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3034 Send Orders for Reprints to reprints@benthamscience.ae Current Pharmaceutical Design, 2016, 22, 3034-3049       no. Metabolic Control of Type 2 Diabetes by Targeting the GLUT4 Glucose Transporter: Intervention Approaches Impact Factor: 3.45 BENTHAM SCIENCE Fahmida Alam1#*, Md. Asiful Islam1#, Md. Ibrahim Khalil1&2 and Siew Hua Gan1* 1 Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia; 2Department of Biochemistry and Molecular Biology, Jahangirnagar University, Savar, Dhaka 1342, Bangladesh Abstract: Type 2 diabetes mellitus (T2DM), the most common form of diabetes, is characterized by insulin resistance in the hepatic and peripheral tissues. Glucose transporter 4 (GLUT4) plays a major role in the pathophysiology of T2DM. Its defective expression or translocation to the peripheral cell plasma membrane in T2DM patients hinders the entrance of glucose into the cell for energy production. In addition to suitable drugs, an appropriate diet and/or exercise can be implemented to target the increase in GLUT4 expression, GLUT4 concentrations and GLUT4 translocation to the cell surface when managing the glucose metabolism of T2DM patients. In this review, we discussed successful intervention strategies that were individually administered or coupled with diet and/or exercise and affected the expression and translocation of GLUT4 in T2DM while reducing the excess glucose load from the blood. Additionally, some potentially good synthetic and natural compounds, which can activate the insulin-independent GLUT4 signaling pathways for the efficient management of T2DM, are highlighted as possible targets or emerging alternative sources for future anti-diabetic drug development. Current Pharmaceutical Design Keywords: Type 2 diabetes mellitus, GLUT4, intervention, exercise, diet, natural compounds. INTRODUCTION Diabetes Mellitus (DM) is the world's most prevalent endocrine disorder, with a worldwide prevalence of 382 million people in 2013 that is projected to reach as high as 592 million by the year 2035 [1]. Type 2 diabetes mellitus (T2DM) is a prominent form of diabetes (90 - 95%) and is characterized by insulin resistance of the hepatic and peripheral tissues, with an insulin secretory defect in pancreatic  cells [2]. There are various contributing factors including physical inactivity, overeating, stress, aging, smoking, obesity, increased cortisol levels, high blood pressure, abnormal sex hormone secretion, alcohol intake and genetic factors. However, insulin resistance is mainly attributed to obesity and physical inactivity, both of which precede and predict T2DM [3]. Therefore, T2DM is considered as a very serious public health problem with enormous socioeconomic burden worldwide [4]. Glucose transporters (GLUTs) are a large cluster of membrane proteins that facilitate the transport of glucose through the cellular plasma membrane. Several GLUTs (such as GLUT 1, 2, 3 and 4) are present in cells to help maintain low blood glucose levels. However, GLUT4 is the only transporter responsible for facilitating glucose transport into the cells in response to insulin, and therefore, is considered as a vital regulator of entire body glucose homeostasis [5, 6]. In normal physiology, when insulin binds on the cell surface insulin receptor (IR), GLUT4 translocates from intracellular environment to the cell surface, docks and fuses with the membrane to facilitate glucose transport into the cell. However, in T2DM patients, GLUT4 is not translocated in the adipose tissues, skeletal and cardiac muscles because of insulin resistance. As a consequence, the metabolic load of insulin increases in the blood and does not actually enter into the cells as a source of energy [7-10]. Researchers have shown that an appropriate diet, regular physical *Address correspondence to these authors at the Human Genome Centre, School of Medical Sciences, Universiti Sains Malaysia, 16150 Kubang Kerian, Kelantan, Malaysia; E-mails: alam.fahmida@yahoo.com; shgan@usm.my # These authors contributed equally to this work. 1873-4286/16 $58.00+.00 exercise and drugs or a combination of these interventions can prevent and resolve insulin resistance. These intervention plans can enhance GLUT4 expression and translocation to the cell membrane and enhance the rate of glucose uptake by the adipose tissues and skeletal and cardiac muscles of T2DM patients [11-13]. Therefore, GLUT4 can be a potential therapeutic target via the effective interventions of diet, exercise or natural compounds for better management strategies in patients with T2DM. In this review, we discussed and compiled all of the scattered data regarding the successful intervention therapies currently available, either when used singly or when coupled with diet and/or exercise, which affects the expression and translocation of GLUT4, in managing T2DM patients. Additionally, we discussed some potential alternative synthetic and natural compounds that can be emerging sources for future drug development targeting GLUT4 in T2DM. METABOLIC CONTROL OF GLUCOSE BY GLUT4 In normal metabolic conditions, the glucose levels in blood are maintained at normal levels (5–6 mM), even following high load of carbohydrate ingestion, via tightly regulated cellular mechanisms. This further prevents severe dysfunctions, such as hypoglycemiarelated unconsciousness and hyperglycemia-related toxicity to the peripheral tissues [5]. It is well established that the muscle and adipose tissues are the main glucose-utilizing centers that contribute to systemic glucose diminution, even in individuals following a carbohydrate-rich diet, with the aim of maintaining normal metabolic homeostasis [14]. Exogenous glucose uptake by the skeletal muscle is regulated by insulin-mediated signaling in the presence of a principal GLUT4 (Gene name, SLC2A4) on cell surface membrane (Fig. 1). This transporter is highly sensitive to insulin. When insulin is absent in the basal state, majority of the muscle GLUT4 resides within small intracellular storage vesicles (named GLUT4 storage vesicles or GSVs) that are excluded from the sarcolemma and T-tubules [15]. From the plasma membrane, these vesicles grasp GLUT4 away through a repulsion mechanism, but in the presence of insulin, they undergo rapid exocytosis in a burst-like manner [16]. © 2016 Bentham Science Publishers Current Pharmaceutical Design, 2016, Vol. 22, No. 20 Metabolic Control of Type 2 Diabetes by Targeting the GLUT4 Glucose Transporter 3035 ȕ cell Pancreas Capillary ȕ cell secretes insulin in the presence of blood glucose Blood Insulin Increased GLUT gene expression Improved insulin sensitivity Cycle ergometer training, Thyroid hormone, Salacia oblonga, Mangiferin extracts, Fenugreek seed extract, Rosiglitazone, Anthocyanin, Tannic acid, Hesperidin, Naringin, Creatine, Alpha-lipoic acid, Vitamin D, Chlorogenic acid and Ferulic acid Aerobic exercise, Anthocyanin, Isoflavones, Myricetin, Cod Protein, Creatine, Chromium, Lipoic acid combined with Glucose in blood Blood glucose and insulin approach towards adipose and muscle tissue Glucose Insulin Plasma membrane Insulin Receptor GLUT4 P GLUT4 storage vesicle P IR Cytoplasm The SNARE ternary complex and Munc18 helps to dock and fuse the GSV with the plasma membrane P13K GLUT4 gene Activate Increased GLUT mRNA expression Increased GLUT4 levels in the membrane GLUT4 mRNA P PKC Akt/PK Short-term and long-term swimming training, Acute exercise, Plumbagin (Plumbago zeylanica L. root), Lingonberry, Rutamarin, Quercetin, Procyanidins Signal GLUT4 storage vesicle GLUT4 protein Increased GLUT4 protein level GLUT4 Increased glucose uptake The exocyst complex, TBC1D4, myosin Glucose intake