Nuclear Factor-Kappa-B Mediates the Advanced Glycation End Product-Induced Repression of Slc2a4 Gene Expression in 3T3-L1 Adipocytes.

Advanced glycated end products (AGEs) are cytotoxic compounds that are mainly increased in diabetes mellitus (DM), kidney failure, inflammation, and in response to the ingestion of AGE-rich diets. AGEs can also impair glycemic homeostasis by decreasing the expression of the Slc2a4 (solute carrier family 2 member 4) gene and its GLUT4 (solute carrier family 2, facilitated glucose transporter member 4) protein in muscle. However, the mechanisms underlying AGE's effect on adipocytes have not been demonstrated yet. This study investigated the effects of AGEs upon Slc2a4/GLUT4 expression in 3T3-L1 adipocytes, as well as the potential role of NFKB (nuclear factor NF-kappa-B) activity in the effects observed. Adipocytes were cultured in the presence of control albumin (CA) or advanced glycated albumin (GA) at concentrations of 0.4, 3.6, and 5.4 mg/mL for 24 h or 72 h. Slc2a4, Rela, and Nfkb1mRNAs were measured by RT-qPCR, GLUT4, IKKA/B, and p50/p65 NFKB subunits using Western blotting, and p50/p65 binding into the Slc2a4 promoter was analyzed by chromatin immunoprecipitation (ChIP) assay. GA at 0.4 mg/mL increased Slc2a4/GLUT4 expression after 24 h and 72 h (from 50% to 100%), but at 5.4 mg/mL, Slc2a4/GLUT4 expression decreased at 72 h (by 50%). Rela and Nfkb1 expression increased after 24 h at all concentrations, but this effect was not observed at 72 h. Furthermore, 5.4 mg/mL of GA increased the p50/p65 nuclear content and binding into Slc2a4 at 72 h. In summary, this study reveals AGE-induced and NFKB-mediated repression of Slc2a4/GLUT4 expression. This can compromise the adipocyte glucose utilization, contributing not only to the worsening of glycemic control in DM subjects but also the impairment of glycemic homeostasis in non-DM subjects under the high intake of AGE-rich foods.

Excess Iodine Consumption Induces Oxidative Stress and Pancreatic Damage Independently of Chemical Form in Male Wistar Rats: Participation of PPAR-γ and C/EBP-ß.

BACKGROUND: Human beings consume different chemical forms of iodine in their diet. These are transported by different mechanisms in the cell. The forms of iodine can be part of thyroid hormones, bind to lipids, be an antioxidant, or be an oxidant, depending on their chemical form. The excessive consumption of iodine has been associated with pancreatic damage and diabetes mellitus type 2, but the association between disease and the chemical form consumed in the diet is unknown. This research analyzes the effect of excessive iodine consumption as Lugol (molecular iodine/potassium iodide solution) and iodate on parameters of pancreatic function, thyroid and lipid profiles, antioxidant and oxidant status, the expression of IR/Akt/P-Akt/GLUT4, and transcription factors PPAR-γ and CEBP-ß. METHODS: Three groups of Wistar rats were treated with 300 µg/L of iodine in drinking water: (1) control, (2) KIO3, and (3) Lugol. RESULTS: Lugol and KIO3 consumption increased total iodine levels. Only KIO3 increased TSH levels. Both induced high serum glucose levels and increased oxidative stress and pancreatic alpha-amylase activity. Insulin levels and antioxidant status decreased significantly. PPAR-γ and C/EBP-ß mRNA expression increased. CONCLUSION: The pancreatic damage, hypertriglyceridemia, and oxidative stress were independent of the chemical form of iodine consumed. These effects depended on PPAR-γ, C/EBP-ß, GLUT-4, and IR.

Flow cytometry protocol for GLUT4-myc detection on cell surfaces.

Insulin and muscle contraction trigger GLUT4 translocation to the plasma membrane, which increases glucose uptake by muscle cells. Insulin resistance and Type 2 diabetes are the result of impaired GLUT4 translocation. Quantifying GLUT4 translocation is essential for comprehending the intricacies of both physiological and pathophysiological processes involved in glucose metabolism. The most commonly used methods for measuring GLUT4 translocation are the ELISA-type assay and the immunofluorescence assay. While some reports suggest that flow cytometry could be useful in quantifying GLUT4 translocation, this technique is not frequently used. Much of our current understanding of the regulation of GLUT4 has been based on experiments using the rat myoblast cell line (L6 cell) which expresses GLUT4 with a myc epitope on the exofacial loop. In the present study, we use the L6-GLUT4myc cell line to develop a flow cytometry-based approach to detect GLUT4 translocation. Flow cytometry offers the advantages of both immunofluorescence and ELISA-based assays. It allows easy identification of separate cell populations in the sample, similar to immunofluorescence, while providing results based on a population-level analysis of multiple individual cells, like an ELISA-based assay. Our results demonstrate a 0.6-fold increase with insulin stimulation compared with basal conditions. Finally, flow cytometry consistently yielded results across different experiments and exhibited sensitivity under the tested conditions.

