Glucose Transport In 3T3-L1 Adipocytes Research Articles

DNA methylation status influences insulin-induced glucose transport in 3T3-L1 adipocytes by mediating p53 expression

Researchers frequently use 3T3-L1 adipocytes as a fat cell line, but the capacity of this line for insulin-mediated glucose transport is lower than that of primary isolated fat cells. In this study, we found that 5-azacytidine (5-aza-C), DNA methyltransferase 1 inhibitor, enhanced insulin-stimulated 2-deoxyglucose (2-DG) transport in 3T3-L1 cells after adipogenic differentiation. We next examined the expression of the genes related to glucose transport and insulin signal transduction. The insulin independent glucose transporter, glucose transporter 1 (GLUT1), showed lower expression in 5-aza-C pre-treated 3T3-L1 adipocytes, than in un-treated control adipocytes, while the expression of insulin dependent transporter GLUT4 remained unchanged. In addition, insulin receptor substrate-1 (IRS-1) was highly expressed in 5-aza-C pre-treated adipocytes. Based on DNA microarray and functional annotation analysis, we noticed that 5-aza-C pretreatment activated the tumor suppressor p53 pathway. We confirmed that in 5-aza-C adipocytes, p53 expression was markedly higher, and the methylation level of CpGs in its promoter region was lower than in un-treated control adipocytes. Moreover, pharmacological inhibition of p53 restored GLUT1 and IRS-1 expression to the same level as in un-treated 3T3-L1 adipocytes, and significantly decreased insulin-mediated 2-DG uptake. These results suggest that glucose transport capacity in adipocytes is influenced by DNA methylation status, and demethylation induced by 5-aza-C increased it possibly through the p53 signaling pathway.

Knockdown of long noncoding RNA dreh facilitates cell surface GLUT4 expression and glucose uptake through the involvement of vimentin in 3T3-L1 adipocytes

Glucose uptake in adipocytes is crucial for regulating systemic metabolism. Long noncoding RNAs (lncRNAs), defined as being transcripts with lengths exceeding 200 nucleotides that are not translated, are recently identified regulators of cellular functions. Previously, we have shown that an lncRNA, “down-regulated expression by hepatitis B virus X” (dreh), is involved in glucose transport in skeletal muscle cells. Here, we aimed to examine the involvement of dreh in glucose transport in 3T3-L1 adipocytes. Expression analysis showed that dreh was expressed in 3T3-L1 fibroblasts and adipocytes. Knockdown of dreh expression using its specific siRNAs lowered the glucose concentration of the medium and facilitated [3H]-2-deoxyglucose transport in adipocytes. Additionally, dreh silencing enhanced the protein expression of glucose transporter (GLUT4) in the plasma membrane of adipocytes. Treatment with siRNA against vimentin attenuated the glucose-lowering effect of dreh depletion. These results suggest that the repression of dreh facilitates glucose transport via increased GLUT4 expression in the plasma membrane through the involvement of vimentin in 3T3-L1 adipocytes. In conclusion, dreh is the first observed lncRNA that regulates glucose transport in adipocytes and could serve as a novel therapeutic target for diabetes by modulating adipocyte function. Considering the new function of dreh, we propose that dreh be renamed “down-regulated expression-related hexose/glucose transport enhancer.”

Effects and mechanisms of iridoid glycosides from Patrinia scabiosaefolia on improving insulin resistance in 3T3-L1 adipocytes

Insulin resistance causes several adverse effects such as hypertension, diabetes and different aspects of cardiovascular diseases. Patrinia scabiosaefolia Fisch. ex Trev. is a traditional Chinese edible herbal, whose n-BuOH extract significantly increased glucose transportin 3T3-L1 adipocytes at the concentration of 12.5 μM. To determine its active constituent, its chemical components and bioactivities were investigated. Two compounds (1–2) could significantly improve insulin resistance in 3T3-L1 adipocytes at concentrations around 25.0 μM (P < 0.001). Compound 2 was more effective to lower the content of glucose content at 12.5 μM (P < 0.001). Compound 1 was a new compound identified by spectroscopic methods. Western-blot experiment demonstrated an upregulation of p-IRS-1, p-Akt, and GLUT4 induced by compounds 1 and 2. Hence, we speculated that compounds 1 and 2 could activate PI3K and Akt signaling by up-regulating of p-IRS-1 which resulted in the activation of PI3K before phosphorylating Akt, ultimately led to translocation of GLUT4. These events finally improve glucose transport. Our results may provide the scientific basis for the development and effective use of P. scabiosaefolia against type 2 diabetes.

