UCP1-dependent brown adipose activation accelerates cardiac metabolic remodeling and reduces initial hypertrophic and fibrotic responses to pathological stress.

Brown adipose tissue (BAT) is correlated to cardiovascular health in rodents and humans, but the physiological role of BAT in the initial cardiac remodeling at the onset of stress is unknown. Activation of BAT via 48 h cold (16°C) in mice following transverse aortic constriction (TAC) reduced cardiac gene expression for LCFA uptake and oxidation in male mice and accelerated the onset of cardiac metabolic remodeling, with an early isoform shift of carnitine palmitoyltransferase 1 (CPT1) toward increased CPT1a, reduced entry of long chain fatty acid (LCFA) into oxidative metabolism (0.59 ± 0.02 vs. 0.72 ± 0.02 in RT TAC hearts, p < .05) and increased carbohydrate oxidation with altered glucose transporter content. BAT activation with TAC reduced early hypertrophic expression of ß-MHC by 61% versus RT-TAC and reduced pro-fibrotic TGF-ß1 and COL3α1 expression. While cardiac natriuretic peptide expression was yet to increase at only 3 days TAC, Nppa and Nppb expression were elevated in Cold TAC versus RT TAC hearts 2.7- and 2.4-fold, respectively. Eliminating BAT thermogenic activation with UCP1 KO mice eliminated differences between Cold TAC and RT TAC hearts, confirming effects of BAT activation rather than autonomous cardiac responses to cold. Female responses to BAT activation were blunted, with limited UCP1 changes with cold, partly due to already activated BAT in females at RT compared to thermoneutrality. These data reveal a previously unknown physiological mechanism of UCP1-dependent BAT activation in attenuating early cardiac hypertrophic and profibrotic signaling and accelerating remodeled metabolic activity in the heart at the onset of cardiac stress.

Expanded polyQ aggregates interact with sarco-endoplasmic reticulum calcium ATPase and Drosophila inhibitor of apoptosis protein1 to regulate polyQ mediated neurodegeneration in Drosophila.

Polyglutamine (polyQ) induced neurodegeneration is one of the leading causes of progressive neurodegenerative disorders characterized clinically by deteriorating movement defects, psychiatric disability, and dementia. Calcium [Ca2+] homeostasis, which is essential for the functioning of neuronal cells, is disrupted under these pathological conditions. In this paper, we simulated Huntington's disease phenotype in the neuronal cells of the Drosophila eye and identified [Ca2+] pump, sarco-endoplasmic reticulum calcium ATPase (SERCA), as one of the genetic modifiers of the neurodegenerative phenotype. This paper shows genetic and molecular interaction between polyglutamine (polyQ) aggregates, SERCA and DIAP1. We present evidence that polyQ aggregates interact with SERCA and alter its dynamics, resulting in a decrease in cytosolic [Ca2+] and an increase in ER [Ca2+], and thus toxicity. Downregulating SERCA lowers the enhanced calcium levels in the ER and rescues, morphological and functional defects caused due to expanded polyQ repeats. Cell proliferation markers such as Yorkie (Yki), Scalloped (Sd), and phosphatidylinositol 3 kinases/protein kinase B (PI3K/Akt), also respond to varying levels of calcium due to genetic manipulations, adding to the amelioration of degeneration. These results imply that neurodegeneration due to expanded polyQ repeats is sensitive to SERCA activity, and its manipulation can be an important step toward its therapeutic measures.

Western Diet Causes Heart Failure With Reduced Ejection Fraction and Metabolic Shifts After Diastolic Dysfunction and Novel Cardiac Lipid Derangements.

Western diet (WD) impairs glucose tolerance and cardiac lipid dynamics, preceding heart failure with reduced ejection fraction (HFrEF) in mice. Unlike diabetic db/db mice with high cardiac triglyceride (TG) and rapid TG turnover, WD mice had high TG but slowed turnover, reducing lipolytic PPAR⍺ activation. WD deranged cardiac TG dynamics by imbalancing synthesis and lipolysis, with low cardiac TG lipase (ATGL), low ATGL co-activator, and high ATGL inhibitory peptide. By 24 weeks of WD, hearts shifted from diastolic dysfunction to diastolic dysfunction with HFrEF with decreases in GLUT4 and exogenous glucose oxidation and elevated ß-hydroxybutyrate dehydrogenase 1 without increasing ketone oxidation.

Textural characteristics and color analyses of 3D printed gluten-free pizza dough and crust.