Iodine Promotes Glucose Uptake through Akt Phosphorylation and Glut-4 in Adipocytes, but Higher Doses Induce Cytotoxic Effects in Pancreatic Beta Cells.

BACKGROUND: Epidemiological clinical reports have shown an association between iodine excess with diabetes mellitus type 2 and higher blood glucose. However, the relationship between iodine, the pancreas, adipose tissue, and glucose transport is unclear. The goal of this study was to analyze the effect of iodine concentrations (in Lugol solution) on glucose transport, insulin secretion, and its cytotoxic effects in mature 3T3-L1 adipocytes and pancreatic beta-TC-6 cells. METHODS: Fibroblast 3T3-L1, mature adipocytes, and pancreatic beta-TC-6 cells were treated with 1 to 1000 µM of Lugol (molecular iodine dissolved in potassium iodide) for 30 min to 24 h for an MTT proliferation assay. Then, glucose uptake was measured with the fluorescent analog 2-NBDG, insulin receptor, Akt protein, p-Akt (ser-473), PPAR-gamma, and Glut4 by immunoblot; furthermore, insulin, alpha-amylase, oxidative stress, and caspase-3 activation were measured by colorimetric methods and the expression of markers of the apoptotic pathway at the RNAm level by real-time PCR. RESULTS: Low concentrations of Lugol significantly induce insulin secretion and glucose uptake in pancreatic beta-TC-6 cells, and in adipose cells, iodine-induced glucose uptake depends on the serine-473 phosphorylation of Akt (p-Akt) and Glut4. Higher doses of Lugol lead to cell growth inhibition, oxidative stress, and cellular apoptosis dependent on PPAR-gamma, Bax mRNA expression, and caspase-3 activation in pancreatic beta-TC-6 cells. CONCLUSIONS: Iodine could influence glucose metabolism in mature adipocytes and insulin secretion in pancreatic beta cells, but excessive levels may cause cytotoxic damage to pancreatic beta cells.

An early onset benign myopathy with glycogen storage caused by a de novo 1.4 Mb-deletion of chromosome 14.

Early onset myopathies are a clinically and histologically heterogeneous monogenic diseases linked to approximately 90 genes. Molecular diagnosis is challenging, especially in patients with a mild phenotype. We describe a 26-year-old man with neonatal hypotonia, motor delay and seizures during infancy, and non-progressive, mild muscular weakness in adulthood. Serum Creatine kinase level was normal. Whole-body muscle MRI showed thin muscles, and brain MRI was unremarkable. A deltoid muscle biopsy showed glycogen storage. WGS revealed a de novo 1.4 Mb-deletion of chromosome 14, confirmed by Array-CGH. This microdeletion causes the loss of ten genes including RALGAPA1, encoding for RalA, a regulator of glucose transporter 4 (GLUT4) expression at the membrane of myofibers. GLUT4 was overexpressed in patient's muscle. Here we highlight the importance to search for chromosomal alterations in the diagnostic workup of early onset myopathies.

Impact of Normal and Overweight Pregnancy in GLUT4 and Glucose-Dependent Vascular Contractility.

INTRODUCTION: Obesity during pregnancy can contribute to hypertensive complications through changes in glucose utilization. We investigated the impact of vascular glucose uptake, GLUT4 density, and endothelium on agonist-induced vasoconstriction in the aortas of overweight pregnant rats. METHODS: Isolated aortic rings with or without endothelium from pregnant or nonpregnant rats fed a standard (SD) or hypercaloric diet (HD) were contracted with phenylephrine or serotonin (10-9 to 10-4M) using standard (11 mm) or without (0 mm) glucose Krebs solution. GLUT4 density in the aortas was measured using the en face method. RESULTS: Aortas from overweight pregnant animals (PHD) showed increased Phe-induced vasoconstriction (p < 0.05 vs. pregnant standard diet [PSD]), which was endothelium-independent. The contraction decreased significantly in the absence of glucose. In contrast, vessels from pregnant SD rats maintained their contraction in glucose-free Krebs solution. 5-HT increases PHD aortic contraction only in the absence of glucose. The fetal aortas from PHD mothers showed blunted vasoconstriction. Overweight significantly reduced GLUT4 expression in maternal and fetal aortas (p < 0.05 vs. PSD). CONCLUSIONS: Aortic contractility is independent of glucose uptake during healthy pregnancy. In contrast, overweight pregnancy increases contractility. This increase depends directly on smooth muscle glucose uptake and inversely on GLUT-4 density. The increased contraction observed in the vasculature of overweight mothers was inverted in the fetal aortas.