by GLUT4 motors (MYO5 and MYO1C), actin and small molecular GTPases help in tethering P GLUT4 storage vesicle Endurance training (treadmill running), Short-term high intensity intermittent swimming training, Resistance training along with head-down bed rest, Strength training, Cycle ergometry, Naringenin, Procyanidins, Alpha-lipoic acid, Pycnogenol, Chromium, Lipoic acid supplement with exercise, Sulfonylurea Anthocyanin, Cyanidin-3-glucoside, Naringenin, Quercetin, Resveratrol, Metformin with exercise, Procyanidins, Gallic acid, Protocatechuic acid, Epigallocatechin gallate, Alpha-lipoic acid, Sulfonylurea Increased GLUT4 translocation Microtubules, actin and kinesin motors help the GSV to approach towards plasma membrane Short-term and long-term swimming training, Endurance training (treadmill walking/running), Acute exercise (cycle ergometer training), High-intensity intermittent exercise training, Plumbagin, Anthocyanin, Isoflavones Troglitazone, Irbesartan, Cinnamaldehyde, Corosolic acid (banaba leaf), Magnolia officinalis, Capparis moonii (fruits, Salacia oblonga , Mangiferin extracts, Pongamia pinata (fruits), Mulberry leaf tea, Anthocyanin, Kaempferitrin, Rutin, Myricetin, Resveratrol, Quercetin, Procyanidins, Gallic acid, Protocatechuic acid, Epigallocatechin gallate, Cod Protein, Chromium, Zinc, Vitamin D, Ginger, Resistance training with dietary protein, Creatine supplementation combined with an exercise Fig. (1). Glucose uptake by the GLUT4 glucose transporter via the insulin-dependent signaling pathway. In muscle and adipose tissues, several cellular mechanisms are initiated for optimal glucose uptake by GLUT4 and glucose utilization. When carbohydrates are ingested, they are slowly broken down into smaller components by different digesting enzymes and are finally converted into glucose, which then enters into the bloodstream via the capillaries. The pancreatic  cells sense the presence of glucose in the blood and secrete insulin. Glucose sensing occurs when blood glucose enters the pancreatic  cell through GLUT2 glucose transporter which is further metabolized by glucokinase enzyme followed by the generation of adenosine triphosphate (ATP). ATP in turn facilitates the interplay of K+ and Ca2+ channels leading to the release of insulin via exocytosis into the blood stream. The canonical PI3K (phosphoinositide 3-kinase)-Akt pathway is activated when insulin engages with its surface receptors on the myocytes and adipocytes) [16]. Several signaling molecules are activated in a cascade-like manner. In brief, the binding of insulin with its receptor (a heterotetramer with two
and two  subunits) persuades a conformational change in the receptor and activates its tyrosine-kinase domain. Upon activation, the receptor recruits and phosphorylates the insulin receptor substrate (IRS) family of proteins (IRS-1 and IRS2) [17]. The tyrosine-phosphorylated IRS proteins display binding sites for several effector molecules - such as PI3K. PI3K then targets the serine/threonine kinase Akt /protein kinase B (PKB) and protein kinase C (PKC) isoforms. In the inner leaflet of the plasma membrane, PI3K activates Akt by producing polyphosphoinositides, which in turns act as the docking site of Akt. This leads to its close proximity to its upstream regulatory kinase, phosphatidylinositol-dependent kinase-1. Even though the activation mechanism of PKC isoforms is unclear, the recruitment of PKC into intracellular membranes might be involved and indeed, the presence of these isoforms were confirmed in intracellular GLUT4-containing vesicles [17]. However, activation of the PI3K-Akt pathway by insulin signaling is adequate to activate the exocytosis of GSVs into the plasma membrane [16]. Up to 50% of GLUT4 is mobilized from the intracellular membrane storage sites into the cell plasma membrane by this process [18]. To direct the GSVs towards the plasma membrane, microtubules, actin (found in adipocytes) and insulinregulated kinesin motors are important for the delivery of GSVs into the cell cortex [19]. The exocyst complex (a tethering apparatus at the plasma membrane) participates in a meaningful interaction by engaging and capturing the GSVs at the cell surface via different small GTPases (RAL, ARF6, TC10, Rab8, Rab10, Rab11, Rho and CDC42) [20, 21]. It has been reported that the TBC1D4 (160 kDa protein with tether-like features) has the ability to bind to GLUT4 vesicles as well as the plasma membrane, although the molecular details of these interactions have not yet been elucidated [22-24]. Additionally, myosin motors (MYO5 and MYO1C) were observed on GLUT4 vesicles as well as at the plasma membrane and make linkages between the GCVs, actin and the plasma membrane [25, 26]. After tethering, the vesicle docks with the plasma membrane by forming a ternary SNARE complex between VAMP2 (v-SNARE) on the GSV and syntaxin-4 and SNAP23 (t-SNAREs) on the plasma membrane. In the presence of Sec1/Munc18-like (SM) protein, the complex then induces the fusion of GSV lipid bilayer and plasma membrane [27]. Upon fusion, the number of GLUT4 transporters expressed on the cell surface increases, thus increasing the glucose uptake. Thus, GLUT4 is one of the main glucose removal mediator from the circulation and is a vital glucose homeostasis regulator of the entire body. 3036 Current Pharmaceutical Design, 2016, Vol. 22, No. 20 GLUT4: A POSSIBLE TARGET FOR DIABETES MANAGEMENT Despite the acute regulation of GLUT4 expression by insulin, the gene expression of GLUT4 can be either hormonally or metabolically regulated [28, 29]. Several studies have focused on GLUT4 expression in insulin-resistant conditions due to its crucial role in regulating glucose transport. In addition to the genetic and environmental factors, an individual’s lifestyle, particularly the ingestion of a high-fat diet, can contribute to insulin resistance, causing T2DM and obesity. A high-fat diet can induce impaired glucose tolerance and a condition resembling T2DM in certain mouse models [30]. Based on the hypothesis that a moderate increase of GLUT4 expression might correct the impaired glucose tolerance in a tissuespecific manner, an experiment was conducted to observe the effects on impaired glucose tolerance. To test this hypothesis, transgenic mice with a 14 kb GLUT4 mini-gene (7 kb of 5'-flanking and 1 kb of 3'-flanking sequence containing all exons and introns of the GLUT4 gene along with a small foreign DNA tag) were fed a high fat diet. Surprisingly, low-level expression of tissue-specific GLUT4 mini-gene prevented the glycemic impairments as well as hyperglycemia [31]. It is assumed that some defects in the signal transduction cascade, beginning with insulin binding to its receptor through the translocation of GLUT4 to cell surface, may be responsible for insulin resistance in muscle. Nevertheless, it is plausible that GLUT4 was overexpressed despite the defect in the signaling cascade, which reduces the glucose tolerance. Another study reported an opposite result, where the selective overexpression of GLUT4 in the adipocytes of transgenic mice [containing an P2 (fatty acid binding protein) promoter/enhancer] on a low-fat diet failed to improve glucose tolerance [32]. The researchers assumed that this may be due to insulin resistance in the skeletal muscle and liver, where the transgene is not expressed. Other studies using transgenic mice [such as genetically diabetic mice, mice with the insertion of a GLUT4 minigene into the genomic DNA, mice with an P2 promoter/enhancer ligated to the human GLUT4 gene, and streptozotocin (STZ)-induced diabetic mouse model] have shown that the over expression of GLUT4 (1) alleviates insulin resistance by translocating