Differential regulation of GLUT1 activity in human corneal limbal epithelial cells and fibroblasts

The corneal epithelial tissue is a layer of rapidly growing cells that are highly glycolytic and express GLUT1 as the major glucose transporter. It has been shown that GLUT1 in L929 fibroblast cells and other cell lines can be acutely activated by a variety agents. However, the acute regulation of glucose uptake in corneal cells has not been systematically investigated. Therefore, we examined glucose uptake in an immortalized human corneal–limbal epithelial (HCLE) cell line and compared it to glucose uptake in L929 fibroblast cells, a cell line where glucose uptake has been well characterized. We report that the expression of GLUT1 in HCLE cells is 6.6-fold higher than in L929 fibroblast cells, but the HCLE cells have a 25-fold higher basal rate of glucose uptake. Treatment with agents that interfere with mitochondrial metabolism, such as sodium azide and berberine, activate glucose uptake in L929 cells over 3-fold, but have no effect on glucose uptake HCLE cells. Also, agents known to react with thiols, such cinnamaldehyde, phenyarsine oxide and nitroxyl stimulate glucose uptake in L929 cells 3–4-fold, but actually inhibit glucose uptake in HCLE cells. These data suggest that in the fast growing HCLE cells, GLUT1 is expressed at a higher concentration and is already highly activated at basal conditions. These data support a model for the acute activation of GLUT1 that suggests that the activity of GLUT1 is enhanced by the formation of an internal disulfide bond within GLUT1 itself.     

High density lipoprotein (HDL) promotes glucose uptake in adipocytes and glycogen synthesis in muscle cells

Background

High density lipoprotein (HDL) was reported to decrease plasma glucose and promote insulin secretion in type 2 diabetes patients. This investigation was designed to determine the effects and mechanisms of HDL on glucose uptake in adipocytes and glycogen synthesis in muscle cells.

Methods and Results

Actions of HDL on glucose uptake and GLUT4 translocation were assessed with 1-[³H]-2-deoxyglucose and plasma membrane lawn, respectively, in 3T3-L1 adipocytes. Glycogen analysis was performed with amyloglucosidase and glucose oxidase-peroxidase methods in normal and palmitate-treated L6 cells. Small interfering RNA was used to observe role of scavenger receptor type I (SR-BI) in glucose uptake of HDL. Corresponding signaling molecules were detected by immunoblotting. HDL stimulated glucose uptake in a time- and concentration-dependent manner in 3T3-L1 adipocytes. GLUT4 translocation was significantly increased by HDL. Glycogen deposition got enhanced in L6 muscle cells paralleling with elevated glycogen synthase kinase3 (GSK3) phosphorylation. Meanwhile, increased phosphorylations of Akt-Ser473 and AMP activated protein kinase (AMPK) α were detected in 3T3-L1 adipocytes. Glucose uptake and Akt-Ser473 activation but not AMPK-α were diminished in SR-BI knock-down 3T3-L1 cells.

Conclusions

HDL stimulates glucose uptake in 3T3-L1 adipocytes through enhancing GLUT4 translocation by mechanisms involving PI3K/Akt via SR-BI and AMPK signaling pathways, and increases glycogen deposition in L6 muscle cells through promoting GSK3 phosphorylation.     

Osthole ameliorates hepatic fibrosis and inhibits hepatic stellate cell activation

Background

Hepatic fibrosis is a dynamic process which ultimately leads to cirrhosis in almost patients with chronic hepatic injury. However, progressive fibrosis is a reversible scarring response. Activation of hepatic stellate cells (HSCs) is the prevailing process during hepatic fibrosis. Osthole is an active component majorly contained in the fruit of Cnidium monnieri (L.) Cusson. This present study investigated the therapeutic effects of osthole on rat liver fibrosis and HSC activation.