A new methodology was developed to print pizza dough with a gluten free flour blend or commercial gluten whole wheat flour using extrusion-based 3-D printing technology. Their physical properties were compared to commercially available pizza dough and crust. The optimized nozzle size, print speed, ingredient flow speed, and line thickness for the 3-D printing of pizza dough were: 0.04 cm, 800 cm/minutes, 1.8, and 0.34 cm, respectively. The printed gluten-free pizza dough required 120 min of fermentation to obtain a comparable color and textural profile (P < 0.05) to that of the gluten whole wheat flour dough fermented for 60 min. The 3-D printed gluten free, whole-wheat pizza and commercially available wheat flour dough and standard crusts demonstrated identical Δ E ab ∗ values of 0.14 and 0.13, respectively with brownness index (BI) values of 1.47 and 1.62, respectively. Textural profile analysis (TPA) of 3-D printed gluten free and whole wheat pizza dough, crust and the commercial standard wheat flour pizza dough and crust demonstrated significant (P < 0.05) correlations in terms of hardness, fracturability, adhesiveness, springiness, cohesiveness, chewiness, and resilience. An optimized method was developed to prepare gluten-free pizza dough and crust with similar functional properties to that of gluten whole wheat flour dough and crust.

Phenotypic and transcriptomic analysis reveals early stress responses in transgenic rice expressing Arabidopsis DREB1a.

Overexpression of Arabidopsis dehydration response element binding 1a (DREB1a) is a well-known approach for developing salinity, cold and/or drought stress tolerance. However, understanding of the genetic mechanisms associated with DREB1a expression in rice is generally limited. In this study, DREB1a-associated early responses were investigated in a transgenic rice line harboring cold-inducible DREB1a at a gene stacked locus. Although the function of other genes in the stacked locus was not relevant to stress tolerance, this study demonstrates DREB1a can be co-localized with other genes for multigenic trait enhancement. As expected, the transgenic lines displayed improved tolerance to salinity stress and water withholding as compared with non-transgenic controls. RNA sequencing and transcriptome analysis showed upregulation of complex transcriptional networks and metabolic reprogramming as DREB1a expression led to the upregulation of multiple transcription factor gene families, suppression of photosynthesis, and induction of secondary metabolism. In addition to the detection of previously described mechanisms such as production of protective molecules, potentially novel pathways were also revealed. These include jasmonate, auxin, and ethylene signaling, induction of JAZ and WRKY regulons, trehalose synthesis, and polyamine catabolism. These genes regulate various stress responses and ensure timely attenuation of the stress signal. Furthermore, genes associated with heat stress response were downregulated in DREB1a expressing lines, suggesting antagonism between heat and dehydration stress response pathways. In summary, through a complex transcriptional network, multiple stress signaling pathways are induced by DREB1a that presumably lead to early perception and prompt response toward stress tolerance as well as attenuation of the stress signal to prevent deleterious effects of the runoff response.

Targeting TOR and SnRK1 Genes in Rice with CRISPR/Cas9.

Genome targeting with CRISPR/Cas9 is a popular method for introducing mutations and creating knock-out effects. However, limited information is currently available on the mutagenesis of essential genes. This study investigated the efficiency of CRISPR/Cas9 in targeting rice essential genes: the singleton TARGET OF RAPAMYCIN (OsTOR) and the three paralogs of the Sucrose non-fermenting-1 (SNF1)-related kinase 1 (OsSnRK1α), OsSnRK1αA, OsSnRK1αB and OsSnRK1αC. Strong activity of constitutively expressed CRISPR/Cas9 was effective in creating mutations in OsTOR and OsSnRK1α genes, but inducible CRISPR/Cas9 failed to generate detectable mutations. The rate of OsTOR mutagenesis was relatively lower and only the kinase domain of OsTOR could be targeted, while mutations in the HEAT region were unrecoverable. OsSnRK1α paralogs could be targeted at higher rates; however, sterility or early senescence was observed in >50% of the primary mutants. Additionally, OsSnRK1αB and OsSnRK1αC, which bear high sequence homologies, could be targeted simultaneously to generate double-mutants. Further, although limited types of mutations were found in the surviving mutants, the recovered lines displayed loss-of-function or knockdown tor or snrk1 phenotypes. Overall, our data show that mutations in these essential genes can be created by CRISPR/Cas9 to facilitate investigations on their roles in plant development and environmental response in rice.

Novel Stem Cells and Nucleic Acid-Based Vaccine Trials Against Viral Outbreak: A Systematic Evaluation During COVID-2019 Pandemic.

