Insulin Resistance In Hepatocytes Research Articles

Celastrol attenuates mitochondrial dysfunction and inflammation in palmitate-mediated insulin resistance in C3A hepatocytes

Accumulating evidence indicates that mitochondrial dysfunction-induced inflammation is among the convergence points for the greatest hallmarks of hepatic insulin resistance. Celastrol, an anti-inflammatory compound from the root of Tripterygium Wilfordii has been reported to mitigate insulin resistance and inflammation in animal disease models. Nevertheless, the specific mechanistic actions of celastrol in modulating such improvements at the cellular level remain obscure. The present study sought to explore the mechanistic roles of celastrol upon insulin resistance induced by palmitate in C3A human hepatocytes. The hepatocytes exposed to palmitate (0.75mM) for 48h exhibited reduced both basal and insulin-stimulated glucose uptake, mitochondrial dysfunction, leading to increased mitochondrial oxidative stress with diminished fatty acid oxidation. Elevated expressions of nuclear factor-kappa B p65 (NF-κB p65), c-Jun NH(2)-terminal kinase (JNK) signaling pathways and the amplified release of pro-inflammatory cytokines including IL-8, IL-6, TNF-α and CRP were observed following palmitate treatment. Consistently, palmitate reduced and augmented phosphorylated Tyrosine-612 and Serine-307 of insulin receptor substrate-1 (IRS-1) proteins, respectively in hepatocytes. However, celastrol at the optimum concentration of 30nM was able to reverse these deleterious occasions and protected the cells from mitochondrial dysfunction and insulin resistance. Importantly, we presented evidence for the first time that celastrol efficiently prevented palmitate-induced insulin resistance in hepatocytes at least, via improved mitochondrial functions and insulin signaling pathways. In summary, the present investigation underlines a conceivable mechanism to elucidate the cytoprotective potential of celastrol in attenuating mitochondrial dysfunction and inflammation against the development of hepatic insulin resistance.

Low-Dose Dihydrotestosterone Drives Metabolic Dysfunction via Cytosolic and Nuclear Hepatic Androgen Receptor Mechanisms.

Androgen excess in women is associated with metabolic dysfunction (e.g., obesity, hyperinsulinemia, insulin resistance, and increased risk of type 2 diabetes) and reproductive dysfunction (e.g., polycystic ovaries, amenorrhea, dysregulated gonadotropin release, and infertility). We sought to identify the effects of androgen excess on glucose metabolic dysfunction and the specific mechanisms of action by which androgens are inducing pathology. We developed a mouse model that displayed pathophysiological serum androgen levels with normal body mass/composition to ensure that the phenotypes were directly from androgens and not an indirect consequence of obesity. We performed reproductive tests, metabolic tests, and hormonal assays. Livers were isolated and examined via molecular, biochemical, and histological analysis. Additionally, a low-dose dihydrotestosterone (DHT) cell model using H2.35 mouse hepatocytes was developed to study androgen effects on hepatic insulin signaling. DHT mice demonstrated impaired estrous cyclicity; few corpora lutea in the ovaries; glucose, insulin, and pyruvate intolerance; and lowered hepatic insulin action. Mechanistically, DHT increased hepatic androgen-receptor binding to phosphoinositide-3-kinase (PI3K)-p85, resulting in dissociation of PI3K-p85 from PI3K-p110, leading to reduced PI3K activity and decreased p-AKT and, thus, lowered insulin action. DHT increased gluconeogenesis via direct transcriptional regulation of gluconeogenic enzymes and coactivators. The hepatocyte model recapitulated the in vivo findings. The DHT-induced hepatocyte insulin resistance was reversed by the androgen-receptor antagonist, flutamide. These findings present a phenotype (i.e., impaired glucose tolerance and disrupted glucose metabolism) in a lean hyperandrogenemia model (low-dose DHT) and data to support 2 molecular mechanisms that help drive androgen-induced impaired glucose metabolism.

Macrophage-Mediated Effects of Airborne Fine Particulate Matter (PM2.5) on Hepatocyte Insulin Resistance in Vitro.

