Glucose Uptake In Human Adipocytes Research Articles

Effect of Origanum vulgare L. essential oil on insulin-induced PI3K/Akt signaling and glucose uptake in human adipocytes treated with palmitic acid

Searchable abstracts of presentations at key conferences in endocrinology ISSN 1470-3947 (print) | ISSN 1479-6848 (online)

Interleukin-33 inhibits glucose uptake in human adipocytes and its expression in adipose tissue is elevated in insulin resistance and type 2 diabetes.

Interleukin-33 (IL-33) is associated with obesity-related inflammation. We aim to investigate IL-33 expression in subcutaneous adipose tissue (SAT) in type 2 diabetes (T2D) subjects and its effects on human adipocyte glucose uptake. Expression of IL-33 was analysed in SAT from cohort studies including subjects with and without obesity and T2D and correlated with insulin resistance and obesity markers. Magnetic resonance imaging (MRI) of tissue fat volumes was performed. We investigated the effects of IL-33 treatment on ex vivo adipocyte glucose uptake. T2D subjects had higher IL-33 gene and protein expression in SAT than the control subjects. IL-33 mRNA expression was positively correlated with markers of dysglycemia (e.g. HbA1c), insulin resistance (e.g. HOMA-IR) and adiposity (BMI, visceral adipose tissue volume, liver and pancreas fat %). In multiple linear regression analyses, insulin resistance and T2D status were the strongest predictors of IL-33, independent of BMI. IL-33 mRNA expression was negatively correlated with expression of genes regulating adipocyte glucose uptake, lipid storage, and adipogenesis (e.g.glucose transporter 1 and 4 (GLUT1/4), fatty acid binding protein 4 (FABP4), and PPARG). Additionally, incubation of SAT with IL-33 reduced adipocyte glucose uptake and GLUT4 gene and protein expression. Our findings suggest that T2D subjects have higher IL-33 gene and protein expressionin SATthan control subjects, which is associated with insulin resistance and reduced gene expression of lipid storage and adipogenesis markers. IL-33 may reduce adipocyte glucose uptake. This opens up a potential pharmacological route for reversing insulin resistance in T2D and prediabetes.

Methylamine Activates Glucose Uptake in Human Adipocytes Without Overpassing Action of Insulin or Stimulating its Secretion in Pancreatic Islets.

Background: Methylamine, a natural soluble amine present in foods, is known to be a substrate of primary amine oxidase (PrAO) widely expressed in animal tissues. Methylamine has been reported to activate glucose transport in fat cells and to facilitate glucose disposal in rabbits but the interests and limits of such insulin-mimicking actions have not been further explored. This work aimed to perform a preclinical study of the inter-individual variations of these biological properties to study the putative link between PrAO activity and insulin resistance. Methods: Methylamine was tested on human adipocyte preparations and in rabbit pancreatic islets to determine its influence on glucose uptake and insulin release, respectively. PrAO activity and related responses were determined in adipose tissues obtained from two cohorts of non-obese and obese women. Results: Adipose tissue PrAO activity was negatively correlated with insulin resistance in high-risk obese women. PrAO-dependent activation of glucose uptake was negatively correlated with body mass index and reflected the decrease of insulin responsiveness of human fat cells with increasing obesity. Methylamine exhibited antilipolytic properties in adipocytes but was unable to directly activate insulin secretion in isolated pancreatic islets. Conclusions: PrAO activation by its substrates, e.g., methylamine, increases glucose utilization in human adipocytes in a manner that is linked to insulin responsiveness. Methylamine/PrAO interaction can therefore contribute to adipose tissue enlargement but should be considered as potentially useful for diabetes prevention since it could limit lipotoxicity and facilitate glucose handling, at the expense of favoring healthy fat accumulation.