Aster koraiensis extract lowers postprandial glucose in normoglycemic and high-fat-diet-induced obese mice.

The Aster koraiensis extract (ASKO) is a newly developed dietary herbal supplement. In this study, the potent blood glucose-lowering activity of ASKO in vitro and in vivo was investigated. In an in vitro glucose uptake assay, ASKO was found to enhance glucose transport in 3T3-L1 adipocytes. Oral administration of ASKO significantly reduced glucose levels in normoglycemic mice during oral glucose tolerance tests (OGTTs). In a long-term efficacy study, 4weeks of oral ASKO treatment significantly attenuated blood glucose levels during OGTTs in diet-induced obese (DIO) mice. ASKO also enhanced plasma insulin levels after glucose loading, leading to a reduction in blood glucose levels. In addition, ASKO normalized glucose transporter-4 mRNA expression in the muscles of DIO mice. These results indicate that ASKO has postprandial glucose-lowering effects and could be beneficial in the management of prediabetes or type 2 diabetes mellitus.

β-catenin is important for the development of an insulin responsive pool of GLUT4 glucose transporters in 3T3-L1 adipocytes

GLUT4 is unique among specialized glucose transporters in being exclusively expressed in muscle and adipocytes. In the absence of insulin the distribution of GLUT4 is preferentially intracellular and insulin stimulation results in the movement of GLUT4 containing vesicles to the plasma membrane. This process is responsible for the insulin stimulation of glucose uptake in muscle and fat. While signalling pathways triggering the translocation of GLUT4 are well understood, the mechanisms regulating the intracellular retention of GLUT4 are less well understood. Here we report a role for β-catenin in this process. In 3T3-L1 adipocytes in which β-catenin is depleted, the levels of GLUT4 at and near the plasma membrane rise in unstimulated cells while the subsequent increase in GLUT4 at the plasma membrane upon insulin stimulation is reduced. Small molecule approaches to acutely activate or inhibit β-catenin give results that support the results obtained with siRNA and these changes are accompanied by matching changes in glucose transport into these cells. Together these results indicate that β-catenin is a previously unrecognized regulator of the mechanisms that control the insulin sensitive pool of GLUT4 transporters inside these adipocyte cells.

In vitro modulation of pancreatic insulin secretion, extrapancreatic insulin action and peptide glycation by Curcuma longa aqueous extracts

Objective: Medicinal, edible, and aromatic plants have been used as folk remedies in traditional treatments worldwide. This study investigates the antidiabetic efficacy and action mode of Curcuma longa Linn. (Zingiberaceae). Methods: Effects of aqueous extracts (AEs) of C. longa on insulin secretion and action were studied using the insulin-secreting BRIN-BD11 and the adipocyte-like 3T3-L1 cell lines, respectively. In vitro models were employed to evaluate effects on starch digestion using α-amylase/amyloglucosidase and protein glycation. Results: C. longa AEs stimulated basal insulin output and potentiated glucose-stimulated insulin secretion concentration-dependently in the clonal pancreatic beta cell line, BRIN-BD11 (P < 0.001). The insulin secretory activity of plant extract was abolished in the absence of extracellular Ca 2+ and by inhibitors of cellular Ca 2+ uptake, diazoxide and verapamil (P < 0.001). Furthermore, the extract increased insulin secretion in depolarized cells and augmented insulin secretion triggered by 3-isobutyl-1-methylxanthine, tolbutamide, and glibenclamide. C. longa AEs lacked insulin mimetic activity but enhanced insulin-stimulated glucose transport in 3T3-L1 adipocytes by 370% (P < 0.001). Similar to aminoguanidine, C. longa AEs (1-50 mg/ml) effected concentration-dependent inhibition of protein glycation (24-70% inhibition, P < 0.001) in vitro. In bioassays of enzymatic starch digestion, C. longa AEs lacked inhibitory effects on α-amylase and α-glucosidase, unlike acarbose, the classical reference drug. Conclusion: This study has revealed that water soluble bioactive principles in C. longa AEs stimulate basal- and potentiate glucose evoked-insulin secretion, enhance insulin action and inhibit insulin glycation, but not starch digestion. Future work assessing the use of C. longa AEs as dietary adjunct or as a source of active antidiabetic agents may provide new opportunities for the combinatorial treatment/prevention of diabetes.