a high level of the GLUT4 protein to the cell surface, leading to a substantial improvement in glycemic control [33]; (2) increases the systemic glucose disposal by increasing the translocation of GLUT4 to the plasma membrane [34]; (3) increases glucose metabolism in all major pathways to maintain metabolic homeostasis [35]; (4) increases both the basal and insulinstimulated glucose uptake and disposal [36, 37]; and (5) insulin action improves with reduced basal plasma glucose levels [38]. From these studies, it is plausible that alterations in GLUT4 expression or activity might be a potential target in the treatment of T2DM (Fig. 1). Therefore, either genetic manipulation or pharmacologic intervention directed at increasing the GLUT4 levels at the plasma membrane may be a beneficial therapy for individuals with T2DM because GLUT4 expression can improve glycemic control, even in the presence of severe insulin resistance and pancreatic defects. THE EFFECT OF INTERVENTIONS ON GLUT4 USING DIETARY COMPOUNDS Polyphenols Effect of polyphenols on GLUT4 is now being highlighted in recent articles [39]. Anthocyanins [ANTs (water-soluble plant pigments)] are widely available in many dietary items, such as cereals, beans, fruits (blueberries, bilberries, or black currants), vegetables and red wine. Therefore, large amounts of ANTs from plant-based diets are ingested on a daily basis [40, 41]. ANTs from dietary bilberry extract (containing 375 g ANT/kg) can ameliorate hyperglycemia and insulin sensitivity in diabetic mice by significantly acti- Alam et al. vating AMPK (adenosine monophosphate-dependent protein kinase) in the white adipose tissue and skeletal muscle. The activation considerably increases the expression of the GLUT4 protein, resulting in enhanced glucose uptake into these tissues via an insulin-independent mechanism [42]. In another study, the STZ-treated diabetic rats exhibited lower GLUT4 expression in heart and skeletal muscle tissues, which was significantly improved by ANT (from the black soybean seed coat) compared to glibenclamide (anti-diabetic drug), with increased translocation of GLUT4 and enhanced uptake of glucose. In addition, IR phosphorylation is activated by ANT, further suggesting better utilization of glucose by the tissues [43]. Thus, the antidiabetic effects of ANTs suggest their potential usability as drugs to regulate diabetes. A recent study reported that the treatment of insulin-resistant 3T3-L1 cells with a black soybean koji extract [(BSK), high-quality protein containing fermented product of black soybean, isoflavones and ANTs] could ameliorate obesity-associated insulin resistance and increase glucose utilization by upregulating the GLUT4 protein levels [44]. Therefore, the authors suggested that BSK might be an effective source for treating obesity-induced insulin resistance. However, BSK active compounds need further investigation to identify the detailed molecular mechanisms of this product. Cyanidin-3-glucoside (C3G), which belongs to the ANT family and is present in black beans, is also an important component that can improve insulin resistance in 3T3-L1 adipocytes via the upregulation of GLUT4 gene expression. However, an additional study on insulin resistance using 3T3-L1 cells and an anti-fat effect using the diabetic model mice is warranted to verify these findings in vivo [45]. C3G (50
mol/L) and its metabolite protocatechuic acid [(PCA), 100
mol/L] also enhance glucose uptake and translocation of GLUT4 because they exert insulin-like activity via PPAR (peroxisome proliferator-activated receptor gamma) activation in 3T3-L1 cells and human omental adipocytes [46]. For the investigation of human adipocyte biology, 3T3-L1 cell line was found to be the most suitable model as it showed similar responses to polyphenol treatment [46]. Another study using diabetic mice reported that C3G ameliorates hyperglycemia and insulin sensitivity by significantly up-regulating GLUT4 and down-regulating retinol binding protein 4 expression in the white adipose tissue [47]. Therefore, based on the evidence of the good biological activities of C3G and PCA, it can be suggested that these polyphenols can be used as dietary bioactive compounds against the pathological conditions associated with insulin resistance. Kaempferitrin (present in some plant leaves) and rutin (found in most citrus fruits, berries, including mulberry and cranberries, buckwheat and asparagus) have been confirmed to affect GLUT4 translocation in adipocytes and skeletal muscle cells by stimulating Akt synthesis and phosphorylation [48, 49]. Another study yielded similar results, where kaempferitrin stimulated GLUT4 translocation and synthesis in adipocytes through the insulin signaling pathway [50]. The dietary intake of myricetin (a natural flavonol from medical plants, vegetables, fruits, berries, red wine and tea) from foods is approximately 0.98 - 1.10 mg/day [51, 52]. Myricetin has been shown to improve insulin sensitivity by phosphorylating IR/IRS-1 and PI3K/Akt, which can subsequently affect the translocation of GLUT4 in the soleus muscles of fructose chow-fed rats [53, 54]. Naringenin (a flavonoid normally present in tomatoes and tomato-based products) from a Canna indica plant extract can enhance the uptake of glucose and increase the levels of plasma membrane GLUT1 and GLUT4 in L8 muscle cells [55]. In addition, the effects of naringenin on GLUT4 translocation or activity have been reported in a recent study [56]. Tannic acid (found in green tea, black beans, red beans, fruits, such as apricots, grape, cherries, peaches, berries and dates, and spices, such as cinnamon, cumin, Metabolic Control of Type 2 Diabetes by Targeting the GLUT4 Glucose Transporter oregano and turmeric) is a major component of tannin (polyphenol), which induces GLUT4 by activating the insulin-mediated signaling pathway in adipocytes [57, 58]. The most common flavonol, quercetin, is present in various fruits (apples and berries), vegetables, tea, and wine, with the highest concentrations found in onion [59]. Quercetin and procyanidins have been reported to possess anti-diabetic properties because they up-regulate the levels of the GLUT4 mRNA and promote the translocation of GLUT4 to the cell membrane of adipocytes and skeletal muscle cells [5, 60, 61]. In vitro studies have indicated the roles of quercetin and resveratrol (found in red wine, mulberries, grapes, peanuts, and legumes) in increasing glucose uptake in adipocytes and muscle cells by inducing GLUT4 translocation, mainly via AMP-activated protein kinase [62, 63]. Hesperidin (present in oranges and mandarins) and naringin (present in grapefruit) can increase GLUT4 expression in adipocytes by activating hepatic PPAR
