Glucose Uptake In Muscle Cells Research Articles

Isoform-specific AMPK association with TBC1D1 is reduced by a mutation associated with severe obesity.

AMP-activated protein kinase (AMPK) is a key regulator of cellular and systemic energy homeostasis which achieves this through the phosphorylation of a myriad of downstream targets. One target is TBC1D1 a Rab-GTPase-activating protein that regulates glucose uptake in muscle cells by integrating insulin signalling with that promoted by muscle contraction. Ser237 in TBC1D1 is a target for phosphorylation by AMPK, an event which may be important in regulating glucose uptake. Here, we show AMPK heterotrimers containing the α1, but not the α2, isoform of the catalytic subunit form an unusual and stable association with TBC1D1, but not its paralogue AS160. The interaction between the two proteins is direct, involves a dual interaction mechanism employing both phosphotyrosine-binding (PTB) domains of TBC1D1 and is increased by two different pharmacological activators of AMPK (AICAR and A769962). The interaction enhances the efficiency by which AMPK phosphorylates TBC1D1 on its key regulatory site, Ser237. Furthermore, the interaction is reduced by a naturally occurring R125W mutation in the PTB1 domain of TBC1D1, previously found to be associated with severe familial obesity in females, with a concomitant reduction in Ser237 phosphorylation. Our observations provide evidence for a functional difference between AMPK α-subunits and extend the repertoire of protein kinases that interact with substrates via stabilisation mechanisms that modify the efficacy of substrate phosphorylation.

Site-specific glycations of apolipoprotein A-I lead to differentiated functional effects on lipid-binding and on glucose metabolism

Prolonged hyperglycemia in poorly controlled diabetes leads to an increase in reactive glucose metabolites that covalently modify proteins by non-enzymatic glycation reactions. Apolipoprotein A-I (apoA-I) of high-density lipoprotein (HDL) is one of the proteins that becomes glycated in hyperglycemia. The impact of glycation on apoA-I protein structure and function in lipid and glucose metabolism were investigated.ApoA-I was chemically glycated by two different glucose metabolites (methylglyoxal and glycolaldehyde). Synchrotron radiation and conventional circular dichroism spectroscopy were used to study apoA-I structure and stability. The ability to bind lipids was measured by lipid-clearance assay and native gel analysis, and cholesterol efflux was measured by using lipid-laden J774 macrophages. Diet induced obese mice with established insulin resistance, L6 rat and C2C12 mouse myocytes, as well as INS-1E rat insulinoma cells, were used to determine in vivo and in vitro glucose uptake and insulin secretion.Site-specific, covalent modifications of apoA-I (lysines or arginines) led to altered protein structure, reduced lipid binding capability and a reduced ability to catalyze cholesterol efflux from macrophages, partly in a modification-specific manner. The stimulatory effects of apoA-I on the in vivo glucose clearance were negatively affected when apoA-I was modified with methylglyoxal, but not with glycolaldehyde. The in vitro data showed that both glucose uptake in muscle cells and insulin secretion from beta cells were affected. Taken together, glycation modifications impair the apoA-I protein functionality in lipid and glucose metabolism, which is expected to have implications for diabetes patients with poorly controlled blood glucose.

Attenuation of FFA‐induced skeletal muscle insulin resistance by rosemary extract

