Knockdown of NYGGF4 (PID1) rescues insulin resistance and mitochondrial dysfunction induced by FCCP in 3T3-L1 adipocytes

NYGGF4 is a recently identified gene that is involved in obesity-associated insulin resistance. Previous data from this laboratory have demonstrated that NYGGF4 overexpression might contribute to the development of insulin resistance (IR) and to mitochondrial dysfunction. Additionally, NYGGF4 knockdown enhanced insulin sensitivity and mitochondrial function in 3T3-L1 adipocytes. We designed this study to determine whether silencing of NYGGF4 in 3T3-L1 adipocytes could rescue the effect of insulin sensitivity and mitochondrial function induced by the cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP), a mitochondrion uncoupler, to ascertain further the mechanism of NYGGF4 involvement in obesity-associated insulin resistance. We found that 3T3-L1 adipocytes, incubated with 5 μM FCCP for 12 h, had decreased levels of insulin-stimulated glucose uptake and had impaired insulin-stimulated GLUT4 translocation. Silencing also diminished insulin-stimulated tyrosinephosphorylation of IRS-1 and serine phosphorylation of Akt. This phenomenon contrasts with the effect of NYGGF4 knockdown on insulin sensitivity and describes the regulatory function of NYGGF4 in adipocytes insulin sensitivity. We next analyzed the mitochondrial function in NYGGF4-silenced adipocytes incubated with FCCP. NYGGF4 knockdown partly rescued the dissipation of mitochondrial mass, mitochondrial DNA, intracellular ATP synthesis, and intracellular reactive oxygen species (ROS) production occurred following the addition of FCCP, as well as inhibition of mitochondrial transmembrane potential (ΔΨm) in 3T3-L1 adipocytes incubated with FCCP. Collectively, our results suggested that addition of silencing NYGGF4 partly rescued the effect of insulin resistance and mitochondrial dysfunction in NYGGF4 silenced 3T3-L1 adipocytes incubated with FCCP, which might explain the involvement of NYGGF4-induced IR and the development of NYGGF4 in mitochondrial function.     

Curcumin is a direct inhibitor of glucose transport in adipocytes

Curcumin has been reported to inhibit insulin signaling and translocation of GLUT4 to the cell surface in 3T3-L1 adipocytes. We have investigated the effect of curcumin on insulin signaling in primary rat adipocytes. Curcumin (20 μM) inhibited both basal and insulin-stimulated glucose transport (2-deoxyglucose uptake), but had no effect on insulin inhibition of lipolysis. Dose–response experiments demonstrated that curcumin (0–100 μM) inhibited basal and insulin-stimulated glucose transport, but even at the highest concentration tested did not affect lipolysis. Inhibition was equal in cells that had been pre-incubated with curcumin and in cells to which curcumin was added immediately before the glucose transport assay. Similarly, time-course experiments revealed that the inhibitory effect of curcumin was evident at the earliest time point tested (30 s). Thus it is unlikely that inhibition of insulin signaling or of translocation of GLUT4 to the cell surface is involved in the inhibitory effect of curcumin. Curcumin did not affect the stimulatory action of insulin on phosphorylation of Akt at serine 473. We conclude that curcumin is a direct inhibitor of glucose transporters in rat adipocytes.     

Effects of major tanshinones isolated from Danshen (Salvia miltiorrhiza) on rat CYP1A2 expression and metabolism of model CYP1A2 probe substrates

This study explored the effects of Danshen on metabolism/pharmacokinetics of model CYP1A2 substrates and hepatic CYP1A2 expression in rats. The effects of Danshen and tanshinones on CYP1A2 activity was determined by metabolism of model substrates in vitro (phenacetin) and in vivo (caffeine). HPLC was used to determine model substrates/metabolites. The effect of Danshen on CYP1A2 expression was determined by Western blot. Tanshinones (1.25–50 μM) competitively inhibited phenacetin O-deethylation in vitro. Inhibition kinetics studies showed the K_i values were in the order: dihydrotanshinone (3.64 μM), cryptotanshinone (4.07 μM), tanshinone I (22.6 μM) and tanshinone IIA (23.8 μM), furafylline (35.8 μM), a CYP1A2 inhibitor. The Ki of Danshen extract (mainly tanshinones) was 72 μg/ml. Acute Danshen extract treatment (50–200 mg/kg, i.p.) decreased metabolism of caffeine to paraxanthine, with overall decrease in caffeine clearance (14–22%); increase in AUC (11–25%) and plasma T_1/2 (12–16%). Danshen treatment with (100 mg/kg/day, i.p. or 200 mg/kg/day, p.o.) for three or fourteen days showed similar pharmacokinetic changes of the CYP1A2 probe substrate without affecting CYP1A2 expression. This study demonstrated that major tanshinones competitively inhibited the metabolism of model CYP1A2 probe substrates but had no effect on rat CYP1A2 expression.     