Postmortem Brains from Subjects with Diabetes Mellitus Display Reduced GLUT4 Expression and Soma Area in Hippocampal Neurons: Potential Involvement of Inflammation.

Diabetes mellitus (DM) is an important risk factor for dementia, which is a common neurodegenerative disorder. DM is known to activate inflammation, oxidative stress, and advanced glycation end products (AGEs) generation, all capable of inducing neuronal dysfunctions, thus participating in the neurodegeneration progress. In that process, disturbed neuronal glucose supply plays a key role, which in hippocampal neurons is controlled by the insulin-sensitive glucose transporter type 4 (GLUT4). We investigated the expression of GLUT4, nuclear factor NF-kappa B subunit p65 [NFKB (p65)], carboxymethyllysine and synapsin1 (immunohistochemistry), and soma area in human postmortem hippocampal samples from control, obese, and obese+DM subjects (41 subjects). Moreover, in human SH-SY5Y neurons, tumor necrosis factor (TNF) and glycated albumin (GA) effects were investigated in GLUT4, synapsin-1 (SYN1), tyrosine hydroxylase (TH), synaptophysin (SYP) proteins, and respective genes; NFKB binding activity in the SLC2A4 promoter; effects of increased histone acetylation grade by histone deacetylase 3 (HDAC3) inhibition. Hippocampal neurons (CA4 area) of obese+DM subjects displayed reduced GLUT4 expression and neuronal soma area, associated with increased expression of NFKB (p65). Challenges with TNF and GA decreased the SLC2A4/GLUT4 expression in SH-SY5Y neurons. TNF decreased SYN1, TH, and SYP mRNAs and respective proteins, and increased NFKB binding activity in the SLC2A4 promoter. Inhibition of HDAC3 increased the SLC2A4 expression and the total neuronal content of CRE-binding proteins (CREB/ICER), and also counterbalanced the repressor effect of TNF upon these parameters. This study revealed reduced postmortem human hippocampal GLUT4 content and neuronal soma area accompanied by increased proinflammatory activity in the brains of DM subjects. In isolated human neurons, inflammatory activation by TNF reduced not only the SLC2A4/GLUT4 expression but also the expression of some genes related to neuronal function (SYN1, TH, SYP). These effects may be related to epigenetic regulations (H3Kac and H4Kac status) since they can be counterbalanced by inhibiting HDAC3. These results uncover the improvement in GLUT4 expression and/or the inhibition of HDAC3 as promising therapeutic targets to fight DM-related neurodegeneration.

The effects of sucrose and arsenic on muscular insulin signaling pathways differ between the gastrocnemius and quadriceps muscles.

Introduction: Insulin resistance in muscle can originate from a sedentary lifestyle, hypercaloric diets, or exposure to endocrine-disrupting pollutants such as arsenic. In skeletal muscle, insulin stimulates glucose uptake by translocating GLUT4 to the sarcolemma. This study aimed to evaluate the alterations induced by sucrose and arsenic exposure in vivo on the pathways involved in insulinstimulated GLUT4 translocation in the quadriceps and gastrocnemius muscles. Methods: Male Wistar rats were treated with 20% sucrose (S), 50 ppm sodium arsenite (A), or both (A+S) in drinking water for 8 weeks. We conducted an intraperitoneal insulin tolerance (ITT) test on the seventh week of treatment. The quadriceps and gastrocnemius muscles were obtained after overnight fasting or 30 min after intraperitoneal insulin injection. We assessed changes in GLUT4 translocation to the sarcolemma by cell fractionation and abundance of the proteins involved in GLUT4 translocation by Western blot. Results: Male rats consuming S and A+S gained more weight than control and Atreated animals. Rats consuming S, A, and A+S developed insulin resistance assessed through ITT. Neither treatments nor insulin stimulation in the quadriceps produced changes in GLUT4 levels in the sarcolemma and Akt phosphorylation. Conversely, A and A+S decreased protein expression of Tether containing UBX domain for GLUT4 (TUG), and A alone increased calpain-10 expression. All treatments reduced this muscle's protein levels of VAMP2. Conversely, S and A treatment increased basal GLUT4 levels in the sarcolemma of the gastrocnemius, while all treatments inhibited insulin-induced GLUT4 translocation. These effects correlated with lower basal levels of TUG and impaired insulin-stimulated TUG proteolysis. Moreover, animals treated with S had reduced calpain-10 protein levels in this muscle, while A and A+S inhibited insulin-induced Akt phosphorylation. Conclusion: Arsenic and sucrose induce systemic insulin resistance due to defects in GLUT4 translocation induced by insulin. These defects depend on which muscle is being analyzed, in the quadriceps there were defects in GLUT4 retention and docking while in the gastrocnemius the Akt pathway was impacted by arsenic and the proteolytic pathway was impaired by arsenic and sucrose.

