2-DOG Uptake Research Articles

P300 or CBP are required for calorie restriction-mediated enhancement of skeletal muscle insulin sensitivity

A calorie-restricted diet robustly improves skeletal muscle insulin sensitivity and is a cornerstone lifestyle intervention for preventing and treating clinical hyperglycemia. While studies demonstrate that insulin-stimulated activation of phosphatidylinositol 3-kinase (PI3K) and RAC-beta serine/threonine-protein kinase (AKT2) are fundamental to enhanced muscle insulin sensitivity after calorie restriction (CR), the downstream signaling steps remain to be fully elucidated. We have previously demonstrated that the lysine acetyltransferases, E1A binding protein (p300) or cAMP response element binding protein binding protein (CBP) are required for insulin-stimulated glucose uptake by skeletal muscle, at both sub-maximal and maximal concentrations of insulin. Moreover, p300/CBP are phosphorylated by AKT, which increases their acetyltransferase activity. Considering these findings together, the objective of this study was to determine whether p300 or CBP are required for the ability of CR to enhance skeletal muscle insulin sensitivity. We hypothesized that combined inhibition of p300 and CBP acetyltransferase activity would prevent CR-induced enhancement of skeletal muscle insulin sensitivity. To address this objective, 8-week-old male C57BL/6NJ mice were either fed ad libitum (AL; n=16) or were fed a CR diet (60% of AL intake; n=16) diet for 20 days. Then, an ex vivo [3H]-2-deoxyglucose (2DOG) approach was used to assess basal and insulin-stimulated (60μU/mL [0.36nM]) 2DOG uptake in paired extensor digitorum longus (EDL) and soleus from fasted (4 h) mice. To define the role of p300/CBP acetyltransferase activity, paired muscles from AL- or CR-fed mice were pre-incubated for 60 minutes with either DMSO (experimental control; n=8/dietary group [AL-DMSO, CR-DMSO]) or the p300/CBP acetyltransferase activity inhibitor, ip300w (25mM; n=8/dietary group [AL-ip300w, CR-ip300w]). As expected, insulin-stimulated 2DOG uptake (i.e. Insulin 2DOG minus basal 2DOG) in DMSO incubated muscles was ~2.3-fold and ~3.3-fold higher in the soleus and EDL, respectively, from CR- vs. AL-fed mice. Remarkably, ip300w not only blocked insulin-stimulated 2DOG uptake in AL-fed mice, but it also abrogated the insulin-sensitizing effects of CR on muscle insulin sensitivity. To further define the importance of p300 and CBP to CR-induced enhancement of skeletal muscle insulin sensitivity, we studied mice with tamoxifen-inducible and skeletal muscle-specific knockout of CBP and heterozygous (HZ) loss of p300 (referred to as, i-mCKO/PHZ) or knockout of p300 and HZ loss of CBP (referred to as, i-mPKO/CHZ). In these mice, the AL diet or 20 days of CR was initiated 3 weeks after ~10 week-old mice were dosed with tamoxifen (5 consecutive days, 2 mg/day); Cre recombinase-negative littermates, who were also dosed with tamoxifen, were the experimental controls (referred to as wildtype [WT]). Interestingly, oral glucose tolerance and EDL insulin sensitivity were comparably enhanced by CR in WT and both i-mCKO/PHZ and i-mPKO/CHZ mice, thus suggesting that just one allele of p300 or CBP is suffcient for CR to enhance skeletal muscle insulin sensitivity. Taken together, these results demonstrate that p300 or CBP are essential for the ability of calorie restriction to enhance skeletal muscle insulin sensitivity. This work was supported by a grant from the National Institutes of Health (R56 DK1311211), and the Dr. Maryam Ahmadian Memorial Summer Fellowship Program (L.M.L.) and the UC San Diego Medical Scientist Training Program (L.S.F.). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Time-of-day does not impact skeletal muscle contractile function, fatigability, or contraction-stimulated glucose uptake