Fine particulate matter (PM2.5) pollution poses significant health risks worldwide, including metabolic syndrome-related diseases with the characteristic feature of insulin resistance. However, the mechanism and influencing factors of this effect are poorly understood. In this serial in vitro study, we aimed at testing the hypothesis that macrophage-mediated effects of PM2.5 on hepatic insulin resistance depend on its chemical composition. Mouse macrophages were exposed to PM2.5 that had been collected during summer or winter in Beijing, which represented different compositions of PM2.5. Thereafter, hepatocytes were treated with macrophage-conditioned medium (CM). PM2.5 induced interleukin-6, tumor necrosis factor-α, and monocyte chemoattractant protein-1 expression and secretion in macrophages, particularly after winter PM2.5 exposure. Correspondingly, winter CM weakened hepatocellular insulin-stimulated glucose consumption. Further investigation revealed that the normal insulin pathway was suppressed in winter CM-treated hepatocytes, with increased phosphorylation of insulin receptor substrate 1 at serine residue 307 (Ser307) and decreased phosphorylation of protein kinase B (PKB/AKT) and forkhead box transcription factor O1 (FoxO1). Moreover, c-Jun N-terminal kinase, a key moderator of the sensitivity response to insulin stimulation, was activated in hepatocytes treated with winter CM. Although further studies are warranted, this preliminary study suggested an association between PM composition and insulin resistance, thus contributing to our understanding of the systemic toxicity of PM2.5.

Prunin is a highly potent flavonoid from Prunus davidiana stems that inhibits protein tyrosine phosphatase 1B and stimulates glucose uptake in insulin-resistant HepG2 cells.

Prunin is the main flavonoid in Prunus davidiana stems and improves hyperglycemia and hyperlipidemia in streptozotocin-induced diabetic rats. The aim of this study was to investigate the in vitro anti-diabetic potential of prunin via the inhibition of protein tyrosine phosphatase 1B (PTP1B), α-glucosidase, peroxynitrite (ONOO-)-mediated tyrosine nitration, and stimulation of glucose uptake in insulin-resistant hepatocytes. In addition, a molecular docking simulation was performed to predict specific prunin binding modes during PTP1B inhibition. Prunin showed strong inhibitory activity against PTP1B, with an IC50 value of 5.5±0.29µM, and significant inhibitory activity against α-glucosidase, with an IC50 value of 317±2.12µM. Moreover, a kinetics study revealed that prunin inhibited PTP1B (K i=8.66) and α-glucosidase (K i=189.56) with characteristics typical of competitive and mixed type inhibitors, respectively. Docking simulations showed that prunin selectively inhibited PTP1B by targeting its active site and exhibited good binding affinity, with a docking score of -9kcal/mol. Furthermore, prunin exhibited dose-dependent inhibitory activity against ONOO--mediated tyrosine nitration and stimulated glucose uptake by decreasing PTP1B expression level in insulin-resistant HepG2 cells. These results indicate that prunin has significant potential as a selective PTP1B inhibitor and may possess anti-diabetic properties by improving insulin resistance.

Fucosterol activates the insulin signaling pathway in insulin resistant HepG2 cells via inhibiting PTP1B.

Insulin resistance is a characteristic feature of type 2 diabetes mellitus (T2DM) and is characterized by defects in insulin signaling. This study investigated the modulatory effects of fucosterol on the insulin signaling pathway in insulin-resistant HepG2 cells by inhibiting protein tyrosine phosphatase 1B (PTP1B). In addition, molecular docking simulation studies were performed to predict binding energies, the specific binding site of fucosterol to PTP1B, and to identify interacting residues using Autodock 4.2 software. Glucose uptake was determined using a fluorescent D-glucose analogue and the glucose tracer 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxyglucose, and the signaling pathway was detected by Western blot analysis. We found that fucosterol enhanced insulin-provoked glucose uptake and conjointly decreased PTP1B expression level in insulin-resistant HepG2 cells. Moreover, fucosterol significantly reduced insulin-stimulated serine (Ser307) phosphorylation of insulin receptor substrate 1 (IRS1) and increased phosphorylation of Akt, phosphatidylinositol-3-kinase, and extracellular signal- regulated kinase 1 at concentrations of 12.5, 25, and 50µM in insulin-resistant HepG2 cells. Fucosterol inhibited caspase-3 activation and nuclear factor kappa B in insulin-resistant hepatocytes. These results suggest that fucosterol stimulates glucose uptake and improves insulin resistance by downregulating expression of PTP1B and activating the insulin signaling pathway. Thus, fucosterol has potential for development as an anti-diabetic agent.

