Insulin-induced Glucose Uptake Research Articles

Anti-Inflammatory and Anti-Adipocyte Dysfunction Effects of Ficus lindsayana Latex and Root Extracts.

Low-grade chronic inflammation and adipocyte dysfunction are prominent risk factors of insulin resistance and type 2 diabetes mellitus (T2DM) in obesity. Thus, prevention of inflammation and adipocyte dysfunction could be one possible approach to mitigate T2DM development. Several Ficus species have been used in traditional medicine for ameliorating inflammation and T2DM. Our previous studies reported biological effects of Ficus lindsayana including antioxidant, anti-cancer, and anti-α-glucosidase activities. Further, this study therefore investigated whether F. lindsayana latex (FLLE) and root (FLRE) extracts inhibit inflammation-stimulated insulin resistance in adipocytes and inflammation in macrophages. FLLE and FLRE (200 µg/mL) had no significant cytotoxicity for macrophages, adipocytes, and blood cells (PBMCs and RBCs). FLRE had a total flavonoid content about three times higher than FLLE, while both had similar levels of total phenolic content. FLRE showed higher abilities than FLLE in suppressing inflammation in both macrophages and adipocytes and reversing the inflammation-induced insulin resistance in adipocytes. In TNF-α-induced adipocytes, FLRE significantly improved insulin-induced glucose uptake and insulin-suppressed lipolysis, while FLLE only significantly improved glucose uptake. Moreover, FLRE and FLLE remarkably reduced chemoattractant (MCP-1) but improved adipogenic (PPARγ and CEBPα) gene expression, leading to the promotion of adipogenesis and the suppression of insulin resistance. In LPS-induced macrophages, FLRE, but not FLLE, significantly inhibited LPS-induced NO production. Moreover, FLRE significantly reduced LPS-stimulated iNOS, COX-2, IL-1β, IL-6, and TNF-α gene expression. These results may provide the potential data for the development of this plant, especially the root part, as an alternative medicine, functional ingredient, or food supplement for the prevention of inflammation and obesity-associated insulin resistance, as well as T2DM.

Leucine-973 is a crucial residue differentiating insulin and IGF-1 receptor signaling.

Insulin and IGF-1 receptors (IR and IGF1R) are highly homologous and share similar signaling systems, but each has a unique physiological role, with IR primarily regulating metabolic homeostasis and IGF1R regulating mitogenic control and growth. Here, we show that replacement of a single amino acid at position 973, just distal to the NPEY motif in the intracellular juxtamembrane region, from leucine, which is highly conserved in IRs, to phenylalanine, the highly conserved homologous residue in IGF1Rs, resulted in decreased IRS-1/PI3K/Akt/mTORC1 signaling and increased Shc/Gab1/MAPK cell cycle signaling. As a result, cells expressing L973F-IR exhibited decreased insulin-induced glucose uptake, increased cell growth, and impaired receptor internalization. Mice with knockin of the L973F-IR showed similar alterations in signaling in vivo, and this led to decreased insulin sensitivity, a modest increase in growth, and decreased weight gain when mice were challenged with a high-fat diet. Thus, leucine-973 in the juxtamembrane region of the IR acts as a crucial residue differentiating IR signaling from IGF1R signaling.

25-Hydroxyvitamin D Increases Insulin-Stimulated Glucose Uptake by Enhancing Adipocyte Differentiation.

Vitamin D and its receptor (vitamin D receptor; VDR) regulate calcium homeostasis in mammals. Recently, studies have shown that serum concentrations of 25-hydroxyvitamin D (25VD) are negatively associated with insulin resistance and the incidence of type 2 diabetes. In adipose tissues, glucose transporter 4 (GLUT4) contributes to insulin-stimulated glucose uptake; however, the effect of 25VD on glucose uptake in adipocytes remains unclear. We examined the role of 25VD in glucose uptake and the differentiation of adipose-derived stromal cells. Insulin-stimulated glucose uptake in adipocytes was increased by treatment with 25VD and decreased by VDR knockdown. The expression levels of GLUT4 were upregulated by 25VD treatment. 25VD exposure increased the expression of adipocyte differentiation-related genes including peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding proteins through VDR, thereby enhancing the formation of mature adipocytes. Moreover, 25VD increased the expression levels of 11β-hydroxysteroid dehydrogenase 1 (HSD11B1), which catalyzes the conversion of cortisone to cortisol in a concentration-dependent manner. 25VD-stimulated adipocyte differentiation was suppressed by HSD11B1 knockdown. Cortisone together with 25VD enhanced adipocyte differentiation, whereas synthesized glucocorticoid dexamethasone-induced adipocyte differentiation is not promoted by 25VD. Overall, these results indicate that 25VD stimulates adipocyte differentiation through the induction of HSD11B1 expression, leading to increased insulin-induced glucose uptake in adipocytes.