Time-of-day impacts different aspects of mammalian physiology, such as the sleep/wake cycle, blood pressure, and body temperature. These time-of-day changes, or “circadian cycles”, are regulated by a central clock in the central suprachiasmatic nucleus of the hypothalamus and peripheral clocks located in other tissues, such as the liver, heart, and skeletal muscle. Interestingly, a growing body of data suggests that exercise performance and capacity also vary by time-of-day, with chronobiological effects on skeletal muscle metabolism and contractile function (i.e., force production) being proposed as important mediators. However, to date, no studies have specifically investigated the effect of time-of-day on skeletal muscle contractile function or glucose metabolism. The objective of this work was to assess ex vivo skeletal muscle contractile function, fatigability, and contraction-stimulated glucose uptake in the extensor digitorum longus (EDL) and soleus from 13-week-old female C57BL/6NJ mice at four different times-of-day (Light cycle time-points: Zeitgeber Time [ZT] ZT1 and ZT7; Dark cycle time-points: ZT13 and ZT19). We used an ex vivo approach as it allowed us to study skeletal muscle in isolation, and thus, independent of other factors that might influence contractile function or glucose metabolism (e.g., nerve function, humoral factors, etc.). We hypothesized that skeletal muscle contractile function, fatigability and contraction-stimulated glucose uptake would not be different by time-of-day. To control for meal-timing, mice were orally gavaged with dextrose (2 g/kg) and then fasted (3 hours) before contractile testing, which consisted of a force-frequency test (1-120 Hz, 0.3 ms pulse, 35 volts; 300 ms and 400 ms train for EDL and soleus, respectively) followed by a 15 minute fatigue test in which the muscles were repeatedly stimulated (EDL: 100 Hz stimulation every 15 seconds; Soleus: 30 Hz stimulation every 2 seconds). Glucose uptake was measured during the fatigue test using the [3H]-2-deoxyglucose (2DOG) uptake approach, with the contralateral muscle being the rested control. Significantly, contractile function, including peak twitch tension and maximal tetanic tension (EDL — ZT1: 281±13; ZT7: 300±21; ZT13: 261±28; ZT19: 280±40 kPa, P = 0.7661, one-way ANOVA; Soleus — ZT1: 117±16; ZT7: 115±18; ZT13: 117±11; ZT19: 111±10 kPa, P = 0.9923, one-way ANOVA) were not different by time-of-day. Similarly, fatigability, as measured as the tension·time integral during the 15 min fatigue test was not different by time-of-day (EDL — ZT1: 640±89; ZT7: 686±92; ZT13: 708±110; ZT19: 683±142 kPa·min, P = 0.9803, one-way ANOVA; Soleus — ZT1: 245±42; ZT7: 261±35; ZT13: 200±41; ZT19: 244±34 kPa·min, P = 0.7019, one-way ANOVA). Finally, while contraction significantly increased 2DOG uptake above basal by ~1.6-fold and ~1.8-fold in the soleus and EDL, respectively, this increase was not different by time-of-day. Taken together, these results demonstrate that time-of-day does not impact skeletal muscle contractile function, fatigability, or contraction-stimulated glucose uptake, regardless of muscle type. By extension, skeletal muscle, per se, is unlikely to be a contributing factor to the documented effects of time-of-day on whole-body exercise performance. This was funded by the UC San Diego Medical Scientist Training Program and NIH R21 AG067495. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

The tyrosine phosphorylation of GRK2 is responsible for activated D2R-mediated insulin resistance

Dopamine D2 receptor (D2R) plays a key role in the regulation of glucose homeostasis by stimulating the secretion of many glucoregulatory hormones. Insulin resistance (IR) is associated with the pathogenesis of metabolic disorders which occurs when PI3K/Akt signaling pathway is downregulated. However, the potential involvement of D2R in insulin resistance remains unclear. In the present study, we investigated the regulation of glucose transport by D2-like receptors and discovered that activation of D2R, but not D3R or D4R, suppressed insulin-induced 2-DOG uptake and Glut4 membrane translocation in a GRK2- and Src-dependent manner. Further study revealed that activation of D2R inhibits insulin-induced phosphorylation of Akt at Thr308 and Ser473, which are hallmarks of its kinase activity, by increasing the interaction of tyrosine phosphorylated GRK2 with Akt and then preventing Akt from interacting with PDK1. Thus, this study demonstrates that Src mediated GRK2 tyrosine phosphorylation is an essential physiological event that mediates the roles of D2R in insulin resistance.