Insulin-induced cytokine production in macrophages causes insulin resistance in hepatocytes.

Overweight and obesity are associated with hyperinsulinemia, insulin resistance, and a low-grade inflammation. Although hyperinsulinemia is generally thought to result from an attempt of the β-cell to compensate for insulin resistance, there is evidence that hyperinsulinaemia itself may contribute to the development of insulin resistance and possibly the low-grade inflammation. To test this hypothesis, U937 macrophages were exposed to insulin. In these cells, insulin induced expression of the proinflammatory cytokines IL-1β, IL-8, CCL2, and OSM. The insulin-elicited induction of IL-1β was independent of the presence of endotoxin and most likely mediated by an insulin-dependent activation of NF-κB. Supernatants of the insulin-treated U937 macrophages rendered primary cultures of rat hepatocytes insulin resistant; they attenuated the insulin-dependent induction of glucokinase by 50%. The cytokines contained in the supernatants of insulin-treated U937 macrophages activated ERK1/2 and IKKβ, resulting in an inhibitory serine phosphorylation of the insulin receptor substrate. In addition, STAT3 was activated and SOCS3 induced, further contributing to the interruption of the insulin receptor signal chain in hepatocytes. These results indicate that hyperinsulinemia per se might contribute to the low-grade inflammation prevailing in overweight and obese patients and thereby promote the development of insulin resistance particularly in the liver, because the insulin concentration in the portal circulation is much higher than in all other tissues.

Blockade of Sphingosine 1-Phosphate Receptor 2 Signaling Attenuates High-Fat Diet-Induced Adipocyte Hypertrophy and Systemic Glucose Intolerance in Mice

Sphingosine 1-phosphate (S1P) is known to regulate insulin resistance in hepatocytes, skeletal muscle cells, and pancreatic β-cells. Among its 5 cognate receptors (S1pr1-S1pr5), S1P seems to counteract insulin signaling and confer insulin resistance via S1pr2 in these cells. S1P may also regulate insulin resistance in adipocytes, but the S1pr subtype(s) involved remains unknown. Here, we investigated systemic glucose/insulin tolerance and phenotypes of epididymal adipocytes in high-fat diet (HFD)-fed wild-type and S1pr2-deficient (S1pr2(-/-)) mice. Adult S1pr2(-/-) mice displayed smaller body/epididymal fat tissue weights, but the differences became negligible after 4 weeks with HFD. However, HFD-fed S1pr2(-/-) mice displayed better scores in glucose/insulin tolerance tests and had smaller epididymal adipocytes that expressed higher levels of proliferating cell nuclear antigen than wild-type mice. Next, proliferation/differentiation of 3T3-L1 and 3T3-F442A preadipocytes were examined in the presence of various S1pr antagonists: JTE-013 (S1pr2 antagonist), VPC-23019 (S1pr1/S1pr3 antagonist), and CYM-50358 (S1pr4 antagonist). S1P or JTE-013 treatment of 3T3-L1 preadipocytes potently activated their proliferation and Erk phosphorylation, whereas VPC-23019 inhibited both of these processes, and CYM-50358 had no effects. In contrast, S1P or JTE-013 treatment inhibited adipogenic differentiation of 3T3-F442A preadipocytes, whereas VPC-23019 activated it. The small interfering RNA knockdown of S1pr2 promoted proliferation and inhibited differentiation of 3T3-F442A preadipocytes, whereas that of S1pr1 acted oppositely. Moreover, oral JTE-013 administration improved glucose tolerance/insulin sensitivity in ob/ob mice. Taken together, S1pr2 blockade induced proliferation but suppressed differentiation of (pre)adipocytes both in vivo and in vitro, highlighting a novel therapeutic approach for obesity/type 2 diabetes.

