Mediated Glucose Uptake Research Articles

Abstract Tu091: Mechanism of beta1-adrenergic receptor signaling by IgG3 subclass of autoantibodies

Autoimmune response to self-antigens results in autoantibodies (AAbs), wherein AAbs against extra cellular loop (ECL) 2 of β1-adrenergic receptor (β1AR) is known to underlie dilated cardiomyopathy (DCM). Contrarily, recent studies show that patients with β1AR AAbs belonging to the IgG3 subclass have beneficial outcomes. However, the signaling mechanisms that underlie the beneficial outcomes are not well understood. We have previously shown that IgG3(+) β1AR AAbs facilitates uniquely biased β1AR signaling in response to β-blocker compared to non-IgG3(+) β1AR AAbs. Since IgG3(+) β1AR AAbs are associated with favorable patient outcomes, it is critical to determine the mechanistic basis of the unique beneficial signaling pathway that may have therapeutic potential. HEK 293 cells stably expressing human β1AR (HEK-β1AR) was generated and treated with β1AR AAbs. Minimal changes in phosphorylation of β1AR was observed following IgG3(+) β1AR AAbs, while significant phosphorylation of β1ARs was observed with non-IgG3(+) β1AR AAbs. To dissect the diverse signaling responses mediated by IgG3(+) and non-IgG(+) β1AR AAbs, HEK-β1AR cells were treated with IgG3(+) or non-IgG3(+) β1AR AAbs. β1ARs were immunoprecipitated and subjected to mass-spectrometry analysis to identify unique co-immunoprecipitating proteins that could complex with receptor in presence of IgG3(+) or non-IgG3(+) β1AR AAbs. One of the proteins that was differentially recruited to the β1AR complex was Insulin Receptor Substrate 4 (IRS4). Traditionally, IRS4 is a signaling hub downstream of insulin/insulin growth factor receptors mediating glucose uptake. The differential interaction of IRS4 with β1AR in the presence of IgG3(+) vs. non-IgG3(+) β1AR AAbs reflects that IRS4 could be a key signaling regulator of uniquely biased signal transduction pathway in the presence of IgG3(+) vs. non-IgG3(+) β1AR AAbs. Mechanisms underlying this unique IRS4 mediated signaling modulation will be discussed.

Inhibition of Sodium-Glucose Cotransporter-2 during Serum Deprivation Increases Hepatic Gluconeogenesis via the AMPK/AKT/FOXO Signaling Pathway.

Sodium-dependent glucose cotransporter 2 (SGLT2) mediates glucose reabsorption in the renal proximal tubules, and SGLT2 inhibitors are used as therapeutic agents for treating type 2 diabetes mellitus. This study aimed to elucidate the effects and mechanisms of SGLT2 inhibition on hepatic glucose metabolism in both serum deprivation and serum supplementation states. Huh7 cells were treated with the SGLT2 inhibitors empagliflozin and dapagliflozin to examine the effect of SGLT2 on hepatic glucose uptake. To examine the modulation of glucose metabolism by SGLT2 inhibition under serum deprivation and serum supplementation conditions, HepG2 cells were transfected with SGLT2 small interfering RNA (siRNA), cultured in serum-free Dulbecco's modified Eagle's medium for 16 hours, and then cultured in media supplemented with or without 10% fetal bovine serum for 8 hours. SGLT2 inhibitors dose-dependently decreased hepatic glucose uptake. Serum deprivation increased the expression levels of the gluconeogenesis genes peroxisome proliferator-activated receptor gamma co-activator 1 alpha (PGC-1α), glucose 6-phosphatase (G6pase), and phosphoenolpyruvate carboxykinase (PEPCK), and their expression levels during serum deprivation were further increased in cells transfected with SGLT2 siRNA. SGLT2 inhibition by siRNA during serum deprivation induces nuclear localization of the transcription factor forkhead box class O 1 (FOXO1), decreases nuclear phosphorylated-AKT (p-AKT), and p-FOXO1 protein expression, and increases phosphorylated-adenosine monophosphate-activated protein kinase (p-AMPK) protein expression. However, treatment with the AMPK inhibitor, compound C, reversed the reduction in the protein expression levels of nuclear p- AKT and p-FOXO1 and decreased the protein expression levels of p-AMPK and PEPCK in cells transfected with SGLT2 siRNA during serum deprivation. These data show that SGLT2 mediates glucose uptake in hepatocytes and that SGLT2 inhibition during serum deprivation increases gluconeogenesis via the AMPK/AKT/FOXO1 signaling pathway.

DHA promotes healthy adipose tissue remodeling and carbohydrate utilization in grass carp (Ctenopharyngodon idellus) and largemouth bass (Micropterus salmoides)