, which was in accord with the finding of another study, where procyanidins (found in grape seed) was observed to increase the amount of insulin-sensitive GLUT4 and found to stimulate glucose uptake in adipose tissues [64, 65]. Gallic acid (which can be found in both green and black teas, as well as in blueberries) from sea buckthorn leaf extracts promotes glucose uptake in 3T3-L1 adipocytes by inducing the translocation of GLUT4 in a PI3K-dependent manner, but not through the activation of AMPK [66]. Epigallocatechin gallate (found in apple skin, plums, onions, carob flour, hazelnuts, and green tea) has also been suggested to increase glucose uptake and promote translocation of GLUT4 to the plasma membrane in skeletal muscle cells [67, 68]. Cod Protein Carbohydrates and lipids play some important and promising roles in glucose metabolism by modulating insulin action. However, the effect of dietary proteins on metabolic homeostasis is not as well studied. A study conducted on high-fat-fed obese rats confirmed that insulin resistance in skeletal muscles can be prevented by feeding the animals with cod protein [69]. Cod protein modulates insulin sensitivity by selectively increasing the translocation of the GLUT4 transporters to the T-tubules of muscle cells. Additionally, cod protein protects the insulin-stimulated PI3-kinase activity from the deleterious effect of fat ingestion, and subsequently prevented insulin resistance. Therefore, the detailed molecular mechanisms of how dietary cod protein improves insulin signaling to PI3kinase/Akt needs to be identified so that novel and targeted therapeutic tools can be developed for insulin resistance. Creatine Creatine is a natural amine that is mainly found in meat and fish. It is partly synthesized from plant or animal protein-containing foods by the pancreas, kidneys and liver in the human body. During digestion, creatine is released from the food into the blood stream and is then transported to the skeletal muscles, brain and testes for absorption [12]. Globally, creatine is one of the most used nutritional supplements due to its efficacy in improving anaerobic power and stimulating protein synthesis, thus enhancing athletes’ fitness [70]. A number of studies reported the beneficial roles of creatine supplements (CrS) for managing T2DM, such as (a) improved glucose intolerance [71] and (b) improved insulin sensitivity [72]. These findings spurred the investigations on CrS to delineate its therapeutic role in diabetes treatment. Several studies have investigated the effects of CrS on GLUT4 expression for T2DM management. It has been demonstrated that 3 weeks of CrS increases GLUT4 gene expression in the rat skeletal muscles [73], although there were some contradictory outcomes. For example, the results from animal studies demonstrated that 5 days of CrS treatment failed to alter the muscle GLUT4 content [74], while a 48-hour CrS treatment did not successfully alter GLUT4 translocation and glucose uptake, although the GLUT4 Current Pharmaceutical Design, 2016, Vol. 22, No. 20 3037 concentrations were increased [75]. In humans, a 6-week intake of CrS did not change the GLUT4 mRNA expression in the muscle or the total GLUT4 concentration, but muscle glycogen was stimulated following creatine ingestion [76]. Due to the discrepancies in these findings, more studies are warranted to provide a clearer picture on the effects and mechanisms of CrS on GLUT4 expression. However, based on our current understanding, the molecular mechanisms of CrS that contribute to the increase in GLUT4 expression are as follows: (a) the expression of the PKB mRNA is assumed to promote GLUT4 translocation to the sarcolemma [77] and (b) the enhancement of the nuclear levels and DNA binding activity of the transcription factors (myocyte enhancer factor 2 isoforms) that regulate GLUT4 gene expression in muscle [73]. Therefore, future studies need to investigate the influence of CrS on the transcription factors that regulate GLUT4 in the insulin signaling pathway for T2DM management. Alpha-Lipoic Acid Alpha-lipoic acid (LA) is an antioxidant that is naturally produced in the body [78]. Very low amounts of LA are found in many foods, such as asparagus, spinach, broccoli, potatoes, tomatoes, garden peas, carrots, brussels sprouts, wheat, rice bran and beets. Red meat, particularly organ meat (kidney and liver), has high amounts of LA [79, 80]. Several lines of evidence have highlighted the benefits of LA in the prevention and treatment of diabetes. Oxidative stress has been widely observed in diabetes [81, 82]. Oxidative stress can impair the insulin-stimulated translocation of GLUT4 and activation of PKB in 3T3-L1 adipocytes. LA is reported to confer the ability to maintain the intracellular redox state, thus providing a partial protection against the impairments induced by oxidative stress [83-85]. In cultured adipocytes, LA treatment can protect the IR from oxidative damage, without damaging its functional integrity [86]. In L6 muscle cells, micromolar concentrations of LA can protect the insulin signaling system from oxidative stress [87]. Evidence suggests that the treatment of diabetic animals and humans with LA improves glucose metabolism [88]. In a cell culture with L6 GLUT4myc myoblasts, Konrad et al. [89] demonstrated that LA can increase the GLUT4 concentrations on the plasma membrane and stimulate glucose uptake in L6 GLUT4-myc myotubes, similar to the action of insulin. They suggested that by translocating and regulating the intrinsic activity of GLUT4, LA stimulates glucose uptake by mimicking the action of insulin. Several studies supported their outcomes and stated the beneficial role of LA in improving insulin-stimulated glucose uptake in T2DM patients. LA increases GLUT4 gene expression as well as its translocation from the internal pools to the plasma membrane via T2DM insulin signaling pathway [90, 91]. This appears to be mediated via increases in the kinase activity of the IR, IRS-1, phosphatidylinositol 3-kinase and PKB, thus suggesting that LA directly influences the early steps in the insulin signaling pathway [92]. The direct involvement of LA in reducing hyperglycemic conditions has made LA a unique potential drug for T2DM management. Chromium Chromium (Cr) is found in a variety of foods including whole grain, nuts, broccoli, high-bran cereals, egg yolks, meat, green beans, brewer’s yeast, wine and beer. It is available as a dietary supplement in mineral products and various types of multivitamins [93]. Decreased dietary intake of Cr is associated with many of the abnormalities (including glucose intolerance, increased body fat and elevated total cholesterol) that are related to insulin resistance. The presence of Cr in its active form can increase insulin’s sensitivity, and, thus, it may resolve insulin resistance as well as the associated defects. Therefore, Cr supplements might be beneficial to the insulin-resistant T2DM patients who are Cr-deficient due to a poor 3038 Current Pharmaceutical Design, 2016, Vol. 22, No. 20 diet [94]. Furthermore, the effects of Cr on normal insulin secretion, which can stimulate the translocation of GLUT4 vesicles to the cell membrane, have been reported. For example, two different studies conducted on the skeletal muscle of high sucrose diet-fed mice and diabetic rats found that supplementation with Cr increased the GLUT4 levels in the membrane [95, 96]. In addition, it has also been reported that Cr increases GLUT4 membrane translocation in myocardial tissues [97]. The mechanism of Cr-mediated GLUT4 translocation to the plasma membrane was further investigated using cultured adipocytes [98]. The experiments resulted in an elevation of GLUT4 at the plasma membrane and increased insulin-stimulated glucose transport across the cell membrane. However, in vitro studies suggested that Cr regulates GLUT4 translocation independently, without the involvement of insulin signaling through the IR, IRS-1, PI3K or Akt proteins. Instead, Cr can increase membrane fluidity by decreasing the membrane cholesterol levels [98]. Therefore, the exact mechanisms behind the beneficial effect of Cr on GLUT4 have not been elucidated. Zinc Zinc (Zn) is considered as an essential trace element, with multiple controlling roles, including insulin synthesis, secretion and signaling, as well as glucose transport. In humans, Zn is the second most common trace metal (after iron) found in all tissues and fluids [99]. The richest zinc food sources include oysters and meat (e.g., beef, lamb, veal and pork) [100]. The recommended dietary intake of Zn is 8 mg/day for women or 11 mg/day for men [101]. Normally, cellular zinc