TRAF1 knockdown alleviates palmitate-induced insulin resistance in HepG2 cells through NF-κB pathway

High-fat diet (HFD) and inflammation are key contributors to insulin resistance (IR) and Type 2 diabetes mellitus (T2DM). With HFD, plasma free fatty acids (FFAs) can activate the nuclear factor-κB (NF-κB) in target tissues, then initiate negative crosstalk between FFAs and insulin signaling. However, the molecular link between IR and inflammation remains to be identified. We here reported that tumor necrosis factor receptor-associated factor 1 (TRAF1), an adapter in signal transduction, was involved in the onset of IR in hepatocytes. TRAF1 was significantly up-regulated in insulin-resistant liver tissues and palmitate (PA)-treated HepG2 cells. In addition, we showed that depletion of TRAF1 led to inhibition of the activity of NF-κB. Given the fact that the activation of NF-κB played a facilitating role in IR, the phosphorylation of Akt and GSK3β was also analyzed. We found that depletion of TRAF1 markedly reversed PA-induced attenuation of the phosphorylation of Akt and GSK3β in the cells. The accumulation of lipid droplets in hepatocyte and expression of two key gluconeogenic enzymes, PEPCK and G6Pase, were also determined and found to display a similar tendency with the phosphorylation of Akt and GSK3β. Glucose uptake assay indicated that knocking down TRAF1 blocked the effect of PA on the suppression of glucose uptake. These data implicated that TRAF1 knockdown might alleviate PA-induced IR in HepG2 cells through NF-κB pathway.

4-Hydroxyisoleucine improves insulin resistance in HepG2 cells by decreasing TNF-α and regulating the expression of insulin signal transduction proteins.

Previous studies have indicated that 4-hydroxy-isoleucine (4-HIL) improves insulin resistance, however, the underlying mechanisms remain to be elucidated. In the present study, the molecular mechanisms underlying how 4-HIL improves insulin resistance in hepatocytes were examined. HepG2 cells were co-cultured with insulin and a high glucose concentration to obtain insulin-resistant (IR) HepG2 cells. Insulin sensitivity was determined by measuring the glucose uptake rate. The IR HepG2 cells were treated with different concentrations of 4-HIL to determine its effect on IR Hep2 cells. The levels of tumor necrosis factor-α (TNF-α) were measured by an enzyme-linked immunosorbent assay and protein levels of TNF-α converting enzyme (TACE)/tissue inhibitor of metalloproteinase 3 (TIMP3), insulin receptor substrate (IRS)-1, IRS-2, phosphorylated (p)-IRS-1 (Ser307) and glucose transporter type 4 (GLUT4) were measured by western blot analysis. The results of the present study demonstrated that insulin-induced glucose uptake was reduced in IR HepG2 cells; however, this reduction was reversed by 4-HIL in a dose-dependent manner. 4-HIL achieved this effect by downregulating the expression of TNF-α and TACE, and upregulating the expression of TIMP3 in IR HepG2 cells. In addition, 4-HIL increased the expression of the insulin transduction regulators IRS-1 and GLUT4, and decreased the expression of p-IRS-1 (Ser307), without affecting the expression of IRS-2. The present study suggests that 4-HIL improved insulin resistance in HepG2 cells by the following mechanisms: 4-HIL reduced TNF-α levels by affecting the protein expression of the TACE/TIMP3 system and 4-HIL stimulated the expression of IRS-1 and GLUT4, but inhibited the expression of p-IRS-1 (Ser307).

Role of hepatocyte S6K1 in palmitic acid-induced endoplasmic reticulum stress, lipotoxicity, insulin resistance and in oleic acid-induced protection

The excess of saturated free fatty acids, such as palmitic acid, that induces lipotoxicity in hepatocytes, has been implicated in the development of non-alcoholic fatty liver disease also associated with insulin resistance. By contrast, oleic acid, a monounsaturated fatty acid, attenuates the effects of palmitic acid. We evaluated whether palmitic acid is directly associated with both insulin resistance and lipoapoptosis in mouse and human hepatocytes and the impact of oleic acid in the molecular mechanisms that mediate both processes. In human and mouse hepatocytes palmitic acid at a lipotoxic concentration triggered early activation of endoplasmic reticulum (ER) stress-related kinases, induced the apoptotic transcription factor CHOP, activated caspase 3 and increased the percentage of apoptotic cells. These effects concurred with decreased IR/IRS1/Akt insulin pathway. Oleic acid suppressed the toxic effects of palmitic acid on ER stress activation, lipoapoptosis and insulin resistance. Besides, oleic acid suppressed palmitic acid-induced activation of S6K1. This protection was mimicked by pharmacological or genetic inhibition of S6K1 in hepatocytes. In conclusion, this is the first study highlighting the activation of S6K1 by palmitic acid as a common and novel mechanism by which its inhibition by oleic acid prevents ER stress, lipoapoptosis and insulin resistance in hepatocytes.