Elevated blood free fatty acids (FFAs), as seen in obesity, impair insulin action in muscle leading to insulin resistance and Type 2 diabetes mellitus. Serine phosphorylation of the insulin receptor substrate (IRS) reduces insulin‐stimulated tyrosine phosphorylation leading to insulin resistance. A number of serine/threonine kinases including JNK, mTOR and p70 6SK have been implicated in serine phosphorylation of IRS‐1 and insulin resistance. Activation of the energy sensor AMP‐activated protein kinase (AMPK) increases glucose uptake and has become an important target to counteract insulin resistance. We reported recently that rosemary extract (RE) increased skeletal muscle cell glucose uptake and activated AMPK. However, its effect on FFA‐induced insulin resistant muscle cells has never been elucidated. In this study, we investigated the effect of RE in palmitate‐induced insulin resistant L6 muscle cells. Glucose uptake was measured using [3H]‐2‐deoxy‐D‐glucose and the signaling molecules involved were investigated by immunoblotting.Exposure of L6 myotubes to the FFA palmitate (P) (0.2 mM, 16 hours) resulted in significant reduction in insulin‐stimulated glucose uptake (I: 201±1.21% and P+I: 117±15.6% of control, P<0.001 respectively) indicating insulin resistance. Importantly, exposure to RE resulted in significant restoration of insulin‐stimulated glucose uptake (179±10.5% of control, P<0.001). Treatment with palmitate increased serine phosphorylation of IRS‐1 and significantly decreased the insulin‐stimulated phosphorylation of Akt and these effects were completely abolished in the presence of RE. Treatment with palmitate increased the phosphorylation/activation of JNK, mTOR and p70 6SK whereas RE completely abolished these effects. RE increased the phosphorylation of AMPK even in the presence of palmitate. Overall, our data indicate that treatment with RE attenuates the FFA‐induced insulin resistance. RE and RE polyphenols may have potent effects against insulin resistance and deserve further study.Support or Funding InformationSupported by a Natural Sciences and Engineering Research Council of Canada (NSERC) grant to ET.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Screening plant derived dietary phenolic compounds for bioactivity related to cardiovascular disease

The potential health benefits of phenolic acids found in food and beverages has been suggested from a number of large population studies. However, the mechanism of how these compounds may exert biological effects is less well established. It is also now recognised that many complex polyphenols in the diet are metabolised to simple phenolic acids which can be taken up in the circulation. In this paper a number of selected phenolic compounds have been tested for their bioactivity in two cell culture models. The expression and activity of endothelial nitric oxide synthase (eNOS) in human aortic endothelial cells and the uptake of glucose in muscle cells. Our data indicate that while none of the compounds tested had a significant effect on eNOS expression or activation in endothelial cells, several of the compounds increased glucose uptake in muscle cells. These compounds also enhanced the translocation of the glucose transporter GLUT4 to the plasma membrane, which may explain the observed increase in cellular glucose uptake. These results indicate that simple cell culture models may be useful to help understand the bioactivity of phenolic compounds in relation to cardiovascular protection.

Ginsenoside Rb1 exerts antidiabetic action on C2C12 muscle cells by leptin receptor signaling pathway

Context: Ginsenoside Rb1 improves insulin sensitivity and glucose uptake in muscle cells via different signaling pathways; however, it is not clear that it has any effect on leptin signaling in skeletal muscle.Objectives: The aim of this study was to investigate the effect of ginsenoside Rb1 on leptin receptors expression and main signaling pathways of leptin (STAT3, PI3 kinase and ERK kinase) in C2C12 skeletal muscle cells.Materials and methods: C2C12 myotubes were incubated with various concentrations of Rb1 (0.1, 1 and 10 μM) for different incubation times (1–12 h). Leptin receptors expression and GLUT-4 translocation were analyzed using realtime PCR and western blot analyses, respectively. PI3 and ERK kinases were blocked using their specific inhibitors (wortmannin and PD98059) in the presence and absence of RB1 to determine the main signaling pathway related to leptin receptor activation in C2C12 cells.Results: Rb1 could maximally stimulate both leptin receptors (OBRa and OBRb) mRNA and protein expression and phosphorylation of STAT3, PI3K and ERK2 in C2C12 myotubes at 10 μM for 3 h. Rb1 induced GLUT4 translocation was inhibited by the silencing of OBRb mRNA, demonstrated that glucose uptake was mediated via leptin receptor activation. GLUT4 recruitment to the cell surface induced by Rb1 was inhibited by wortmannin, an inhibitor of PI3K in combination with OBRb siRNA, but not by PD98059 an ERK2 kinase-1 inhibitor, indicating that GLUT4 translocation induced by Rb1 was associated with the leptin receptor upregulation and subsequent activation of PI3K.Conclusions: Our results suggest that Rb1 promote translocation of GLUT4 by upregulation of leptin receptors and activation of PI3K.