levels are tightly regulated, and if disturbed, can lead to diabetes mellitus [102]. A severe Zn deficiency can affect insulin secretion in the pancreatic islets, which leads to the development of hyperglycemia [103]. In 3T3-L1 adipocytes, it has been reported that Zn(opt)2 [bis(1oxy-2-pyridine-thiolato)Zn(II)] stimulates Akt/PKB phosphorylation more strongly and at a faster rate (within 5 min) than insulinstimulated Akt/PKB phosphorylation. The activated Akt/PKB help the translocation of GLUT4 vesicles to the plasma membrane within 30 min [7]. Thus, it is concluded that Zn(opt)2 has an insulinomimetic activity that helps translocating GLUT4 protein from the cytosol to the plasma membrane by activating the insulin signaling cascade through Akt/PKB phosphorylation [104]. Similar results were obtained with Zn(alx)2 [Zn(II) complex with allixin found in garlic] and Zn(tanm)2 [Zn(II)-thioallixin-N-methyl, a Zn(II) complex with the allixin-derivative] in another study [105]. These studies indicate that the stimulation of GLUT4 translocation is a key insulinomimetic function of Zn. Based on its molecular mechanism; we can propose that Zn would be a beneficial agent in the treatment of type 2 diabetes. Vitamin D Some recent experiments have revealed that vitamin D has the potential to decrease the blood glucose level by upregulating GLUT4 in T2DM [106]. In 2015, it was reported that vitamin D increases GLUT4 expression in STZ-induced T2DM Wistar rat adipocytes [107]. GLUT4 translocation was observed in cultured adipocytes (murine 3T3L1 fibroblast cell) [108] as well as in the skeletal muscle of STZ-induced T2DM Webster mice [109]. Tamilselvan et al. [110] reported that the anti-diabetic activity of vitamin D occurs via increasing GLUT4 gene expression in the muscle cells (L6-myotubes). Therefore, vitamin D is expected to be another possible emerging therapy to target GLUT4 in T2DM management. THE EFFECTS OF EXERCISE INTERVENTIONS ON GLUT4 Glucose uptake in skeletal muscle is generally mediated by insulin via translocation of the GLUT4 glucose transporter to the plasma membrane. The insulin-stimulated glucose uptake by Alam et al. GLUT4 is impaired in the skeletal muscles due to insulin resistance in T2DM. The contractile activity of skeletal muscles during physical exercise is a potent therapeutic for T2DM management, as it induces an increase in GLUT4 expression in skeletal muscle, thus helping to improve glucose transport capacity and insulin sensitivity [111]. It is reported that exercise-induced glucose uptake is normal (or near normal) in the muscle tissues of T2DM patients [112]. Several studies have suggested that the glucose uptake due to exercise is mediated by an insulin-independent mechanism [113]. For example, insulin signaling involves IR phosphorylation, IRS1/2 tyrosine phosphorylation and phosphatidylinositol 3-kinase activation [114, 115]. However, exercise has no effect on these activities [114, 116]. Therefore, it is plausible that exercise leads to adaptations that induce glucose transport by activating some molecular signals that could bypass the insulin signaling defects in skeletal muscle. Exercise leads to changes in gene expression, greater blood flow, and increased signaling, as well as changes in GLUT4 protein exocytosis and endocytosis [117]. The effects of exercise on GLUT4 and glucose uptake have been established in many studies (Table 1). It is thought that acute exercise increases the uptake of glucose skeletal muscle by stimulating the translocation of GLUT4 and activating distinct proximal signaling mechanisms. However, the detailed mechanisms by which the activated signaling pathways increase glucose uptake and/or GLUT4 translocation have not been reported. AS160 is a Rab GTPase-activating protein and is a substrate for the Akt kinase [118]. AS160 is phosphorylated on multiple phospho-Akt-substrate (PAS) sites by the Akt kinase in response to insulin, which is important for GLUT4 trafficking towards the cell surface [118]. Furthermore, defects in insulin action on AS160 may impair GLUT4 trafficking in T2DM [119]. Based on both human [120, 121] and animal [115] studies, prolonged exercise has been shown to increase AS160 PAS phosphorylation. In skeletal muscle, combination of 5-Aminoimidazole-4-carboxamide ribonucleotide [an analog of adenosine monophosphate (AMP) that is capable of stimulating AMPK activity] with exercise can enhance the AMPK mediated phosphorylation of AS160 (PAS site) [122]. It is reported that uptake of glucose facilitated by exercise was inhibited significantly due to the alterations of the AS160 domain responsible for calmodulin-binding, but not the insulin-dependent glucose uptake [123]. Therefore, exercise can regulate the phosphorylation and binding of calmodulin to AS160 to promote GLUT4 translocation and glucose uptake. TBC1D1 is a protein that regulates transportation of glucose provoked by insulin using a PAS-independent approach [124]. Distinctive TBC1D1 mutations can regulate glucose uptake differentially by the stimulation of both insulin and exercise through distinct phosphorylation sites [124, 125]. Therefore, TBC1D can serve as a possible molecular link between the signaling pathways that converge on GLUT4 translocation and skeletal muscle glucose uptake due to exercise. Protein kinases which are dependent on Ca2+/Calmodulin, serve as vital constituents of GLUT4 mediated skeletal muscle glucose uptake in response to exercise. The activation of protein kinase II which occurs in response to raised levels of cytosolic Ca2+, can upregulate GLUT4 during muscle contraction [111]. In rats, contraction mediated glucose transportation was reduced after incubating the skeletal muscle with the Ca2+/Calmodulin inhibitor (KN-93) [126]. Liver kinase B-1 (LKB-1) is the upstream kinase of AMPK (an element important in cellular metabolism that maintains energy homeostasis) and directly phosphorylates and activates AMPK [127]. Nevertheless, the contribution of LKB-1 in glucose uptake as a result of exercise is debated. For example, glucose uptake in LKB-1 knockout (KO) mice was reported to be impaired even after exercise by one study [128]; whereas partial inhibition of exercise- Metabolic Control of Type 2 Diabetes by Targeting the GLUT4 Glucose Transporter Table 1. No. of Study Current Pharmaceutical Design, 2016, Vol. 22, No. 20 3039 The effect of different exercises on GLUT4 from human and animal studies. Type of Exercise Type of Study Number of Individuals (n) Effect on Age Duration and Intensity Length GLUT4 Year, References Human studies T2DM patients (n=9): 1 Acute exercise (cycle ergometry) 6 M and 3 F 60 minutes 5 M and 4 F T2DM patients: HIT 53 ± 4 years Case control Control (n=9): 2 T2DM: - Control: 3 4 5 6 7 8 Case control Cycle exercise training - Acute exercise Bicycle ergometer training Strenuous treadmill exercise Strength training - Case control 54.9 - 70.1 years Cycle ergometer training Each session with 10 x 60 s cycling bouts (Total 6 sessions), HRmax (90%), 75 min / week Continuous moderate intensity - 3 days/week (60 minutes at 55% Wmax); Control (n=7) Control: 54 ± 4 years Intermittent high-intensity 2 days/week (6 x 5 minute bouts at 70% Wmax) Healthy but untrained subjects (n=12) 20.9 - 22.6 years 16 repetitions of the exercise were performed for 6 min at ~90% VO2peak, once per hour Healthy, physically active but untrained individuals (n=6) 21.2 - 24.4 years Obese T2DM (n=8): 6 M and 2 F and obese nonT2DM (n=7): 6 M and 1 F T2DM: 42.4 - 46.8 years and nonT2DM: 43.8 - 51.6 years Semi-balanced, randomized, 3way crossover Healthy adult horses (n=7) - Caucasian T2DM patients (n=10) and male healthy controls subjects [M (n=7)] T2DM (n=6): 4 M and 2 F 9 (60% and 66%, p < 0.05). T2DM: 47 ± 2 years T2DM patients with obesity (n=7) Case control Non-T2DM (n=16): 6 M and 10 F 2012, [168] 48 ± 3 years (n=8) Continuous and high-intensity intermittent training (cycle ergometry) - GLUT4 expression