The Effect of Butyric Acid on Gene Expression of GLUT2 and IRS1 on Human Hepatocytes in vitro

Diabetes mellitus type 2 (DMT2) affects hundreds of millions of people globally and costs billions of dollars each year. The importance of research toward prevention and treatment of this disease cannot be overestimated. Butyric acid is a short chain fatty acid that has been shown in mice and in intestinal cell culture studies to increase insulin sensitivity at the level of gene expression. However, little or no work has been reported on its effects on human liver cells. The present study determined the changes in gene expression of glucose transporter 2 (GLUT2) and insulin receptor substrate 1 (IRS1) on insulin shocked THLE-2 human liver cells exposed in vitro to the following concentrations of butyric acid in mg/ml: 0.05, 0.1, and 1.0. GLUT2 and IRS1 had increases in expression at doses of butyric acid previously found to be nontoxic in human serum. This work indicates that more studies involving the effects of butyric acid on gene expression of insulin resistant human hepatocytes are warranted.

TRAM1 protect HepG2 cells from palmitate induced insulin resistance through ER stress-JNK pathway

Excess serum free fatty acids (FFAs) are fundamental to the pathogenesis of insulin resistance. Chronic endoplasmic reticulum (ER) stress is a major contributor to obesity-induced insulin resistance in the liver. With high-fat feeding (HFD), FFAs can activate chronic endoplasmic reticulum (ER) stress in target tissues, initiating negative crosstalk between FFAs and insulin signaling. However, the molecular link between insulin resistance and ER stress remains to be identified. We here reported that translocating chain-associated membrane protein 1 (TRAM1), an ER-resident membrane protein, was involved in the onset of insulin resistance in hepatocytes. TRAM1 was significantly up-regulated in insulin-resistant liver tissues and palmitate (PA)-treated HepG2 cells. In addition, we showed that depletion of TRAM1 led to hyperactivation of CHOP and GRP78, and the activation of downstream JNK pathway. Given the fact that the activation of ER stress played a facilitating role in insulin resistance, the phosphorylation of Akt and GSK-3β was also analyzed. We found that depletion of TRAM1 markedly attenuated the phosphorylation of Akt and GSK-3β in the cells. Moreover, application with JNK inhibitor SP600125 reversed the effect of TRAM1 interference on Akt phosphorylation. The accumulation of lipid droplets and expression of two key gluconeogenic enzymes, PEPCK and G6Pase, were also determined and found to display a similar tendency with the phosphorylation of Akt. Glucose uptake assay indicated that knocking down TRAM1 augmented PA-induced down-regulation of glucose uptake, and inhibition of JNK using SP600125 could block the effect of TRAM1 on glucose uptake. These data implicated that TRAM1 might protect HepG2 cells against PA-induced insulin resistance through alleviating ER stress.

MiR-301a Mediates the Effect of IL-6 on the AKT/GSK Pathway and Hepatic Glycogenesis by Regulating PTEN Expression

