Fluorescence Microscopy-Based Quantitation of GLUT4 Translocation

Postprandial insulin-stimulated glucose uptake into target tissue is crucial for the maintenance of normal blood glucose homeostasis. This step is rate-limited by the number of facilitative glucose transporters type 4 (GLUT4) present in the plasma membrane. Since insulin resistance and impaired GLUT4 translocation are associated with the development of metabolic disorders such as type 2 diabetes, this transporter has become an important target of antidiabetic drug research. The application of screening approaches that are based on the analysis of GLUT4 translocation to the plasma membrane to identify substances with insulinomimetic properties has gained global research interest in recent years. Here, we review methods that have been implemented to quantitate the translocation of GLUT4 to the plasma membrane. These methods can be broadly divided into two sections: microscopy-based technologies (e.g., immunoelectron, confocal or total internal reflection fluorescence microscopy) and biochemical and spectrometric approaches (e.g., membrane fractionation, photoaffinity labeling or flow cytometry). In this review, we discuss the most relevant approaches applied to GLUT4 thus far, highlighting the advantages and disadvantages of these approaches, and we provide a critical discussion and outlook into new methodological opportunities.

Knockout of Syntaxin-4 in 3T3-L1 adipocytes reveals new insight into GLUT4 trafficking and adiponectin secretion

Adipocytes are key to metabolic regulation, exhibiting insulin-stimulated glucose transport which is underpinned by the insulin-stimulated delivery of glucose transporter-4 (GLUT4)- containing vesicles to the plasma membrane where they dock and fuse increasing cell surface GLUT4 levels. Adipocytokines such as adiponectin are secreted via a similar mechanism. We used genome editing to knockout Syntaxin-4 a protein reported to mediate GLUT4-vesicle fusion with the plasma membrane in 3T3-L1 adipocytes. Syntaxin-4 knockout reduced insulin-stimulated glucose transport and adiponectin secretion by ∼50% and reduced GLUT4 levels. Ectopic expression of HA-GLUT4-GFP showed that Syntaxin-4 knockout cells retain significant GLUT4 translocation capacity demonstrating that Syntaxin-4 is dispensable for insulin-stimulated GLUT4 translocation. Analysis of recycling kinetics revealed only a modest reduction in the exocytic rate of GLUT4 in knockout cells, and little effect on endocytosis. These analyses demonstrate that Syntaxin-4 is not always rate limiting for GLUT4 delivery to the cell surface. In sum, we show that Syntaxin-4 knockout results in reduced insulin-stimulated glucose transport, depletion of cellular GLUT4 levels and inhibition of adiponectin secretion but has only modest effects on the translocation capacity of the cells.

A Novel Partial PPARγ Agonist Has Weaker Lipogenic Effect in Adipocytes and Stimulates GLUT4 Translocation in Skeletal Muscle Cells via AMPK-Dependent Signaling

<b><i>Introduction:</i></b> Peroxisome proliferator-activated receptor gamma (PPARγ) agonists are highly effective in treating insulin resistance. However, associated side effects such as weight gain due to increase in adipogenesis and lipogenesis hinder their clinical use. The aim of the study was to design and synthesize novel partial PPARγ agonists with weaker lipogenic effect in adipocytes and enhanced glucose transporter 4 (GLUT4) translocation stimulatory effect in skeletal muscle cells. <b><i>Methods:</i></b> Novel partial PPARγ agonists (GS1, GS2, and GS3) were designed and screened to predict their binding interactions with PPARγ by molecular docking. The stability of the docked ligand-PPARγ complex was studied by molecular dynamics (MD) simulation. The cytotoxicity of synthesized compounds was tested in 3T3-L1 adipocytes and L6 myoblasts by MTT assay. The lipogenic effect was investigated in 3T3-L1 adipocytes using oil red O staining and GLUT4 translocation stimulatory effect in L6-GLUT4<i>myc</i> myotubes by an antibody-coupled colorimetric assay. <b><i>Results:</i></b> The molecular docking showed the binding interactions between designed agonists and PPARγ. MD simulation demonstrated good stability between the GS2-PPARγ complex. GS2 and GS3 did not show any significant effect on cell viability up to 80 or 100 μM concentration. Pioglitazone treatment significantly increased intracellular lipid accumulation in adipocytes compared to control. However, this effect was significantly less in GS2- and GS3-treated conditions compared to pioglitazone at 10 μM concentration, indicating weaker lipogenic effect. Furthermore, GS2 significantly stimulated GLUT4 translocation to the plasma membrane in a dose-dependent manner via the AMPK-dependent signaling pathway in skeletal muscle cells. <b><i>Conclusion:</i></b> GS2 may be a promising therapeutic agent for the treatment of insulin resistance and type 2 diabetes mellitus without adiposity.