Proteomic study on the protective mechanism of fibroblast growth factor 21 to ischemia–reperfusion injury

Fibroblast growth factor (FGF)-21 is a novel regulator of insulin-independent glucose transport in 3T3-L1 adipocytes and has glucose and triglyceride lowering effects in rodent models of diabetes. In this study, we found that FGF-21 can significantly attenuate ischemia-reperfusion (I/R) induced damage in H9c2 cells (rat heart). However, it is unclear which signal transduction pathway is involved in the cardioprotective effect of FGF-21. Thus, this study was designed to investigate the potential mechanism induced by FGF-21. The results showed that FGF-21 treatment prevented the oxidative stress and apoptosis associated with I/R damage by reducing the levels of superoxide anions, inhibiting glycogen synthase kinase (GSK) 3β by activating Akt phosphorylation, and recovering the levels of ATP synthase pyruvate kinase isozymes M1 and protein kinase C, thereby improving energy supply. In summary, we conclude that FGF-21 protects H9c2 cells against I/R injury mainly through the Akt-GSK-3β-caspase-3 dependent pathway, preventing oxidative stress, and recovery of the energy supply.

Regulation of Glucose Transport by ROCK1 Differs from That of ROCK2 and Is Controlled by Actin Polymerization

A role of Rho-associated coiled-coil-containing protein kinase (ROCK)1 in regulating whole-body glucose homeostasis has been reported. However, cell-autonomous effects of ROCK1 on insulin-dependent glucose transport in adipocytes and muscle cells have not been elucidated. To determine the specific role of ROCK1 in glucose transport directly, ROCK1 expression in 3T3-L1 adipocytes and L6 myoblasts was biologically modulated. Here, we show that small interfering RNA-mediated ROCK1 depletion decreased insulin-induced glucose transport in adipocytes and myoblasts, whereas adenovirus-mediated ROCK1 expression increased this in a dose-dependent manner, indicating that ROCK1 is permissive for glucose transport. Inhibition of ROCK1 also impaired glucose transporter 4 translocation in 3T3-L1 adipocytes. Importantly, the ED₅₀ of insulin for adipocyte glucose transport was reduced when ROCK1 was expressed, leading to hypersensitivity to insulin. These effects are dependent on actin cytoskeleton remodeling, because inhibitors of actin polymerization significantly decreased ROCK1's effect to promote insulin-stimulated glucose transport. Unlike ROCK2, ROCK1 binding to insulin receptor substrate (IRS)-1 was not detected by immunoprecipitation, although cell fractionation demonstrated both ROCK isoforms localize with IRS-1 in low-density microsomes. Moreover, insulin's ability to increase IRS-1 tyrosine 612 and serine 632/635 phosphorylation was attenuated by ROCK1 suppression. Replacing IRS-1 serine 632/635 with alanine reduced insulin-stimulated phosphatidylinositol 3-kinase activation and glucose transport in 3T3-L1 adipocytes, indicating that phosphorylation of these serine residues of IRS-1, which are substrates of the ROCK2 isoform in vitro, are crucial for maximal stimulation of glucose transport by insulin. Our studies identify ROCK1 as an important positive regulator of insulin action on glucose transport in adipocytes and muscle cells.

Resistin Exacerbates Insulin Resistance under the Condition of Low Adiponectin in 3T3-L1 Adipocytes