Insulin-mimetic and insulin-sensitizing activities of a pentacyclic triterpenoid insulin receptor activator

Five pentacyclic triterpenoids isolated from Campsis grandiflora were tested for insulin-mimetic and insulin-sensitizing activity. The compounds enhanced the activity of insulin on tyrosine phosphorylation of the IR (insulin receptor) beta-subunit in CHO/IR (Chinese-hamster ovary cells expressing human IR). Among the compounds tested, CG7 (ursolic acid) showed the greatest enhancement and CG11 (myrianthic acid) the least. We characterized the effect of CG7 further, and showed that it acted as an effective insulin-mimetic agent at doses above 50 mug/ml and as an insulin-sensitizer at doses as low as 1 mug/ml. Additional experiments showed that CG7 increased the number of IRs that were activated by insulin. This indicates that a major mechanism by which CG7 enhances total IR auto-phosphorylation is by promoting the tyrosine phosphorylation of additional IRs. CG7 not only potentiated insulin-mediated signalling (tyrosine phosphorylation of the IR beta-subunit, phosphorylation of Akt and glycogen synthase kinase-3beta), but also enhanced the effect of insulin on translocation of glucose transporter 4 in a classical insulin-sensitive cell line, 3T3-L1 adipocytes. The results of the present study demonstrate that a specific pentacyclic triterpenoid, CG7, exerts an insulin-sensitizing effect as an IR activator in CHO/IR cells and adipocytes. The enhancement of insulin activity by CG7 may be useful for developing a new class of specific IR activators for treatment of Type 1 and Type 2 diabetes.     

Tanshinones as selective and slow-binding inhibitors for SARS-CoV cysteine proteases

In the search for anti-SARS-CoV, tanshinones derived from Salvia miltiorrhiza were found to be specific and selective inhibitors for the SARS-CoV 3CL^pro and PL^pro, viral cysteine proteases. A literature search for studies involving the seven isolated tanshinone hits showed that at present, none have been identified as coronaviral protease inhibitors. We have identified that all of the isolated tanshinones are good inhibitors of both cysteine proteases. However, their activity was slightly affected by subtle changes in structure and targeting enzymes. All isolated compounds (1–7) act as time dependent inhibitors of PL^pro, but no improved inhibition was observed following preincubation with the 3CL^pro. In a detail kinetic mechanism study, all of the tanshinones except rosmariquinone (7) were identified as noncompetitive enzyme isomerization inhibitors. However, rosmariquinone (7) showed a different kinetic mechanism through mixed-type simple reversible slow-binding inhibition. Furthermore, tanshinone I (5) exhibited the most potent nanomolar level inhibitory activity toward deubiquitinating (IC₅₀ = 0.7 μM). Additionally, the inhibition is selective because these compounds do not exert significant inhibitory effects against other proteases including chymotrysin, papain, and HIV protease. These findings provide potential inhibitors for SARS-CoV viral infection and replication.     

A Role for Protein Kinase Bβ/Akt2 in Insulin-Stimulated GLUT4 Translocation in Adipocytes