Spexin: A novel regulator of adipogenesis and fat tissue metabolism

Spexin (SPX, NPQ) is a novel peptide involved in the regulation of energy metabolism. SPX inhibits food intake and reduces body weight. In obese humans, SPX is the most down-regulated gene in fat. Therefore, SPX might be involved in the regulation of lipid metabolism. Here, we study the effects of SPX on lipolysis, lipogenesis, glucose uptake, adipogenesis, cell proliferation and survival in isolated human adipocytes or murine 3T3-L1 cells. SPX and its receptors, GALR2 and GALR3, are present at mRNA and protein levels in murine 3T3-L1 cells and human adipocytes. SPX inhibits adipogenesis and down-regulates mRNA expression of proadipogenic genes such as Pparγ, C/ebpα, C/ebpβ and Fabp4. SPX stimulates lipolysis by increasing the phosphorylation of hormone sensitive lipase (HSL). Simultaneously, SPX inhibits lipogenesis and glucose uptake in human adipocytes and murine 3T3-L1 cells. SPX has no effect on murine 3T3-L1 cell proliferation and viability. Moreover, our research showed that the SPX effect on adipocytes metabolism is mediated via GALR2 and GALR3 receptors. SPX is a novel regulator of lipid metabolism in murine 3T3-L1 and human adipocytes.

Calcineurin is an important factor involved in glucose uptake in human adipocytes

Calcineurin inhibitors are used in immunosuppressive therapy applied after transplantation, but they are associated with major metabolic side effects including the development of new onset diabetes. Previously, we have shown that the calcineurin inhibiting drugs tacrolimus and cyclosporin A reduce adipocyte and myocyte glucose uptakes by reducing the amount of glucose transporter type 4 (GLUT4) at the cell surface, due to an increased internalization rate. However, this happens without alteration in total protein and phosphorylation levels of key proteins involved in insulin signalling or in the total amount of GLUT4. The present study evaluates possible pathways involved in the altered internalization of GLUT4 and consequent reduction of glucose uptake provoked by calcineurin inhibitors in human subcutaneous adipose tissue. Short- and long-term treatments with tacrolimus, cyclosporin A or another CNI deltamethrin (herbicide) decreased basal and insulin-dependent glucose uptake in adipocytes, without any additive effects observed when added together. However, no tacrolimus effects were observed on glucose uptake when gene transcription and protein translation were inhibited. Investigation of genes potentially involved in GLUT4 trafficking showed only a small effect on ARHGEF11 gene expression (p < 0.05). In conlusion, the specific inhibition of calcineurin, but not that of protein phosphatases, decreases glucose uptake in human subcutaneous adipocytes, suggesting that calcineurin is an important regulator of glucose transport. This inhibitory effect is mediated via gene transcription or protein translation; however, expression of genes potentially involved in GLUT4 trafficking and endocytosis appears not to be involved in these effects.

Natriuretic peptides promote glucose uptake in a cGMP-dependent manner in human adipocytes

Robust associations between low plasma level of natriuretic peptides (NP) and increased risk of type 2 diabetes (T2D) have been recently reported in humans. Adipose tissue (AT) is a known target of NP. However it is unknown whether NP signalling in human AT relates to insulin sensitivity and modulates glucose metabolism. We here show in two European cohorts that the NP receptor guanylyl cyclase-A (GC-A) expression in subcutaneous AT was down-regulated as a function of obesity grade while adipose NP clearance receptor (NPRC) was up-regulated. Adipose GC-A mRNA level was down-regulated in prediabetes and T2D, and negatively correlated with HOMA-IR and fasting blood glucose. We show for the first time that NP promote glucose uptake in a dose-dependent manner. This effect is reduced in adipocytes of obese individuals. NP activate mammalian target of rapamycin complex 1/2 (mTORC1/2) and Akt signalling. These effects were totally abrogated by inhibition of cGMP-dependent protein kinase and mTORC1/2 by rapamycin. We further show that NP treatment favoured glucose oxidation and de novo lipogenesis independently of significant gene regulation. Collectively, our data support a role for NP in blood glucose control and insulin sensitivity by increasing glucose uptake in human adipocytes. This effect is partly blunted in obesity.