Carnosic acid as a component of rosemary extract stimulates skeletal muscle cell glucose uptake via AMPK activation.

Compounds that increase the activity of the energy sensor AMP-activated kinase (AMPK) have the potential to regulate blood glucose levels. Although rosemary extract (RE) has been reported to activate AMPK and reduce blood glucose levels in vivo, the chemical components responsible for these effects are not known. In the present study, we measured the levels of the polyphenol carnosic acid (CA) in RE and examined the effects and the mechanism of action of CA on glucose transport system in muscle cells. High performance liquid chromatography (HPLC) was used to measure the levels of CA in RE. Parental and GLUT4myc or GLUT1myc overexpressing L6 rat myotubes were used. Glucose uptake was assessed using [3 H]-2-deoxy-d-glucose. Total and phosphorylated levels of Akt and AMPK were measured by immunoblotting. Plasma membrane GLUT4myc and GLUT1myc levels were examined using a GLUT translocation assay. Statistics included analysis of variance (ANOVA) followed by Tukey's post-hoc test. At concentrations found in rosemary extract, CA stimulated glucose uptake in L6 myotubes. At 2.0μmol/L CA a response (226±9.62% of control, P=.001), similar to maximum insulin (201±7.86% of control, P=.001) and metformin (213±10.74% of control, P=.001) was seen. Akt phosphorylation was not affected by CA while AMPK and ACC phosphorylation was increased and the CA-stimulated glucose uptake was significantly reduced by the AMPK inhibitor compound C. Plasma membrane GLUT4 or GLUT1 glucose transporter levels were not affected by CA. Our study shows increased muscle cell glucose uptake and AMPK activation by low CA concentrations, found in rosemary extract, indicating that CA may be responsible for the antihyperglycemic properties of rosemary extract seen in vivo.

5,7-Dihydroxy-6-geranylflavanone improves insulin sensitivity through PPARα/γ dual activation.

In the present study, we demonstrate that 5,7-dihydroxy-6-geranylflavanone (DGF) isolated from Amorpha fruticosa (A. fruticosa) is a novel peroxisome proliferator-activated receptor (PPAR)α/γ dual agonist which may be used to improve insulin sensitivity. The extract from A. fruticosa increased the transcriptional activity of both PPARα and PPARγ which was, in part, driven by the active ingredient DGF. Treatment with DGF markedly enhanced the adipogenesis of 3T3-L1 preadipocytes, which was comparable to the effect of the PPARγ agonist, troglitazone. In addition, DGF was found to enhance fatty acid oxidation and glucose utilization through the dual activation of PPARα/γ. In addition treatment with DGF led to an improvement in insulin sensitivity, resulting in enhanced glucose uptake in muscle cells. The findings of our study data suggest that DGF may be used as potential therapeutic agent in the treatment of type 2 diabetes and related metabolic disorders by enhancing glucose and lipid metabolism.

Abstract P5-05-05: Inhibition of enhanced glucose uptake and glycolysis by KU-55933 as a novel strategy against aggressive breast cancer