Insulin stimulates glucose uptake into muscle and fat cells by promoting the translocation of glucose transporter 4 (GLUT4) to the cell surface. Phosphatidylinositide 3-kinase (PI3K) has been implicated in this process. However, the involvement of protein kinase B (PKB)/Akt, a downstream target of PI3K in regulation of GLUT4 translocation, has been controversial. Here we report that microinjection of a PKB substrate peptide or an antibody to PKB inhibited insulin-stimulated GLUT4 translocation to the plasma membrane by 66 or 56%, respectively. We further examined the activation of PKB isoforms following treatment of cells with insulin or platelet-derived growth factor (PDGF) and found that PKBbeta is preferentially expressed in both rat and 3T3-L1 adipocytes, whereas PKBalpha expression is down-regulated in 3T3-L1 adipocytes. A switch in growth factor response was also observed when 3T3-L1 fibroblasts were differentiated into adipocytes. While PDGF was more efficacious than insulin in stimulating PKB phosphorylation in fibroblasts, PDGF did not stimulate PKBbeta phosphorylation to any significant extent in adipocytes, as assessed by several methods. Moreover, insulin, but not PDGF, stimulated the translocation of PKBbeta to the plasma membrane and high-density microsome fractions of 3T3-L1 adipocytes. These results support a role for PKBbeta in insulin-stimulated glucose transport in adipocytes.     

Niacin bound chromium treatment induces myocardial Glut-4 translocation and caveolar interaction via Akt,AMPK and eNOS phosphorylation in streptozotocin induced diabetic rats after ischemia-reperfusion injury

Diabetes, one of the major risk factors of metabolic syndrome culminates in the development of Ischemic Heart Disease (IHD). Refined diets that lack micronutrients, mainly trivalent chromium (Cr³⁺) have been identified as the contributor in the rising incidence of diabetes. We investigated the effect of niacin-bound chromium (NBC) during ischemia/reperfusion (IR) injury in streptozotocin induced diabetic rats. Rats were randomized into: Control (Con); Diabetic (Dia) and Diabetic rats fed with NBC (Dia + NBC). After 30 days of treatment, the isolated hearts were subjected to 30 min of global ischemia followed by 2 h of reperfusion. NBC treatment demonstrated significant increase in left ventricular functions and significant reduction in infarct size and cardiomyocyte apoptosis in Dia + NBC compared with Dia. Increased Glut-4 translocation to the lipid raft fractions was also observed in Dia + NBC compared to Dia. Reduced Cav-1 and increased Cav-3 expression along with phosphorylation of Akt, eNOS and AMPK might have resulted in increased Glut-4 translocation in Dia + NBC. Our results indicate that the cardioprotective effect of NBC is mediated by increased activation of AMPK, Akt and eNOS resulting in increased translocation of Glut-4 to the caveolar raft fractions thereby alleviating the effects of IR injury in the diabetic myocardium.     

(+)-Episesamin inhibits adipogenesis and exerts anti-inflammatory effects in 3T3-L1 (pre)adipocytes by sustained Wnt signaling,down-regulation of PPARγ and induction of iNOS

Obesity and its associated health risks still demand for effective therapeutic strategies. Drugs and compositions derived from Oriental medicine such as green tea polyphenols attract growing attention. Previously, an extract from the Japanese spice bush Lindera obtusiloba (L. obtusiloba) traditionally used for treatment of inflammation and prevention of liver damage was shown to inhibit adipogenesis. Aiming for the active principle of this extract (+)-episesamin was identified, isolated and applied in adipogenic research using 3T3-L1 (pre)adipocytes, an established cell line for studying adipogenesis. With an IC₅₀ of 10 μM (+)-episesamin effectively reduced the growth of 3T3-L1 preadipocytes and decreased hormone-induced 3T3-L1 differentiation as shown by reduced accumulation of intracellular lipid droplets and diminished protein expression of GLUT-4 and vascular endothelial growth factor. Mechanistically, the presence of (+)-episesamin during hormone-induced differentiation provoked a reduced phosphorylation of ERK1/2 and β-catenin along with a reduced protein expression of peroxisome proliferator-activated receptor γ and a strongly increased protein expression of iNOS. Treatment of mature adipocytes with (+)-episesamin resulted in a reduction of intracellular stored lipid droplets and induced the proapoptotic enzymes caspases-3/-7. Besides interfering with adipogenesis, (+)-episesamin showed anti-inflammatory activity by counteracting the lipopolysaccharide- and tumor necrosis factor α-induced secretion of interleukin 6 by 3T3-L1 preadipocytes. In conclusion, (+)-episesamin seems to be the active drug in the L. obtusiloba extract being responsible for the inhibition of adipogenesis and, thus, should be evaluated as a novel potential complementary treatment for obesity.     