Adipocytokines such as resistin, TNF-α, and adiponectin, which are adipocyte-derived peptides, play important roles in glucose metabolism. Resistin and TNF-α have been implicated as factors associated with the development of insulin resistance in obesity. In contrast, adiponectin has been shown to improve insulin sensitivity in insulin resistance. However, the interaction among these adipocytokines is still unknown. In this study, we investigated the mechanism of the effects of these 3 adipocytokines (resistin, adiponectin, and TNF-α) on glucose transport in 3T3-L1 adipocytes. Glucose uptake was evaluated by 2-[3H] deoxy-glucose (DOG) uptake assay in 3T3-L1 adipocytes. Resistin and adiponectin secretion were analyzed by western blotting. Adenovirus-mediated overexpression of resistin inhibited insulin-stimulated 2-DOG uptake by only 15% compared with control cells. In contrast, pretreatment of cells with 10 ng/mL TNF-α for 3 hrs did not inhibit insulin stimulated 2-DOG uptake compared with control cells, whereas overexpression of resistin led to a ~ 40% decrease in insulin stimulated 2-DOG uptake following pretreatment with TNF-α. TNF-α has been shown to suppress the expression and secretion of adiponectin from adipocytes. Therefore, we speculated that this potentiating effect of resistin might be caused by the reduction in adiponectin secretion. We confirmed that the secretion of adiponectin was decreased by ~ 50% in TNF-α treated cells compared to control cells. Furthermore, overexpression of adiponectin prevented this additive effect of resistin and TNF-α. In conclusion, these results suggest that: (1) TNF-α enhances the action of resistin via the reduction of adiponectin, 2) Resistin may cause severe insulin resistance under low adiponectin levels.

IL-6 induces lipolysis and mitochondrial dysfunction, but does not affect insulin-mediated glucose transport in 3T3-L1 adipocytes

Interleukin-6 (IL-6) has emerged as an important cytokine involved in the regulation of metabolism. However, the role of IL-6 in the etiology of obesity and insulin resistance is not fully understood. Mitochondria are key organelles of energy metabolism, and there is growing evidence that mitochondrial dysfunction plays a crucial role in the pathogenesis of obesity-associated insulin resistance. In this study, we determined the direct effect of IL-6 on lipolysis in adipocytes, and the effects of IL-6 on mitochondrial function were investigated. We found that cells treated with IL-6 displayed fewer lipids and an elevated glycerol release rate. Further, IL-6 treatment led to decreased mitochondrial membrane potential, decreased cellular ATP production, and increased intracellular ROS levels. The mitochondria in IL-6-treated cells became swollen and hollow with reduced or missing cristae. However, insulin-stimulated glucose transport was unaltered. PGC-1α, NRF1, and mtTFA mRNA levels were markedly increased, and the mitochondrial contents were also increased. Our results demonstrate that IL-6 can exert a direct lipolytic effect and induce mitochondrial dysfunction. However, IL-6 did not affect insulin sensitivity in adipocytes in vitro. We deduce that in these cells, enhanced mitochondrial biogenesis might play a compensatory role in glucose transport.

Differential regulation of NHE1 phosphorylation and glucose uptake by inhibitors of the ERK pathway and p90RSK in 3T3-L1 adipocytes

Insulin stimulates trafficking of GLUT4 to the cell surface for glucose uptake into target cells, and phosphorylation of Ser703 of the Na+/H+ exchanger NHE1, which activates proton efflux. The latter has been proposed to facilitate optimal glucose uptake into cardiomyocytes. We found that the insulin-stimulated phosphorylation of Ser703 of NHE1 is mediated by p90RSK but not directly coupled to glucose uptake in 3T3-L1 adipocytes in the short-term. Inhibiting Erk1/2 activation prevented NHE1 phosphorylation but not glucose uptake in 3T3-L1 adipocytes. In contrast, both NHE1 phosphorylation and insulin-stimulated uptake of glucose into 3T3-L1 adipocytes were blocked by inhibitors of the N-terminal kinase domain of p90RSK, namely BI-D1870 and SL0101, but not the FMK inhibitor of the C-terminal kinase domain of p90RSK, though in our hands FMK did not inhibit p90RSK in 3T3-L1 adipocytes. Further experiments were consistent with phosphorylation of AS160 by PKB/Akt mediating insulin-stimulated trafficking of GLUT4 to the plasma membrane. BI-D1870 and SL0101 however, inhibited glucose uptake without blocking GLUT4 translocation. While BI-D1870 partially inhibited insulin-stimulated PKB activation in these cells, this only partially inhibited AS160 phosphorylation and did not block GLUT4 trafficking, suggesting that p90RSK might regulate glucose transport after GLUT4 translocation. Moreover, BI-D1870 also prevented PMA-induced glucose transport in 3T3-L1 adipocytes further suggesting a role for p90RSK in regulating uptake of glucose into the cells. Kinetic experiments are consistent with SL0101 being a direct competitor of 2-deoxyglucose entry into cells, and this compound might also inhibit uptake of glucose into cells via inhibiting p90RSK, as revealed by comparison with the inactive form of the inhibitor. Taken together, we propose that BI-D1870 and SL0101 might exert their inhibitory effects on glucose uptake in 3T3-L1 adipocytes at least partially through a p90RSK dependent step after GLUT4 becomes associated with the plasma membrane.