Abstract The ability of cancer cells to produce large amounts of lactate through aerobic glycosis (Warburg effect) is coupled to high rates of glucose uptake. Enhanced glucose uptake and glycolysis are closely correlated to increased breast tumor aggressiveness and poor prognosis. However, despite the importance of glucose uptake in supplying energy and preventing apoptosis of cancer cells, the majority of current efforts in searching for therapeutic agents targeting glucose metabolism have been aimed at modulating activities of different metabolic enzymes that are involved in glycolysis. Very limited studies have been done in developing novel therapeutic agents against glucose uptake in breast cancer cells. Ataxia-telangiectasia (A-T) is a monogenic, autosomal recessive disorder characterized by cerebellar ataxia and oculocutaneous telangiectasias. The gene mutated in this disease, ATM (A-T, mutated), encodes a 370-kDa protein kinase. Although ATM is traditionally considered to be a nuclear protein that functions as a signal transducer in the cellular response to DNA damage, it is now known that ATM is also present in the cytoplasm and has important cytoplasmic functions. We previously discovered that ATM activates Akt, a main regulator of glucose uptake, by stimulating its phosphorylation at Ser473 following insulin treatment. We also found that ATM participates in insulin-mediated glucose uptake in muscle cells, and KU-55933, a specific inhibitor of ATM, strongly inhibits this process. Recently, we found that KU-55933 inhibits cell proliferation by inducing apoptosis in MDA-MB-231, a triple-negative breast cancer cell line. We have also found that KU-55933 inhibits migration of MDA-MB-231 by a cell invasion assay. Furthermore, we found that these cancer cells exhibit enhanced glucose uptake in response to insulin and the addition of KU-55933 leads to a dramatic reduction of insulin-mediated glucose uptake in these cells. To further test whether KU-55933's ability to induce apoptosis is linked to its inhibition of glucose uptake, we performed a cell death ELISA assay in MDA-MB-231 cells treated with KU-55933 and different concentrations of glucose. Our results show that KU-55933 induces apoptosis of MDA-MB-231 cells, resulting in a similar degree of cell death as glucose starvation, while cells treated with glucose in conjunction with KU-55933 have decreased apoptosis. Moreover, we performed a cell migration assay and found that KU-55933 strongly inhibits the migration of MDA-MB-231 cells (similar to that caused by glucose starvation), which is almost fully rescued by the extra glucose supplemented in the cell culture medium. We have also established a positional isotope labeling-based targeted metabolomics method that can directly measure the conversion from glucose to lactate through glycolysis in cancer cells. Our results show strong production of lactate from glucose in MDA-MB-231 cells even under normal aerobic growth conditions, and KU-55933 strongly inhibits this process. Our findings may lead to the development of KU-55933 and its analogs as a new generation of therapeutic agents against aggressive breast cancer. Citation Format: Yang D-Q, Harris B, Jiang S, Li Y, Freund D, Hegeman A, Cleary M. Inhibition of enhanced glucose uptake and glycolysis by KU-55933 as a novel strategy against aggressive breast cancer. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-05-05.

A Novel Partial PPARα/γ Dual Agonist SN159 Improves Insulin Sensitivity

We here demonstrate that (E)‐1‐(3‐aminophenyl)‐3‐(5‐bromo‐4‐hydroxy‐2‐methoxyphenyl)prop‐2‐en‐1‐one (SN159) is a novel peroxisome proliferator‐activated receptor (PPAR) partial agonist that improves insulin sensitivity. SN159 interacted directly with PPARα and PPARγ, which were confirmed by LanthaScreen ligand binding assay and molecular docking study. SN159 treatment leads to a significant improvement of insulin sensitivity, resulting in enhancing glucose uptake in muscle cells. SN159 stimulated adipogenic differentiation of 3T3‐L1 preadipocytes, however, the effects were much weaker than those of PPARγ agonist troglitazone. In parallel, SN159 increased weakly the transcriptional activities of PPARα/γ, compared to the positive control. Furthermore, PPARγ activation and adipogenic differentiation by troglitazone were significantly reduced by treatment with SN159, indicating that SN159 is a partial agonist of PPARs. SN159 was able to enhance fatty acid oxidation and glucose utilization through the dual activation of PPARα/γ. Taken together, these results suggest that SN159 is a novel PPAR partial agonist, which can be used as potential therapeutic agents against type 2 diabetes and related metabolic disorders by enhancing glucose and lipid metabolism.

AMPK, a metabolic sensor, is involved in isoeugenol-induced glucose uptake in muscle cells.