Synthesis and biological evaluation of novel bis-aromatic amides as novel PTP1B inhibitors

A series of bis-aromatic amides was designed, synthesized, and evaluated as a new class of inhibitors with IC₅₀ values in the micromolar range against protein tyrosine phosphatase 1B (PTP1B). Among them, compound 15 displayed an IC₅₀ value of 2.34 ± 0.08 μM with 5-fold preference over TCPTP. More importantly, the treatment of CHO/HIR cells with compound 15 resulted in increased phosphorylation of insulin receptor (IR), which suggested extensive cellular activity of compound 15. These results provided novel lead compounds for the design of inhibitors of PTP1B as well as other PTPs.     

Interleukin-4 Restores Insulin Sensitivity in Lipid-Induced Insulin-Resistant Adipocytes

Obesity and latent inflammation in adipose tissue significantly contribute to the development of insulin resistance (IR) and type 2 diabetes. Here we studied whether the antiinflammatory interleukin-4 (IL-4) can restore insulin sensitivity in cultured 3T3-L1 adipocytes. The activity of key components of the insulin signaling cascade was assessed by immunoblotting using phospho-specific antibodies to insulin receptor substrate IRS1 (Tyr612), Akt (Thr308 and Ser473), and AS160 (Ser318) protein that regulates translocation of the GLUT4 glucose transporter to the plasma membrane. IR was induced in mature adipocytes with albumin-conjugated palmitate. IR significantly reduced phosphorylation levels of all the above-mentioned proteins. Addition of IL-4 to the culturing medium during IR induction led to a dose-dependent stimulation of the insulin-promoted phosphorylation of IRS1, Akt, and AS160. At the optimal concentration of 50 ng/ml, IL-4 fully restored activation of the insulin cascade in IR cells, but it did not affect insulin signaling activation in the control cells. IL- 4 neither upregulated expression of key adipogenesis markers GLUT4 and PPARγ nor caused lipid accumulation in the adipocytes. These results demonstrate that IL-4 can restore insulin sensitivity in adipocytes via mechanisms not associated with induced adipogenesis or de novo formation of lipid depots.     

α-Lipoic acid ameliorates impaired glucose uptake in LYRM1 overexpressing 3T3-L1 adipocytes through the IRS-1/Akt signaling pathway

Overexpression of the Homo sapiens LYR motif containing 1 (LYRM1) causes mitochondrial dysfunction and induces insulin resistance in 3T3-L1 adipocytes. α-Lipoic acid (α-LA), a dithiol compound with antioxidant properties, improves glucose transport and utilization in 3T3-L1 adipocytes. The aim of this study was to investigate the direct effects of α-LA on reactive oxygen species (ROS) production and insulin sensitivity in LYRM1 overexpressing 3T3-L1 adipocytes and to explore the underlying mechanism. Pretreatment with α-LA significantly increased both basal and insulin-stimulated glucose uptake and insulin-stimulated GLUT4 translocation, while intracellular ROS levels in LYRM1 overexpressing 3T3-L1 adipocytes were decreased. These changes were accompanied by a marked upregulation in expression of insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt following treatment with α-LA. These results indicated that α-LA protects 3T3-L1 adipocytes from LYRM1-induced insulin resistance partially via its capacity to restore mitochondrial function and/or increase phosphorylation of IRS-1 and Akt.     

Knockdown of LYRM1 Rescues Insulin Resistance and Mitochondrial Dysfunction Induced by FCCP in 3T3-L1 Adipocytes