P-67: Use of recombinant adenovirus-mediated gene transfer to investigate the effects of dominant negative ras on the activation of PI3 kinase and glucose transport in 3T3L1 adipocytes

P-67: Use of recombinant adenovirus-mediated gene transfer to investigate the effects of dominant negative ras on the activation of PI3 kinase and glucose transport in 3T3L1 adipocytes

The atypical kinase Cdk5 is activated by insulin, regulates the association between GLUT4 and E-Syt1, and modulates glucose transport in 3T3-L1 adipocytes

Here, we report that Cdk5 activation is stimulated by insulin and plays a key role in the regulation of GLUT4-mediated glucose uptake in 3T3-L1 adipocytes. Insulin activation of Cdk5 requires PI3K signaling. Insulin-activated Cdk5 phosphorylates E-Syt1, a 5 C2-domain protein-related to the synaptotagmins that is induced during adipocyte differentiation. Phosphorylated E-Syt1 associates with GLUT4, an event inhibited by the Cdks inhibitor roscovitine. Cdk5 silencing inhibits glucose uptake by 3T3-L1 adipocytes. These studies elucidate a previously unknown activity of Cdk5 and demonstrate the involvement of this kinase in the regulation of insulin-dependent glucose uptake in adipocytes.

Over-expression of NYGGF4 inhibits glucose transport in 3T3-L1 adipocytes via attenuated phosphorylation of IRS-1 and Akt

NYGGF4 is a novel gene that is abundantly expressed in the adipose tissue of obese patients. The purpose of this study was to investigate the effects of NYGGF4 on basal and insulin-stimulated glucose uptake in mature 3T3-L1 adipocytes and to understand the underlying mechanisms. 3T3-L1 preadipocytes transfected with either an empty expression vector (pcDNA3.1Myc/His B) or an NYGGF4 expression vector were differentiated into mature adipocytes. Glucose uptake was determined by measuring 2-deoxy-D-[3H]glucose uptake into the adipocytes. Immunoblotting was performed to detect the translocation of insulin-sensitive glucose transporter 4 (GLUT4). Immunoblotting also was used to measure the phosphorylation and total protein contents of insulin signaling proteins such as the insulin receptor (IR), insulin receptor substrate (IRS)-1, Akt, ERK1/2, p38, and JNK. NYGGF4 over-expression in 3T3-L1 adipocytes reduced insulin-stimulated glucose uptake and impaired insulin-stimulated GLUT4 translocation. It also diminished insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt without affecting the phosphorylation of IR, ERK1/2, p38, and JNK. NYGGF4 regulates the functions of IRS-1 and Akt, decreases GLUT4 translocation and reduces glucose uptake in response to insulin. These observations highlight the potential role of NYGGF4 in glucose homeostasis and possibly in the pathogenesis of obesity.

Oleanolic acid derivative NPLC441 potently stimulates glucose transport in 3T3-L1 adipocytes via a multi-target mechanism

The natural product oleanolic acid (OA) has been discovered to exhibit varied pharmacological functions including anti-inflammation, anti-tumor and anti-diabetes, while appropriate synthetic oleanolic acid derivatives seem to possess more potent activities. Here we identified a new oleanolic acid derivative, 3-β-(2-carboxybenzoyloxy)-oleanolic acid (NPLC441), which functioned as a competitive PTP1B inhibitor and enhanced insulin-stimulated phosphorylation of IR and AKT in HepG2 cells. As an RXRα antagonist, it could selectively activate LXRα:RXRα heterodimer and increase the promoter activities of ABCA1 and ABCG1 genes in transient transfection assays. Quantitative RT-PCR and Western blot analyses suggested that NPLC441 could up-regulate GLUT4 expression in 3T3-L1 adipocytes, and such effect was further proved to be dependent on LXRα:RXRα activation. Moreover, 2-deoxyglucose uptake technology-based characterization demonstrated that this compound could stimulate glucose uptake in 3T3-L1 adipocytes. Finally, NPLC441 was observed to be able to suppress 11β-HSD 1 expression in HepG2 cells, following the discovery that activation of LXRα:RXRα could repress the expression of 11β-HSD 1. Compared with NPLC441, OA showed no effects on the transactivation of either LXRα:RXRα heterodimer or RXRα-LBD. Our work is thus expected to provide a new insight into the anti-diabetic application for oleanolic acid derivatives via multi-target mechanism, and NPLC441 could be used as a potential lead compound for further research.