Background/Aims: IL-6 has been implicated in the pathogenesis of insulin resistance. MiR-301a plays an important role in various biological and pathological processes, including cellular development and differentiation, inflammation, apoptosis and cancer. However, whether miR-301a mediates IL-6-induced insulin resistance in hepatocytes remains unknown. Methods: The activation of AKT/GSK pathway and the level of glycogenesis were examed in NCTC 1469 cells transfected miR-301a mimics and inhibitor. Using computational miRNA target prediction database, PTEN was a target of miR-301a. The effect of miR-301a on PTEN expression was evaluated using Luciferase assay and western blot. A PTEN-specific siRNA was used to further determine the effect of PTEN on IL-6-induced insulin resistance. Results: In vivo and in vitro treatment with IL-6 was led to down-regulation of miR-301a, accompanied by impairment of theAKT/GSK pathway and glycogenesis. Importantly, over-expression of miR-301a rescued IL-6-induced decreased activation of the AKT/GSK pathway and hepatic glycogenesis. In contrast, down-regulation of miR-301a induced impaired phosphorylation of AKT and GSK, accompanied by reduced glycogenesis in hepatocytes. Moreover, our results indicate that suppression of PTEN, a target of miR-301a, diminished the effect of IL-6 on the AKT/GSK pathway and hepatic glycogenesis. Conclusion: We present novel evidence of the contribution of miR-301a to IL-6-induced insulin resistance by direct regulation of PTEN expression.

Hepatoprotection by curcumin and sulforaphane against tBHP-induced oxidative damage

Background and aim: Antioxidants have recently been of keen therapeutic interest, due to the causative role of oxidative stress in induction and progression of liver, pancreatic and neurodegenerative diseases. Most plant-derived antioxidants are key components of vegetables and spices which are part of daily meals. Curcumin (from Asian spice turmeric) and sulforaphane (abundant in cruciferous vegetables such as broccoli), are reported to have antioxidant potential [1, 2] and were assessed in the current study for their cytoprotective activity in HepG2 liver cells. Methods: Radical scavenging activities of curcumin and sulforaphane were investigated against 2,2-diphenyl-1-picrylhydrazyl (DPPH). Using HepG2 liver cells, cytoprotection against tert-butyl hydroperoxide (tBHP)-induced cytotoxicity was evaluated in 5h co-exposure and 20h pre-exposure protocols with phytochemicals. Cell viability was determined in Neutral Red assay. Quercetin served as a positive phyto-antioxidant standard [3]. All phytochemicals were dissolved in DMSO to final solution of 10 mg/ml. Results and Discussion: Radical scavenging activity of quercetin was stronger than curcumin, but not significantly more potent in 5h co-exposure with tBHP. Sulforaphane was not cytoprotective in 5h protocol, this it lacked antiradical activity. Unlike quercetin, both phytochemicals were cytotoxic at high concentrations (Table 1). Conclusion: Sulforaphane, curcumin and quercetin could protect HepG2 cells via indirectly inducing intrinsic cytoprotective mechanisms during 20h pre-exposure protocol. Sulforophane does not act as a free radical scavenger, whilst curcumin and quercetin have antiradical properties, acting as direct and indirect antioxidants. The cytoprotective activities of quercetin, curcumin and sulforaphane suggest that these may be strong therapeutic phyto-antioxidants in preventing oxidative damage. Keywords: Curcumin, Antioxidant, Quercetin, Sulforaphane, HepG2

MiRNA-145 is involved in the development of resistin-induced insulin resistance in HepG2 cells

BackgroundResistin is associated with insulin resistance, and determining its developmental and molecular mechanisms may help the development of novel treatments. MicroRNAs (miRNAs) are involved in many physiological and pathological processes as negative regulators. However, it remains unclear whether miRNAs play a role in resistin-induced insulin resistance. We performed mouse liver miRNA microarrays to analyze the differences in expression between resistin-treated and control mice. Resistin upregulated miR-145 both in vivo and in vitro. Therefore, we aimed to study whether miR-145 played a role in resistin-induced insulin resistance. Methods and resultsWe transfected HepG2 cells, and used miR-145 mimics and inhibitors to assess the role of miR-145 in resistin-induced insulin resistance. The overexpression of miR-145 inhibited glucose uptake in HepG2 cells, diminished the phosphorylation of Akt and IRS-1, and induced insulin resistance in hepatocytes. Next, a study of transcriptional regulation revealed that p65 was essential for the upregulation of miR-145 by resistin, and chromatin immunoprecipitation (ChIP) confirmed that p65 could bind to the promoter region of miR-145. ConclusionmiR-145 plays a role in the development of resistin-induced insulin resistance via the p65 pathway.

Roles of Vitamin A Metabolism in the Development of Hepatic Insulin Resistance.