Hyperuricemia contributes to glucose intolerance of hepatic inflammatory macrophages and impairs the insulin signaling pathway via IRS2-proteasome degradation.

AimNumerous reports have demonstrated the key importance of macrophage-elicited metabolic inflammation in insulin resistance (IR). Our previous studies confirmed that hyperuricemia or high uric acid (HUA) treatment induced an IR state in several peripheral tissues to promote the development of type 2 diabetes mellitus (T2DM). However, the effect of HUA on glucose uptake and the insulin sensitivity of macrophages and its mechanism is unclear.MethodsTo assess systemic IR, we generated hyperuricemic mice by urate oxidase knockout (UOX-KO). Then, glucose/insulin tolerance, the tissue uptake of 18F-fluorodeoxyglucose, body composition, and energy balance were assessed. Glucose uptake of circulating infiltrated macrophages in the liver was evaluated by glucose transporter type 4 (GLUT-4) staining. Insulin sensitivity and the insulin signaling pathway of macrophages were demonstrated using the 2-NBDG kit, immunoblotting, and immunofluorescence assays. The immunoprecipitation assay and LC-MS analysis were used to determine insulin receptor substrate 2 (IRS2) levels and its interacting protein enrichment under HUA conditions.ResultsCompared to WT mice (10 weeks old), serum uric acid levels were higher in UOX-KO mice (WT, 182.3 ± 5.091 μM versus KO, 421.9 ± 45.47 μM). Hyperuricemic mice with metabolic disorders and systemic IR showed inflammatory macrophage recruitment and increased levels of circulating proinflammatory cytokines. HUA inhibited the nuclear translocation of GLUT-4 in hepatic macrophages, restrained insulin-induced glucose uptake and glucose tolerance, and blocked insulin IRS2/PI3K/AKT signaling. Meanwhile, HUA mediated the IRS2 protein degradation pathway and activated AMPK/mTOR in macrophages. LC-MS analysis showed that ubiquitination degradation could be involved in IRS2 and its interacting proteins to contribute to IR under HUA conditions.ConclusionThe data suggest that HUA-induced glucose intolerance in hepatic macrophages contributed to insulin resistance and impaired the insulin signaling pathway via IRS2-proteasome degradation

Rumen-protected methionine during heat stress alters mTOR, insulin signaling, and 1-carbon metabolism protein abundance in liver, and whole-blood transsulfuration pathway genes in Holstein cows

We investigated effects of rumen-protected Met (RPM) during a heat stress (HS) challenge on (1) hepatic abundance of mTOR, insulin, and antioxidant signaling proteins, (2) enzymes in 1-carbon metabolism, and (3) innate immunity. Holstein cows (n = 32; mean ± standard deviation, 184 ± 59 d in milk) were randomly assigned to 1 of 2 environmental groups, and 1 of 2 diets [total mixed ration (TMR) with RPM (Smartamine M; 0.105% dry matter as top-dress) or TMR without (CON); n = 16/diet] in a split-plot crossover design. There were 2 periods with 2 phases. During phase 1 (9 d), all cows were in thermoneutral conditions (TN; temperature-humidity index = 60 ± 3) and fed ad libitum. During phase 2 (9 d), half the cows (n = 8/diet) were exposed to HS using electric heat blankets. The other half (n = 8/diet) remained in TN, but was pair-fed to HS counterparts. After a 14-d washout and 7-d adaptation period, the study was repeated (period 2) and environmental treatments were inverted relative to phase 2, but dietary treatments were the same. Blood was collected on d 6 of each phase 2 to measure immune function and isolate whole-blood RNA. Liver biopsies were performed at the end of each period for cystathione β-synthase (CBS) and methionine adenosyltransferase activity, glutathione concentration, and protein abundance. Data were analyzed using PROC MIXED in SAS. Abundance of CUL3, inhibitor of antioxidant responses, tended to be downregulated by HS suggesting increased oxidative stress. Heat-shock protein 70 abundance was upregulated by HS. Phosphorylated mTOR abundance was greater overall with RPM, suggesting an increase in pathway activity. An environment × diet (E × D) effect was observed for protein kinase B (AKT), whereas there was a tendency for an interaction for phosphorylated AKT. Abundance of AKT was upregulated in CON cows during HS versus TN, this was not observed in RPM cows. For phosphorylated AKT, tissue from HS cows fed CON had greater abundance compared with all other treatments. The same effect was observed for EIF2A (translation initiation) and SLC2A4 (insulin-induced glucose uptake). An E × D effect was observed for INSR due to upregulation in CON cows during HS versus TN cows fed CON or RPM. There was an E × D effect for CBS, with lower activity in RPM versus CON cows during HS. The CON cows tended to have greater CBS during HS versus TN. An E × D effect was observed for methionine adenosyltransferase, with lower activity in RPM versus CON during HS. Although activity increased in CON during HS versus TN, RPM cows tended to have greater activity during TN. Neutrophil and monocyte oxidative burst and monocyte phagocytosis decreased with HS. An (E × D) effect was observed for whole-blood mRNA abundance of CBS, SOD1 and CSAD; RPM led to upregulation during TN versus HS. Regardless of diet, CDO1, CTH, and SOD1 decreased with HS. Although HS increased hepatic HSP70 and seemed to alter antioxidant signaling, feeding RPM may help cows maintain homeostasis in mTOR, insulin signaling, and 1-carbon metabolism. Feeding RPM also may help maintain whole-blood antioxidant response during HS, which is an important aspect of innate immune function.