Enhancing insulin sensitivity by dual PPARγ partial agonist, β-catenin inhibitor: Design, synthesis of new αphthalimido-o-toluoyl2-aminothiazole hybrids

AimsPartial PPARγ agonists attracted substantially heightened interest as safer thiazolidinediones alternatives. On the other hand, Wnt/β-catenin antagonists have been highlighted as promising strategy for type 2 diabetes management via up-regulating PPARγ gene expression. We aimed at synthesizing novel partial PPARγ agonists with β-catenin inhibitory activity which could enhance insulin sensitivity and avoid the side effects of full PPARγ agonists. Main methodsWe synthesized novel series of α-phthlimido-o-toluoyl-2-aminothiazoles hybrids for evaluating their antidiabetic activity and discovering its mechanistic pathway. We assessed effect of the new hybrids on PPARγ activation using a luciferase reporter assay system. Moreover, intracellular triglyceride levels, gene levels of c/EBPα, PPARγ and PPARγ targets including GLUT4, adiponectin, aP2 were measured in 3T3-L1 cells. Uptake of 2-DOG together with PPARγ and β-catenin protein levels were evaluated in 3T3-L1cells. In addition, molecular docking studies with PPARγ LBD, physicochemical properties and structure activity relationship of the novel hybrids were also studied. Key findingsThree of the synthesized hybrids showed partial PPARγ agonistic activity and distinct PPARγ binding pattern. These compounds modulated PPARγ gene expression and PPARγ target genes; and increased glucose uptake in 3T3-L1 and slightly induced adipogenesis compared to rosiglitazone. Moreover, these compounds reduced β-catenin protein level which reflected in increased both PPARγ gene and protein levels that leads to improved insulin sensitivity and increased GLUT4 and adiponectin gene expression. SignificanceOur synthesized compounds act as novel partial PPARγ agonists and β-catenin inhibitors that have potent insulin sensitizing activity and mitigate the lipogenic side effects of TZDs.

Short-Term Physical Inactivity Induces Diacylglycerol Accumulation and Insulin Resistance in Muscle via Lipin1 Activation

Background: Physical inactivity impairs muscle insulin sensitivity. However, its mechanism is unclear. Methods: To model physical inactivity, we applied 24-h hind-limb cast immobilization (HCI) to mice with normal or high fat diet (HFD), and evaluated intramyocellular lipids and the insulin signaling pathway in the soleus muscle. Findings: While 2-wk HFD alone did not alter intramyocellular diacylglycerol (IMDG) accumulation, HCI alone increased it by 1.9-fold and HCI after HFD further increased it by 3.3-fold. Parallel to this, we found increased PKCe activity, reduced insulin-induced 2-deoxy-glucose (2-DOG) uptake, and reduced phosphorylation of IRβ and Akt, key molecules for insulin signaling pathway. Lipin1, which converts phosphatidic acid to diacylglycerol, showed enhanced by HCI, and dominant-negative lipin1 expression in muscle prevented HCI-induced IMDG accumulation and impaired insulin-induced 2-DOG uptake. Further, 24-h leg cast immobilization in human increased lipin1 expression. Interpretation: Even short-term immobilization increases IMDG and impairs insulin sensitivity in muscle via enhanced lipin1 activity. Funding Statement: This work was supported by High Technology Research Center Grant, Strategic Research Foundation at Private Universities and KAKENHI (Grant-in-Aid for Scientific Research (C)) (15K01729) from the Ministry of Education, Culture, Sports, Science, and Technology of Japan. Declaration of Interests: All authors declare no conflict of interest. Ethics Approval Statement: All subjects gave written informed consent to participate in the study, which was approved by the ethics committee of Juntendo University. This study was carried out in accordance with the principles outlined in the Declaration of Helsinki. The present study complied with the principles and guidelines of the Japanese Council on Animal Care, and it was also approved by the Committee for Animal Research of Juntendo University.

Calorie Restriction-Induced Increase in Skeletal Muscle Insulin Sensitivity Is Not Prevented by Overexpression of the p55α Subunit of Phosphoinositide 3-Kinase.

Introduction: The Phosphoinositide 3-kinase (PI3K) signaling pathway plays an important role in skeletal muscle insulin-stimulated glucose uptake. While whole-body and tissue specific knockout (KO) of individual or combinations of the regulatory subunits of PI3K (p85α, p55α, and p50α or p85β); increase insulin sensitivity, no study has examined whether increasing the expression of the individual regulatory subunits would inhibit insulin action in vivo. Therefore, the objective of this study was to determine whether skeletal muscle-specific overexpression of the p55α regulatory subunit of PI3K impairs skeletal muscle insulin sensitivity, or prevents its enhancement by caloric restriction.Methods: We developed a novel “floxed” mouse that, through the Cre-LoxP approach, allows for tamoxifen (TMX)-inducible and skeletal muscle-specific overexpression of the p55α subunit of PI3K (referred to as, ‘p55α-mOX’). Beginning at 10 weeks of age, p55α-mOX mice and their floxed littermates (referred to as wildtype [WT]) either continued with free access to food (ad libitum; AL), or were switched to a calorie restricted diet (CR; 60% of AL intake) for 20 days. We measured body composition, whole-body energy expenditure, oral glucose tolerance and ex vivo skeletal muscle insulin sensitivity in isolated soleus and extensor digitorum longus muscles using the 2-deoxy-glucose (2DOG) uptake method.Results: p55α mRNA and protein expression was increased ∼2 fold in muscle from p55α-mOX versus WT mice. There were no differences in energy expenditure, total activity, or food intake of AL-fed mice between genotypes. Body weight, fat and lean mass, tissue weights, and fasting glucose and insulin were comparable between p55α-mOX and WT mice on AL, and were decreased equally by CR. Interestingly, overexpression of p55α did not impair oral glucose tolerance or skeletal muscle insulin signaling or sensitivity, nor did it impact the ability of CR to enhance these parameters.Conclusion: Skeletal muscle-specific overexpression of p55α does not impact skeletal muscle insulin action, suggesting that p85α and/or p50α may be more important regulators of skeletal muscle insulin signaling and sensitivity.