Isoeugenol exerts various beneficial effects on human health. However, the mechanisms underlying these effects are poorly understood. In this study, we observed that isoeugenol activated AMP-activated protein kinase (AMPK) and increased glucose uptake in rat L6 myotubes. Isoeugenol-induced increase in intracellular calcium concentration and glucose uptake was inhibited by STO-609, an inhibitor of calcium/calmodulin-dependent protein kinase kinase (CaMKK). Isoeugenol also increased the phosphorylation of protein kinase C-α (PKCα). Chelation of calcium with BAPTA-AM blocked isoeugenol-induced AMPK phosphorylation and glucose uptake. Isoeugenol stimulated p38MAPK phosphorylation that was inhibited after pretreatment with compound C, an AMPK inhibitor. Isoeugenol also increased glucose transporter type 4 (GLUT4) expression and its translocation to the plasma membrane. GLUT4 translocation was not observed after the inhibition of AMPK and CaMKK. In addition, isoeugenol activated the Akt substrate 160 (AS160) pathway, which is downstream of the p38MAPK pathway. Knockdown of the gene encoding AS160 inhibited isoeugenol-induced glucose uptake. Together, these results indicate that isoeugenol exerts beneficial health effects by activating the AMPK/p38MAPK/AS160 pathways in skeletal muscle.

Cafestol, a Bioactive Substance in Coffee, Stimulates Insulin Secretion and Increases Glucose Uptake in Muscle Cells: Studies in Vitro.

Diet and exercise intervention can delay or prevent development of type-2-diabetes (T2D), and high habitual coffee consumption is associated with reduced risk of developing T2D. This study aimed to test whether selected bioactive substances in coffee acutely and/or chronically increase insulin secretion from β-cells and improve insulin sensitivity in skeletal muscle cells. Insulin secretion from INS-1E rat insulinoma cells was measured after acute (1-h) and long-term (72-h) incubation with bioactive substances from coffee. Additionally, we measured uptake of radioactive glucose in human skeletal muscle cells (SkMC) after incubation with cafestol. Cafestol at 10(-8) and 10(-6) M acutely increased insulin secretion by 12% (p < 0.05) and 16% (p < 0.001), respectively. Long-term exposure to 10(-10) and 10(-8) M cafestol increased insulin secretion by 34% (p < 0.001) and 68% (p < 0.001), respectively. Caffeic acid also increased insulin secretion acutely and chronically. Chlorogenic acid, trigonelline, oxokahweol, and secoisolariciresinol did not significantly alter insulin secretion acutely. Glucose uptake in SkMC was significantly enhanced by 8% (p < 0.001) in the presence of 10(-8) M cafestol. This newly demonstrated dual action of cafestol suggests that cafestol may contribute to the preventive effects on T2D in coffee drinkers and be of therapeutic interest.

Involvement of mTOR in Type 2 CRF Receptor Inhibition of Insulin Signaling in Muscle Cells.

Type 2 corticotropin-releasing factor receptor (CRFR2) is expressed in skeletal muscle and stimulation of the receptor has been shown to inhibit the effect of insulin on glucose uptake in muscle cells. Currently, little is known about the mechanisms underlying this process. In this study, we first showed that both in vivo and in vitro CRFR2 expression in muscle was closely correlated with insulin sensitivity, with elevated receptor levels observed in insulin resistant muscle cells. Stimulation of CRFR2 by urocortin 2 (Ucn 2), a CRFR2-selective ligand, in C2C12 myotubes greatly attenuated insulin-induced glucose uptake. The inhibitory effect of CRFR2 signaling required cAMP production and is involved the mammalian target of rapamycine pathway, as rapamycin reversed the inhibitory effect of CRFR2 stimulation on insulin-induced glucose uptake. Moreover, stimulation of CRFR2 failed to inhibit glucose uptake in muscle cells induced by platelet-derived growth factor, which, similar to insulin, signals through Akt-mediated pathway but is independently of insulin receptor substrate (IRS) proteins to promote glucose uptake. This result argues that CRFR2 signaling modulates insulin's action likely at the levels of IRS. Consistent with this notion, Ucn 2 reduced insulin-induced tyrosine phosphorylation of IRS-1, and treatment with rapamycin reversed the inhibitory effect of Ucn 2 on IRS-1 and Akt phosphorylation. In conclusion, the inhibitory effect of CRFR2 signaling on insulin action is mediated by cAMP in a mammalian target of rapamycine-dependent manner, and IRS-1 is a key nodal point where CRFR2 signaling modulates insulin-stimulated glucose uptake in muscle cells.

Increased skeletal muscle glucose uptake by rosemary extract through AMPK activation.