LYR motif-containing 1 (LYRM1) was recently discovered to be involved in adipose tissue homeostasis and obesity-associated insulin resistance. We previously demonstrated that LYRM1 overexpression might contribute to insulin resistance and mitochondrial dysfunction. Additionally, knockdown of LYRM1 enhanced insulin sensitivity and mitochondrial function in 3T3-L1 adipocytes. We investigated whether knockdown of LYRM1 in 3T3-L1 adipocytes could rescue insulin resistance and mitochondrial dysfunction induced by the cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP), a mitochondrion uncoupler, to further ascertain the mechanism by which LYRM1 is involved in obesity-associated insulin resistance. Incubation of 3T3-L1 adipocytes with 1 µM FCCP for 12 h decreased insulin-stimulated glucose uptake, reduced intracellular ATP synthesis, increased intracellular reactive oxygen species (ROS) production, impaired insulin-stimulated Glucose transporter type 4 (GLUT4) translocation, and diminished insulin-stimulated tyrosine phosphorylation of Insulin receptor substrate-1 (IRS-1) and serine phosphorylation of Protein Kinase B (Akt). Knockdown of LYRM1 restored insulin-stimulated glucose uptake, rescued intracellular ATP synthesis, reduced intracellular ROS production, restored insulin-stimulated GLUT4 translocation, and rescued insulin-stimulated tyrosine phosphorylation of IRS-1 and serine phosphorylation of Akt in FCCP-treated 3T3-L1 adipocytes. This study indicates that FCCP-induced mitochondrial dysfunction and insulin resistance are ameliorated by knockdown of LYRM1.     

Fluvoxamine and sigma-1 receptor agonists dehydroepiandrosterone (DHEA)-sulfate induces the Ser473-phosphorylation of Akt-1 in PC12 cells

AimsThe expression of brain-derived neurotrophic factor (BDNF) may be a downstream target of a variety of antidepressant treatments, and selective serotonin reuptake inhibitors (SSRIs) are used clinically for the treatment of depression. BDNF binds to and activates tyrosine kinases receptor (TrkB) to exert its effects. TrkB, after activation by ligands, stimulates phosphoinositide 3-kinase (PI3K). The downstream target of PI3K is Akt-1, a serine-threonine kinase. BDNF has signaling through the PLC-?IP₃/Ca²⁺ pathway. Furthermore, the PLC-?γ/IP₃/Ca²⁺ pathway is regulated by the sigma-1 receptors. Here, we examined whether fluvoxamine (FLV) activated Akt-1 and increased phosphorylation of Akt-1 via sigma-1 receptor in PC12 cells.Main methodsWe examined the effect of the SSRI, FLV and BDNF on the phosphorylation levels of serine-threonine kinase Akt-1 in PC12 cells using immunoblotting techniques.Key findingsTreatment with 10 μM and 100 μM FLV of PC12 cells stimulated a 2.4- and 3.8-fold maximal increase in Ser⁴⁷³-phosphorylated Akt-1 levels at 40 min, respectively. Treatment with 50 ng/ml BDNF also stimulated Ser⁴⁷³ -phosphorylated Akt-1 by 2.6-fold with a maximal increase at 5 min. In addition, the phosphorylation induced by FLV and BDNF was blocked by LY294002, a selective inhibitor of PI3K. The sigma-1 receptor agonists dehydroepiandrosterone (DHEA)-sulfate also stimulated a 2.1-fold increase in the level of Ser473-phosphorylated Akt-1.SignificanceThis study demonstrates that fluvoxamine treatment rapidly increased phosphorylation of Akt-1. And BDNF activated Akt-1 phosphorylation by the TrkB/PI3K/Akt-1 pathway. We conclude that the phosphorylation of Akt-1, downstream of PI3K, was the key to their antidepressant effects.     

The insulin sensitizing effect of homoisoflavone-enriched fraction in Liriope platyphylla Wang et Tang via PI3-kinase pathway

In the present study, we screened candidates for enhancing insulin action, using glucose uptake as an indicator, from Liriope platyphylla Wang et Tang (LPWT) extract, Liliaceae, in 3T3-L1 adipocytes. The mechanism of insulin sensitizing action in the fractions was also investigated. LPWT extract with 70% MeOH was sequentially separated with Diaion HP-20 and silica gel column chromatography. The 9:1 fraction from silica gel column chromatography increased glucose uptake with 1 ng/mL up to glucose uptake with 50 ng/mL insulin. The 9:1 fraction, determined as homoisoflavone-enriched fraction, worked as an insulin sensitizer. It increased insulin stimulated glucose uptake in 3T3-L1 adipocytes, insulin responsive cells, through increased glucose transporter 4 (GLUT4) contents in the plasma membrane. GLUT4 translocation was increased through insulin receptor substrate 1 (IRS1)-PI3 kinase-Akt signaling mechanism. Thus, homoisoflavone-enriched fraction in LPWT extract played an important role as an insulin sensitizer in adipocytes.     