in both groups immediately after exercise 60 min at ~40% VO2 peak (low) or 27 ± 2 min at ~80% 2 weeks 4 weeks - 8 hours/day T2DM Patients: 60 - 64 years and controls: 59 - 63 years One leg three times per week (each session total 30 min) T2DM patients: 4149 years and controls: 34 - 38 years 3 times/week (60% of VO2 peak for 20 minutes), which increased to 4 times/week (70% of VO2 peak for 45 minutes) GLUT4 expression significantly
in skeletal muscle and adipose tissue (36% and 20%, p < 0.05) after exercise intervention compared to baseline. 16 hours GLUT4, but not GLUT1,
(p < 0.05) after repetitive sessions of heavy exercise. - Exercise bouts at ~40 and 80% of the VO2 peak
the GLUT4 mRNA and GLUT4 protein in human skeletal muscle to a similar extent, despite the differences in exercise intensity and duration. Short term exercise (7 consecutive days) The GLUT4 protein content
in obese T2DM patients (p < 0.05), but not in obese non-T2DM subjects following chronic exercise. 3 days The GLUT4 expression in skeletal muscle increased within hours after exercise; however, carbohydrate meals did not enhance GLUT4 expression in muscles. 6 weeks GLUT4
(40%) in the trained muscle of T2DM subjects (p < 0.05), but the 13% increase in the control subjects did not achieve statistical significance. 8 weeks GLUT4 protein
by 22 ± 10% and 38 ± 8% in the diabetic and non-diabetic subjects, respectively (p < 0.05), before and after training. VO2 peak (high) Acute bout of exercise (60 min at 75% VO2 peak) and chronic short term exercise (1 hour at 75% VO2 peak) GLUT4 protein content
after training [(~369%) (p < 0.05)]. 2011, [169] 2011, [170] 2008, [171] 2006, [172] 2006, [133] 2005, [173] 2004, [174] 2004, [163] 3040 Current Pharmaceutical Design, 2016, Vol. 22, No. 20 Alam et al. (Table 1) Contd…. No. of Study Type of Exercise Type of Study Number of Individuals (n) Effect on Age Duration and Intensity Length GLUT4 Year, References Control group: 6° head-down tilt at all times throughout bed rest, except for showering every other day Young subjects 19 days GLUT4 content in the VL muscle of the control group significantly  after 19 days of bed rest (p < 0.05), whereas it significantly
during 19 days of bed rest plus isometric training (p < 0.01) in the training group. 1999, [134] 7 – 10 days The GLUT4 immunoreactivity in muscle
[98% (p < 0.001)] after training. 1995, [175] 7 days The GLUT4 protein content
[~2.8 ± 0.5-fold (p < 0.05)] in human skeletal muscle. 1995, [176] 9 weeks GLUT4 protein content
(p < 0.05) in all groups during training (to 0.43 ± 0.03, 0.40 ± 0.03 and 0.57 ± 0.08 arbitrary units, respectively). 2 days The GLUT4 mRNA expression
2-fold and protein expression increased 50% in the epitrochlearis muscle. 14 weeks The GLUT4 protein concentration
in the skeletal muscle by 1.8-fold after exercise in previously sedentary middle-aged men. 15 weeks Lean-PX & obese-PX showed hyperglycemia with  GLUT4 levels [65% & 62%, (p < 0.05)] compared to lean and obese CTLs, respectively, whereas the obese-PX treated with exercise showed reduced hyperglycemia with (2-fold)
GLUT4 levels compared to the obese CTL. [M (n=9)] 10 Resistance training along with headdown bed rest Training group: RCT 2 groups: control (n=4) or 18 - 26 years resistance training group (n=5) 11 Cycle ergometer - Healthy subjects (n=8): 4 M and 4 F 29 - 33 years exercise 12 Short-term exercise (cycle ergometry) - ergometer bicycle training NIDDM subjects: 7 (M) Case control Healthy controls: 8 (M) Young subjects: 5 14 Swimming 15 Endurance training (overground and/or treadmill walking and/or running) 16 Endurance training (treadmill running, only obese pancreatomized (PX) ~ 25 years 2 hours/day at 65-70% of peak O2 uptake 1 hour/day, Wmax (55%) [M (n=7)] One-legged 13 Sedentary individual: Remained at bed rest, except during resistance (30 isometric maximal voluntary contractions for 3 sec each; leg-press exercise was used to recruit the extensor muscles of the ankle, knee, and hip) training once in the morning Case control Case control Specific pathogenfree rats (F) Previously sedentary middle-aged Caucasian men for the exercise group [Case (n = 13)]; NIDDM: 56 - 60 years; Healthy: 58 - 60 years; Young: 22 24 years - 40 - 65 years Sedentary men without exercise [Control (n=7)] RCT Lean and obese Zucker rats- divided into shamoperated control (CTL) or 90% PX groups 6 days/week, 30 min/day 10 min/day Weeks 1 - 3 (30 min 3 days/week at 70-80% of MHR); weeks 4 - 7 (40 min 4 days/week at 70 - 80% MHR); weeks 7 - 14 (45 min 4 days/week at 80-85% MHR) 2 hours/day, 5 days/week, 5 weeks old 15% grade, at 1518 m/min (Only obese-PX) 1994, [177] 1994, [178] 1993, [179] 1993, [180] Current Pharmaceutical Design, 2016, Vol. 22, No. 20 Metabolic Control of Type 2 Diabetes by Targeting the GLUT4 Glucose Transporter 3041 (Table 1) Contd…. No. of Study Type of Exercise Type of Study Number of Individuals (n) Effect on Age Duration and Intensity Length GLUT4 Year, References Animal studies 1 Swimming training RCT Two groups of Wistar rats (M): exercise group and sedentary control group - 10 min/day for 2 days followed by 2 x 3 hour long swimming sessions separated by a 45 min long rest for 3 days, one group was fasted and the other was fed either 5 days GLUT4 mRNA (~3-fold) and protein levels (~2-fold)
in the epitrochlearis muscle after exercise (within 18 h). However, this exercise-induced result was completely reversed () in rats fed a high-carbohydrate diet (within 42 hour). In contrast, the increase in the GLUT4 protein was still present 66 hours after exercise in the muscles of rats fed the carbohydrate-free diet. 60 min No significant difference was detected in the muscle GLUT4 protein or mRNA content before and after exercise. chow or lard ad libitum 2 Running on treadmills Balanced, randomized, 3way crossover fashion study Healthy adult Thoroughbred horses (n=6): 4 mares and 2 geldings - HIT: 8-10 x 20 sec swimming bouts with a 10 sec pause Sprague-Dawley rats (M) 3 Short-term high intensity intermittent swimming training RCT 3 trials separated by 7 day intervals 3 groups: HIT (n=16), 3-4 weeks RHT (n=15), old RHT: 5 x17 min swimming bouts with 3 min of rest between bouts 8 days LIT: 6 hours/day in 2 x 3 hour bouts separated by 45 min of rest SST: 2 x 3 hour/day, 5 days Specific pathogenfree female Wistar 4 SST, LST and treadmill training RCT LST: 2 x 3 h/day, 5 days/wk rats - Treadmill training: 2 x 10 min/day 2 groups: exercise training or sedentary control groups for 3 days followed by 60 min at 30 m/min on a 2003, [182] 2001, [183] The GLUT4 content after HIT did not differ from that after RHT (66% higher in the trained rats than in the controls). LIT (n=8) and sedentary controls (n =16) The GLUT4 content in the epitrochlearis muscle was significantly
by 83 and 91% after training in the HIT and LIT groups, respectively, compared to the control rats. 2003, [181] SST (5 days), LST (5 week) and treadmill run (5 week) SST and LST increases the GLUT4 mRNA by 48% and 60%, respectively; both training types increase the GLUT4 protein (30%), whereas treadmill run training produces a transient increase in the GLUT4 protein (35%) that is completely reversed after the last bout of exercise (at 48 hour). 2000, [184] 15% incline for 3 weeks 5 6 Cycle ergometer training Swimming training - 6 F and 4 M RCT Three groups of Wistar rats (F): 1 day of exercise, 5 days of exercise, and the sedentary controls 20.2 ± 2.6 years - - 6 hours/day 60 min GLUT4 gene expression