A novel botanical formula prevents diabetes by improving insulin resistance

BackgroundType 2 diabetes mellitus (T2DM) is a major risk factor for cardiovascular disease, and the prevalence has increased significantly in recent decades to epidemic proportions in China. Individually, fenugreek (Trigonella foenum graecum) seed, mulberry (Morus alba L.) leaf and American ginseng (Panax quinquefolius) root can improve glycemia in various animal models and humans with impaired glucose metabolism and T2DM. The aim of this study was to design an optimized botanical formula containing these herbal extracts as a nutritional strategy for the prevention of insulin resistance and T2DM.MethodsCell-free α-amylase and α-glucosidase enzyme assays were used to determine inhibitory potential of extracts. Glucose uptake was examined in differentiated human adipocytes using radiolabeled 2-deoxyglucose. Male Sprague Dawley rats were divided and glycemia balanced into 5 groups: two controls (naïve and model) and three doses of the botanical test formula containing standardized fenugreek seed, mulberry leaf and American ginseng extracts (42.33, 84.66 and 169.33 mg/kg BW). Insulin resistance and T2DM was induced by feeding animals a high fat diet and with an alloxan injection. Glucose tolerance was examined by measuring serum glucose levels following an oral glucose load.ResultsFenugreek seed and mulberry leaf dose dependently inhibited α-amylase (IC50 = 73.2 μg/mL) and α-glucosidase (IC50 = 111.8 ng/mL), respectively. All three botanical extracts improved insulin sensitivity and glucose uptake in human adipocytes, which lead to the design of an optimized botanical test formula. In a rat model of insulin resistance and T2DM, the optimized botanical test formula improved fasting serum glucose levels, fasting insulin resistance and the development of impaired glucose tolerance. The reduction in epididymal adipose tissue GLUT4 and PDK1 expression induced by high fat diet and alloxan was blunted by the botanical test formula.ConclusionsA novel botanical formula containing standardized extracts of mulberry leaf, fenugreek seed and American ginseng at a ratio of 1:1.3:3.4 prevented the development of insulin resistance, impaired glucose tolerance and T2DM. Given the rising need for effective non-drug targeting of insulin resistance and progression to T2DM, complementary and alternative nutritional strategies without intolerable side effects could have meaningful impact on metabolic health and diabetes risks.

Salt-inducible kinase 2 and -3 are downregulated in adipose tissue from obese or insulin-resistant individuals: implications for insulin signalling and glucose uptake in human adipocytes

Aims/hypothesisSalt-inducible kinases (SIKs) are related to the metabolic regulator AMP-activated protein kinase (AMPK). SIK2 is abundant in adipose tissue. The aims of this study were to investigate the expression of SIKs in relation to human obesity and insulin resistance, and to evaluate whether changes in the expression of SIKs might play a causal role in the development of disturbed glucose uptake in human adipocytes.MethodsSIK mRNA and protein was determined in human adipose tissue or adipocytes, and correlated to clinical variables. SIK2 and SIK3 expression and phosphorylation were analysed in adipocytes treated with TNF-α. Glucose uptake, GLUT protein levels and localisation, phosphorylation of protein kinase B (PKB/Akt) and the SIK substrate histone deacetylase 4 (HDAC4) were analysed after the SIKs had been silenced using small interfering RNA (siRNA) or inhibited using a pan-SIK-inhibitor (HG-9-91-01).ResultsWe demonstrate that SIK2 and SIK3 mRNA are downregulated in adipose tissue from obese individuals and that the expression is regulated by weight change. SIK2 is also negatively associated with in vivo insulin resistance (HOMA-IR), independently of BMI and age. Moreover, SIK2 protein levels and specific kinase activity display a negative correlation to BMI in human adipocytes. Furthermore, SIK2 and SIK3 are downregulated by TNF-α in adipocytes. Silencing or inhibiting SIK1–3 in adipocytes results in reduced phosphorylation of HDAC4 and PKB/Akt, less GLUT4 at the plasma membrane, and lower basal and insulin-stimulated glucose uptake in adipocytes.Conclusion/interpretationThis is the first study to describe the expression and function of SIKs in human adipocytes. Our data suggest that SIKs might be protective in the development of obesity-induced insulin resistance, with implications for future treatment strategies.

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

ObjectiveEvidence 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.MethodsZAG 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.ResultsZAG 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.ConclusionsZAG 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.

Inflamed macrophage microvesicles induce insulin resistance in human adipocytes.