Hot topic: Ceramide inhibits insulin sensitivity in primary bovine adipocytes

In nonruminants, the sphingolipid ceramide inhibits insulin sensitivity by inactivating protein kinase B (AKT) within the insulin-signaling pathway. We have established that ceramide accrual develops with impaired systemic insulin action in ruminants during the transition from gestation to lactation, dietary palmitic acid supplementation, or controlled nutrient restriction. We hypothesized that ceramide promotes AKT inactivation and antagonizes insulin sensitivity in primary bovine adipocytes. Stromal-vascular cells were grown from bovine adipose tissue explants and cultured in differentiation media. To modify ceramide supply, we treated differentiated adipocytes with (1) myriocin, an inhibitor of de novo ceramide synthesis, or (2) cell-permeable C2:0-ceramide. Insulin-stimulated AKT activation (i.e., phosphorylation) and 2-deoxy-D-[3H]-glucose (2DOG) uptake were measured. Treatment of adipocytes with myriocin consistently decreased concentrations of ceramide, monohexosylceramide, and lactosylceramide. The insulin-stimulated ratio of phosphorylated AKT to total AKT was increased with myriocin but decreased with C2:0-ceramide. Moreover, adipocyte insulin-stimulated 2DOG uptake was decreased with C2:0-ceramide and increased with myriocin. We conclude that ceramide inhibits insulin-stimulated glucose uptake by downregulating AKT activation in primary bovine adipocytes.

Synthesis, crystal structure, and insulin-like activity of dimeric vanadium complex derived from 4-bromo-N'-(2-hydroxy-5-methoxybenzylidene)benzohydrazide

ABSTRACTVanadium complexes have been proved to possess interesting insulin-like activity. Reaction of VO(acac)2 with 4-bromo-N’-(2-hydroxy-5-methoxybenzylidene)benzohydrazide (H2L) in methanol afforded a new dimeric vanadium(V) complex, [V2O2L2(OMe)2]. Structure of the complex has been characterized by physicochemical methods and single-crystal X-ray diffraction. The complex crystallizes as the triclinic space group P, with unit cell dimensions a = 7.5830(9) Å, b = 9.1136(11) Å, c = 13.3512(13) Å, α = 71.420(2)°, β = 76.640(2)°, γ = 82.433(2)°, V = 849.23(17) Å3, Z = 2, R1 = 0.0903, wR2 = 0.1910, S = 1.037. X-ray analysis indicated that the V atoms in the complex are in octahedral coordination. The distance between the two V atoms is 3.327(2) Å. The complex has interesting insulin-like activity. The presence of the complex in the incubation medium stimulated glucose uptake, with 2-DOG uptake value of 0.67 nmol/3 min.

A Kluyveromyces marxianus 2-deoxyglucose-resistant mutant with enhanced activity of xylose utilization.

Thermotolerant ethanologenic yeast Kluyveromyces marxianus is capable of fermenting various sugars including xylose but glucose represses to hamper the utilization of other sugars. To acquire glucose repression-defective strains, 33 isolates as 2-deoxyglucose (2-DOG)-resistant mutants were acquired from about 100 colonies grown on plates containing 2-DOG, which were derived from an efficient strain DMKU 3-1042. According to the characteristics of sugar consumption abilities and cell growth and ethanol accumulation along with cultivation time, they were classified into three groups. The first group (3 isolates) utilized glucose and xylose in similar patterns along with cultivation to those of the parental strain, presumably due to reduction of the uptake of 2-DOG or enhancement of its export. The second group (29 isolates) showed greatly delayed utilization of glucose, presumably by reduction of the uptake or initial catabolism of glucose. The last group, only one isolate, showed enhanced utilization ability of xylose in the presence of glucose. Further analysis revealed that the isolate had a single nucleotide mutation to cause amino acid substitution (G270S) in RAG5 encoding hexokinase and exhibited very low activity of the enzyme. The possible mechanism of defectiveness of glucose repression in the mutant is discussed in this paper. [Int Microbiol 18(4):235-244 (2015)].