The effects of intracellular calcium depletion on insulin signaling in 3T3-L1 adipocytes

We have examined the requirement for intracellular calcium (Ca(2+)) in insulin signal transduction in 3T3-L1 adipocytes. Using the Ca(2+) chelator 1,2- bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid, sodium (BAPTA-AM), we find both augmentation and inhibition of insulin signaling phenomena. Pretreatment of cells with 50 microM BAPTA-AM did not affect tyrosine phosphorylation of insulin receptor substrate (IRS)1/2 or insulin receptor (IR)beta. The decreased mobility of IRS1 normally observed after chronic stimulation with insulin, due to serine phosphorylation, was completely eliminated by Ca(2+) chelation. Correlating with decreased insulin-induced serine phosphorylation of IRS1, phosphotyrosine-mediated protein-protein interactions involving p85, IRS1, IRbeta, and phosphotyrosine-specific antibody were greatly enhanced by pretreatment of cells with BAPTA-AM. As a result, insulin-mediated, phosphotyrosine-associated PI3K activity was also enhanced. BAPTA-AM pretreatment inhibited other insulin-induced phosphorylation events including phosphorylation of Akt, MAPK (ERK1 and 2) and p70 S6K. Phosphorylation of Akt on threonine-308 was more sensitive to Ca(2+) depletion than phosphorylation of Akt on serine-473 at the same insulin dose (10 nM). In vitro 3'-phosphatidylinositol-dependent kinase 1 activity was unaffected by BAPTA-AM. Insulin-stimulated insulin-responsive glucose transporter isoform translocation and glucose uptake were both inhibited by calcium depletion. In summary, these data demonstrate a positive role for intracellular Ca(2+) in distal insulin signaling events, including initiation/maintenance of Akt phosphorylation, insulin-responsive glucose transporter isoform translocation, and glucose transport. A negative role for Ca(2+) is also indicated in proximal insulin signaling steps, in that, depletion of intracellular Ca(2+) blocks IRS1 serine/threonine phosphorylation and enhances insulin-stimulated protein-protein interaction and PI3K activity.     

A highly sensitive enzyme-linked immunosorbent assay for quantification of adipocytokines secreted by mouse adipocytes

The mouse preadipocyte 3T3-L1 line is the most useful cell line for the study of adipocytes. Adipocytes secrete adipocytokines, and abnormal adipocytokine production can cause the metabolic syndrome. Although it is important to understand the characteristics of mouse adipocytokine secretion, it is difficult to quantify these products because they are produced in low concentrations. Here, we developed a highly sensitive enzyme-linked immunosorbent assay (ELISA) for detecting the concentrations of mouse adipocytokines, such as TNFα and leptin. In this method, the antigen was sandwiched by using goat- and rabbit-derived polyclonal antibodies, and the fluorescence intensity produced in the reaction with 4-methylumbelliferyl-β-galacto-sidase pyranosidase (MUG) was measured. TNFα and leptin could be measured at concentrations as low as approximately 1 pg/ml. By using our ELISA method, we also measured the concentrations of TNFα and leptin in mouse 3T3-L1 preadipocytes and adipocytes. The differentiation of preadipocytes into adipocytes enhanced TNFα production and secretion and reduced the leptin production. The amount of TNFα produced in the adipocytes was 3.0 ng/mg-protein; this amount was considerably higher than that of leptin.     

Study of insulin resistance in cybrid cells harboring diabetes-susceptible and diabetes-protective mitochondrial haplogroups