The Exocyst Is Required for CD36 Fatty Acid Translocase Trafficking and Free Fatty Acid Uptake in Skeletal Muscle Cells.

In obesity, chronic membrane-localization of CD36 free fatty acid (FFA) translocase, but not other FFA transporters, enhances FFA uptake and intracellular lipid accumulation. This ectopic lipid accumulation promotes insulin resistance by inhibiting insulin-induced GLUT4 glucose transporter trafficking and glucose uptake. GLUT4 and CD36 cell surface delivery is triggered by insulin- and contraction-induced signaling, which share conserved downstream effectors. While we have gathered detailed knowledge on GLUT4 trafficking, the mechanisms regulating CD36 membrane delivery and subsequent FFA uptake in skeletal muscle are not fully understood. The exocyst trafficking complex facilitates the docking of membrane-bound vesicles, a process underlying the controlled surface delivery of fuel transporters. The exocyst regulates insulin-induced glucose uptake via GLUT4 membrane trafficking in adipocytes and skeletal muscle cells and plays a role in lipid uptake in adipocytes. Based on the high degree of conservation of the GLUT4 and CD36 trafficking mechanisms in adipose and skeletal muscle tissue, we hypothesized that the exocyst also contributes to lipid uptake in skeletal muscle and acts through the targeted plasma membrane delivery of CD36 in response to insulin and contraction. Here, we show that the exocyst complex is necessary for insulin- and contraction-induced CD36 membrane trafficking and FFA uptake in muscle cells.

203-LB: The Role of the Exocyst Trafficking Complex in CD36 Fatty Acid Translocase Trafficking in Skeletal Muscle Cells

Skeletal muscle free fatty acid (FFA) uptake correlates with the amount of fatty acid translocase CD36 on the cell surface. In obesity, chronic membrane-localization of CD36, but not other FFA transporters, augments FFA uptake and intracellular lipid accumulation. This ectopic lipid accumulation then causes insulin resistance (IR) by inhibiting GLUT4 glucose transporter trafficking and glucose uptake in response to insulin. GLUT4 and CD36 cell-surface delivery is stimulated by insulin and contraction signaling, sharing conserved downstream effectors. The eight-protein exocyst complex is responsible for the targeted docking of membrane-bound vesicles, a process underlying cell-surface delivery of fuel transporters. The exocyst indeed regulates insulin-induced glucose uptake via GLUT4 trafficking to the membrane in adipocytes and skeletal muscle. In addition, it controls lipid uptake in adipocytes, but we do not know which of the FFA-transporting proteins the exocyst targets during this process. Based on the high degree of conservation of the fuel transporter trafficking mechanisms in adipose and skeletal muscle tissue, we hypothesized that the exocyst contributes to lipid uptake in skeletal muscle via the targeted trafficking of CD36. Our data shows that the exocyst plays a role in FFA uptake in skeletal muscle cells. Using an L6 GLUT4myc myoblast model we demonstrate that the exocyst is recruited to CD36 vesicles upon insulin and contraction signaling. We also show that disrupted exocyst activity leads to an impaired CD36 surface delivery and decreased FFA uptake in skeletal myoblasts. We are the first to demonstrate that the exocyst is necessary for insulin- and contraction-induced CD36 membrane trafficking and FFA metabolism in muscle cells. Further studies will continue to investigate the mechanism of exocyst-regulated FFA uptake and lipid metabolism in skeletal muscle to help our understanding of ectopic lipid accumulation leading to IR. Disclosure N. K. Nakamura: None. D. S. Tokunaga: None. H. Y. J. Ha: None. N. Polgar: None.