Results

We established the thioacetamide (TAA)-model of Sprague–Dawley (SD) rats to induce hepatic fibrosis. Rats were divided into three groups: control, TAA, and TAA + osthole (10 mg/kg). In vivo, osthole significantly reduced liver injury by diminishing levels of plasma AST and ALT, improving histological architecture, decreasing collagen and α-SMA accumulation, and improving hepatic fibrosis scores. Additionally, osthole reduced the expression of fibrosis-related genes significantly. Osthole also suppressed the production of fibrosis-related cytokines and chemokines. Moreover, nuclear translocation of p65 was significantly suppressed in osthole-treated liver. Osthole also ameliorated TAA-induced injury through reducing cellular oxidation. Osthole showed inhibitory effects in inflammation-related genes and chemokines production as well. In vitro, we assessed osthole effects in activated HSCs (HSC-T6 and LX-2). Osthole attenuated TGF-β1-induced migration and invasion in HSCs. Furthermore, osthole decreased TNF-α-triggered NF-κB activities significantly. Besides, osthole alleviated TGF-β1- or ET-1-induced HSCs contractility.

Conclusions

Our study demonstrated that osthole improved TAA-caused liver injury, fibrogenesis and inflammation in rats. In addition, osthole suppressed HSCs activation in vitro significantly.

Electronic supplementary material

The online version of this article (doi:10.1186/s12929-015-0168-5) contains supplementary material, which is available to authorized users.     

Shikonin increases glucose uptake in skeletal muscle cells and improves plasma glucose levels in diabetic Goto-Kakizaki rats

Background

There is considerable interest in identifying compounds that can improve glucose homeostasis. Skeletal muscle, due to its large mass, is the principal organ for glucose disposal in the body and we have investigated here if shikonin, a naphthoquinone derived from the Chinese plant Lithospermum erythrorhizon, increases glucose uptake in skeletal muscle cells.

Methodology/Principal Findings

Shikonin increases glucose uptake in L6 skeletal muscle myotubes, but does not phosphorylate Akt, indicating that in skeletal muscle cells its effect is medaited via a pathway distinct from that used for insulin-stimulated uptake. Furthermore we find no evidence for the involvement of AMP-activated protein kinase in shikonin induced glucose uptake. Shikonin increases the intracellular levels of calcium in these cells and this increase is necessary for shikonin-mediated glucose uptake. Furthermore, we found that shikonin stimulated the translocation of GLUT4 from intracellular vesicles to the cell surface in L6 myoblasts. The beneficial effect of shikonin on glucose uptake was investigated in vivo by measuring plasma glucose levels and insulin sensitivity in spontaneously diabetic Goto-Kakizaki rats. Treatment with shikonin (10 mg/kg intraperitoneally) once daily for 4 days significantly decreased plasma glucose levels. In an insulin sensitivity test (s.c. injection of 0.5 U/kg insulin), plasma glucose levels were significantly lower in the shikonin-treated rats. In conclusion, shikonin increases glucose uptake in muscle cells via an insulin-independent pathway dependent on calcium.

Conclusions/Significance

Shikonin increases glucose uptake in skeletal muscle cells via an insulin-independent pathway dependent on calcium. The beneficial effects of shikonin on glucose metabolism, both in vitro and in vivo, show that the compound possesses properties that make it of considerable interest for developing novel treatment of type 2 diabetes.     

Methylene blue stimulates 2-deoxyglucose uptake in L929 fibroblast cells

Methylene blue (MB), a common cell stain, has been shown to inhibit nitric oxide synthase and guanylate cyclase, which has led to the recent use of MB in nitric oxide signaling studies. This study documents the effects of MB on 2-deoxyglucose (2DG) uptake in L929 fibroblast cells where uptake is controlled by a single glucose transporter, GLUT 1. MB significantly activates cytochalasin B-inhibitable glucose transport in a dose dependent fashion within 10 min. A maximal stimulation of up to 800% was achieved by 50 microM MB after a 45-min exposure. The Vmax of transport increased without a change in the Km, which was accomplished without a significant change in the GLUT 1 content. The reduced form of MB, did not stimulate 2DG uptake and potassium ferricyanide, an extracellular redox agent, prevented both the staining and stimulatory effects of MB suggesting MB is reduced at the cell surface before it enters L929 cells. Phenylarsine oxide did not block cell staining as noted in other cells lines, but it did inhibit both basal and MB-stimulated 2DG uptake. Likewise, methyl-beta-cyclodextrin, an agent used to remove membrane cholesterol, blocked both the staining and stimulatory effects of MB. The AMP analog, AICAR, inhibited rather than activated basal 2DG uptake, and it did not alter MB-stimulated uptake suggesting that AMP kinase activation is not critical to the MB effect. Wortmannin, an inhibitor of PI kinase, had no effect on MB-stimulated 2DG uptake. These data provide additional insight into the acute regulation of GLUT 1 transport activity in L929 cells.     