Insulin Sensitization by PPARγ and GLUT-4 Overexpression/Translocation Mediates the Antidiabetic Effect of Plantago australis.

Plantago australis Lam. Subsp. hirtella (Kunth) Rahn is a medicinal plant used as a diuretic, anti-inflammatory, antibacterial, throat cancer treatment and for the control of diabetes. P. australis was collected in the state of Morelos, México. The hydroalcoholic extract (HAEPa) of P. australis was obtained by maceration and concentrated in vacuo. Once dry, it was evaluated through an oral glucose tolerance test (OGTT) in normoglycemic mice and in a non-insulin-dependent diabetic mice model. The expression of PPARγ and GLUT-4 mRNA was determined by rt-PCR, and GLUT-4 translocation was confirmed by confocal microscopy. The toxicological studies were conducted in accordance with the guidelines suggested by the OECD, sections 423 and 407, with some modifications. HAEPa significantly decreased glycemia in OGTT curves, as well as in the experimental diabetes model compared to the vehicle group. In vitro tests showed that HAEPa induced an α-glucosidase inhibition and increased PPARγ and GLUT-4 expression in cell culture. The LD50 of HAEPa was greater than 2000 mg/kg, and sub-chronic toxicity studies revealed that 100 mg/kg/day for 28 days did not generate toxicity. Finally, LC-MS analysis led to the identification of verbascoside, caffeic acid and geniposidic acid, and phytochemical approaches allowed for the isolation of ursolic acid, which showed significant PPARγ overexpression and augmented GLUT-4 translocation. In conclusion, HAEPa induced significant antidiabetic action by insulin sensitization through PPARγ/GLUT-4 overexpression.

Reactive Oxygen and Nitrogen Species (RONS) and Cytokines-Myokines Involved in Glucose Uptake and Insulin Resistance in Skeletal Muscle.

Insulin resistance onset in skeletal muscle is characterized by the impairment of insulin signaling, which reduces the internalization of glucose, known as glucose uptake, into the cell. Therefore, there is a deficit of intracellular glucose, which is the main source for energy production in the cell. This may compromise cellular viability and functions, leading to pathological dysfunction. Skeletal muscle fibers continuously generate reactive oxygen and nitrogen species (RONS). An excess of RONS produces oxidative distress, which may evoke cellular damage and dysfunction. However, a moderate level of RONS, which is called oxidative eustress, is critical to maintain, modulate and regulate cellular functions through reversible interactions between RONS and the components of cellular signaling pathways that control those functions, such as the facilitation of glucose uptake. The skeletal muscle releases peptides called myokines that may have endocrine and paracrine effects. Some myokines bind to specific receptors in skeletal muscle fibers and might interact with cellular signaling pathways, such as PI3K/Akt and AMPK, and facilitate glucose uptake. In addition, there are cytokines, which are peptides produced by non-skeletal muscle cells, that bind to receptors at the plasma membrane of skeletal muscle cells and interact with the cellular signaling pathways, facilitating glucose uptake. RONS, myokines and cytokines might be acting on the same signaling pathways that facilitate glucose uptake in skeletal muscle. However, the experimental studies are limited and scarce. The aim of this review is to highlight the current knowledge regarding the role of RONS, myokines and cytokines as potential signals that facilitate glucose uptake in skeletal muscle. In addition, we encourage researchers in the field to lead and undertake investigations to uncover the fundamentals of glucose uptake evoked by RONS, myokines, and cytokines.

Sexual dimorphism in the molecular mechanisms of insulin resistance during a critical developmental window in Wistar rats.