BackgroundCytokines secreted by adipose tissue macrophages (ATMs) significantly alter adipocyte function, inducing inflammatory responses and decreasing insulin sensitivity. However, little relevant information is available regarding the role of microvesicles (MVs) derived from ATMs in macrophage-adipocyte crosstalk.MethodsMVs were generated by stimulation of M1 or M2 phenotype THP-1 macrophages and incubated with human primary mature adipocytes and differentiated adipocytes. Subsequently, insulin-stimulated phosphorylation of Akt (pAkt) and glucose uptake were determined. Glucose transporter 4 (GLUT4) translocation and nuclear translocation of nuclear factor (NF)-kappa B were also analyzed in treated adipocytes.ResultsM1 macrophage-derived MVs (M1 MVs) significantly reduced protein abundance of insulin-induced Akt phosphorylation in human primary mature adipocytes and differentiated adipocytes, when compared with the same concentration of M2 macrophage-derived MVs (M2 MVs). In contrast to M2 MVs, which enhanced the insulin-induced glucose uptake measured by 2-NBDG, M1 MVs decreased this effect in treated adipocytes. M1 MVs treatment also brought about a significant increase in the nuclear translocation of nuclear factor (NF)-kappa B, coupled with a decrease in pAkt level and GLUT4 translocation compared with M2 MVs-treated adipocytes. These effects were reversed by BAY 11–7085, a NF- kappa B specific inhibitor.ConclusionsMVs derived from proinflammatory (M1) macrophages may, at least in part, contribute to the pathogenesis of obesity-induced insulin resistance, reducing insulin signal transduction and decreasing glucose uptake in human adipocytes, through NF-kappa B activation. Therefore, these MVs may be potential therapy candidates for the management of type 2 diabetes mellitus.Electronic supplementary materialThe online version of this article (doi:10.1186/s12986-015-0016-3) contains supplementary material, which is available to authorized users.

Omentin, an adipokine with insulin-sensitizing properties, is negatively associated with insulin resistance in normal gestation.

Omentin, a newly identified adipokine, enhances insulin mediated glucose uptake in human adipocytes, thus, inducing systemic insulin-sensitizing effect. The aims of this study were to determine whether circulating maternal omentin levels are associated with insulin resistance indices and to assess which compartment, maternal, fetal, or placental, is the source of omentin in maternal circulation. Fasting serum glucose, insulin, and omentin were determined in 25 healthy pregnant women at the third trimester, before and 3 days after elective cesarean section. Cord blood omentin was measured in the 25 term neonates. Homeostasis model assessment (HOMA) was used to evaluate insulin sensitivity before and after delivery. Antepartum maternal omentin levels were negatively correlated with insulin levels (r=-0.41, P=0.04) and positively correlated with insulin sensitivity (HOMA%S; r=0.4, P=0.04). Postpartum omentin levels were negatively correlated with maternal body mass index (r=-0.44, P=0.02). Median maternal omentin levels was comparable before and after delivery (57.2, inter-quartile range: 38.2-76.2 ng/mL vs. 53.4, 39.8-69.4 ng/mL, respectively, P=0.25) and highly correlated (r=0.83, P<0.001). Antepartum maternal and neonatal omentin levels did not differ significantly (fetal: 62.2, 44.3-74.2 ng/mL, P=0.77) and did not correlate (P=0.6). Circulating maternal omentin levels are correlated with insulin resistance indices, suggesting that this adipokine may play a role in metabolic adaptations of normal gestation. The strong correlation between anteparum and postpartum maternal omentin levels, as well as the lack of association between maternal and neonatal omentin levels, suggest that placental or fetal compartments are unlikely as the main source of circulating maternal omentin.

LXR is a negative regulator of glucose uptake in human adipocytes

Obesity increases the risk of developing type 2 diabetes mellitus, characterised by impaired insulin-mediated glucose uptake in peripheral tissues. Liver X receptor (LXR) is a positive regulator of adipocyte glucose transport in murine models and a possible target for diabetes treatment. However, the levels of LXRα are increased in obese adipose tissue in humans. We aimed to investigate the transcriptome of LXR and the role of LXR in the regulation of glucose uptake in primary human adipocytes. The insulin responsiveness of human adipocytes differentiated in vitro was characterised, adipocytes were treated with the LXR agonist GW3965 and global transcriptome profiling was determined by microarray, followed by quantitative RT-PCR (qRT-PCR), western blot and ELISA. Basal and insulin-stimulated glucose uptake was measured and the effect on plasma membrane translocation of glucose transporter 4 (GLUT4) was assayed. LXR activation resulted in transcriptional suppression of several insulin signalling genes, such as AKT2, SORBS1 and CAV1, but caused only minor changes (<15%) in microRNA expression. Activation of LXR impaired the plasma membrane translocation of GLUT4, but not the expression of its gene, SLC2A4. LXR activation also diminished insulin-stimulated glucose transport and lipogenesis in adipocytes obtained from overweight individuals. Furthermore, AKT2 expression was reduced in obese adipose tissue, and AKT2 and SORBS1 expression was inversely correlated with BMI and HOMA index. In contrast to murine models, LXR downregulates insulin-stimulated glucose uptake in human adipocytes from overweight individuals. This could be due to suppression of Akt2, c-Cbl-associated protein and caveolin-1. These findings challenge the idea of LXR as a drug target in the treatment of diabetes.