Hydroxylamine acutely activates glucose uptake in L929 fibroblast cells

Nitroxyl (HNO) has a unique, but varied, set of biological properties including beneficial effects on cardiac contractility and stimulation of glucose uptake by GLUT1. These biological effects are largely initiated by HNO's reaction with cysteine residues of key proteins. The intracellular production of HNO has not yet been demonstrated, but the small molecule, hydroxylamine (HA), has been suggested as possible intracellular source. We examined the effects of this molecule on glucose uptake in L929 fibroblast cells. HA activates glucose uptake from 2 to 5-fold within two minutes. Prior treatment with thiol-active compounds, such as iodoacetamide (IA), cinnamaldehyde (CA), or phenylarsine oxide (PAO) blocks HA-activation of glucose uptake. Incubation of HA with the peroxidase inhibitor, sodium azide, also blocks the stimulatory effects of HA. This suggests that HA is oxidized to HNO by L929 fibroblast cells, which then reacts with cysteine residues to exert its stimulatory effects. The data suggest that GLUT1 is acutely activated in L929 cells by modification of cysteine residues, possibly the formation of a disulfide bond within GLUT1 itself.     

Induction of apoptosis by Polygonatum odoratum lectin and its molecular mechanisms in murine fibrosarcoma L929 cells

Background

The Galanthus nivalis agglutinin (GNA)-related lectins have been reported to bear antiproliferative and apoptosis-inducing activities in cancer cells; however, the precise mechanisms by which GNA-related lectins induce cell death are still only rudimentarily understood.

Methods

In the present study, Polygonatum odoratum lectin (designated POL), a mannose-binding specific GNA-related lectin, possessed a remarkable antiproliferative activity toward murine fibrosarcoma L929 cells. And, this lectin induced L929 cell apoptosis in a caspase-dependent manner. In addition, POL treatment increased the levels of FasL and Fas-Associated protein with Death Domain (FADD) proteins and resulted in caspase-8 activation. Also, POL treatment caused mitochondrial transmembrane potential collapse and cytochrome c release, leading to activations of caspase-9 and caspase-3. Moreover, POL treatment enhanced tumor necrosis factor α (TNFα)-induced L929 cell apoptosis.

Results

Our data demonstrate for the first time that this lectin induces apoptosis through both death-receptor and mitochondrial pathways, as well as amplifies TNFα-induced L929 cell apoptosis.

General significance

These inspiring findings would provide new molecular basis for further understanding cell death mechanisms of the Galanthus nivalis agglutinin (GNA)-related lectins in future cancer investigations.     

Glucagon like Peptide-1-induced glucose metabolism in differentiated human muscle satellite cells is attenuated by hyperglycemia

Background

Glucagon like peptide-1 (GLP-1) stimulates insulin secretion from the pancreas but also has extra-pancreatic effects. GLP-1 may stimulate glucose uptake in cultured muscle cells but the mechanism is not clearly defined. Furthermore, while the pancreatic effects of GLP-1 are glucose-dependent, the glucose-dependency of its extra-pancreatic effects has not been examined.

Methods

Skeletal muscle satellite cells isolated from young (22.5±0.97 yr), lean (BMI 22.5±0.6 kg/m²), healthy males were differentiated in media containing either 22.5 mM (high) or 5 mM (normal) glucose for 7 days in the absence or presence of insulin and/or various GLP-1 concentrations. Myocellular effects of GLP-1, insulin and glucose were assessed by western-blot, glucose uptake and glycogen synthesis.