Stimulation of the energy sensor AMP-activated kinase (AMPK) has been viewed as a targeted approach to increase glucose uptake by skeletal muscle and control blood glucose homeostasis. Rosemary extract (RE) has been reported to activate AMPK in hepatocytes and reduce blood glucose levels in vivo but its effects on skeletal muscle are not known. In the present study, we examined the effects of RE and the mechanism of regulation of glucose uptake in muscle cells. RE stimulated glucose uptake in L6 myotubes in a dose- and time-dependent manner. Maximum stimulation was seen with 5 μg/mL of RE for 4 h (184% ± 5.07% of control, p < 0.001), a response comparable to maximum insulin (207% ± 5.26%, p < 0.001) and metformin (216% ± 8.77%, p < 0.001) stimulation. RE did not affect insulin receptor substrate 1 and Akt phosphorylation but significantly increased AMPK and acetyl-CoA carboxylase phosphorylation. Furthermore, the RE-stimulated glucose uptake was significantly reduced by the AMPK inhibitor compound C, but remained unchanged by the PI3K inhibitor, wortmannin. RE did not affect GLUT4 or GLUT1 glucose transporter translocation in contrast with a significant translocation of both transporters seen with insulin or metformin treatment. Our study is the first to show a direct effect of RE on muscle cell glucose uptake by a mechanism that involves AMPK activation.

The Negative Impact of Insulin Therapy for Acute Hyperglycemia Secondary to Glucose Load on Plasma Amino Acid Profiles in a Rat Model of Sepsis

Background: In critical illnesses, insulin therapy under overfed conditions with an excessive glucose infusion may cause metabolic disturbances in skeletal muscle mainly through muscle cell glucose uptake and the inhibition of physiological protein breakdown. The aim of this study was to examine the potential negative aspects of insulin therapy in a rat model of sepsis. Materials and Methods: Male Sprague-Dawley rats underwent cecal ligation and puncture (CLP) or sham surgery. A pre-established continuous intravenous glucose infusion was initiated immediately after surgery. Rats with sepsis were divided into four groups (n = 7 in each group) based on target blood glucose (BG) levels: a no glucose (NG) group (100-150 mg/dl), moderate glucose (MG) group (200-300 mg/dl), high glucose (HG) group (>300 mg/dl), and the hyperinsulinemia (HI) group, which received the same glucose infusion as the HG group with the insulin infusion (200-300 mg/dl). The sham group underwent sham surgery and received the same glucose infusion as the HG group. All rats were sacrificed 9 h after surgery, and blood samples were collected to measure plasma amino acid (AA) profiles. To examine survival rates in the 48 h following CLP, the HG, MG, and HI groups were newly prepared according to the aforementioned experimental design. Results: Plasma levels of the branched-chain AAs, glutamine, arginine, citrulline, and alanine among the septic groups slightly and inversely decreased with the amount of glucose infused, and HI had significantly lower values (p < 0.01). A strong correlation was observed among the AAs. Plasma 3-methylhistidine concentrations were the highest in the HI group. The survival rate of the HI group was greater than that of the HG, but did not reach the level of the MG group. Conclusion: In critical illnesses, insulin therapy under overfed conditions may impair the physiological supply of AAs and conditionally essential AA starvation, such as glutamine and arginine, and may have an adverse impact on the prognosis of patients.

Anti-diabetic phytochemicals that promote GLUT4 translocation via AMPK signaling in muscle cells

Skeletal muscles are the largest tissue in our body and play an important role in maintaining glucose homeostasis. Cultured L6 myotubes and C2C12 myotubes are useful to construct simple glucose uptake assay systems, to screen various phytochemicals that promote glucose uptake, and to clarify their modes of actions. In skeletal muscles, insulin promotes glucose uptake by activating phosphatidylinositol-3 kinase (PI3K) and Akt, leading to increased translocation of glucose transporter 4 (GLUT4) to the plasma membrane. Another GLUT4 translocation promoter is 5′ adenosine monophosphate-activated protein kinase (AMPK). In mammalian cells, AMPK activated by an increase in AMP/ATP ratio acts as an energy sensor. AMPK is activated by exercise/contraction in muscle cells and compound such as metformin, this resulting in stimulation of GLUT4 translocation to plasma membrane and hence glucose uptake in skeletal muscles. Thus, studies on novel compounds that activate skeletal muscle glucose uptake and AMPK would be useful for the development of new treatment of insulin resistance and type 2 diabetes (T2D). Here we review the current knowledge of phytochemicals in foods and beverages that promote GLUT4 translocation via AMPK signaling in muscle cells, and their effects on glucose metabolism in mainly T2D model mice. Phytochemicals that have potential to stimulate glucose uptake in muscle cells are suggested to be anti-diabetic.