Angiotensin II Inhibits Insulin Receptor Signaling in Adipose Cells

Angiotensin II (Ang II) is a critical regulator of insulin signaling in the cardiovascular system and metabolic tissues. However, in adipose cells, the regulatory role of Ang II on insulin actions remains to be elucidated. The effect of Ang II on insulin-induced insulin receptor (IR) phosphorylation, Akt activation, and glucose uptake was examined in 3T3-L1 adipocytes. In these cells, Ang II specifically inhibited insulin-stimulated IR and insulin receptor substrate-1 (IRS-1) tyrosine-phosphorylation, Akt activation, and glucose uptake in a time-dependent manner. These inhibitory actions were associated with increased phosphorylation of the IR at serine residues. Interestingly, Ang II-induced serine-phosphorylation of IRS was not detected, suggesting that Ang II-induced desensitization begins from IR regulation itself. PKC inhibition by BIM I restored the inhibitory effect of Ang II on insulin actions. We also found that Ang II promoted activation of several PKC isoforms, including PKCα/βI/βII/δ, and its association with the IR, particularly PKCβII, showed the highest interaction. Finally, we also found a similar regulatory effect of Ang II in isolated adipocytes, where insulin-induced Akt phosphorylation was inhibited by Ang II, an effect that was prevented by PKC inhibitors. These results suggest that Ang II may lead to insulin resistance through PKC activation in adipocytes.

Hydrogen sulfide disrupts insulin-induced glucose uptake in L6 skeletal muscle cells

Hydrogen sulfide (H2S) has been known for its toxicity. However, recent studies have focused on the mechanisms involved in endogenous production and function. To date, the H2S role in insulin signaling and glucose homeostasis is unclear. This uncertainty is even more evident in skeletal muscle, a physiological niche highly relevant for regulating glycemia in response to insulin. This study aimed to investigate the role of H2S on insulin signaling and glucose uptake in the L6 skeletal muscle cell line. We evaluated the endogenous synthesis with the fluorescent dye, 7-azido-4-methyl coumarin (7-AzMC). Glucose restriction-induced an increase in the endogenous levels of H2S, likely through stimulation of cystathionine γ-lyase activity, as its specific inhibitor, PAG (5 mM) prevented this increase, and mRNA levels of CSE decreased with glucose and amino acid restriction. Exogenous H2S reduced insulin-induced glucose uptake at 0.5 up to 24 h, an effect dissociated from the level of Akt phosphorylation. Our results show that glucose restriction induces endogenous production of H2S via CSE. In addition, H2S disrupts insulin-induced glucose uptake independent of the Akt pathway. These results suggest that H2S antagonism over insulin-induced glucose uptake could help maintain the plasmatic glucose levels in conditions that provoke hypoglycemia, which could serve as an H2S-regulated mechanism for maintaining glucose plasmatic levels through the inhibition of the skeletal muscle insulin-depended glucose uptake.

Effect of Exogenous Hydrogen Sulfide and Polysulfide Donors on Insulin Sensitivity of the Adipose Tissue.

Hydrogen sulfide (H2S) and inorganic polysulfides are important signaling molecules; however, little is known about their role in adipose tissue. We examined the effect of H2S and polysulfides on insulin sensitivity of the adipose tissue in rats. Plasma glucose, insulin, non-esterified fatty acids, and glycerol were measured after administration of H2S and the polysulfide donors, Na2S and Na2S4, respectively. In addition, the effect of Na2S and Na2S4 on insulin-induced glucose uptake and inhibition of lipolysis was studied in adipose tissue explants ex vivo. Na2S and Na2S4 administered in vivo at a single dose of 100 μmol/kg had no effect on plasma glucose and insulin concentrations. In addition, Na2S and Na2S4 did not modify the effect of insulin on plasma glucose, fatty acids, and glycerol concentrations. Na2S and Na2S4had no effect on the antilipolytic effect of insulin in adipose tissue explants ex vivo. The effect of insulin on 2-deoxyglucose uptake by adipose tissue was impaired in obese rats which was accompanied by lower insulin-induced tyrosine phosphorylation of IRS-1 and Akt. Na2S4, but not Na2S, improved insulin signaling and increased insulin-stimulated 2-deoxyglucose uptake by adipose tissue of obese rats. The results suggest that polysulfides may normalize insulin sensitivity, at least in the adipose tissue, in obesity/metabolic syndrome.