Glucose and Fatty Acid Metabolism in Placental Explants From Pregnancies Complicated With Gestational Diabetes Mellitus.

Placental metabolism is an important mechanism for the regulation of fetal growth and long-term health of the newborns. In this study, we investigated the effects of maternal metabolic environment on human placental fatty acid and glucose metabolism. We used placental explants from uncomplicated pregnancies or pregnancies complicated with gestational diabetes mellitus (GDM), undergoing vaginal delivery (VD) or cesarean section (CS). Fatty acid oxidation (FAO) and glucose uptake (2-DOG) were similar in both modes of delivery in normal and GDM pregnancies. However, placental explants from GDM exhibited 40% to 50% reduced FAO capacity compared to control placentas in women undergoing VD or CS. In contrast, 2-DOG uptake was 2- to 3-fold higher in placental explants from GDM compared to control placentas in women undergoing VD or CS, respectively. In conclusion, ex vivo placental fuel selection is influenced by maternal GDM, but placental metabolic characteristics are not altered by the mode of delivery.

Knockout of STAT3 in skeletal muscle does not prevent high-fat diet-induced insulin resistance.

ObjectiveIncreased signal transducer and activator of transcription 3 (STAT3) signaling has been implicated in the development of skeletal muscle insulin resistance, though its contribution, in vivo, remains to be fully defined. Therefore, the aim of this study was to determine whether knockout of skeletal muscle STAT3 would prevent high-fat diet (HFD)-induced insulin resistance.MethodsWe used Cre-LoxP methodology to generate mice with muscle-specific knockout (KO) of STAT3 (mKO). Beginning at 10 weeks of age, mKO mice and their wildtype/floxed (WT) littermates either continued consuming a low fat, control diet (CON; 10% of calories from fat) or were switched to a HFD (60% of calories from fat) for 20 days. We measured body composition, energy expenditure, oral glucose tolerance and in vivo insulin action using hyperinsulinemic-euglycemic clamps. We also measured insulin sensitivity in isolated soleus and extensor digitorum longus muscles using the 2-deoxy-glucose (2DOG) uptake technique.ResultsSTAT3 protein expression was reduced ∼75–100% in muscle from mKO vs. WT mice. Fat mass and body fat percentage did not differ between WT and mKO mice on CON and were increased equally by HFD. There were also no genotype differences in energy expenditure or whole-body fat oxidation. As determined, in vivo (hyperinsulinemic-euglycemic clamps) and ex vivo (2DOG uptake), skeletal muscle insulin sensitivity did not differ between CON-fed mice, and was impaired similarly by HFD.ConclusionsThese results demonstrate that STAT3 activation does not underlie the development of HFD-induced skeletal muscle insulin resistance.

Hepatocyte growth factor is elevated in amniotic fluid from obese women and regulates placental glucose and fatty acid metabolism

IntroductionTo evaluate the impact of the pro-inflammatory cytokine hepatocyte growth factor (HGF) on the regulation of glucose and lipid placental metabolism. MethodsHGF levels were quantified in amniotic fluid and placenta from control and obese women. 2-deoxy-glucose (2-DOG) uptake, glycolysis, fatty acid oxidation (FAO), fatty acid esterification, de novo fatty acid synthesis, triglyceride levels and carnitine palmitoyltransferase activities (CPT) were measured in placental explants upon addition of pathophysiological HGF levels. ResultsIn obese women, total- and -activated-HGF levels in amniotic fluid were elevated ∼24%, and placental HGF levels were ∼3-fold higher than in control women. At a similar dose to that present in amniotic fluid of obese women, HGF (30 ng/mL) increased Glut-1 levels and 2-DOG uptake by ∼25–30% in placental explants. HGF-mediated effect on 2-DOG uptake was dependent on the activation of phosphatidylinositol 3-kinase signaling pathway. In addition, HGF decreased ∼20% FAO, whereas esterification and de novo fatty acid synthesis increased ∼15% and ∼25% respectively, leading to 2-fold triglyceride accumulation in placental explants. In parallel, HGF reduced CPT-I activity ∼70%. DiscussionHGF is a cytokine elevated in amniotic fluid and placental tissue of obese women, which through its ability to stimulate 2-DOG uptake and metabolism impairs FAO and enhances esterification and de novo fatty acid synthesis, leading to accumulation of placental triglycerides.