BACKGROUND: Insulin resistance (IR) is a condition in which the response of organs to insulin is impaired. IR is an early marker of metabolic dysfunction. However, IR also appears in physiological contexts during critical developmental windows. The molecular mechanisms of physiological IR are largely unknown in both sexes. Sexual dimorphism in insulin sensitivity is observed since early stages of development. We propose that during periods of accelerated growth, such as around weaning, at postnatal day 20 (p20) in rats, the kinase S6K1 is overactivated and induces impairment of insulin signaling in its target organs. This work aimed to characterize IR at p20, determine its underlying mechanisms, and identify whether sexual dimorphism in physiological IR occurs during this stage. METHODS: We determined systemic insulin sensitivity through insulin tolerance tests, glucose tolerance tests, and blood glucose and insulin levels under fasting and fed conditions at p20 and adult male and female Wistar rats. Furthermore, we quantified levels of S6K1 phosphorylated at threonine 389 (T389) (active form) and its target IRS1 phosphorylated at serine 1101 (S1101) (inhibited form). In addition, we assessed insulin signal transduction by measuring levels of Akt phosphorylated at serine 473 (S473) (active form) in white adipose tissue and skeletal muscle through western blot. Finally, we determined the presence and function of GLUT4 in the plasma membrane by measuring the glucose uptake of adipocytes. Results were compared using two-way ANOVA (With age and sex as factors) and one-way ANOVA with post hoc Tukey's tests or t-student test in each corresponding case. Statistical significance was considered for P values < 0.05. RESULTS: We found that both male and female p20 rats have elevated levels of glucose and insulin, low systemic insulin sensitivity, and glucose intolerance. We identified sex- and tissue-related differences in the activation of insulin signaling proteins in p20 rats compared to adult rats. CONCLUSIONS: Male and female p20 rats present physiological insulin resistance with differences in the protein activation of insulin signaling. This suggests that S6K1 overactivation and the resulting IRS1 inhibition by phosphorylation at S1101 may modulate to insulin sensitivity in a sex- and tissue-specific manner. Video Abstract.

Insulin regulates the synthesis of carbohydrates, lipids and proteins differently between males, and females. One of its primary functions is maintaining adequate blood glucose levels favoring glucose entry in muscle and adipose tissue after food consumption. Insulin resistance (IR) is a condition in which the response of organs to insulin is impaired. IR is frequently associated with metabolic dysfunction such as inflammation, obesity, or type 2 diabetes. However, physiological IR develops in healthy individuals during periods of rapid growth, pregnancy, or aging by mechanisms not fully understood. We studied the postnatal development, specifically around weaning at postnatal day 20 (p20) of Wistar rats. In previous works, we identified insulin resistance during this period in male rats. This work aimed to characterize IR at p20, determine its underlying mechanisms, and identify whether sexual dimorphism in physiological IR occurs during this stage. We found that p20 rats of both sexes have elevated blood glucose and insulin levels, low systemic insulin sensitivity, and glucose intolerance. We identified differences in insulin-regulated protein activation (S6K1, IRS1, Akt, and GLUT4) between sexes in different tissues and adipose tissue depots. Studying these mechanisms and their differences between males and females is essential to understanding insulin actions and their relationship with the possible development of metabolic diseases in both sexes.

Gain-of-Function Dynamin-2 Mutations Linked to Centronuclear Myopathy Impair Ca2+-Induced Exocytosis in Human Myoblasts.

Gain-of-function mutations of dynamin-2, a mechano-GTPase that remodels membrane and actin filaments, cause centronuclear myopathy (CNM), a congenital disease that mainly affects skeletal muscle tissue. Among these mutations, the variants p.A618T and p.S619L lead to a gain of function and cause a severe neonatal phenotype. By using total internal reflection fluorescence microscopy (TIRFM) in immortalized human myoblasts expressing the pH-sensitive fluorescent protein (pHluorin) fused to the insulin-responsive aminopeptidase IRAP as a reporter of the GLUT4 vesicle trafficking, we measured single pHluorin signals to investigate how p.A618T and p.S619L mutations influence exocytosis. We show here that both dynamin-2 mutations significantly reduced the number and durations of pHluorin signals induced by 10 µM ionomycin, indicating that in addition to impairing exocytosis, they also affect the fusion pore dynamics. These mutations also disrupt the formation of actin filaments, a process that reportedly favors exocytosis. This altered exocytosis might importantly disturb the plasmalemma expression of functional proteins such as the glucose transporter GLUT4 in skeletal muscle cells, impacting the physiology of the skeletal muscle tissue and contributing to the CNM disease.

4-Hydroxybenzoic Acid and ß-Sitosterol from Cucurbita ficifolia Act as Insulin Secretagogues, Peroxisome Proliferator-Activated Receptor-Gamma Agonists, and Liver Glycogen Storage Promoters: In Vivo, In Vitro, and In Silico Studies.