Results

We firstly show that the GLP-1 receptor protein is expressed in differentiated human muscle satellite cells (myocytes). Secondly, we show that in 5 mM glucose media, exposure of myocytes to GLP-1 results in a dose dependent increase in glucose uptake, GLUT4 amount and subsequently glycogen synthesis in a PI3K dependent manner, independent of the insulin signaling cascade. Importantly, we provide evidence that differentiation of human satellite cells in hyperglycemic (22.5 mM glucose) conditions increases GLUT1 expression, and renders the cells insulin resistant and interestingly GLP-1 resistant in terms of glucose uptake and glycogen synthesis. Hyperglycemic conditions did not affect the ability of insulin to phosphorylate downstream targets, PKB or GSK3. Interestingly we show that at 5 mM glucose, GLP-1 increases GLUT4 protein levels and that this effect is abolished by hyperglycemia.

Conclusions

GLP-1 increases glucose uptake and glycogen synthesis into fully-differentiated human satellite cells in a PI3-K dependent mechanism potentially through increased GLUT4 protein levels. The latter occurs independently of the insulin signaling pathway. Attenuation of both GLP-1 and insulin-induced glucose metabolism by hyperglycemia is likely to occur downstream of PI3K.     

Secretion and uptake of TAT-fusion proteins produced by engineered mammalian cells

Background

Intracellular signaling can be regulated by the exogenous addition of physiological protein inhibitors coupled to the TAT protein transduction domain. Thus far experiments have been performed with purified inhibitors added exogenously to cells in vitro or administered in vivo. Production of secretable TAT-fusion proteins by engineered mammalian cells, their uptake, and route of entry has not been thoroughly investigated. Such methodology, if established, could be useful for transplantation purposes.

Methods

Secretion of TAT-fusion proteins from transfected mammalian cells was achieved by means of a signal peptide. Cell uptake and subcellular localization of TAT-fusion proteins were determined by immunoblotting and confocal microscopy.

Results

Engineered TAT-fusion proteins were secreted with variable efficiency depending on the nature of the protein fused to the TAT peptide. Secreted proteins were able to transduce unmanipulated cells. Their mechanism of entry into cells partly involves lipid rafts and a portion of the internalised protein is directed to the Golgi.

Conclusions

Generation of secretable TAT-coupled inhibitors of signaling pathways, able to transduce other cells can be achieved.

General significance

These results provide key information that will assist in the design of TAT-inhibitors and engineered cells in order to regulate cell function within tissues.     

Inhibition of glucose uptake and suppression of glucose transporter 1 mRNA expression in L929 cells by tumour necrosis factor-alpha

Recombinant human tumour necrosis factor-alpha (rhTNF-alpha) arrested the growth and suppressed glucose uptake of mouse fibrosarcoma L929 cells in vitro. When the cells were treated with rhTNF-alpha for 24 hours, the mRNA level of glucose transporter 1 (GLUT 1), which is the only GLUT found to be present in L929 cells in our study, was suppressed in a dose-dependent manner. Since the growth of tumour cells depends mainly on glucose catabolism, our findings may indicate that rhTNF-alpha inhibits L929 cells growth by lowering the glucose transport through suppression of GLUT 1 mRNA expression in the cells.     

3β-taraxerol of Mangifera indica, a PI3K dependent dual activator of glucose transport and glycogen synthesis in 3T3-L1 adipocytes

Background

The present study focuses on identifying and developing an anti-diabetic molecule from plant sources that would effectively combat insulin resistance through proper channeling of glucose metabolism involving glucose transport and storage.

Methods

Insulin-stimulated glucose uptake formed the basis for isolation of a bioactive molecule through column chromatography followed by its characterization using NMR and mass spectroscopic analysis. Mechanism of glucose transport and storage was evaluated based on the expression profiling of signaling molecules involved in the process.

Results

The study reports (i) the isolation of a bioactive compound 3β-taraxerol from the ethyl acetate extract (EAE) of the leaves of Mangifera indica (ii) the bioactive compound exhibited insulin-stimulated glucose uptake through translocation and activation of the glucose transporter (GLUT4) in an IRTK and PI3K dependent fashion. (iii) the fate of glucose following insulin-stimulated glucose uptake was ascertained through glycogen synthesis assay that involved the activation of PKB and suppression of GSK3β.