Fasting potentiates insulin-mediated glucose uptake in rested and prior-contracted rat skeletal muscle.

A single bout of exercise can potentiate the effect of insulin on skeletal muscle glucose uptake via activation of the AMPK-TBC1 domain family member 4 (TBC1D4) pathway, which suggests a positive correlation between AMPK activation and insulin sensitization. In addition, prolonged fasting in rodents is known to upregulate and thereby synergistically enhance the effect of exercise on muscle AMPK activation. Therefore, fasting may potentiate the insulin-sensitizing effect of exercise. In the present study, we mimicked exercise by in situ muscle contraction and evaluated the effect of a 36-h fast on muscle contraction-induced insulin sensitization. Male Wistar rats weighing 150-170 g were allocated to either a 36-h fasting or feeding group. The extensor digitorum longus (EDL) muscles were electrically contracted via the common peroneal nerve for 10 min followed by a 3-h recovery period. EDL muscles were dissected and incubated in the presence or absence of submaximal insulin. Our results demonstrated that acute muscle contraction and 36 h of fasting additively upregulated AMPK pathway activation. Insulin-stimulated muscle glucose uptake and site-specific TBC1D4 phosphorylation were enhanced by prior muscle contraction in 36-h-fasted rats, but not in fed rats. Moreover, enhanced insulin-induced muscle glucose uptake and Akt phosphorylation due to 36 h of fasting were associated with a decrease in tribbles homolog 3 (TRB3), a negative regulator of Akt activation. In conclusion, fasting and prior muscle contraction synergistically enhance insulin-stimulated TBC1D4 phosphorylation and glucose uptake, which is associated with augmented AMPK pathway activation in rodents.NEW & NOTEWORTHY In this study, we revealed that 36 h of fasting additively upregulated acute muscle contraction-induced AMPK pathway activation in rats. Besides, fasting and muscle contraction synergistically enhanced insulin-stimulated site-specific TBC1D4 phosphorylation and glucose uptake, which was associated with augmented AMPK pathway activation. These results contribute to understanding the regulation of muscle insulin sensitivity.

Peripheral Dopamine Directly Acts on Insulin-Sensitive Tissues to Regulate Insulin Signaling and Metabolic Function

Dopamine is a key regulator of glucose metabolism in the central nervous system. However, dopamine is also present in the periphery and may have direct effects on insulin-sensitive tissues. Dopamine receptor 2 (D2R) agonist bromocriptine is a FDA-approved drug for type 2 diabetes. Herein, we explored the role of peripheral dopamine and its receptors in regulating glucose uptake and metabolism on insulin-sensitive tissues. Peripheral dopamine effect in [3H]2-deoxyglucose uptake in insulin-sensitive tissues was tested in vivo in rats. Direct effects on [3H]2-deoxyglucose uptake, insulin receptor phosphorylation, and regulation of metabolic function were tested ex vivo in the liver, soleus muscle, and white and brown adipose tissues. Bromocriptine and the antagonists domperidone, D2R antagonist, and haloperidol, antagonist of both dopamine receptor 1 (D1R) and D2R, were used to disclose dopamine receptors’ involvement.Peripheral dopamine increases glucose uptake in vivo. Ex vivo, only dopamine increased glucose uptake in the soleus, while bromocriptine increased it in the liver; the effects were reverted by haloperidol and domperidone, respectively. In adipose tissue, domperidone reverted dopamine- and bromocriptine-mediated potentiation of insulin-induced glucose uptake, but in turn increased the insulin receptor, Akt, AMPK, HSL, ACC, and ACL, phosphorylation. In the soleus muscle, AMPK-phosphorylation increased with bromocriptine and dopamine whose effects were suppressed by domperidone and haloperidol.In conclusion, peripheral dopamine stimulates glucose uptake with its receptors being differentially involved in glucose uptake in insulin-sensitive tissues. Dopamine also has a role in lipid metabolism in white adipose tissue. Altogether, these results suggest that peripheral modulation of the dopaminergic system should be further evaluated as a putative therapeutic approach for metabolic disorders.