Glucose-dependent insulinotropic polypeptide induces cytokine expression, lipolysis, and insulin resistance in human adipocytes

Obesity-related insulin resistance is linked to a chronic state of systemic and adipose tissue-derived inflammation. Glucose-dependent insulinotropic polypeptide (GIP) is an incretin hormone also acting on adipocytes. We investigated whether GIP affects inflammation, lipolysis, and insulin resistance in human adipocytes. Human subcutaneous preadipocyte-derived adipocytes, differentiated in vitro, were treated with human GIP to analyze mRNA expression and protein secretion of cytokines, glycerol, and free fatty acid release and insulin-induced glucose uptake. GIP induced mRNA expression of IL-6, IL-1β, and the IL-1 receptor antagonist IL-1Ra, whereas TNFα, IL-8, and monocyte chemotactic protein (MCP)-1 remained unchanged. Cytokine induction involved PKA and the NF-κB pathway as well as an autocrine IL-1 effect. Furthermore, GIP potentiated IL-6 and IL-1Ra secretion in the presence of LPS, IL-1β, and TNFα. GIP induced lipolysis via activation of hormone-sensitive lipase and was linked to NF-κB activation. Finally, chronic GIP treatment impaired insulin-induced glucose uptake possibly due to the observed impaired translocation of glucose transporter GLUT4. In conclusion, GIP induces an inflammatory and prolipolytic response via the PKA -NF-κB-IL-1 pathway and impairs insulin sensitivity of glucose uptake in human adipocytes.

Nucleotide-binding Oligomerization Domain-1 Ligand Induces Inflammation and Attenuates Glucose Uptake in Human Adipocytes

To investigate the effects of stimulant for nucleotide-binding oligomerization domain 1 (NOD1) on secretion of proinflammatory chemokine ÷ cytokines and insulin-dependent glucose uptake in human differentiated adipocytes. Adipose tissues were obtained from patients undergoing liposuction. Stromal vascular cells were extracted and differentiated into adipocytes.A specific ligand for NOD1, was administered to human adipocytes in culture. Nuclear factor-κB transcriptional activity and proinflammatory chemokine ÷ cytokines production were determined by reporter plasmid assay and enzyme-linked immunosorbent assay, respectively.Insulin-stimulated glucose uptake was measured by 2-deoxy-D-[³H] glucose uptake assay. Furthermore, chemokine ÷ cytokine secretion and glucose uptake in adipocytes transfected with small interfering RNA (siRNA) targeting NOD1 upon stimulation of NOD1 ligand were analyzed. Nuclear factor-κB transcriptional activity and monocyte chemoattractant protein-1 (MCP-1), interleukin (IL)-6, and IL-8 secretion in human adipocytes were markedly increased stimulated with NOD1 ligand (all P<0.01).Insulin-induced glucose uptake was decreased upon the activation of NOD1 (P<0.05).NOD1 gene silencing by siRNA reduced NOD1 ligand-induced MCP-1,IL-6, and IL-8 release and increased insulin-induced glucose uptake (all P<0.05). NOD1 activation in adipocytes might be implicated in the onset of insulin resistance.