Atrophy of White Adipose Tissue Accompanied with Decreased Insulin-Stimulated Glucose Uptake in Mice Lacking the Small GTPase Rac1 Specifically in Adipocytes

Insulin stimulates glucose uptake in adipose tissue and skeletal muscle by inducing plasma membrane translocation of the glucose transporter GLUT4. Although the small GTPase Rac1 is a key regulator downstream of phosphoinositide 3-kinase (PI3K) and the protein kinase Akt2 in skeletal muscle, it remains unclear whether Rac1 also regulates glucose uptake in white adipocytes. Herein, we investigated the physiological role of Rac1 in white adipocytes by employing adipocyte-specific rac1 knockout (adipo-rac1-KO) mice. Subcutaneous and epididymal white adipose tissues (WATs) in adipo-rac1-KO mice showed significant reductions in size and weight. Actually, white adipocytes lacking Rac1 were smaller than controls. Insulin-stimulated glucose uptake and GLUT4 translocation were abrogated in rac1-KO white adipocytes. On the other hand, GLUT4 translocation was augmented by constitutively activated PI3K or Akt2 in control, but not in rac1-KO, white adipocytes. Similarly, to skeletal muscle, the involvement of another small GTPase RalA downstream of Rac1 was demonstrated. In addition, mRNA levels of various lipogenic enzymes were down-regulated in rac1-KO white adipocytes. Collectively, these results suggest that Rac1 is implicated in insulin-dependent glucose uptake and lipogenesis in white adipocytes, and reduced insulin responsiveness due to the deficiency of Rac1 may be a likely explanation for atrophy of WATs.

Activation of the NLRP3 Inflammasome Increases the IL-1β Level and Decreases GLUT4 Translocation in Skeletal Muscle during Insulin Resistance

Low-grade chronic inflammation plays a pivotal role in the pathogenesis of insulin resistance (IR), and skeletal muscle has a central role in this condition. NLRP3 inflammasome activation pathways promote low-grade chronic inflammation in several tissues. However, a direct link between IR and NLRP3 inflammasome activation in skeletal muscle has not been reported. Here, we evaluated the NLRP3 inflammasome components and their role in GLUT4 translocation impairment in skeletal muscle during IR. Male C57BL/6J mice were fed with a normal control diet (NCD) or high-fat diet (HFD) for 8 weeks. The protein levels of NLRP3, ASC, caspase-1, gasdermin-D (GSDMD), and interleukin (IL)-1β were measured in both homogenized and isolated fibers from the flexor digitorum brevis (FDB) or soleus muscle. GLUT4 translocation was determined through GLUT4myc-eGFP electroporation of the FBD muscle. Our results, obtained using immunofluorescence, showed that adult skeletal muscle expresses the inflammasome components. In the FDB and soleus muscles, homogenates from HFD-fed mice, we found increased protein levels of NLRP3 and ASC, higher activation of caspase-1, and elevated IL-1β in its mature form, compared to NCD-fed mice. Moreover, GSDMD, a protein that mediates IL-1β secretion, was found to be increased in HFD-fed-mice muscles. Interestingly, MCC950, a specific pharmacological NLRP3 inflammasome inhibitor, promoted GLUT4 translocation in fibers isolated from the FDB muscle of NCD- and HFD-fed mice. In conclusion, we found increased NLRP3 inflammasome components in adult skeletal muscle of obese insulin-resistant animals, which might contribute to the low-grade chronic metabolic inflammation of skeletal muscle and IR development.

Deuterium-depleted water stimulates GLUT4 translocation in the presence of insulin, which leads to decreased blood glucose concentration

Deuterium (D) is a stable isotope of hydrogen (H) with a mass number of 2. It is present in natural waters in the form of HDO, at a concentration of 16.8 mmol/L, equivalent to 150 ppm. In a phase II clinical study, deuterium depletion reduced fasting glucose concentration and insulin resistance. In this study, we tested the effect of subnormal D-concentration on glucose metabolism in a streptozotocin (STZ)-induced diabetic rat model. Animals were randomly distributed into nine groups to test the effect of D2O (in a range of 25–150 ppm) on glucose metabolism in diabetic animals with or without insulin treatment. Serum glucose, fructose amine-, HbA1c, insulin and urine glucose levels were monitored, respectively. After the 8-week treatment, membrane-associated GLUT4 fractions from the soleus muscle were estimated by Western blot technique. Our results indicate that, in the presence of insulin, deuterium depletion markedly reduced serum levels of glucose, -fructose amine, and –HbA1c, in a dose-dependent manner. The optimal concentration of deuterium was between 125 and 140 ppm. After a 4-week period of deuterium depletion, the highest membrane-associated GLUT4 content was detected at 125 ppm. These data suggest that deuterium depletion dose-dependently enhances the effect of insulin on GLUT4 translocation and potentiates glucose uptake in diabetic rats, which explains the lower serum glucose, -fructose amine, and –HbA1c concentrations. Based on our experimental data, deuterium-depleted water could be used to treat patients with metabolic syndrome (MS) by increasing insulin sensitivity. These experiments indicate that naturally occurring deuterium has an impact on metabolic regulations.