The current Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) outbreak, the cause of coronavirus disease (COVID-19), has influenced health globally. So far, there are no established management options and prophylaxis for those who have been exposed to SARS-CoV-2, and those who develop COVID-19. Documented scientific evidences in similar viral outbreaks in past suggested few therapy regimens. These rather have not shown promising results in management of current pandemic. So, in the current review, we are exploring novel treatment strategies and therapies that are being explored and are in clinical and preclinical stages of research. To explore more about the same, we directed our search towards stem cell based, DNA based, or RNA based vaccines against COVID-19 under development by various universities, institutes or pharmaceutical companies. The current scientific literature and database search were performed by exploring various Trials registry (NIH: https://clinicaltrials.gov/ and https://www.coronavirus.gov) and Chinese clinical trial registry http://www.chictr.org.cn/) and for preclinical trials various University, Institutions, Pharmaceutical companies websites and news bulletins along with google search were checked routinely from 3rd March 2020 to 16 May 2020. The term "Stem Cell therapy and COVID-19", "Mesenchymal stem cell and corona 2019 virus", "DNA Vaccines and COVID-19, RNA Vaccines and COVID-19" and "Cell-based therapy with SARS-CoV-2, University/Institutions and COVID-19 research" were used. The vaccine trials (Stem Cells/DNA/RNA) which were cancelled were not included in this review. Similarly, few others like repurposing of drugs, Nano Vaccines, other miscellaneous trials of Herbs, Music therapy etc., were also excluded. In the present review, we have included the various novel therapies like stem cell therapy, DNA or RNA vaccines which are under development and if proven successful may have a lasting impact on the health industry.

Fructose-induced AGEs-RAGE signaling in skeletal muscle contributes to impairment of glucose homeostasis.

Increased fructose intake has been linked to the development of dyslipidemia, obesity and impaired glucose tolerance. Due to its specific metabolic fate, fructose impairs normal lipid and carbohydrate metabolism and facilitates the non-enzymatic glycation reaction leading to enhanced accumulation of advanced glycation end products (AGEs). However, the formation of fructose-AGEs under in vivo setup and its tissue specific accumulation is less explored. Here, we investigated the impact of high fructose on AGEs accumulation in skeletal muscle and its causal role in impaired glucose homeostasis. In L6 rat skeletal muscle cells, chronic exposure to fructose induced AGEs accumulation and the cellular level of the receptor for AGEs (RAGE) and the effect was prevented by pharmacological inhibition of glycation. Under in vivo settings, Sprague Dawley rats exposed to 20% fructose in drinking water for 16 weeks, displayed increased fasting glycemia, impaired glucose tolerance, decreased skeletal muscle Akt (Ser-473) phosphorylation, and enhanced triglyceride levels in serum, liver and gastrocnemius muscle. We also observed a high level of AGEs in serum and gastrocnemius muscle of fructose-supplemented animals, associated with methylglyoxal accumulation and up regulated expression of RAGE in gastrocnemius muscle. Treatment with aminoguanidine inhibited fructose-induced AGEs accumulation and normalized the expression of RAGE and Dolichyl-Diphosphooligosaccharide-Protein Glycosyltransferase (DDOST) in gastrocnemius muscle. Inhibition of AGEs-RAGE axis counteracted fructose-mediated glucose intolerance without affecting energy metabolism. These data reveal diet-derived AGEs accumulation in skeletal muscle and the implication of tissue specific AGEs in metabolic derangement, that may open new perspectives in pathogenic mechanisms and management of metabolic diseases.

Nod1-mediated lipolysis promotes diacylglycerol accumulation and successive inflammation via PKCδ-IRAK axis in adipocytes.

Chronic inflammation contributes to obesity mediated metabolic disturbances, including insulin resistance. Obesity is associated with altered microbial load in metabolic tissues that can contribute to metabolic inflammation. Different bacterial components such as, LPS, peptidoglycans have been shown to underpin metabolic disturbances through interaction with host innate immune receptors. Activation of Nucleotide-binding oligomerization domain-containing protein 1 (Nod1) with specific peptidoglycan moieties promotes insulin resistance, inflammation and lipolysis in adipocytes. However, it was not clear how Nod1-mediated lipolysis and inflammation is linked. Here, we tested if Nod1-mediated lipolysis caused accumulation of lipid intermediates and promoted cell autonomous inflammation in adipocytes. We showed that Nod1-mediated lipolysis caused accumulation of diacylglycerol (DAG) and activation of PKCδ in 3T3-L1 adipocytes, which was prevented with a Nod1 inhibitor. Nod1-activated PKCδ caused downstream stimulation of IRAK1/4 and was associated with increased expression of proinflammatory cytokines such as, IL-1ß, IL-18, IL-6, TNFα and MCP-1. Pharmacological inhibition or siRNA mediated knockdown of IRAK1/4 attenuated Nod1-mediated activation of NF-κB, JNK, and the expression of proinflammatory cytokines. These results reveal that Nod1-mediated lipolysis promoted accumulation of DAG, which engaged PKCδ and IRAK1/4 to augment inflammation in 3T3-L1 adipocytes.