General significance

This study demonstrates the dual activity of 3β-taraxerol and the ethyl acetate extract of Mangifera indica as a glucose transport activator and stimulator of glycogen synthesis. 3β-taraxerol can be validated as a potent candidate for managing the hyperglycemic state.     

Azoxystrobin,a mitochondrial complex III Qo site inhibitor,exerts beneficial metabolic effects in vivo and in vitro

Background

Several anti-diabetes drugs exert beneficial effects against metabolic syndrome by inhibiting mitochondrial function. Although much progress has been made toward understanding the role of mitochondrial function inhibitors in treating metabolic diseases, the potential effects of these inhibitors on mitochondrial respiratory chain complex III remain unclear.

Methods

We investigated the metabolic effects of azoxystrobin (AZOX), a Q_o inhibitor of complex III, in a high-fat diet-fed mouse model with insulin resistance in order to elucidate the mechanism by which AZOX improves glucose and lipid metabolism at the metabolic cellular level.

Results

Acute administration of AZOX in mice increased the respiratory exchange ratio. Chronic treatment with AZOX reduced body weight and significantly improved glucose tolerance and insulin sensitivity in high-fat diet-fed mice. AZOX treatment resulted in decreased triacylglycerol accumulation and down-regulated the expression of genes involved in liver lipogenesis. AZOX increased glucose uptake in L6 myotubes and 3T3-L1 adipocytes and inhibited de novo lipogenesis in HepG2 cells. The findings indicate that AZOX-mediated alterations to lipid and glucose metabolism may depend on AMP-activated protein kinase (AMPK) signaling.

Conclusions

AZOX, a Q_o inhibitor of mitochondrial respiratory complex III, exerts whole-body beneficial effects on the regulation of glucose and lipid homeostasis in high-fat diet-fed mice.

General significance

These findings provide evidence that a Q_o inhibitor of mitochondrial respiratory complex III could represent a novel approach for the treatment of obesity.     

Rab4b Is a Small GTPase Involved in the Control of the Glucose Transporter GLUT4 Localization in Adipocyte

Background

Endosomal small GTPases of the Rab family, among them Rab4a, play an essential role in the control of the glucose transporter GLUT4 trafficking, which is essential for insulin-mediated glucose uptake. We found that adipocytes also expressed Rab4b and we observed a consistent decrease in the expression of Rab4b mRNA in human and mice adipose tissue in obese diabetic states. These results led us to study this poorly characterized Rab member and its potential role in glucose transport.

Methodology/Principal Findings

We used 3T3-L1 adipocytes to study by imaging approaches the localization of Rab4b and to determine the consequence of its down regulation on glucose uptake and endogenous GLUT4 location. We found that Rab4b was localized in endosomal structures in preadipocytes whereas in adipocytes it was localized in GLUT4 and in VAMP2-positive compartments, and also in endosomal compartments containing the transferrin receptor (TfR). When Rab4b expression was decreased with specific siRNAs by two fold, an extent similar to its decrease in obese diabetic subjects, we observed a small increase (25%) in basal deoxyglucose uptake and a more sustained increase (40%) in presence of submaximal and maximal insulin concentrations. This increase occurred without any change in GLUT4 and GLUT1 expression levels and in the insulin signaling pathways. Concomitantly, GLUT4 but not TfR amounts were increased at the plasma membrane of basal and insulin-stimulated adipocytes. GLUT4 seemed to be targeted towards its non-endosomal sequestration compartment.

Conclusion/Significance

Taken our results together, we conclude that Rab4b is a new important player in the control of GLUT4 trafficking in adipocytes and speculate that difference in its expression in obese diabetic states could act as a compensatory effect to minimize the glucose transport defect in their adipocytes.     

Insulin sensitization via partial agonism of PPARγ and glucose uptake through translocation and activation of GLUT4 in PI3K/p-Akt signaling pathway by embelin in type 2 diabetic rats

Background

The present study was aimed at isolating an antidiabetic molecule from a herbal source and assessing its mechanism of action.