Insulin secretion and GLUT4 expression are two critical events in glucose regulation. The receptors G-protein-coupled receptor 40 (GPR40) and peroxisome proliferator-activated receptor-gamma (PPARγ) modulate these processes, and they represent potential therapeutic targets for new antidiabetic agent's design. Cucurbita ficifolia fruit is used in traditional medicine for diabetes control. Previous studies demonstrated several effects: a hypoglycemic effect mediated by an insulin secretagogue action, antihyperglycemic effect, and promoting liver glycogen storage. Anti-inflammatory and antioxidant effects were also reported. Moreover, some of its phytochemicals have been described, including d-chiro-inositol. However, to understand these effects integrally, other active principles should be investigated. The aim was to perform a chemical fractionation guided by bioassay to isolate and identify other compounds from C. ficifolia fruit that explain its hypoglycemic action as insulin secretagogue, its antihyperglycemic effect by PPARγ activation, and on liver glycogen storage. Three different preparations of C. ficifolia were tested in vivo. Ethyl acetate fraction derived from aqueous extract showed antihyperglycemic effect in an oral glucose tolerance test and was further fractioned. The insulin secretagogue action was tested in RINm5F cells. For the PPARγ activation, C2C12 myocytes were treated with the fractions, and GLUT4 mRNA expression was measured. Chemical fractionation resulted in the isolation and identification of ß-sitosterol and 4-hydroxybenzoic acid (4-HBA), which increased insulin secretion, GLUT4, PPARγ, and adiponectin mRNA expression, in addition to an increase in glycogen storage. 4-HBA exhibited an antihyperglycemic effect, while ß-sitosterol showed hypoglycemic effect, confirming the wide antidiabetic related results we found in our in vitro models. An in silico study revealed that 4-HBA and ß-sitosterol have potential as dual agonists on PPARγ and GPR40 receptors. Both compounds should be considered in the development of new antidiabetic drug development.

El tejido adiposo, ¿solo un almacén de energía? / Adipose Tissue, Just an Energy Storage?

Resumen Cuando los alimentos cubren los requerimientos energéticos, el organismo almacena el exceso de calorías como glucógeno en el hígado y el músculo, y los triacilgliceroles en el tejido adiposo. Morfológica y funcionalmente se clasifica en blanco y pardo. El pardo tiene gran cantidad de mitocondrias, almacena los triacilgliceroles en vacuolas y disipa la energía en forma de calor; el blanco almacena energía en gotas lipídicas que ocupan la mayor parte de su volumen. Después de la ingesta de alimentos se libera insulina, lo que hace que externen GLUT4 para absorber glucosa. Los quilomicrones o las lipoproteínas de muy baja densidad (VLDL) transportan los triacilgliceroles a los depósitos de tejido adiposo. Durante el ayuno, por acción del glucagón, se liberan enzimas que degradarán a los tri, di y monogliceroles para liberar a los ácidos grasos. El tejido adiposo libera citocinas pro y antiinflamatorias, así como leptina, adiponectina que regulan el apetito y la saciedad. La proteína cinasa activada por AMP se activa como respuesta a una baja en la cantidad de energía de la célula y le ayuda a mantener un balance energético. En el adipocito promueve la degradación de los triacilgliceroles para liberar a los ácidos grasos que se emplearán como fuente energética. Se requiere de mayor cantidad de estudios para conocer más sobre la función del tejido adiposo como regulador del metabolismo y no solo como almacén de energía.

Abstract When food meets energy requirements, the body stores in the liver and in the muscle the excess of calories as glycogen and triacylglycerols in the adipose tissue. Morphologically and functionally, it is classified into white and brown tissues. Brown tissue has many large mitochondria and stores triacylglycerols in vacuoles and dissipates energy as heat; white tissue stores energy as lipid droplets that occupy most of the adipocyte's volume. After food intake insulin is released, which causes GLUT4 externalization into the cellular membrane to absorb glucose. Chylomicrons or VLDL transport triacylglycerols to adipose tissue depots. During fasting, by the action of glucagon, enzymes are released that will degrade tri-, di- and mono-glycerols to release fatty acids. Adipose tissue releases pro and anti-inflammatory cytokines, as well as leptin and adiponectin that regulate appetite and satiety. AMPK is activated in response to a decrease in the cell's energy and helps it to maintain its energetic balance. In the adipocyte, it promotes the degradation of triacylglycerols releasing fatty acids to be used as an energy source. More studies are needed to learn more about the function of adipose tissue as a regulator of the metabolism and not only as an energy storage.

Effects of Moderate-Intensity Physical Training on Skeletal Muscle Substrate Transporters and Metabolic Parameters of Ovariectomized Rats.