Activated Alpha-2 Macroglobulin Improves Insulin Response via LRP1 in Lipid-Loaded HL-1 Cardiomyocytes.

Activated alpha-2 Macroglobulin (α2M*) is specifically recognized by the cluster I/II of LRP1 (Low-density lipoprotein Receptor-related Protein-1). LRP1 is a scaffold protein for insulin receptor involved in the insulin-induced glucose transporter type 4 (GLUT4) translocation to plasma membrane and glucose uptake in different types of cells. Moreover, the cluster II of LRP1 plays a critical role in the internalization of atherogenic lipoproteins, such as aggregated Low-density Lipoproteins (aggLDL), promoting intracellular cholesteryl ester (CE) accumulation mainly in arterial intima and myocardium. The aggLDL uptake by LRP1 impairs GLUT4 traffic and the insulin response in cardiomyocytes. However, the link between CE accumulation, insulin action, and cardiac dysfunction are largely unknown. Here, we found that α2M* increased GLUT4 expression on cell surface by Rab4, Rab8A, and Rab10-mediated recycling through PI3K/Akt and MAPK/ERK signaling activation. Moreover, α2M* enhanced the insulin response increasing insulin-induced glucose uptake rate in the myocardium under normal conditions. On the other hand, α2M* blocked the intracellular CE accumulation, improved the insulin response and reduced cardiac damage in HL-1 cardiomyocytes exposed to aggLDL. In conclusion, α2M* by its agonist action on LRP1, counteracts the deleterious effects of aggLDL in cardiomyocytes, which may have therapeutic implications in cardiovascular diseases associated with hypercholesterolemia.

High-throughput drug screening models of mature adipose tissues which replicate the physiology of patients’ Body Mass Index (BMI)

Obesity is a complex and incompletely understood disease, but current drug screening strategies mostly rely on immature in vitro adipose models which cannot recapitulate it properly. To address this issue, we developed a statistically validated high-throughput screening model by seeding human mature adipocytes from patients, encapsulated in physiological collagen microfibers. These drop tissues ensured the maintenance of adipocyte viability and functionality for controlling glucose and fatty acids uptake, as well as glycerol release. As such, patients’ BMI and insulin sensitivity displayed a strong inverse correlation: the healthy adipocytes were associated with the highest insulin-induced glucose uptake, while insulin resistance was confirmed in the underweight and severely obese adipocytes. Insulin sensitivity recovery was possible with two type 2 diabetes treatments, rosiglitazone and melatonin. Finally, the addition of blood vasculature to the model seemed to more accurately recapitulate the in vivo physiology, with particular respect to leptin secretion metabolism.

Endothelial Nox5 Expression Modulates Glucose Uptake and Lipid Accumulation in Mice Fed a High-Fat Diet and 3T3-L1 Adipocytes Treated with Glucose and Palmitic Acid.

Obesity is a global health issue associated with insulin resistance and altered lipid homeostasis. It has been described that reactive oxygen species (ROS) derived from nicotinamide adenine dinucleotide phosphate (NADPH) oxidase (NOX) activity are involved in the development of these pathologies. The present study describes the role of endothelial NOX5 expression over adipose tissue by using two experimental systems: NOX5 conditional knock-in mice fed with a high-fat diet and 3T3-L1 adipocytes cultured with conditioned media of NOX5-expressing endothelial cells previously treated with glucose and palmitic acid. Animals expressing NOX5 presented lower body weight gain and less mesenteric and epididymal adipose mass compared to control mice fed with the same diet. NOX5-expressing mice also showed significantly lower glycaemia and improved insulin-induced glucose uptake. In addition, Glut4 and Caveolin 1 (Cav1) expression were significantly increased in the adipose tissue of these animals. Likewise, 3T3-L1 adipocytes treated with conditioned media from NOX5-expressing endothelial cells, incubated with high glucose and palmitic acid, presented a reduction in lipid accumulation and an increase in glucose uptake. Moreover, a significant increase in the expression of Glut4 and Cav1 was also detected in these cells. Taken together, all these data support that, in response to a highly caloric diet, NOX5 endothelial activity may regulate glucose sensitivity and lipid homeostasis in the adipose tissue.