Phillyrin, a natural lignan, attenuates tumor necrosis factor α-mediated insulin resistance and lipolytic acceleration in 3T3-L1 adipocytes.

In obese adipose tissue, tumor necrosis factor-α secreted from macrophages plays an important role in the adipocyte dysfunctions, including insulin resistance, lipolytic acceleration, and changes of adipokines, which promote the development of obesity-related complications. Phillyrin, an active ingredient found in many medicinal plants and certain functional foods, elicits anti-obesity and anti-inflammatory properties in vivo. The aim of the current study was to investigate the role of phillyrin in preventing tumor necrosis factor α-induced insulin resistance or lipolytic acceleration in 3T3-L1 adipocytes. Our results showed that phillyrin partially restored insulin-stimulated 2-DOG uptake, which was reduced by tumor necrosis factor-α, with concomitant restoration in serine phosphorylation of insulin receptor substrate-1 and insulin-stimulated Glut4 translocation to plasma membrane. Phillyrin also dose-dependently prevented tumor necrosis factor α-stimulated adipocyte lipolysis with preserved downregulation of perilipin. The mitogen-activated protein kinases and I kappaB kinase activation was promoted in tumor necrosis factor α-stimulated adipocytes, but pretreatment with 40 µM phillyrin inhibited the phosphorylation of extracellular signal-regulated kinases1/2, stress-activated protein kinase/Jun N-terminal kinase and I kappaB kinase (p<0.05). Moreover, phillyrin could inhibit the expressions of interleukin-6 and monocyte chemoattractant protein-1 induced by tumor necrosis factor-α. Using transwell coculture method with 3T3-L1 adipocytes and RAW 264.7 macrophages, the enhanced productions of tumor necrosis factor-α and free fatty acids in the medium were significantly reduced by phillyrin (p<0.05). These results indicate that phillyrin exerts a beneficial effect on adipocyte dysfunctions induced by tumor necrosis factor-α through suppression of the activation of I kappaB kinase and N-terminal kinase. Phillyrin may have the potential to ameliorate the inflammatory changes and insulin resistance in obese adipose tissue.

Isoform-selective Inhibition of Facilitative Glucose Transporters: ELUCIDATION OF THE MOLECULAR MECHANISM OF HIV PROTEASE INHIBITOR BINDING

Pharmacologic HIV protease inhibitors (PIs) and structurally related oligopeptides are known to reversibly bind and inactivate the insulin-responsive facilitative glucose transporter 4 (GLUT4). Several PIs exhibit isoform selectivity with little effect on GLUT1. The ability to target individual GLUT isoforms in an acute and reversible manner provides novel means both to investigate the contribution of individual GLUTs to health and disease and to develop targeted treatment of glucose-dependent diseases. To determine the molecular basis of transport inhibition, a series of chimeric proteins containing transmembrane and cytosolic domains from GLUT1 and GLUT4 and/or point mutations were generated and expressed in HEK293 cells. Structural integrity was confirmed via measurement of N-[2-[2-[2-[(N-biotinylcaproylamino)ethoxy)ethoxyl]-4-[2-(trifluoromethyl)-3H-diazirin-3-yl]benzoyl]-1,3-bis(mannopyranosyl-4-yloxy)-2-propylamine (ATB-BMPA) labeling of the chimeric proteins in low density microsome fractions isolated from stably transfected 293 cells. Functional integrity was assessed via measurement of zero-trans 2-deoxyglucose (2-DOG) uptake. ATB-BMPA labeling studies and 2-DOG uptake revealed that transmembrane helices 1 and 5 contain amino acid residues that influence inhibitor access to the transporter binding domain. Substitution of Thr-30 and His-160 in GLUT1 to the corresponding positions in GLUT4 is sufficient to completely transform GLUT1 into GLUT4 with respect to indinavir inhibition of 2-DOG uptake and ATB-BMPA binding. These data provide a structural basis for the selectivity of PIs toward GLUT4 over GLUT1 that can be used in ongoing novel drug design.