NOD2 activation induces oxidative stress contributing to mitochondrial dysfunction and insulin resistance in skeletal muscle cells.

Nucleotide-binding oligomerization domain protein-2 (NOD2) activation in skeletal muscle cells has been associated with insulin resistance, but the underlying mechanisms are not yet clear. Here we demonstrate the implication of oxidative stress in the development of mitochondrial dysfunction and insulin resistance in response to NOD2 activation in skeletal muscle cells. Treatment with the selective NOD2 ligand muramyl dipeptide (MDP) increased mitochondrial reactive oxygen species (ROS) generation in L6 myotubes. MDP-induced ROS production was associated with increased levels of protein carbonyls and reduction in citrate synthase activity, cellular ATP level, and mitochondrial membrane potential, as well as altered expression of genes involved in mitochondrial function and metabolism. Antioxidant treatment attenuated MDP-induced ROS production and restored mitochondrial functions. In addition, the presence of antioxidant prevented NOD2-mediated activation of MAPK kinases and the inflammatory response. This was associated with reduced serine phosphorylation of insulin receptor substrate-1 (IRS-1) and improved insulin-stimulated tyrosine phosphorylation of IRS-1 and downstream activation of Akt phosphorylation. These data indicate that oxidative stress plays a role in NOD2 activation-induced inflammatory response and that MDP-induced oxidative stress correlates with impairment of mitochondrial functions and induction of insulin resistance in skeletal muscle cells.

Fructose induces mitochondrial dysfunction and triggers apoptosis in skeletal muscle cells by provoking oxidative stress.

Mitochondrial dysfunction in skeletal muscle has been implicated in the development of insulin resistance, a major characteristic of type 2 diabetes. There is evidence that oxidative stress results from the increased production of reactive oxygen species and reactive nitrogen species leads to mitochondrial dysfunction, tissue damage, insulin resistance, and other complications observed in type 2 diabetes. It has been suggested that intake of high fructose contributes to insulin resistance and other metabolic disturbances. However, there is limited information about the direct effect of fructose on the mitochondrial function of skeletal muscle, the major metabolic determinant of whole body insulin activity. Here, we assessed the effect of fructose exposure on mitochondria-mediated mechanisms in skeletal muscle cells. Exposure of L6 myotubes to high fructose stimulated the production of mitochondrial reactive oxygen species and nitric oxide (NO), and the expression of inducible NO synthase. Fructose-induced oxidative stress was associated with increased translocation of nuclear factor erythroid 2-related factor-2 to the nucleus, decreases in mitochondrial DNA content and mitochondrial dysfunctions, as evidenced by decreased activities of citrate synthase and mitochondrial dehydrogenases, loss of mitochondrial membrane potential, decreased activity of the mitochondrial respiratory complexes, and impaired mitochondrial energy metabolism. Furthermore, positive Annexin-propidium iodide staining and altered expression of Bcl-2 family members and caspases in L6 myotubes indicated that the cells progressively became apoptotic upon fructose exposure. Taken together, these findings suggest that exposure of skeletal muscle cells to fructose induced oxidative stress that decreased mitochondrial DNA content and triggered mitochondrial dysfunction, which caused apoptosis.

Free fatty acid induced impairment of insulin signaling is prevented by the diastereomeric mixture of calophyllic acid and isocalophyllic acid in skeletal muscle cells.