Berberine reverses free-fatty-acid-induced insulin resistance in 3T3-L1 adipocytes through targeting IKKβ

To investigate the effects and molecular mechanisms of berberine on improving insulin resistance induced by free fatty acids (FFAs) in 3T3-L1 adipocytes. The model of insulin resistance in 3T3-L1 adipocytes was established by adding palmic acid (0.5 mmol/L) to the culture medium. Berberine treatment was performed at the same time. Glucose uptake rate was determined by the 2-deoxy-[(3)H]-D-glucose method. The levels of IkB kinase beta (IKKbeta) Ser(181) phosphorylation, insulin receptor substrate-1(IRS-1) Ser(307) phosphorylation, expression of IKKbeta, IRS-1, nuclear transcription factor kappaB p65 (NF-kappaB p65), phosphatidylinositol-3-kinase p85 (PI-3K p85) and glucose transporter 4 (GLUT4) proteins were detected by Western blotting. The distribution of NF-kappaB p65 proteins inside the adipocytes was observed through confocal laser scanning microscopy (CLSM). After the intervention of palmic acid for 24 h, the insulin-stimulated glucose transport in 3T3-L1 adipocytes was inhibited by 67%. Meanwhile, the expression of IRS-1 and PI-3K p85 protein was reduced, while the levels of IKKbeta Ser(181) and IRS-1 Ser(307) phosphorylation, and nuclear translocation of NF-kappaB p65 protein were increased. However, the above indexes, which indicated the existence of insulin resistance, were reversed by berberine although the expression of GLUT4, IKKbeta and total NF-kappaB p65 protein were not changed during this study. Insulin resistance induced by FFAs in 3T3-L1 adipocytes can be improved by berberine. Berberine reversed free-fatty-acid-induced insulin resistance in 3T3-L1 adipocytes through targeting IKKbeta.

Endothelin-1 induction of Glut1 transcription in 3T3-L1 adipocytes involves distinct PKCε- and p42/p44 MAPK-dependent pathways

We have shown previously that chronic exposure to endothelin-1 (ET-1) may stimulate GLUT1-mediated glucose transport in 3T3-L1 adipocytes via both protein kinase C (PKC)- and mitogen-activated protein kinase (p42/p44 MAPK)-dependent pathways. In the present study, by using a luciferase reporter driven by Glut1 promoter and enhancers (pLuc-GT1/E1/E2) and various constitutively active and dominant negative mutants of PKC isoforms, we identified PKCε as the PKC isoform involved. In addition, we provide evidence that there is no direct interaction between ET-1 activated PKCε and MAPK, at least at the kinase activity level. Furthermore, investigations employing deletion mutants of pLuc-GT1/E1/E2 to locate the putative ET-1 responsive sites and inhibitory agents to suppress the activities of putative transcription factors suggested that transcription factors CREB, Sp1 and NF-κB were involved. In summary, the results of this study indicate that ET-1 induction of Glut1 transcription involves distinct PKCε- and MAPK-dependent pathways, as well as downstream transcription factors CREB, Sp1 and NF-κB.

The SUMO Conjugating Enzyme Ubc9 is a Regulator of GLUT4 Turnover and Targeting to the Insulin-Responsive Storage Compartment in 3T3-L1 Adipocytes