Effects of paeoniflorin on tumor necrosis factor-α-induced insulin resistance and changes of adipokines in 3T3-L1 adipocytes

TNFα plays an important role in the adipocyte dysfunction, including lipolysis acceleration, insulin resistance and changes of adipokines. Recently, we showed that paeoniflorin attenuates adipocyte lipolysis and inhibits the phosphorylation of ERK, JNK, IKK stimulated by TNFα. However, the effects of paeoniflorin on adipocytes insulin resistance and changes of adipokines remain unknown. The aim of the current study was to investigate the role of paeoniflorin in preventing insulin resistance or inflammation in 3T3-L1 adipocytes treated with TNFα. Our results showed that paeoniflorin restored insulin-stimulated [3H]2-DOG uptake, which was reduced by TNFα, with concomitant restoration in serine phosphorylation of IRS-1 and insulin-stimulated phosphorylation of AKT in adipocytes. Paeoniflorin attenuated TNFα-mediated suppression of the expressions of PPARγ and PPARγ target genes, and the improvement of paeoniflorin on TNFα-induced insulin resistance was attenuated by GW9662, an antagonist of PPARγ activity. Moreover, paeoniflorin could inhibit the expressions and secretions of IL-6 and MCP-1 from adipocytes induced by TNFα. These results, together with our previous data, indicate that paeoniflorin exerts a beneficial effect on adipocytes to prevent TNFα-induced insulin resistance and inflammatory adipokine release. Our studies provide important evidence for an ability of paeoniflorin in amelioration of TNFα-induced adipocyte dysfunction, which would be helpful to clarify its potential role in the treatment of obesity.

Testosterone Modulates the Expression of Molecules Linked to Insulin Action and Glucose Uptake in Endometrial Cells

Polycystic ovary syndrome (PCOS) is a common hyperandrogenic disorder associated with insulin resistance. Insulin exerts its metabolic function by activating the PI3K/Akt pathway and favoring glucose uptake. Caveolin-1 is a scaffolding protein which increases insulin receptor (IR) stability. Alternatively, activation of IR increases caveolin-1 phosphorylation on tyrosine-14. Furthermore, endometrial tissue from PCOS patients is proposed to be insulin resistant; however, the particular role of testosterone in modulating the metabolic effects of insulin remains unexplored in endometrial stromal cells. To evaluate whether androgens modulate the response to insulin, T-HESCs cells were stimulated with 100 nM testosterone for 24 h and changes in the protein levels of caveolin-1, IR, and Akt were determined by Western blotting (WB). After testosterone treatment, the consequences of acute insulin stimulation were evaluated by WB analysis of phospho-S473Akt and phospho-Y14Caveolin-1, as well as by measuring glucose incorporation analyzing 2-deoxyglucose (2-DOG) uptake. For cells pretreated with testosterone, higher IR, IRS-1, and caveolin-1 protein levels compared with control conditions were detected. However, in testosterone treated cells acute insulin stimulation did not increase phospho-S473Akt and phospho-Y14caveolin-1 levels and reduced 2-DOG uptake was observed compared to control cells. Our results suggest that testosterone may have a detrimental role on the metabolic effects of insulin in endometrial stromal cell cultures. Thus, the high androgen levels in patients with PCOS may favor insulin resistance observed in endometria from these women.

Decreased Phosphorylation of Y14Caveolin-1 in Endometrial Tissue of Polycystic Ovary Syndrome Patients may be Related with an Insulin Resistant State in this Tissue

Endometrial tissue of patients with polycystic ovary syndrome (PCOS) shows an impaired expression of insulin signaling molecules. Tyrosine phosphorylation of the insulin receptor (IR) by insulin promotes glucose uptake by activating the PI3K/Akt pathway. IR stability and function depend on the presence of the protein caveolin-1. Activation of IR increases phosphorylation of Y¹⁴caveolin-1. Since the endometrium of PCOS patients is proposed to be insulin resistant, we evaluated the phosphorylation of IR and caveolin-1 in endometria of patients with insulin resistance (PCOSE-IR) compared to controls (CE). To explore the mechanism associated with this condition, cultured endometrial cells (T-HESC) were exposed to high glucose (25 mM, 24 h), an experimental condition that leads to insulin resistance in other cell types. Endometrial protein levels of phospho-Y⁹⁷²IR, phospho-Y¹⁴caveolin-1 and caveolin-1 were determined by Western blotting. In cultured cells, protein levels of caveolin-1, IR, and Akt were evaluated by Western blotting. After acute insulin stimulation, phospho-S⁴⁷³Akt, phospho-Y¹⁴caveolin-1, and 2-deoxyglucose (2-DOG) uptake were determined. PCOSE-IR samples showed high protein levels of caveolin-1, but reduced phospho-Y¹⁴caveolin-1 compared to CE. No differences were observed for phospho-Y⁹⁷²IR between both groups. Cells pretreated with glucose showed a reduction in protein levels of IR and caveolin-1 and were unable to increase 2-DOG uptake, phospho-S⁴⁷³Akt and phospho-Y¹⁴caveolin-1 after insulin stimulation. In conclusion, in PCOSE-IR the impaired phosphorylation of IR downstream molecules such as phospho-Y¹⁴caveolin-1 suggests a diminished insulin sensitivity in endometria, condition that could be supported in vitro by the ability of T-HESCs to become insulin resistant when they are exposed to high glucose.