The increase in the number of people with obesity- and noninsulin-dependent diabetes mellitus has become a major public health concern. Insulin resistance is a common feature closely associated with human obesity and diabetes. Insulin regulates metabolism, at least in part, via the control of the expression of the hepatic genes involved in glucose and fatty acid metabolism. Insulin resistance is always associated with profound changes of the expression of hepatic genes for glucose and lipid metabolism. As an essential micronutrient, vitamin A (VA) is needed in a variety of physiological functions. The active metablite of VA, retinoic acid (RA), regulates the expression of genes through the activation of transcription factors bound to the RA-responsive elements in the promoters of RA-targeted genes. Recently, retinoids have been proposed to play roles in glucose and lipid metabolism and energy homeostasis. This paper summarizes the recent progresses in our understanding of VA metabolism in the liver and of the potential transcription factors mediating RA responses. These transcription factors are the retinoic acid receptor, the retinoid X receptor, the hepatocyte nuclear factor 4α, the chicken ovalbumin upstream promoter-transcription factor II, and the peroxisome proliferator-activated receptor β/δ. This paper also summarizes the effects of VA status and RA treatments on the glucose and lipid metabolism in vivo and the effects of retinoid treatments on the expression of insulin-regulated genes involved in the glucose and fatty acid metabolism in the primary hepatocytes. I discuss the roles of RA production in the development of insulin resistance in hepatocytes and proposes a mechanism by which RA production may contribute to hepatic insulin resistance. Given the large amount of information and progresses regarding the physiological functions of VA, this paper mainly focuses on the findings in the liver and hepatocytes and only mentions the relative findings in other tissues and cells.

MiR-200s Contribute to Interleukin-6 (IL-6)-induced Insulin Resistance in Hepatocytes

By influencing the activity of the PI3K/AKT pathway, IL-6 acts as an important regulator of hepatic insulin resistance. miR-200s have been shown to control growth by regulating PI3K, but the role of miR-200s in the development of hepatic insulin resistance remains unclear. The present study showed that elevated serum concentration of IL-6 is associated with decreased levels of miR-200s, impaired activation of the AKT/glycogen synthase kinase (GSK) pathway, and reduced glycogenesis that occurred in the livers of db/db mice. As shown in the murine NCTC 1469 hepatocytes and the primary hepatocytes treated with 10 ng/ml IL-6 for 24 h and in 12-week-old male C57BL/6J mice injected with 16 μg/ml IL-6 by pumps for 7 days, IL-6 administration induced insulin resistance through down-regulation of miR-200s. Moreover, IL-6 treatment inhibited the phosphorylation of AKT and GSK and decreased the glycogenesis. The effects of IL-6 could be diminished by suppression of FOG2 expression. We concluded that IL-6 treatment may impair the activities of the PI3K/AKT/GSK pathway and inhibit the synthesis of glycogen, perhaps via down-regulating miR-200s while augmenting FOG2 expression.

The cannabinoid Δ9-tetrahydrocannabivarin (THCV) ameliorates insulin sensitivity in two mouse models of obesity

Background:Cannabinoid type-1 (CB1) receptor inverse agonists improve type 2 diabetes and dyslipidaemia but were discontinued due to adverse psychiatric effects. Δ9-Tetrahydrocannabivarin (THCV) is a neutral CB1 antagonist producing hypophagia and body weight reduction in lean mice. We investigated its effects in dietary-induced (DIO) and genetically (ob/ob) obese mice.Methods:We performed two dose-ranging studies in DIO mice; study 1: 0.3, 1, 2.5, 5 and 12.5 mg kg−1, oral twice daily for 30 days and study 2: 0.1, 0.5, 2.5 and 12.5 mg kg−1, oral, once daily for 45 days. One pilot (study 3: 0.3 and 3 mg kg−1, oral, once daily) and one full dose-ranging (study 4: 0.1, 0.5, 2.5 and 12.5 mg kg−1, oral, once daily) studies in ob/ob mice for 30 days. The CB1 inverse agonist, AM251, oral, 10 mg kg−1 once daily or 5 mg kg−1 twice daily was used as the positive control. Cumulative food and water intake, body weight gain, energy expenditure, glucose and insulin levels (fasting or during oral glucose tolerance tests), plasma high-density lipoprotein and total cholesterol, and liver triglycerides were measured. HL-5 hepatocytes or C2C12 myotubes made insulin-resistant with chronic insulin or palmitic acid were treated with 0, 1, 3 and 10 μℳ THCV or AM251.Results:THCV did not significantly affect food intake or body weight gain in any of the studies, but produced an early and transient increase in energy expenditure. It dose-dependently reduced glucose intolerance in ob/ob mice and improved glucose tolerance and increased insulin sensitivity in DIO mice, without consistently affecting plasma lipids. THCV also restored insulin signalling in insulin-resistant hepatocytes and myotubes.Conclusions:THCV is a new potential treatment against obesity-associated glucose intolerance with pharmacology different from that of CB1 inverse agonists/antagonists.