BKCa channel participates in insulin-induced lipid deposition in adipocytes by increasing intracellular calcium.

Storing energy in the form of triglyceride (TG) is one of the basic functions of adipose tissue. Large-conductance calcium-activated potassium channels (BKCa channels) are expressed in adipose tissue and adipocyte-specific BKCa deficiency resists obesity in mice, but the role of BKCa channels in lipid deposition and the underlying mechanisms have not been elucidated. In the present study, we generated BKCa knockout (KO) rats and performed a transcriptome analysis of adipose tissue. We found that the phosphoinositide 3-kinase (PI3K)-protein kinase B (Akt) signaling pathway, which is important for lipid deposition, exhibited the most notable reduction among various signaling pathways in BKCa KO rats compared to wild-typerats. Insulin-induced TG deposition, glucose uptake, and Akt (Ser473) phosphorylation were significantly reduced in cultured adipocytes differentiated from adipose-derived stem cellsof BKCa KO rats. Furthermore, we found that the insulin-induced increase of intracellular calcium resulting from extracellular calcium influx was significantly impaired in BKCa KO adipocytes. Finally, insulin activated BKCa currents through PI3K, which was independent of Akt and intracellular calcium. The results of this study suggested that BKCa channels participate in the insulin signaling pathway and promote TG deposition by increasing extracellular calcium influx in adipocytes.

A Citrullus colocynthis fruit extract acutely enhances insulin-induced GLUT4 translocation and glucose uptake in adipocytes by increasing PKB phosphorylation

Ethnopharmacological relevanceCitrullus colocynthis (L.) Schrad is a common fruit in traditional medicine and used as remedy against various diseases, especially diabetes. Up to now, its anti-diabetic effects have been fully attributed to its enhancement of pancreatic insulin secretion. Whether C. colocynthis also ameliorates insulin action in peripheral tissues has not been investigated. Aim of the studyIn the present study, using 3T3-L1 adipocytes as cell model, we have investigated whether colocynth fruit extracts affect insulin action. Materials and methodsVarious extracts were prepared from the C. colocynthis fruit and screened using a cell-based 96 well plate GLUT4 translocation assay. Promising extracts were further studied for their effects on glucose uptake and cell viability. The effect on insulin signal transduction was determined by Western blot and the molecular composition was established by LC-MS. ResultsThe ethyl acetate fractions of aqueous non-defatted extracts of seed and pulp, designated Sna1 and Pna1, acutely enhanced insulin-induced GLUT4 translocation. In accordance, both extracts increased insulin-stimulated cellular glucose uptake. Pna1, which displayed greater effects on GLUT4 and glucose uptake than Sna1, was further investigated and was demonstrated to increase GLUT4 translocation without changing the half-maximum dose (ED50) of insulin, nor changing GLUT4 translocation kinetics. At the molecular level, Pna1 was found to enhance insulin-induced PKB phosphorylation without changing phosphorylation of the insulin receptor. Pna1 appeared not to be toxic to cells and, like insulin, restored cell viability during serum starvation. By investigating the molecular composition of Pna1, nine compounds were identified that made up 87% of the mass of the extract, one of which is likely to be responsible for the insulin-enhancing effects of Pna1. ConclusionsThe C. colocynthis fruit possesses insulin-enhancing activity. This activity may explain in part its anti-diabetic effects in traditional medicine. It also identifies the C. colocynthis as a source of a potential novel insulin enhancer that may prove to be useful to reduce hyperglycemia in type 2 diabetes.

Psidium guajava L. leaf extract inhibits adipocyte differentiation and improves insulin sensitivity in 3T3-L1 cells.