immediately after exercise and remained significantly higher than the baseline at 3 hour after the end of exercise (p <0.05). 1 or 5 days The GLUT4 content
2-fold in the epitrochlearis muscle at 16 hour after 1 day of exercise (p <0.05 vs. sedentary rats), with no further increase noted after 5 days of exercise. 2000, [185] 2000, [186] 3042 Current Pharmaceutical Design, 2016, Vol. 22, No. 20 Alam et al. (Table 1) Contd…. No. of Study Type of Exercise 7 Functional electrical stimulation (FES) Ieg cycle ergometer training Age - M (n=4) and F (n=1) with motorcomplete SCI (3 25 years postinjury involving levels C5 - T8) 31 - 50 years 30 minutes, 3 days/ week Case control Two groups of Wistar rats: exercise and control groups 7 weeks old 10 min/day for 2 days, then 6 hours with 2 x 3 bouts/day for 2 days RCT Two groups of Wistar rats [M]: sedentary group & swimming training group Progressive 8 9 swimming program Swimming training Effect on Number of Individuals (n) Type of Study Duration and Intensity GLUT4 Year, References 8 weeks GLUT4
[72% (p < 0.05)] 1999, [187] - The GLUT4 protein concentration
(~2-fold) in triceps muscle of the exercised group compared to the control group. 4 days Total GLUT4 levels
(30%) in swimming training vs. sedentary rats 5 days or 5 weeks The GLUT4 levels were higher (90%) in triceps muscle of the trained (5 days) animals compared to the controls, whereas, the increase in the GLUT4 protein levels was completely reversed after the last exercise bout (within 40 hour) after both 5 days and 5 weeks of training. 5 days In the trained rats, the GLUT4 protein
(85%) in the epitrochlearis muscle at 18 hour after training, and remained ~50% higher than the control group at 42 hour after training. 5 days In the epitrochlearis muscle, the cell surface GLUT4 expression
48% in the insulin-stimulated group and  40% in the hypoxiastimulated group. 18 or 30 weeks Exercise training of obese rats for 18 or 30 weeks
the GLUT4 levels by 1.7 and 2.3-fold, respectively, compared to the sedentary obese rats. Length First day: 1 hour followed by a 2 hour bout - Swimming training RCT 3 groups: sedentary control, 5-day trained & 5-day trained/40-h detrained 6 weeks old Sprague-Dawley rats [M (n=180)] with initial body weights of 80–90 g 11 Swimming training 4 weeks old RCT 2 groups: sedentary group & swimming training group 12 13 Swimming Endurance training (treadmill running) Case control RCT Specific pathogenfree male Wistar rats Lean (Fa/-) and obese (fa/fa) Zucker rats (M) 1998, [189] Last 3 days: 2 x 3 h/day Specific-pathogenfree Wistar rats (F) 10 1999, [188] 6 hours/day 2 hours/day in 4 x 30 min bouts separated by 5 min of rest - 2 times, 3 hours/day 36 weeks old 18 meters/min (15% grade), 5 days/week for gradually increasing durations during the first 2 weeks and at 20 meters/min for 1.5 hours/day thereafter 1998, [190] 1997, [191] 1997, [192] 1990, [193] T2DM = Type 2 diabetes mellitus, Wmax = Peak power output, M = Male, F = Female, HRmax = Maximal heart rate, MHR = Maximum heart rate, VL = Vastus lateralis, SCI = Spinal cord injury, SST = Short-term swim training, LST = Long-term swim training, HIT = High-intensity intermittent exercise training, RHT = Relatively high-intensity intermittent prolonged exercise training, LIT = Low-intensity prolonged exercise training, RCT = Randomized controlled trial,
= Increase,  = Decrease stimulated glucose transport was reported by another study in LKB1 knockout mice [129]. Therefore, acute exercise activates the alternative signaling pathway in skeletal muscle and bypasses the insulin signaling defects, causing enhancement of glucose uptake without insulin stimulation. On the other hand, chronic exercise improves skeletal muscle insulin sensitivity in T2DM patients by specifically restoring the mitochondrial function in skeletal muscle and by increasing the expression of the GLUT4 protein [130]. Metabolic Control of Type 2 Diabetes by Targeting the GLUT4 Glucose Transporter However, the effects of exercise can be reversed within 18-24 hours of the activity. For example, it is reported that the discontinuation of exercise for one week is sufficient to reduce the GLUT4 levels in the heart and in adipocytes [131]. Therefore, detraining can affect the translocation of GLUT4 to the insulinsensitive cell surface. Consequently, the GLUT4 levels return to the baseline levels. To obtain the proper therapeutic effects of exercise, it is necessary to know the total amount, duration, intensity and length of the exercise session to be conducted. Although several opinions exist regarding the essential quantity of exercise for the improvement of metabolism, however, the recommended amount of exercise for T2DM individuals is a moderate intensity exercise ( 210 min) or high-intensity exercise (
125 min) per week [132]. In addition, both resistance and aerobic exercise can be followed in combination. In each week, it is suggestible to perform exercise for minimum 3 days. If possible, exercise performing with moderate and high-intensity can also be adopted in combination [132]. However, in every occasion, it is recommended that exercise programs are planned and delivered by qualified and trained personnel, particularly for T2DM patients. Despite optimistic outputs of experiments conducted on the effect of exercise on GLUT4, many researchers stated some limitations of their studies including limited number of experimental subjects [133] and statistical limitation due to the small number of controls [134]. POTENTIAL SYNTHETIC AND ALTERNATIVE NATURAL COMPOUNDS IN THE REGULATION OF GLUT4 A number of alternative synthetic and natural compounds were shown to significantly affect the metabolic regulation of glucose via GLUT4 translocation. For example, Troglitazone, an insulinsensitizing synthetic compound was reported to fuel the uptake of glucose through activating the translocation of GLUT4 in L6 myotubes [135]. Rosiglitazone (insulin sensitizer) exerts its anti-diabetic effect by increasing GLUT4 expression [136]. In cell surface, Irbesartan (an angiotensin II type 1 receptor inhibitor) induced GLUT4 translocation lead to significant increase in glucose transportation in L6-GLUT4-myc myoblasts as well as in male Zucker rats (genetically obese) [137]. The thyroid hormone (T3) induces GLUT4 gene expression in male Sprague-Dawley rats [138] and is believed to be useful when used in combination with other treatments (for example, combination with -blockers) for T2DM. In an in vivo study using male Wistar rats, Cinnamaldehyde (isolated from the stem bark of Cinnamomum zeylanicum) increased the translocation of GLUT4. It also induced glucose transport across the membranes of skeletal muscle in diabetic rats compared to the untreated diabetic rats [139]. In another in vivo study, Plumbagin, which is isolated from Plumbago zeylanica L. root, was confirmed to enhance the expression of the GLUT4 mRNA and protein in STZ-induced diabetic rats (male albino Wistar rats) [140]. It was also concluded that glucose homeostasis was likely maintained by enhancing GLUT4 translocation. In another study, Miura et al., [141] demonstrated that corosolic acid, which is isolated from banaba leaf, exerts its hypoglycemic activity by increasing GLUT4 translocation in the total muscle membrane of male KK-Ay mice, which have genetically induced T2DM. In a recent study, translocation of GLUT4 was facilitated by Magnolia officinalis in 3T3-L1 adipocytes as well as C2C12 myotubes by activating the signaling pathways related to insulin [142]. Pycnogenol [a procyanidin (a natural flavonoids) enriched extract of Pinus maritima bark] also had found to increase the relative