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

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

Pioglitazone upregulates adiponectin receptor 2 in 3T3-L1 adipocytes

AimsPioglitazone, a full peroxisome proliferator-activated receptor (PPAR)-γ agonist, improves insulin sensitivity by increasing circulating adiponectin levels. However, the molecular mechanisms by which pioglitazone induces insulin sensitization are not fully understood. In this study, we investigated whether pioglitazone improves insulin resistance via upregulation of either 2 distinct receptors for adiponectin (AdipoR1 or AdipoR2) expression in 3T3-L1 adipocytes. Main methodsGlucose uptake was evaluated by 2-[3H] deoxy-glucose uptake assay in 3T3-L1 adipocytes with pioglitazone treatment. AdipoR1 and AdipoR2 mRNA expressions were analyzed by qRT–PCR. Key findingsWe first confirmed that pioglitazone significantly increased insulin-induced 2-deoxyglucose (2-DOG) uptake in 3T3-L1 adipocytes. Next, we investigated the mRNA expression and regulation of AdipoR1 and AdipoR2 after treatment with pioglitazone. Interestingly, pioglitazone significantly induced AdipoR2 expression but it did not affect AdipoR1 expression. In addition, adenovirus-mediated PPARγ expression significantly enhanced the effects of pioglitazone on insulin-stimulated 2-DOG uptake and AdipoR2 expression in 3T3-L1 adipocytes. These data suggest that pioglitazone enhances adiponectin's autocrine and paracrine actions in 3T3-L1 adipocytes via upregulation of PPARγ-mediated AdipoR2 expression. Furthermore, we found that pioglitazone significantly increased AMP-activated protein kinase (AMPK) phosphorylation in insulin-stimulated 3T3-L1 adipocytes, but it did not lead to the phosphorylation of IRS-1, Akt, or protein kinase Cλ/ζ. SignificanceOur results suggest that pioglitazone increases insulin sensitivity, at least partly, by PPARγ-AdipoR2-mediated AMPK phosphorylation in 3T3-L1 adipocytes. In conclusion, the upregulation of AdipoR2 expression may be one of the mechanisms by which pioglitazone improves insulin resistance in 3T3-L1 adipocytes.

Effects of Eicosapentaenoic Acid (EPA) Treatment on Insulin Sensitivity in an Animal Model of Diabetes: Improvement of the Inflammatory Status

In addition to decreased insulin sensitivity, diabetes is a pathological condition associated with increased inflammation. The ω-3 fatty acids have been proposed as anti-inflammatory agents. Thus, the major goal of this study was to analyze the effects of fatty acid supplementation on both insulin sensitivity and inflammatory status in an animal model of type 2 diabetes. Diabetic rats (Goto-Kakizaki model) were treated with eicosapentaenoic acid (EPA) or linoleic acid at 0.5 g/kg body weigh (bw) dose. In vivo incorporation of (14)C-triolein into adipose tissue was improved by the ω-3 administration. In vitro incubations of adipose tissue slices from EPA-treated rats showed an increase in (14)C-palmitate incorporation into the lipid fraction. These observations were linked with a decreased rate of fatty acid oxidation. EPA treatment resulted in a decreased fatty acid oxidation in incubated strips from extensor digitorum longus (EDL) muscles. The changes in lipid utilization were associated with a decrease in insulin plasma concentration, suggesting an improvement in insulin sensitivity. These changes in lipid metabolism were associated with an activation of AMP-activated protein kinase (AMPK) in white adipose tissue. In addition, EPA treatment resulted in a decreased content of peroxisome proliferator-activated receptor-α (PPARα) and PPARδ and in increased GLUT4 expression in skeletal muscle. Moreover, EPA increased 2-deoxy-D-[(14)C]glucose (2-DOG) uptake in C2C12 myotubes, suggesting an improvement in glucose metabolism. Concerning the inflammatory status, EPA treatment resulted in a decreased gene expression for both tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) both in skeletal muscle and adipose tissue. The data suggest that EPA treatment to diabetic rats clearly improves lipid metabolism although the evidences on insulin sensitization are less clear.