BACKGROUND/OBJECTIVESPsidium guajava L. (guava) leaves have been shown to exhibit hypoglycemic and antidiabetic effects in rodents. This study investigated the effects of guava leaf extract on adipogenesis, glucose uptake, and lipolysis of adipocytes to examine whether the antidiabetic properties are mediated through direct effects on adipocytes.MATERIALS/METHODS3T3-L1 cells were treated with 25, 50, 100 µg/mL of methanol extract from guava leaf extract (GLE) or 0.1% dimethyl sulfoxide as a control. Lipid accumulation was evaluated with Oil Red O Staining and AdipoRed assay. Immunoblotting was performed to measure the expression of adipogenic transcription factors, fatty acid synthase (FAS), and AMP-activated protein kinase (AMPK). Glucose uptake under basal or insulin-stimulated condition was measured using a glucose analog 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxy-D-glucose. Lipolysis from fully differentiated adipocytes was measured by free fatty acids release into the culture medium in the presence or absence of epinephrine.RESULTSOil Red O staining and AdipoRed assay have shown that GLE treatment reduced lipid accumulation during adipocyte differentiation. Mitotic clonal expansion, an early essential event for adipocyte differentiation, was inhibited by GLE treatment. GLE inhibited the expression of transcription factors involved in adipocyte differentiation, such as peroxisome proliferator-activated receptor γ (PPARγ), CCAAT/enhancer-binding protein α (C/EBPα), and sterol regulatory element-binding protein-1c (SREBP-1c). FAS expression was also decreased while the phosphorylation of AMPK was increased by GLE treatment. In addition, GLE increased insulin-induced glucose uptake into adipocytes. In lipid-filled mature adipocytes, GLE enhanced epinephrine-induced lipolysis but reduced basal lipolysis dose-dependently.CONCLUSIONSThe results show that GLE inhibits adipogenesis and improves adipocyte function by reducing basal lipolysis and increasing insulin-stimulated glucose uptake in adipocytes, which can be partly associated with antidiabetic effects of guava leaves.

Effect of Pravastatin on Development of Diabetes Mellitus - A Statement of Review

According to the ADA, diabetes mellitus is defined as having fasting blood glucose levels >= 7.0mmol/l. Statins are the lipid-lowering agents used to treat dyslipidemia and act by inhibiting the HMG Co-A reductase enzyme. Very few studies have reported the results of drugs concerning the profile of blood lipids or which have anti-inflammatory activity behaviour on diabetes. Pravastatin is such a drug that shows inherent beneficial pleiotropic effects on plasma lipids and inflammatory actions. Pravastatin remarkably reduced the increased levels of plasma glucose when compared with the control group, subjecting to no change in body weight or food intake in mice. By performing certain insulin tolerance tests using tracers like 2- [3 H]-deoxy glucose (2DG), they concluded that insulin-induced glucose uptake is increased in adipose tissue. Pravastatin is shown to reduce the circulating concentration of the interleukin-6, cytokines, and TNF-alpha. Pravastatin's anti-inflammatory properties interrupt the natural progression from central obesity to insulin resistance mediated by cytokines from the adipose tissue. Pravastatin can significantly impact selective tissue perfusion through restoring the endothelial function and thus benefit the transportation of glucose and insulin.

ITRAQ- and LC-MS/MS-based quantitative proteomics reveals Pqlc2 as a potential regulator of hepatic glucose metabolism and insulin signalling pathway during fasting.

Glucose homeostasis is tightly controlled by balance between glucose production and uptake in liver tissue upon energy shortage condition. Altered glucose homeostasis contributes to the pathophysiology of metabolic disorders including diabetes and obesity. Here, we aimed to analyse the change of proteomic profile upon prolonged fasting in mice with isobaric tag for relative and absolute quantification (iTRAQ) labelling followed by liquid chromatography-mass spectrometry (LC/MS) technology. Adult male mice were fed or fasted for 16hours and liver tissues were collected for iTRAQ labelling followed by LC/MS analysis. A total of 322 differentially expressed proteins were identified, including 189 upregulated and 133 downregulated proteins. Bioinformatics analyses, including Gene Ontology analysis (GO), Kyoto encyclopaedia of genes and genomes analysis (KEGG) and protein-protein interaction analysis (PPI) were conducted to understand biological process, cell component, and molecular function of the 322 differentially expressed proteins. Among 322 hepatic proteins differentially expressed between fasting and fed mice, we validated three upregulated proteins (Pqlc2, Ehhadh and Apoa4) and two downregulated proteins (Uba52 and Rpl37) by western-blotting analysis. In cultured HepG2 hepatocellular cells, we found that depletion of Pqlc2 by siRNA-mediated knockdown impaired the insulin-induced glucose uptake, inhibited GLUT2 mRNA level and suppressed the insulin-induced Akt phosphorylation. By contrast, knockdown of Pqlc2 did not affect the cAMP/dexamethasone-induced gluconeogenesis. In conclusion, our study provides important information on protein profile change during prolonged fasting with iTRAQ- and LC-MS/MS-based quantitative proteomics, and identifies Pqlc2 as a potential regulator of hepatic glucose metabolism and insulin signalling pathway in this process.