Enhancement of glucose uptake in muscular cell by soybean charged peptides isolated by electrodialysis with ultrafiltration membranes (EDUF): Activation of the AMPK pathway

Soy peptides consumption has been associated with beneficial effects in type 2 diabetes patients. However, the peptide fractions responsible for these effects, and their mechanisms of action, have not been identified yet. In this study, we have isolated soybean peptides by electrodialysis with an ultrafiltration membrane (EDUF) at 50V/100kDa, and tested them for their capacity to improve glucose uptake in L6 muscle cells. We observed that these fractions were able to significantly enhance glucose uptake in the presence of insulin. The reported bioactivity would be due to the low molecular weight peptides (300–500Da) recovered. Moreover, we observed that an enhancement of glucose uptake was correlated to the activation of the AMPK enzyme, well known for its capacity to increase glucose uptake in muscle cells. To our knowledge, this is the first time that bioactive peptides with glucose uptake activity have been isolated from a complex soy matrix, and that the implication of AMPK in it is demonstrated.

Z‐2‐(β‐d‐glucopyranosyloxy)‐3‐phenylpropenoic acid, an α‐hydroxy acid from rooibos (Aspalathus linearis) with hypoglycemic activity

The rare enolic phenylpyruvic acid-2-O-glucoside, (PPAG:Z-2-(β-D-glucopyranosyloxy)-3-phenylpropenoic acid), is one of the major constituents of fermented rooibos infusions. 3-Phenylpyruvic acid (2-oxo-3-phenylpropanoic acid), without the sugar moiety and with a keto form instead of an enolic arrangement, has been shown to enhance insulin release and glucose uptake in muscle cells. The purpose of this study was to assess if PPAG has similar activity on glucose metabolism. Preliminary in vitro studies confirmed that PPAG, isolated from rooibos, enhanced glucose uptake. A dose-response study in Chang cells showed that PPAG enhanced glucose uptake in the concentration range 1.0-31.6 μM (EC50 = 3.6 μM). In obese insulin-resistant rats, oral administration of PPAG lowered fasting glucose concentrations and improved oral glucose tolerance values; messenger RNA expression of glucokinase, glucose transporter 1 and 2, insulin receptor, peroxisome proliferator-activated receptor alpha, and suppressor of cytokine signaling 3, were increased in the liver. This suggests that the liver is mainly responsible for PPAG bioactivity. This study describes for the first time that PPAG increases in vitro glucose uptake and improves glucose tolerance in an obese insulin-resistant rat model, suggesting that it has potential as a new class of antidiabetic therapeutics that would contribute to the antidiabetic effect of rooibos.

Anti-diabetic effect of acid hydrolyzed silk peptides via cytoprotection of pancreas β-cells and up-regulation of glucose uptake in muscle cells

In this study, we observed anti-diabetic effects of acid hydrolyzed silk peptides, where the amount of peptides in the total amino acid mixture was strictly regulated. Using in vitro diabetes models, silk peptide-containing amino acid mixtures of 5.60% (G5), 11.30% (G10), 14.50% (G15), and 20.50% (G20) were examined separately in order to determine whether they have biological activities. According to our results, a cytoprotective effect was observed following treatment of interleukin-1β in RINm5f pancreas β-cells. As a consequence, Bax, a pro-apoptotic gene, was downregulated, while Bcl-2, a pro-survival gene, was retained at normal level. Results of the 4’,6-diamidino-2-phentylindole (DAPI) staining assay confirmed that G20 has a better cytoprotective effect. Insulin release from RINm5f cells showed a significant increase following treatment with G5-G20, suggesting that silk peptide effectively regulated and induced insulin production. Single treatment with G5-G20 resulted in enhanced glucose uptake in L6 skeletal muscle cells. In addition, a higher amount of each group inhibited the activity of α-glucosidase. In summary, these data suggest that silk peptide may have an anti-diabetic effect through protection of pancreas β-cells and enhancement of insulin release, which showed a close association with Type 1 diabetes mellitus (DM), and can improve glucose uptake, which was the major target for therapy of Type 2 diabetes. Taken together, we concluded that acid hydrolyzed silk peptides can be used effectively for control of blood sugar metabolism via improvement of the problematic indices of Type 1 and Type 2 DM.