AimThis study aims to elucidate the independent role of mitochondria in the pathogenesis of insulin resistance (IR).MethodsCybrids derived from 143B osteosarcoma cell line and harboring the same nuclear DNA but different mitochondrial haplogroups were studied. Cybrid B4 (the major diabetes-susceptible haplogroup in Chinese population), cybrid D4 (the major diabetes-resistant haplogroup in Chinese population) and cybrid N9 (the diabetes-resistant haplogroup in Japanese population) were cultured in a medium containing 25 mM glucose and stimulated with 0 μM, 0.1 μM, and 1.0 μM insulin. We compared the insulin activation of PI3K–Akt (glucose uptake) and ERK–MAPK (pro-inflammation) signaling pathways, intracellular and mitochondrial oxidative stress (DCF and MitoSOX Red), and their responses to the antioxidant N-acetylcysteine (NAC).ResultsUpon insulin treatment, the translocation of cytoplasmic GLUT1/GLUT4 to the cell membrane in cybrid D4 and N9 cells increased significantly, whereas the changes in B4 cells were not or less significant. On the contrary, the ratio of insulin-induced JNK and P38 to Akt phosphorylation was significantly greater in cybrid B4 cells than in cybrid D4 and N9 cells. The levels of DCF and MitoSOX Red, which are indicative of the oxidative stress, were significantly higher in the B4 cells in basal conditions and after insulin treatment. Following treatment with the antioxidant NAC, cybrid B4 cells showed significantly reduced insulin-induced phosphorylation of P38 and increased GLUT1/GLUT4 translocation to the cell membrane, suggesting that NAC may divert insulin signaling from pro-inflammation to glucose uptake.ConclusionsMitochondria play an independent role in the pathogenesis of IR, possibly through altered production of intracellular ROS.     

Promotion of Salvia miltiorrhiza hairy root growth and tanshinone production by polysaccharide–protein fractions of plant growth-promoting rhizobacterium Bacillus cereus

This study was to examine the effects of polysaccharides from a plant growth-promoting rhizobacterium (PGPR) Bacillus cereus on the growth and tanshinone production of Salvia miltiorrhiza hairy roots. A polysaccharide fraction designated BPS was isolated from the hot water extract of B. cereus cells by ethanol precipitation. BPS applied to the root culture at 100–400 mg l⁻¹ a few days before the stationary growth phase stimulated the tanshinone accumulation of roots by about 7-fold (1.59 mg g⁻¹ versus 0.19 mg g⁻¹) and also notably promoted the root growth (15% increase in biomass). BPS was a polysaccharide–protein complex containing about 27% protein, which is essential for root growth promotion. BPS was separated by ultrafiltration into two molecular weight (MW) fractions, of which the high MW fraction (∼35.8 kDa) with higher protein content (∼31%) promoted the root growth while the lower MW fraction with lower protein content (∼17%) suppressed the growth. The results suggest that the polysaccharide portion of BPS was responsible for stimulating the tanshinone accumulation while the protein portion was responsible for promoting the hairy root growth. Polysaccharides from PGPR are potential sources of active elicitors and growth-promoting agents for plant roots in culture.     

Insulin and Chromium Picolinate Induce Translocation of CD36 to the Plasma Membrane Through Different Signaling Pathways in 3T3-L1 Adipocytes,and with a Differential Functionality of the CD36

Chromium picolinate (CrPic) has been indicated to activate glucose transporter 4 (GLUT4) trafficking to the plasma membrane (PM) to enhance glucose uptake in 3T3-L1 adipocytes. In skeletal and heart muscle cells, insulin directs the intracellular trafficking of the fatty acid translocase/CD36 to induce the uptake of cellular long-chain fatty acid (LCFA). The current study describes the effects of CrPic and insulin on the translocation of CD36 from intracellular storage pools to the PM in 3T3-L1 adipocytes in comparison with that of GLUT4. Immunofluorescence microscopy and immunoblotting revealed that both CD36 and GLUT4 were expressed and primarily located intracellularly in 3T3-L1 adipocytes. Upon insulin or CrPic stimulation, PM expression of CD36 increased in a similar manner as that for GLUT4; the CrPic-stimulated PM expression was less strong than that of insulin. The increase in PM localization for these two proteins by insulin paralleled LCFA ([1-¹⁴C]palmitate) or [³H]deoxyglucose uptake in 3T3-L1 adipocytes. The induction of the PM expression of GLUT4, but not CD36, or substrate uptake by insulin and CrPic appears to be additive in adipocytes. Furthermore, wortmannin completely inhibited the insulin-stimulated translocation of GLUT4 or CD36 and prevented the increased uptake of glucose or LCFA in these cells. Taken together, for the first time, these findings suggest that both insulin and CrPic induce CD36 translocation to the PM in 3T3-L1 adipocytes and that their translocation-inducing effects are not additive. The signaling pathway inducing the translocations is different, apparently resulting in a differential activity of CD36.