MTOR inhibition with rapamycin causes impaired insulin signalling and glucose uptake in human subcutaneous and omental adipocytes

Rapamycin is an immunosuppressive agent used after organ transplantation, but its molecular effects on glucose metabolism needs further evaluation. We explored rapamycin effects on glucose uptake and insulin signalling proteins in adipocytes obtained via subcutaneous (n=62) and omental (n=10) fat biopsies in human donors.At therapeutic concentration (0.01μM) rapamycin reduced basal and insulin-stimulated glucose uptake by 20–30%, after short-term (15min) or long-term (20h) culture of subcutaneous (n=23 and n=10) and omental adipocytes (n=6 and n=7). Rapamycin reduced PKB Ser473 and AS160 Thr642 phosphorylation, and IRS2 protein levels in subcutaneous adipocytes. Additionally, it reduced mTOR–raptor, mTOR–rictor and mTOR–Sin1 interactions, suggesting decreased mTORC1 and mTORC2 formation. Rapamycin also reduced IR Tyr1146 and IRS1 Ser307/Ser616/Ser636 phosphorylation, whereas no effects were observed on the insulin stimulated IRS1–Tyr and TSC2 Thr1462 phosphorylation.This is the first study to show that rapamycin reduces glucose uptake in human adipocytes through impaired insulin signalling and this may contribute to the development of insulin resistance associated with rapamycin therapy.

St. John's Wort inhibits insulin signaling in murine and human adipocytes

Adipocytes are insulin-sensitive cells that play a major role in energy homeostasis. Obesity is the primary disease of fat cells and a major risk factor for the development of Type 2 diabetes, cardiovascular disease, and metabolic syndrome. The use of botanicals in the treatment of metabolic diseases is an emerging area of research. In previous studies, we screened over 425 botanical extracts for their ability to modulate adipogenesis and insulin sensitivity. We identified St. John's Wort (SJW) extracts as inhibitors of adipogenesis of 3T3-L1 cells and demonstrated that these extracts also inhibited insulin-sensitive glucose uptake in mature fat cells. In these follow-up studies we have further characterized the effects of SJW on insulin action in both murine and human fat cells. We have shown that SJW also attenuates insulin-sensitive glucose uptake in human adipocytes. Moreover, SJW inhibits IRS-1 tyrosine phosphorylation in both murine and human fat cells. Botanical extracts are complex mixtures. Many bioactive compounds have been identified in SJW, including hypericin (HI) and hyperforin (HF). We have examined the ability of HI and HF, purified from SJW, to modulate adipocyte development and insulin action in mature adipocytes. Our novel studies indicate that the profound effects of SJW on adipogenesis, IRS-1 activation, and insulin-stimulated glucose uptake are not mediated by HI and/or HF. Nonetheless, we propose that extracts of SJW may contribute to adipocyte related diseases by limiting differentiation of preadipocytes and significantly inducing insulin resistance in mature fat cells.

Monoclonal antibody to the six-transmembrane epithelial antigen of prostate 4 promotes apoptosis and inhibits proliferation and glucose uptake in human adipocytes

We previously identified the six-transmembrane epithelial antigen of prostate (STEAP) 4 as a novel plasma membrane protein that is up-regulated in obese patients and may play a significant role in the development of human obesity. In this study, a STEAP4-specific antibody was used to characterize the biological functions of the STEAP4 protein in human adipocytes. Cell viability assays (Trypan Blue exclusion), CCK-8 assays and cell cycle analysis showed that the STEAP4 antibody inhibited pre-adipocyte proliferation. Morphological observations by electron microscopy and confocal laser microscopy, annexin V-FITC labeling, caspase-3 and caspase-8 activity assays as well as data from quantitative real-time RT-PCR (qPCR) further determined that the STEAP4 antibody could promote apoptosis in pre-adipocytes. Based on quantitative Oil Red O staining and the expression profiles of specific markers, we demonstrated that the STEAP4 antibody did not affect adipogenesis, but the 2-deoxy-d-[3H]-glucose uptake tests showed that it induced the insulin-stimulated glucose uptake in mature human adipocytes. In conclusion, our results demonstrated that the STEAP4 antibody does not influence human adipocyte differentiation, but it is likely that the STEAP4 protein regulates proliferation and apoptosis and plays an important role in modulating the insulin sensitivity of human adipocytes.