Role of RalA downstream of Rac1 in insulin-dependent glucose uptake in muscle cells

The small GTPase RalA has been implicated in glucose uptake in insulin-stimulated adipocytes, although it remains unclear whether RalA has a similar role in insulin signaling in other types of cells. Recently, we have demonstrated that the Rho family GTPase Rac1 has a critical role in insulin-dependent glucose uptake in myoblast culture and mouse skeletal muscle. However, the mechanisms underlying Rac1-dependent glucose uptake, mostly mediated by the plasma membrane translocation of the glucose transporter GLUT4, remain largely unknown. The purpose of this study is to examine the involvement of RalA in Rac1 regulation of the translocation of GLUT4 to the plasma membrane in muscle cells. Ectopic expression of a constitutively activated RalA mutant indeed stimulated GLUT4 translocation, suggesting an important role of RalA also in muscle cells. GLUT4 translocation induced by constitutively activated mutation of Rac1 or more physiologically by upstream Rac1 regulators, such as phosphoinositide 3 kinase and the guanine nucleotide exchange factor FLJ00068, was abrogated when the expression of RalA was downregulated by RNA interference. The expression of constitutively activated Rac1, on the other hand, caused GTP loading and subcellular redistribution of RalA. Collectively, we propose a novel mechanism involving RalA for Rac1-mediated GLUT4 translocation in skeletal muscle cells.

Defining the Contribution of AMP-activated Protein Kinase (AMPK) and Protein Kinase C (PKC) in Regulation of Glucose Uptake by Metformin in Skeletal Muscle Cells

The importance of AMP-activated protein kinase (AMPK) and protein kinase C (PKC) as effectors of metformin (Met) action on glucose uptake (GU) in skeletal muscle cells was investigated. GU in L6 myotubes was stimulated 2-fold following 16 h of Met treatment and acutely enhanced by insulin in an additive fashion. Insulin-stimulated GU was sensitive to PI3K inhibition, whereas that induced by Met was not. Met and its related biguanide, phenformin, stimulated AMPK activation/phosphorylation to a level comparable with that induced by the AMPK activator, 5-amino-1-β-d-ribofuranosyl-imidazole-4-carboxamide (AICAR). However, the increase in GU elicited by AICAR was significantly lower than that induced by either biguanide. Expression of a constitutively active AMPK mimicked the effects of AICAR on GU, whereas a dominant interfering AMPK or shRNA silencing of AMPK prevented AICAR-stimulated GU and Met-induced AMPK signaling but only repressed biguanide-stimulated GU by ∼20%. Consistent with this, analysis of GU in muscle cells from α1(-/-)/α2(-/-) AMPK-deficient mice revealed a significant retention of Met-stimulated GU, being reduced by ∼35% compared with that of wild type cells. Atypical PKCs (aPKCs) have been implicated in Met-stimulated GU, and in line with this, Met and phenformin induced activation/phosphorylation of aPKC in L6 myotubes. However, although cellular depletion of aPKC (>90%) led to loss in biguanide-induced aPKC phosphorylation, it had no effect on Met-stimulated GU, whereas inhibitors targeting novel/conventional PKCs caused a significant reduction in biguanide-induced GU. Our findings indicate that although Met activates AMPK, a significant component of Met-stimulated GU in muscle cells is mediated via an AMPK-independent mechanism that involves novel/conventional PKCs.