An Extract from Wax Apple (Syzygium samarangense (Blume) Merrill and Perry) Effects Glycogenesis and Glycolysis Pathways in Tumor Necrosis Factor-α-Treated FL83B Mouse Hepatocytes

FL83B mouse hepatocytes were treated with tumor necrosis factor-α (TNF-α) to induce insulin resistance to investigate the effect of a wax apple aqueous extract (WAE) in insulin-resistant mouse hepatocytes. The uptake of 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino]-2-deoxyglucose (2 NBDG), a fluorescent D-glucose derivative, was performed, and the metabolism of carbohydrates was evaluated by examining the expression of glycogenesis or glycolysis-related proteins in insulin-resistant hepatocytes. The results show that WAE significantly improves the uptake of glucose and enhances glycogen content in insulin-resistant FL83B mouse hepatocytes. The results from Western blot analysis also reveal that WAE increases the expression of glycogen synthase (GS), hexokinase (HXK), glucose-6-phosphate dehydrogenase (G6PD), phosphofructokinase (PFK) and aldolase in TNF-α treated cells, indicating that WAE may ameliorate glucose metabolism by promoting glycogen synthesis and the glycolysis pathways in insulin-resistant FL83B mouse hepatocytes.

Sutherlandia frutescens prevents changes in diabetes-related gene expression in a fructose-induced insulin resistant cell model

Ethnopharmacological relevanceThe African medicinal plant Sutherlandia frutescens (L.) R.Br. (Fabaceae) is traditionally used to treat diabetes and has been shown to have anti-diabetic properties in animal models. The present study investigated the capacity of an aqueous extract of Sutherlandia frutescens to prevent insulin resistance (a precursor of type 2 diabetes) in a human liver cell culture and to identify genes regulated by Sutherlandia frutescens treatment. Materials and methodsA combination of insulin and fructose was used to generate an in vitro model of insulin resistance in human liver cells to compare untreated control, insulin resistant and Sutherlandia frutescens treated insulin resistant cultures. Insulin resistance and its prevention by Sutherlandia frutescens were measured by glucose uptake, gluconeogenesis and lipid accumulation in the cell cultures. Changes in gene expression were quantified using the RT2ProfilerTM PCR Array of 84 diabetes-related genes. ResultsThe insulin resistant Chang liver cells took up significantly less 2-[3H]-deoxyglucose (p<0.05) than controls, released more glucose into the culture medium (p<0.05) and accumulated more intracellular lipid (p<0.05). Simultaneous treatment with Sutherlandia frutescens prevented development of these insulin resistance parameters (p<0.05). A total of 27 potential gene targets of Sutherlandia frutescens were significantly up or down regulated in the Sutherlandia frutescens treated insulin resistant cells. The gene VAMP3, which plays a role in vesicle transport, was down-regulated by insulin resistance, and up-regulated by Sutherlandia frutescens. Twenty six other genes encoding vesicle transporters, receptors, signalling molecules, transcription factors, and metabolic enzymes were significantly regulated by Sutherlandia frutescens. ConclusionThese results confirm that Sutherlandia frutescens can prevent insulin resistance in hepatocytes. The identified changes in gene expression indicate several potential mechanisms of anti-diabetic action for Sutherlandia frutescens, reflecting the multiple bioactive compounds previously identified in aqueous extracts of Sutherlandia frutescens.