Mechanisms of metformin action on glucose transport and metabolism in human adipocytes

The mechanisms of metformin effects on glucose transport and metabolism were investigated in human adipocytes. Human preadipocytes obtained from surgical biopsies were differentiated in vitro into adipocytes and the effects of metformin on glucose uptake, glucose oxidation and the involved signaling pathways were analyzed. Metformin (1 mM, 24 h) increased glucose uptake 2.3 ± 0.2-fold ( p < 0.001 vs. basal) in human adipocytes, without altering cell viability and oxygen consumption. Metformin did not alter GLUT-1 mRNA expression and protein content but increased GLUT-4 mRNA expression and cellular protein content, leading to increased GLUT-4 protein content in the plasma membrane. Neither basal nor insulin-induced phosphorylation of Akt at Ser-473 and AS160 (Akt substrate of 160 kDa) at Thr-642 were enhanced by metformin. Suppression of metformin-induced AMP-activated protein kinase (AMPK) activity by AMPKα1 silencing, however, reduced metformin-associated GLUT-4 expression and stimulation of glucose uptake. In addition, metformin induced glucose oxidation. In conclusion, activation of AMPKα1 without impairment of cell respiration is crucial for metformin-mediated increase in GLUT-4 protein content and glucose uptake in human adipocytes.

Type 2 diabetes-related bioactivities of coffee: Assessment of antioxidant activity, NF-κB inhibition, and stimulation of glucose uptake

Coffee consumption is correlated with a lower risk for type 2 diabetes mellitus (T2D), though whether differences exist in the T2D-mitigating bioactivities of decaffeinated (RD) and regular (RR) coffee is unclear. We conducted cell-based experiments to determine whether different phenolic levels in RD and RR affect T2D-mitigating bioactivities. The total phenolic content and the chemical antioxidant activity were significantly higher in RD than RR. However, these coffees had comparable cellular antioxidant activity. Both coffees reduced activation of NF-κB, with RR being twice as strong as RD. They also both increased glucose uptake in human adipocytes by 2-fold. Of the bioactivities examined, only chemical antioxidant activity was related to total phenolic levels. The NF-κB inhibition was proportional to chlorogenic acid levels, though chlorogenic acids could not account for the full inhibitory effect of coffee. Thus, a matrix effect may exist, whereby components of coffee work together to provide bioactivities that ameliorate the T2D risk.

Polyphenol-rich grape powder extract (GPE) attenuates inflammation in human macrophages and in human adipocytes exposed to macrophage-conditioned media

Obesity-associated inflammation is characterized by an increased abundance of macrophages (MPhis) in white adipose tissue (WAT), leading to the production of inflammatory cytokines, chemokines and prostaglandins (PGs) that can cause insulin resistance. Grape powder extract (GPE) is rich in phenolic phytochemicals that possess anti-oxidant and anti-inflammatory properties. We examined the ability of GPE to prevent lipopolysaccharide (LPS)-mediated inflammation in human MPhis and silence the cross-talk between human MPhis and adipocytes. We investigated the effect of GPE pretreatment on LPS-mediated activation of mitogen activated protein kinases (MAPKs), nuclear factor kappa B (NF-kappaB) and activator protein-1 (AP-1), and induction of inflammatory genes in human MPhis (that is, differentiated U937 cells). In addition, we determined the effect of GPE pretreatment of MPhis on inflammation and insulin resistance in primary human adipocytes incubated with LPS-challenged MPhi-conditioned medium (MPhi-CM). Pretreatment of MPhis with GPE attenuated LPS-induction of inflammatory cytokines, such as tumor necrosis factor-alpha (TNF-alpha), interleukin-6 (IL-6) and IL-1beta; chemokines, such as IL-8 and interferon-gamma inducible protein-10 (IP-10); and a marker of PG production, cyclooxygenase-2 (COX-2). Grape powder extract also attenuated LPS activation of MAPKs, NF-kappaB and AP-1 (c-Jun), as evidenced by decreased (1) phosphorylation of c-Jun NH(2)-terminal kinase (JNK) and p38; (2) degradation of IkappaBalpha and activation of an NF-kappaB reporter construct; and (3) phosphorylation of c-Jun and Elk-1. Using LPS-challenged MPhi-CM, GPE pretreatment attenuated MPhi-mediated inflammatory gene expression, activation of an NF-kappaB reporter and suppression of insulin-stimulated glucose uptake in human adipocytes. Collectively, these data demonstrate that GPE attenuates LPS-mediated inflammation in MPhis, possibly by decreasing the activation of MAPKs, NF-kappaB and AP-1, and that GPE decreases the capacity of LPS-stimulated MPhis to inflame adipocytes and cause insulin resistance.