Elevated fatty acid levels play a pathogenic role in the development of insulin resistance, associated with type 2 diabetes. Interventions with ability to ameliorate fatty acid-induced insulin resistance might be useful for the management of diabetes. Here, we explored the effect of the diastereomeric mixture of calophyllic acid and isocalophyllic acid (F015) on palmitate-induced insulin resistance in skeletal muscle cells. An incubation of L6 myotubes with palmitate inhibited insulin-stimulated glucose uptake and translocation of GLUT4 to cell surface. Addition of F015 strongly prevented these inhibitions. Furthermore, F015 effectively inhibited the ability of palmitate to reduce insulin-stimulated phosphorylation of IRS-1, AKT and GSK-3ß in L6 myotubes. F015 presented a strong inhibition on palmitate-induced production of reactive oxygen species and associated inflammation, as the activation JNK, ERK1/2 and p38 MAPK were greatly reduced. F015 also inhibited inflammation-stimulated IRS-1 serine phosphorylation and restored insulin-stimulated IRS-1 tyrosine phosphorylation in presence of palmitate, resulted in enhanced insulin sensitivity. Results suggest that F015 inhibits palmitate-induced, reactive oxygen species-associated MAPK kinase activation and restored insulin sensitivity through regulating IRS-1 function. All these indicate F015 to be a potentially therapeutic candidate for insulin resistance and type 2 diabetes.

4-Hydroxyisoleucine ameliorates fatty acid-induced insulin resistance and inflammatory response in skeletal muscle cells.

The 4-hydroxyisoleucine (4-HIL), an unusual amino acid isolated from the seeds of Trigonella foenum-graecum was investigated for its metabolic effects to ameliorate free fatty acid-induced insulin resistance in skeletal muscle cells. An incubation of L6 myotubes with palmitate inhibited insulin stimulated-glucose uptake and -translocation of glucose transporter 4 (GLUT4) to the cell surface. Addition of 4-HIL strongly prevented this inhibition. We then examined the insulin signaling pathway, where 4-HIL effectively inhibited the ability of palmitate to reduce insulin-stimulated phosphorylation of insulin receptor substrate-1 (IRS-1), protein kinase B (PKB/AKT), AKT substrate of 160 kD (AS160) and glycogen synthase kinase 3ß (GSK-3ß) in L6 myotubes. Moreover, 4-HIL presented strong inhibition on palmitate-induced production of reactive oxygen species (ROS) and associated inflammation, as the activation of NF-κB, JNK1/2, ERK1/2 and p38 MAPK was greatly reduced. 4-HIL also inhibited inflammation-stimulated IRS-1 serine phosphorylation and restored insulin-stimulated IRS-1 tyrosine phosphorylation in the presence of palmitate, leading to enhanced insulin sensitivity. These findings suggested that 4-HIL could inhibit palmitate-induced, ROS-associated inflammation and restored insulin sensitivity through regulating IRS-1 function.

PTP1B inhibitors for type 2 diabetes treatment: a patent review (2011 - 2014).

INTRODUCTION: Protein tyrosine phosphatase 1B (PTP1B) plays an important role in the negative regulation of insulin signal transduction pathway and has emerged as novel therapeutic strategy for the treatment of type 2 diabetes. PTP1B inhibitors enhance the sensibility of insulin receptor (IR) and have favorable curing effect for insulin resistance-related diseases. A large number of PTP1B inhibitors, either synthetic or isolated as bioactive agents from natural products, have developed and investigated for their ability to stimulate insulin signaling. AREAS COVERED: This review includes an updated summary (2011 - 2014) of PTP1B inhibitors that have been published in patent applications, with an emphasis on their chemical structure, mode of action and therapeutic outcomes. The usefulness of PTP1B inhibitors as pharmaceutical agents for the treatment of type 2 diabetes is also discussed. EXPERT OPINION: PTP1B inhibitors show beneficial effects to enhance sensibility of IR by restricting the activity of enzyme and have favorable curing effects. However, structural homologies in the catalytic domain of PTP1B with other protein tyrosine phosphatases (PTPs) like leukocyte common antigen-related, CD45, SHP-2 and T-cell-PTP present a challenging task of achieving selectivity. Thus, for therapeutic application of PTP1B inhibitors, highly selective molecules exhibiting desired effects without side effects are expected to find clinical application.

Design and synthesis of lupeol analogues and their glucose uptake stimulatory effect in L6 skeletal muscle cells.

Structure modifications of lupeol at the isopropylene moiety have been described via allylic oxidation using selenium dioxide. The antidiabetic efficacy of lupeol analogues were evaluated in vitro as glucose uptake stimulatory effect in L6 skeletal muscle cells. From all tested compounds, 2, 3, 4b and 6b showed significant stimulation of glucose uptake with respective percent stimulation of 173.1 (p <0.001), 114.1 (p <0.001), 98.3 (p <0.001) and 107.3 (p <0.001) at 10µM concentration. Stimulation of glucose uptake by these compounds is associated with enhanced translocation of glucose transporter 4 (GLUT4) and activation of IRS-1/PI3-K/AKT-dependent signaling pathway in L6 cells. Structure-activity relationship analysis of these analogues demonstrated that the integrity of α,ß-unsaturated carbonyl and acetyl moieties were important in the retention of glucose uptake stimulatory effect. It is therefore proposed that naturally occurring lupeol and their analogues might reduce blood glucose, at least in part, through stimulating glucose utilization by skeletal muscles.