A deficit of estrogen is associated with energy substrate imbalance, raising the risk of metabolic diseases. Physical training (PT) is a potent metabolic regulator through oxidation and storage of substrates transported by GLUT4 and FAT CD36 in skeletal muscle. However, little is known about the effects of PT on these carriers in an estrogen-deficit scenario. Thus, the aim of this study was to determine the influence of 12 weeks of PT on metabolic variables and GLUT4 and FAT CD36 expression in the skeletal muscle of animals energetically impaired by ovariectomy (OVX). The trained animals swam 30 min/day, 5 days/week, at 80% of the critical load intensity. Spontaneous physical activity was measured biweekly. After training, FAT CD36 and GLUT4 expressions were quantified by immunofluorescence in the soleus, as well as muscular glycogen and triglyceride of the soleus, gluteus maximus and gastrocnemius. OVX significantly reduced FAT CD36, GLUT4 and spontaneous physical activity (p < 0.01), while PT significantly increased FAT CD36, GLUT4 and spontaneous physical activity (p < 0.01). PT increased soleus glycogen, and OVX decreased muscular triglyceride of gluteus maximus. Therefore, OVX can cause energy disarray through reduction in GLUT4 and FAT CD36 and their muscle substrates and PT prevented these metabolic consequences, masking ovarian estrogen's absence.

Combination of Aerobic Training and Cocoa Flavanols as Effective Therapies to Reduce Metabolic and Inflammatory Disruptions in Insulin-Resistant Rats: The Exercise, Cocoa, and Diabetes Study.

We aimed to investigate the combined effects of aerobic exercise (EXE) and cocoa flavanol (COCOA) supplementation on performance, metabolic parameters, and inflammatory and lipid profiles in obese insulin-resistant rats. Therefore, 32 male Wistar rats (230-250 g) were fed a high-fat diet and a fructose-rich beverage for 30 days to induce insulin resistance. Next, the rats were randomized into four groups, orally administered placebo solution or COCOA supplementation (45 mg·kg-1), and either remained sedentary or were subjected to EXE on a treadmill at 60% peak velocity for 30 min, for 8 weeks. Blood samples and peripheral tissues were collected and processed to analyze metabolic and inflammatory parameters, lipid profiles, and morphological parameters. Supplementation with COCOA and EXE improved physical performance and attenuated body mass gain, adipose index, and adipocyte area. When analyzed as individual interventions, supplementation with COCOA and EXE improved glucose intolerance and the lipid profile reduced the concentrations of leptin, glucose, and insulin, and reduced homeostasis assessment index (all effects were p < .001 for both interventions), while ameliorated some inflammatory mediators in examined tissues. In skeletal muscles, both COCOA supplementation and EXE increased the expression of glucose transporter (p < .001 and p < .001), and combined intervention showed additive effects (p < .001 vs. COCOA alone or EXE alone). Thus, combining COCOA with EXE represents an effective nonpharmacological strategy to treat insulin resistance; it could prevent Type 2 diabetes mellitus by improving physical performance, glucose metabolism, neuroendocrine control, and lipid and inflammatory mediators in the liver, pancreas, adipose tissue, and skeletal muscle in obese male insulin-resistant rats.

Activation of the NLRP3 Inflammasome Increases the IL-1ß Level and Decreases GLUT4 Translocation in Skeletal Muscle during Insulin Resistance.

Low-grade chronic inflammation plays a pivotal role in the pathogenesis of insulin resistance (IR), and skeletal muscle has a central role in this condition. NLRP3 inflammasome activation pathways promote low-grade chronic inflammation in several tissues. However, a direct link between IR and NLRP3 inflammasome activation in skeletal muscle has not been reported. Here, we evaluated the NLRP3 inflammasome components and their role in GLUT4 translocation impairment in skeletal muscle during IR. Male C57BL/6J mice were fed with a normal control diet (NCD) or high-fat diet (HFD) for 8 weeks. The protein levels of NLRP3, ASC, caspase-1, gasdermin-D (GSDMD), and interleukin (IL)-1ß were measured in both homogenized and isolated fibers from the flexor digitorum brevis (FDB) or soleus muscle. GLUT4 translocation was determined through GLUT4myc-eGFP electroporation of the FBD muscle. Our results, obtained using immunofluorescence, showed that adult skeletal muscle expresses the inflammasome components. In the FDB and soleus muscles, homogenates from HFD-fed mice, we found increased protein levels of NLRP3 and ASC, higher activation of caspase-1, and elevated IL-1ß in its mature form, compared to NCD-fed mice. Moreover, GSDMD, a protein that mediates IL-1ß secretion, was found to be increased in HFD-fed-mice muscles. Interestingly, MCC950, a specific pharmacological NLRP3 inflammasome inhibitor, promoted GLUT4 translocation in fibers isolated from the FDB muscle of NCD- and HFD-fed mice. In conclusion, we found increased NLRP3 inflammasome components in adult skeletal muscle of obese insulin-resistant animals, which might contribute to the low-grade chronic metabolic inflammation of skeletal muscle and IR development.