Methods

Embelin, isolated from Embelia ribes Burm. (Myrsinaceae) fruit, was evaluated for its potential to regulate insulin resistance, alter β-cell dysfunction and modulate key markers involved in insulin sensitivity and glucose transport using high-fat diet (HFD) fed-streptozotocin (STZ) (40 mg/kg)-induced type 2 diabetic rats. Molecular-dockings were performed to investigate the binding modes of embelin into PPARγ, PI3K, p-Akt and GLUT4 active sites.

Results

Embelin (50 mg/kg b wt.) reduced body weight gain, blood glucose and plasma insulin in treated diabetic rats. It further modulated the altered lipid profiles and antioxidant enzymes with cytoprotective action on β-cell. Embelin significantly increased the PPARγ expression in epididymal adipose tissue compared to diabetic control group; it also inhibited adipogenic activity; it mildly activated PPARγ levels in the liver and skeletal muscle. It also regulated insulin mediated glucose uptake in epididymal adipose tissue through translocation and activation of GLUT4 in PI3K/p-Akt signaling cascade. Embelin bound to PPARγ; it disclosed stable binding affinities to the active sites of PI3K, p-Akt and GLUT4.

Conclusions

These findings show that embelin could improve adipose tissue insulin sensitivity without increasing weight gain, enhance glycemic control, protect β-cell from damage and maintain glucose homeostasis in adipose tissue.

General significance

Embelin can be used in the prevention and treatment of type 2 diabetes mellitus caused due to obesity.     

Chronic hypoxia leads to a glycolytic phenotype and suppressed HIF-2 signaling in PC12 cells

Background

Along with other regulators of cell metabolism, hypoxia-inducible factors HIF-1 and HIF-2 differentially regulate cell adaptation to hypoxia. Switches in HIF-1/HIF-2 signaling in chronic hypoxia have not been fully investigated.

Methods

Proliferation, viability, apoptosis, neuronal and bioenergetic markers, mitochondrial function, respiration, glycolysis, HIF signalling, responses to O₂ and glucose deprivation (OGD) were examined using tumor PC12 and SH-SY5Y cells continuously grown at 3% O₂.

Results

Hypoxic PC12 cells (H-cells) exhibit reduced proliferation and histone H4 acetylation, NGF-independent differentiation, activation of AMPK, inhibition of Akt, altered mitochondria and response to NGF. Cellular cytochrome c is increased with no effect on apoptosis. Reduction in respiration has minor effect on cellular ATP which is maintained through activated uptake (GLUT1) and utilization (HK2, PFK2) of glucose. H-cells exhibit resistance to OGD linked to increased glycogen stores. HIF-2alpha protein is decreased without changes in mRNA. Unlike HIF-1alpha, HIF-2alpha is not stabilized pharmacologically or by O₂ deprivation. Capacity for HIF-2alpha stabilization is partly restored when H-cells are cultured at normoxia. In low-respiring SH-SY5Y cells cultured under the same conditions HIF-2alpha stabilization and energy budget are not affected.

Conclusions

In chronically hypoxic PC12 cells glycolytic energy budget, increased energy preservation and low susceptibility to OGD are observed. HIF-2alpha no longer orchestrates adaptive responses to anoxia.

General significance

Demonstrated switch in HIF-1/HIF-2 signaling upon chronic hypoxia can facilitate cell survival in energy crisis, by regulating balance between energy saving and decrease in proliferation, on one hand and active cell growth and tumor expansion, on the other.     

Cloning,characterization, and expression of glucose transporter 2 in the freeze-tolerant wood frog, Rana sylvatica

Background

The essential role of glucose transporter 2 (GLUT2) in glucose homeostasis has been extensively studied in mammals; however, little is known about this important protein in lower vertebrates. The freeze-tolerant wood frog (Rana sylvatica), which copiously mobilizes glucose in response to freezing, represents an excellent system for the study of glucose transport in amphibians.

Methods

GLUT2 was sequenced from northern and southern phenotypes of R. sylvatica, as well as the freeze-intolerant Rana pipiens. These proteins were expressed and functionally characterized in Xenopus oocytes. Abundance of GLUT2 in tissues was analyzed using immunoblotting techniques.