The small ubiquitin-related modifier (SUMO) conjugating enzyme Ubc9 has been shown to upregulate GLUT4 in L6 myoblast cells, although the mechanism of action has remained undefined. Here we investigated the physiological significance of Ubc9 in GLUT4 turnover and subcellular targeting by adenovirus vector-mediated overexpression and by small interfering RNA (siRNA)-mediated gene silencing of Ubc9 in 3T3-L1 adipocytes. Overexpression of Ubc9 resulted in an inhibition of GLUT4 degradation and promoted its targeting to the unique insulin-responsive GLUT4 storage compartment (GSC), leading to an increase in GLUT4 amount and insulin-responsive glucose transport in 3T3-L1 adipocytes. Overexpression of Ubc9 also antagonized GLUT4 downregulation and its selective loss in GSC induced by long-term insulin stimulation. By contrast, siRNA-mediated depletion of Ubc9 accelerated GLUT4 degradation and decreased the amount of the transporter, concurrent with its selective loss in GSC, which resulted in attenuated insulin-responsive glucose transport. Intriguingly, overexpression of the catalytically inactive mutant Ubc9-C93A produced effects indistinguishable from those with wild-type Ubc9, suggesting that Ubc9 regulates GLUT4 turnover and targeting to GSC by a mechanism independent of its catalytic activity. Thus, Ubc9 is a pivotal regulator of the insulin sensitivity of glucose transport in adipocytes.

ArPIKfyve–PIKfyve interaction and role in insulin-regulated GLUT4 translocation and glucose transport in 3T3-L1 adipocytes

Insulin activates glucose transport by promoting translocation of the insulin-sensitive fat/muscle-specific glucose transporter GLUT4 from an intracellular storage compartment to the cell surface. Here we report that an optimal insulin effect on glucose uptake in 3T3-L1 adipocytes is dependent upon expression of both PIKfyve, the sole enzyme for PtdIns 3,5-P 2 biosynthesis, and the PIKfyve activator, ArPIKfyve. Small-interfering RNAs that selectively ablated PIKfyve or ArPIKfyve in this cell type depleted the PtdIns 3,5-P 2 pool and reduced insulin-activated glucose uptake to a comparable degree. Combined loss of PIKfyve and ArPIKfyve caused further PtdIns 3,5-P 2 ablation that correlated with greater attenuation in insulin responsiveness. Loss of PIKfyve–ArPIKfyve reduced insulin-stimulated Akt phosphorylation and the cell surface accumulation of GLUT4 or IRAP, but not GLUT1-containing vesicles, without affecting overall expression of these proteins. ArPIKfyve and PIKfyve were found to physically associate in 3T3-L1 adipocytes and this was insulin independent. In vitro labeling of membranes isolated from basal or insulin-stimulated 3T3-L1 adipocytes documented substantial insulin-dependent increases of PtdIns 3,5-P 2 production on intracellular membranes. Together, the data demonstrate for the first time a physical association between functionally related PIKfyve and ArPIKfyve in 3T3-L1 adipocytes and indicate that the novel ArPIKfyve–PIKfyve–PtdIns 3,5-P 2 pathway is physiologically linked to insulin-activated GLUT4 translocation and glucose transport.

Soluble dietary fibre fraction ofTrigonella foenum-graecum(fenugreek) seed improves glucose homeostasis in animal models of type 1 and type 2 diabetes by delaying carbohydrate digestion and absorption, and enhancing insulin action

Trigonella foenum-graecum (fenugreek) seeds have been documented as a traditional plant treatment for diabetes. In the present study, the antidiabetic properties of a soluble dietary fibre (SDF) fraction of T. foenum-graecum were evaluated. Administration of SDF fraction (0 x 5 g/kg body weight) to normal, type 1 or type 2 diabetic rats significantly improved oral glucose tolerance. Total remaining unabsorbed sucrose in the gastrointestinal tract of non-diabetic and type 2 diabetic rats, following oral sucrose loading (2 x 5 g/kg body weight) was significantly increased by T. foenum-graecum (0 x 5 g/kg body weight). The SDF fraction suppressed the elevation of blood glucose after oral sucrose ingestion in both non-diabetic and type 2 diabetic rats. Intestinal disaccharidase activity and glucose absorption were decreased and gastrointestinal motility increased by the SDF fraction. Daily oral administration of SDF to type 2 diabetic rats for 28 d decreased serum glucose, increased liver glycogen content and enhanced total antioxidant status. Serum insulin and insulin secretion were not affected by the SDF fraction. Glucose transport in 3T3-L1 adipocytes and insulin action were increased by T. foenum-graecum. The present findings indicate that the SDF fraction of T. foenum-graecum seeds exerts antidiabetic effects mediated through inhibition of carbohydrate digestion and absorption, and enhancement of peripheral insulin action.