Biological activity of 2α,3ß,23-trihydroxyolean-12-ene on glucose homeostasis.

We studied the effect and the mechanisms of action of 2α,3ß,23-trihydroxyolean-12-ene (THO), from Croton heterodoxus Baill. (Euphorbiaceae), in glucose uptake in hyperglycemic rats. The effect of in vivo pretreatment with THO in hyperglycemic rats was analyzed. The in vitro effects of THO were observed in adipocytes and in adipose tissue. THO reduced glycemia, in part by increasing serum insulin and augmenting the disposal of glucose as glycogen in hepatocytes but did not change the serum concentration of glucagon-like peptide-1. THO increased glucose uptake in adipocytes and in adipose tissue by a mechanism dependent on phosphatidylinositol 3-kinase vesicular traffic and on the process of vesicle fusion at the plasma membrane in regions containing cholesterol, indicating the involvement of glucose transporter-4 (GLUT4). This triterpene may act solely via the activation and translocation of GLUT4 (rather than via nuclear actions, such as upregulation of GLUT4 synthesis), since THO did not alter the amount of GLUT4 mRNA or the content of GLUT4. Consistent with these data, the stimulatory effect of this triterpene on the quantity of GLUT4 in the membrane fraction was dependent upon p38 phosphorylation. In this experimental model, orally administered 10 mg/kg THO did not modulate extracellular serum lactate dehydrogenase. In conclusion, THO decreases hyperglycemia by increasing serum insulin and hepatic glycogen content. The THO mechanism of action on adipose tissue for glucose uptake is suggested to be via GLUT4 translocation stimulation mediated by a p38-dependent mechanism. THO is a potential antihyperglycemic agent that acts in a target tissue for glucose homeostasis.

Complexin-2 redistributes to the membrane of muscle cells in response to insulin and contributes to GLUT4 translocation.

Insulin stimulates glucose uptake in muscle cells by rapidly redistributing vesicles containing GLUT4 glucose transporters from intracellular compartments to the plasma membrane (PM). GLUT4 vesicle fusion requires the formation of SNARE complexes between vesicular VAMP and PM syntaxin4 and SNAP23. SNARE accessory proteins usually regulate vesicle fusion processes. Complexins aide in neuro-secretory vesicle-membrane fusion by stabilizing trans-SNARE complexes but their participation in GLUT4 vesicle fusion is unknown. We report that complexin-2 is expressed and homogeneously distributed in L6 rat skeletal muscle cells. Upon insulin stimulation, a cohort of complexin-2 redistributes to the PM. Complexin-2 knockdown markedly inhibited GLUT4 translocation without affecting proximal insulin signalling of Akt/PKB phosphorylation and actin fiber remodelling. Similarly, complexin-2 overexpression decreased maximal GLUT4 translocation suggesting that the concentration of complexin-2 is finely tuned to vesicle fusion. These findings reveal an insulin-dependent regulation of GLUT4 insertion into the PM involving complexin-2.

Estrogen and Glycemic Homeostasis: The Fundamental Role of Nuclear Estrogen Receptors ESR1/ESR2 in Glucose Transporter GLUT4 Regulation.

Impaired circulating estrogen levels have been related to impaired glycemic homeostasis and diabetes mellitus (DM), both in females and males. However, for the last twenty years, the relationship between estrogen, glycemic homeostasis and the mechanisms involved has remained unclear. The characterization of estrogen receptors 1 and 2 (ESR1 and ESR2) and of insulin-sensitive glucose transporter type 4 (GLUT4) finally offered a great opportunity to shed some light on estrogen regulation of glycemic homeostasis. In this manuscript, we review the relationship between estrogen and DM, focusing on glycemic homeostasis, estrogen, ESR1/ESR2 and GLUT4. We review glycemic homeostasis and GLUT4 expression (muscle and adipose tissues) in Esr1-/- and Esr2-/- transgenic mice. We specifically address estradiol-induced and ESR1/ESR2-mediated regulation of the solute carrier family 2 member 4 (Slc2a4) gene, examining ESR1/ESR2-mediated genomic mechanisms that regulate Slc2a4 transcription, especially those occurring in cooperation with other transcription factors. In addition, we address the estradiol-induced translocation of ESR1 and GLUT4 to the plasma membrane. Studies make it clear that ESR1-mediated effects are beneficial, whereas ESR2-mediated effects are detrimental to glycemic homeostasis. Thus, imbalance of the ESR1/ESR2 ratio may have important consequences in metabolism, highlighting that ESR2 hyperactivity assumes a diabetogenic role.