Results

GLUT2s cloned from these anurans encoded proteins with high sequence homologies to known vertebrate GLUT2s and had similar transport properties, although, notably, transport of the glucose analog 3-O-methyl-d-glucose (3-OMG) was strongly inhibited by 150 mM urea. Proteins from all study subjects had similar affinity constants (~ 12 mM) and other kinetic properties; however, GLUT2 abundance in liver was 3.5-fold greater in northern R. sylvatica than in the southern conspecific and R. pipiens.

Conclusion

Our results indicate that amphibian GLUT2s are structurally and functionally similar to their homologs in other vertebrates, attesting to the conserved nature of this transport protein. The greater abundance of this protein in the northern phenotype of R. sylvatica suggests that these transporters contribute importantly to freezing survival.

General significance

This study provides the first functional characterization of any GLUT isoform from an anuran amphibian and novel insights into the role of these proteins in glucose homeostasis and cryoprotectant mobilization in freeze-tolerant vertebrates.     

Zinc-α2-Glycoprotein Modulates AKT-Dependent Insulin Signaling in Human Adipocytes by Activation of the PP2A Phosphatase

Objective

Evidence from mouse models suggests that zinc-α2-glycoprotein (ZAG) is a novel anti-obesity adipokine. In humans, however, data are controversial and its physiological role in adipose tissue (AT) remains unknown. Here we explored the molecular mechanisms by which ZAG regulates carbohydrate metabolism in human adipocytes.

Methods

ZAG action on glucose uptake and insulin action was analyzed. β1 and β2-adrenoreceptor (AR) antagonists and siRNA targeting PP2A phosphatase were used to examine the mechanisms by which ZAG modulates insulin sensitivity. Plasma levels of ZAG were measured in a lean patient cohort stratified for HOMA-IR.

Results

ZAG treatment increased basal glucose uptake, correlating with an increase in GLUT expression, but induced insulin resistance in adipocytes. Pretreatment of adipocytes with propranolol and a specific β1-AR antagonist demonstrated that ZAG effects on basal glucose uptake and GLUT4 expression are mediated via β1-AR, whereas inhibition of insulin action is dependent on β2-AR activation. ZAG treatment correlated with an increase in PP2A activity. Silencing of the PP2A catalytic subunit abrogated the negative effect of ZAG on insulin-stimulated AKT phosphorylation and glucose uptake but not on GLUT4 expression and basal glucose uptake. ZAG circulating levels were unchanged in a lean patient cohort stratified for HOMA-IR. Neither glucose nor insulin was associated with plasma ZAG.

Conclusions

ZAG inhibits insulin-induced glucose uptake in human adipocytes by impairing insulin signaling at the level of AKT in a β2-AR- and PP2A-dependent manner.     

Nitroxyl (HNO) acutely activates the glucose uptake activity of GLUT1

Nitroxyl (HNO) is a molecule of significant interest due to its unique pharmacological properties, particularly within the cardiovascular system. A large portion of HNO biological effects can be attributed to its reactivity with protein thiols, where it can generate disulfide bonds. Evidence from studies in erythrocytes suggests that the activity of GLUT1 is enhanced by the formation of an internal disulfide bond. However, there are no reports that document the effects of HNO on glucose uptake. Therefore, we examined the acute effects of Angeli’s salt (AS), a HNO donor, on glucose uptake activity of GLUT1 in L929 fibroblast cells. We report that AS stimulates glucose uptake with a maximum effective concentration of 5.0 mM. An initial 7.2-fold increase occurs within 2 min, which decreases and plateaus to a 4.0-fold activation after 10 min. About 60% of the 4.0-fold activation recovers within 10 min, and 40% remains after an hour. The activation is blocked by the pretreatment of cells with thiol-reactive compounds, iodoacetamide (0.75 mM), cinnamaldehyde (2.0 mM), and phenylarsine oxide (10 μM). The effects of AS are not additive to the stimulatory effects of other acute activators of glucose uptake in L929 cells, such as azide (5 mM), berberine (50 μM), or glucose deprivation. These data suggest that GLUT1 is acutely activated in L929 cells by the formation of a disulfide bond, likely within GLUT1 itself.