Synthesis of novel triterpene and N-allylated/N-alkylated niacin hybrids as α-glucosidase inhibitors.

Diabetes mellitus is a metabolic disorder characterized by chronic hyperglycemia. α-Glucosidase (EC 3.2.1.20) inhibitors interfere with enzymatic action to slow down the liberation of d-glucose from oligosaccharides and disaccharides, resulting in delayed glucose absorption and decreased postprandial plasma glucose levels. In continuation of our drug discovery program on antidiabetic agents, we synthesized novel N-allylated/N-alkylated niacin and α-amyrin (4-9) and lupeol (12-16) hybrids and tested for their α-glucosidase inhibiting activity. Compounds 4-9 showed better activity profile than the marketed α-glucosidase inhibitor i.e. acarbose. Compound 4 possess the highest inhibitory action with IC50 of 5 µM. Kinetic and CD studies revealed that 4 inhibited the α-glucosidase in a noncompetitive manner and caused conformational changes in secondary structure of the enzyme protein.

Diastereomeric mixture of calophyllic acid and isocalophyllic acid stimulates glucose uptake in skeletal muscle cells: involvement of PI-3-kinase- and ERK1/2-dependent pathways.

The diastereomeric mixture of calophyllic acid and isocalophyllic acid (F015) isolated from the leaves of Calophyllum inophyllum was investigated for the metabolic effect on glucose transport in skeletal muscle cells. In L6 myotubes, F015 dose-dependently stimulated glucose uptake by increasing translocation of glucose transporter4 (GLUT4) to plasma membrane without affecting their gene expression. The effects on glucose uptake were additive to insulin. Inhibitors analyses revealed that F015-induced glucose uptake was dependent on the activation of phosphatidylinositol-3-kinase (PI-3-K) and extracellular signal-regulated kinases 1 and 2 (ERK1/2), while independent to the activation of 5'AMP-activated kinase (AMPK). F015 significantly increased the phosphorylation of AKT, AS160 and ERK1/2, account for the augmented glucose transport capacity in L6 myotubes. Furthermore, F015 improved glucose tolerance and enhanced insulin sensitivity in skeletal muscle of dexamethasone-induced insulin resistant mice. Our findings demonstrate that F015 activates glucose uptake in skeletal muscle cells through PI-3-K- and EKR1/2-dependent mechanisms and can be a potential lead for the management of diabetes and obesity.

4-Hydroxyisoleucine stimulates glucose uptake by increasing surface GLUT4 level in skeletal muscle cells via phosphatidylinositol-3-kinase-dependent pathway.

PURPOSE: To determine the effect of 4-Hydroxyisoleucine (4-HIL), an unusual amino acid isolated from the seeds of Trigonella foenum-graecum, on glucose uptake and the translocation of glucose transporter 4 (GLUT4) to plasma membrane in skeletal muscle cells and to investigate the underlying mechanisms of action. METHODS: Rat skeletal muscle cells (L6-GLUT4myc) were treated with 4-HIL, and the effect on glucose uptake was determined by measuring the incorporation of radio-labeled 2-deoxy-[(3)H]-D-glucose (2-DG) into the cell. Translocation of GLUT4myc to plasma membrane was measured by an antibody-coupled colorimetric assay. RESULTS: The prolonged exposure (16 h) of L6-GLUT4myc myotubes to 4-HIL caused a substantial increase in the 2-DG uptake and GLUT4 translocation to the cell surface, without changing the total amount of GLUT4 and GLUT1. Cycloheximide treatment reversed the effect of 4-HIL on GLUT4 translocation to the basal level suggesting the requirement of new protein synthesis. The 4-HIL-induced increase in GLUT4 translocation was completely abolished by wortmannin, and 4-HIL significantly increased the basal phosphorylation of AKT (Ser-473), but did not change the mRNA expression of AKT, IRS-1, GLUT4, and GSK3ß. CONCLUSION: Results suggest that 4-HIL stimulates glucose uptake in L6-GLUT4myc myotubes by enhancing translocation of GLUT4 to the cell surface in a PI-3-kinase/AKT-dependent mechanism.