cardiovascular diseases. Mechanisms for vitamin D deficiency associated with inherited gene polymorphisms are also presented, and its relevance in the immunoregulatory function and insulin resistance development is addressed. Although important advances in the field have been reached, the underlying mechanisms involved in these processes require further investigation. G. Zhang et al., studying white rabbits, modified a protocol for the development of a new model of human atherosclerosis and vulnerable plaque, allowing a novel approach for related studies. Several markers of the disease were used in order to validate the model, including structural alterations, triacylglycerol and LDL-cholesterol serum levels, inflammatory parameters, and oxidative stress. This pattern of atherosclerosis for rabbits is very close to that in humans with several advantages when compared to other animal models, showing potential applicability and clinical relevance. C. Leandro et al. focused on gestational insulin resistance and low insulin secretion induced by undernutrition during pregnancy. They found that low-protein diet during gestation impairs the glucose-stimulated insulin secretion and glucose tolerance. In contrast, a program of pregestational and gestational moderate physical training improved the glucose-stimulated insulin secretion and partially prevented the effects of perinatal undernutrited rats. Thus, well-controlled programs of moderate physical training, which have well-characterized systemic anti-inflammatory effects, starting at early pregnancy, are a potential tool for preventing insulin resistance and gestational diabetes mellitus during late pregnancy, as well as for increasing insulin secretion stimulated by glucose and avoiding deleterious effects on the offspring. A. Alfadda and R. Sallam discussed in their review article the biological functions of reactive oxygen species (ROS) in several physiological and pathological processes. The importance of physiological ROS production on normal vascular diameter regulation, cellular oxygen sensing, immune system function, skeletal muscle physiology, cellular signaling pathways, and gene expression control is presented. On the other hand, the effects of excessive ROS production on the development of diseases, including cancer, diabetes mellitus, obesity, chronic inflammation, cardiovascular diseases, and metabolic syndrome are also discussed. This review provides recent findings and advances in this area for understanding the functions of ROS on different targets during various conditions in health and disease.