abundance of GLUT4 in fully differentiated 3T3-L1 adipocytes [143]. Additionally, the fruits of Capparis moonii were found to increase the mRNA expression of GLUT4, causing increased glucose uptake in L6 cells [144]. Current Pharmaceutical Design, 2016, Vol. 22, No. 20 3043 In the skeletal muscle of L6 rat, Girón et al. [145] suggested that the Salacia oblonga and mangiferin extracts may exert their anti-diabetic effects through enhancing the expression and translocation of GLUT4 via the 5' AMPK and PPAR pathways. Khan et al. [146] successfully identified at least four compounds (1, 5, 6 and 7) from Kigelia pinnata that stimulated significant translocation activity of GLUT4 in the skeletal muscle cell surface, without affecting cell viability. Jaiswal et al. [147] identified that Karanjin (obtained from Pongamia pinata fruits) improved the GLUT4 translocating activity toward the cell surface of L6 myotubes through the AMPK pathway stimulation. In L6 myotubes, chlorogenic and ferulic acids exerted higher glucose uptake by increased GLUT4 expression via PI3-K independent and dependent pathways, respectively [148]. In 2014, an in vivo investigation on Fenugreek seed extract confirmed that it may be a potent inducer of GLUT4 expression, as it has been found to improve T2DM in STZ-induced T2DM male Sprague-Dawley rats compared to the control group [149]. Mulberry (Morusalba L.) leaf tea, which is a popular drink in Asian nations, has been reported to have anti-diabetic effects by stimulating GLUT4 translocation and glucose uptake in the adipocytes of STZ-induced Sprague-Dawley diabetic male rats [150]. Recently, Lingonberry (Vccinium vitisidaea L.) in diabetic mice model induced by diet, reported to exhibit anti-diabetic activities in skeletal muscle via enhanced expression of GLUT4 [151]. In an in vitro study Rani et al. [152] observed that Ginger (Zingiber officinale) has the potential to express or transport GLUT4 receptors from internal vesicles. Rutamarin, a natural compound, was identified as the inducer of GLUT4 expression. Eventually, GLUT4 translocation capability of rutamarin was also confirmed to maintain glucose homeostasis in diet-induced obese mice through the PI3K-Akt/PKB pathways [153]. A recent study (2015) revealed that Kaempferol (a natural flavonoid) is another potential compound to restore GLUT4 activity in T2DM obese mice [154]. The successful natural compounds could be evaluated further for their effects on GLUT4 in combination with popular antidiabetic drugs. If they could produce synergistic effect on GLUT4, potential ant-diabetic drugs from the useful chemical component of those natural compounds can be developed in future targeting GLUT4. To be noted, although many bioactive compounds present in natural products are therapeutically useful, investigations into the chemical components from a natural remedy is often challenging [155]. THE EFFECTS OF COMBINED INTERVENTIONS ON GLUT4 Many researchers have shown that, occasionally, the combined interventions of diet, exercise or drugs in T2DM patients can significantly improve the glucose uptake compared to individual interventions, due to increased insulin activity and GLUT4 expression and translocation. For example, a study revealed that exercise can improve the expression of IRS-1 (65% - 90%) and significantly decrease the fasting blood glucose level (p < 0.05) of patients treated with metformin and/or sulfonylurea [156]. It is plausible that the glucose uptake is mediated by the increased expression or translocation of GLUT4 [133]. Another study confirmed that a combination of diet (high fiber, low-fat) and daily aerobic exercise ameliorates insulin sensitivity, likely by increasing the patients’ GLUT4 concentrations [157]. When a combined intervention of diet (lipoic acid) and exercise was applied, an in vivo study reported increased levels of GLUT4, with glucose lowering effects (26 - 32%), and a significant improvement in insulin sensitivity (29 - 30%) in Zucker rats compared to the sedentary controls [158]. Furthermore, a study on nine postmenopausal Greek women with T2DM (seven were treated with sulfonylurea and two were under dietary control) showed a significant improvement (p < 0.001) of glucose uptake and insulin action 3044 Current Pharmaceutical Design, 2016, Vol. 22, No. 20 [159], which were believed to be mediated by GLUT4, after a 16week exercise program [160]. In addition, an in vivo study by Smith et al., [161] found that a combined intervention with metformin and exercise can significantly improve insulin-stimulated glucose transport via GLUT4 in female Zucker diabetic fatty rats, further confirming that a combined intervention is superior to individual interventions. It has been reported that resistance training in combination with a moderately high amount of dietary proteins can enhance the development of insulin signaling proteins in older persons (61 ± 1 year) [162]. It is plausible that this phenomenon is the consequence of enhanced GLUT translocation [12, 163]. A double-blind, randomly assigned trial (12 weeks) conducted in 2011 [12] revealed that creatine supplementation combined with an exercise program can improve glycemic control in T2DM patients by increasing GLUT4 recruitment and translocation to the sarcolemma comparing to placebo [12]. In addition, several randomized controlled trial studies have concluded that a combination of diet and exercise significantly improved the plasma glucose levels in Finnish [164], Japanese-American [165], British [166] and Dutch [167] patients with T2DM, possibly due to the enhanced expression and translocation of GLUT4. CONCLUSION The management of T2DM remains a major challenge to clinicians, researchers and patients. In addition to using appropriate medications, proper diet and/or exercise can be potential strategies to increase GLUT4 expression and translocation when managing glucose metabolism in patients with T2DM. In addition to conventional drugs, some potential synthetic and alternative natural compounds that activate the insulin-dependent or -independent GLUT4 signaling pathways may be new, emerging target sources of future drug development for T2DM. ABBREVIATIONS AMP = Adenosine monophosphate AMPK = Adenosine monophosphate-dependent protein kinase ANTs = Anthocyanins BSK = Black soybean koji extract C3G = Cyanidin-3-glucoside Cr = Chromium CrS = Creatine supplements GLUT4 = Glucose Transporter 4 GLUTs = Glucose transporters GSVs = GLUT4 storage vesicles IR = Insulin receptor IRS = Insulin receptor substrate LA = Alpha-lipoic acid LKB-1 = Liver kinase B-1 PAS = Phospho-Akt-substrate PCA = Protocatechuic acid PI3K = Phosphoinositide 3-kinase PKB = Protein kinase B PKC = Protein kinase C PPAR
= Peroxisome proliferator-activated receptor gamma STZ = Streptozotocin T2DM = Type 2 diabetes mellitus Zn = Zinc = [bis(1-oxy-2-pyridine-thiolato)Zn(II)] Zn(opt)2 Alam et al. CONFLICT OF INTEREST The authors confirm that this article content has no conflict of interest. ACKNOWLEDGEMENTS We would like to acknowledge Universiti Sains Malaysia (USM) for Research University (RU) grant (1001/PPSP/812115). We would also like to acknowledge USM Global Fellowship (2014/ 2015) and USM Vice-Chancellor Award (2015/2016) awarded to Fahmida Alam and Md. Asiful Islam, respectively, to pursue their PhD. REFERENCES [1] [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] [12] [13] [14] [15] [16] [17] [18] [19] [20] [21] Guariguata L, Whiting D, Hambleton I, Beagley J, Linnenkamp U, Shaw J. Global estimates of diabetes prevalence for 2013 and projections for 2035. Diabetes Res Clin Practice 2014; 103: 137-49. Nolan CJ, Damm P, Prentki M. 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