A xanthene derivative, DS20060511, attenuates glucose intolerance by inducing skeletal muscle-specific GLUT4 translocation in mice

Reduced glucose uptake into the skeletal muscle is an important pathophysiological abnormality in type 2 diabetes, and is caused by impaired translocation of glucose transporter 4 (GLUT4) to the skeletal muscle cell surface. Here, we show a xanthene derivative, DS20060511, induces GLUT4 translocation to the skeletal muscle cell surface, thereby stimulating glucose uptake into the tissue. DS20060511 induced GLUT4 translocation and stimulated glucose uptake into differentiated L6-myotubes and into the skeletal muscles in mice. These effects were completely abolished in GLUT4 knockout mice. Induction of GLUT4 translocation by DS20060511 was independent of the insulin signaling pathways including IRS1-Akt-AS160 phosphorylation and IRS1-Rac1-actin polymerization, eNOS pathway, and AMPK pathway. Acute and chronic DS20060511 treatment attenuated the glucose intolerance in obese diabetic mice. Taken together, DS20060511 acts as a skeletal muscle-specific GLUT4 translocation enhancer to facilitate glucose uptake. Further studies of DS20060511 may pave the way for the development of novel antidiabetic medicines.

Identification of Insulin-Mimetic Plant Extracts: From an In Vitro High-Content Screen to Blood Glucose Reduction in Live Animals

Type 2 diabetes mellitus (T2DM) is linked to insulin resistance and a loss of insulin sensitivity, leading to millions of deaths worldwide each year. T2DM is caused by reduced uptake of glucose facilitated by glucose transporter 4 (GLUT4) in muscle and adipose tissue due to decreased intracellular translocation of GLUT4-containing vesicles to the plasma membrane. To treat T2DM, novel medications are required. Through a fluorescence microscopy-based high-content screen, we tested more than 600 plant extracts for their potential to induce GLUT4 translocation in the absence of insulin. The primary screen in CHO-K1 cells resulted in 30 positive hits, which were further investigated in HeLa and 3T3-L1 cells. In addition, full plasma membrane insertion was examined by immunostaining of the first extracellular loop of GLUT4. The application of appropriate inhibitors identified PI3 kinase as the most important signal transduction target relevant for GLUT4 translocation. Finally, from the most effective hits in vitro, four extracts effectively reduced blood glucose levels in chicken embryos (in ovo), indicating their applicability as antidiabetic pharmaceuticals or nutraceuticals.

Mitofusion is required for MOTS‐c induced GLUT4 translocation

MOTS‐c (mitochondrial ORF of the twelve S-c) is a 16-amino-acid mitochondrial peptide that has been shown to counter insulin resistance and alleviate obesity in vivo. However, the mechanisms involved in the pharmacological action of MOTS-c remain elusive. Based on the ability of MOTS-c to improve insulin resistance and promote cold adaptation, we hypothesized that MOTS-c might play a role in boosting the number of mitochondria in a cell. We found that treatment of mammalian cells with MOTS‐c increased protein levels of TFAM, COX4, and NRF1, which are markers for mitochondrial biogenesis. However, flow cytometry analysis using MitoTracker Green revealed a sharp reduction in the mitochondrial count after MOTS‐c treatment. We then anticipated possible synchronized activation of mitofusion/mitochondrial fusion by MOTS‐c following the onset of mitochondrial biogenesis. This was confirmed after a significant increase in protein levels two GTPases, OPA1, and MFN2, both vital for the fusion of mammalian mitochondria. Finally, we found that inhibition of the two GTPases by TNFα abrogated the ability of MOTS‐c to prompt GLUT4 translocation and glucose uptake. Similar results were obtained by siRNA KD of MFN2 as well. Our results reveal for the first time a pathway that links mitofusion to MOTS-c-induced GLUT4 translocation.