Due to the wide usage of high-carbohydrate diets in aquaculture, improving the ability of fish to utilize high-carbohydrate diets is one of the important issues in aquaculture. Adipose tissue is an essential organ for storing energy and plays a vital role in glucose homeostasis. In this study, RNA sequencing reveals that docosahexaenoic acid (DHA) promoted the expression of the glucose metabolism related-genes in grass carp adipocytes, raising the hypothesis that DHA may attenuate the adverse effect of high-carbohydrate diets on fish via promoting glucose metabolism in adipose tissue. To verify this hypothesis, a total of 180 juvenile grass carp (14.24 ± 0.43 g) and a total of 180 largemouth bass (40.03 ± 0.12 g) were randomly distributed into 18 tanks (200 L), respectively, and fed with high-carbohydrate diets containing DHA. The results showed that high-carbohydrate diets promoted glycolysis and inhibited lipolysis and fatty acid β-oxidation, leading to the excessive accumulation of abdominal fat and adipose tissue inflammation. After feeding high-carbohydrate diets supplementation with DHA, glucose transporter type 4 (GLUT4) mediated-glucose uptake was increased in adipose tissue, resulting in significantly decreased blood glucose in grass carp and largemouth bass (P < 0.05). And then, the glucose was converted to triglyceride through the glucose-6-phosphate dehydrogenase (G6PD) mediated-pentose phosphate pathway, de novo synthesis of fatty acids and triglyceride (TG) synthesis. Subsequently, the resulting TG was broken down through lipolysis and fatty acid β-oxidation. These reprogrammed metabolic processes inhibited the excessive accumulation of abdominal fat. In addition, DHA attenuated inflammation in adipose tissue, and further reduced the levels of inflammatory cytokines in serum (P < 0.05), indicating that DHA promotes healthy adipose tissue remodeling. Ultimately, these benefits improved the growth performance of grass carp and largemouth bass fed with high-carbohydrate diets. Taken together, our study indicates that adipose tissue is an important target for improving the negative effects of high-carbohydrate diets, and DHA could promote healthy adipose tissue remodeling and improve carbohydrate utilization in fish by reprogramming the glucose and lipid metabolism in adipose tissues.

An In Vitro Pharmacognostical Study on Gluconeogenesis and Glucose

Traditional medicine over 60% of the world’s population used for health care name as Mukia maderaspatana (L.) M. Roem. (Cucurbitaceae) (Mukia) is extensively important medicine as an anti-inflammatory plant. It is rich in content of phenolics that exerts various medicinal properties. Mukia extract and its derivatives phenolics such as quercetin and phloroglucinol are investigated for their in vitro anti-inflammatory activity. Materials used was Quercetin, phloroglucinol, and methanol which extract of the dried whole plant (0.55 and 0.6 mg/ml) were studied for the inhibition of gluconeogenesis and glucose Phenolics of Mukia were analyzed by HPLC and UV-spectroscopy. Results obtained were Glucose (1.5 mg/g/h) was synthesized from pyruvate, the synthesis was whole inhibited by insulin (1 U/ml). Quercetin at 0.35 and 0.6 mg/ml caused 60% and 90% inhibition (0.43 mg/g/h and 0.12 mg/g/h glucose). Addition of insulin did not increase inhibition. Phloroglucinol inhibited 100% glucose production with or without insulin. Mukia (0.20 mg/ml) inhibited gluconeogenesis (0.65 mg/g/h) by 40%, and with insulin, inhibition increased to 50% (0.59 mg/g/h). At 0.6 mg/ml, glucose synthesis was stimulated by1.3-fold, but with insulin gets inhibited by 90% (0.12 mg/g/h glucose). Mukia possessed no effect on glucose uptake, in case it potentiated the activity of insulin mediated glucose uptake (153.82 ± 12.30 mg/dl/g/30 min) compared with insulin control (122.41 ± 9.14 mg/dl/g/30 min) (p &lt; 0.02). HPLC analysis revealed the presence of phenolics. Results were concluded scientific rationale for the use of Mukia in medicine as an anti-inflammatory nutraceutical.

Isoliquiritigenin inhibits gastric cancer growth through suppressing GLUT4 mediated glucose uptake and inducing PDHK1/PGC-1α mediated energy metabolic collapse

BackgroundIsoliquiritigenin (ISL), a natural flavonoid, has anti-tumor activity. But, the understanding of the impact and molecular mechanism of ISL on the growth of gastric cancer (GC) remains limited. PurposeThe study was to explore the tumor suppressive effect of ISL on GC growth both in vitro and in vivo, meanwhile, clarify its molecular mechanisms. MethodsCell viability was detected by cell counting kit-8 (CCK-8) assay. Apoptotic cells in vitro were monitored by Hoechst 33,342 solution. Protein expression was assessed by Western blot. Reactive oxygen species (ROS) level was evaluated by utilizing 2′,7′- dichlorofluorescin diacetate (DCFH-DA). Lactic acid level was detected with L-lactate assay kit. Glucose uptake was monitored with fluorescently tagged glucose 2-[N-(7-nitrobenz-2-oxa-1,3-diaxol-4-yl)amino]-2-deoxyglucose (2-NBDG). Glycolytic proton efflux rate (GlycoPER) was evaluated by glycolytic rate assay kit. Oxygen consumption rate (OCR) was conducted by mito stress test kit. A nude mouse model of gastric cancer cell xenograft was established by subcutaneous injection with MGC803 cells. Pathological changes were evaluated by using H&E staining. Cell apoptosis in vivo was evaluated by terminal deoxy-nucleotide transferase mediated dUTP nick end labeling (TUNEL) assay. ResultsISL remarkably suppressed GC growth and increased cell apoptosis. It regulated apoptosis-related and metabolism-related protein expression both in vitro and in vivo. ISL blocked glucose uptake and suppressed production and secretion of lactic acid, which was accompanied with suppressed mitochondrial oxidative phosphorylation (OXPHOS) and glycolysis but increased ROS accumulation. Overexpression of peroxisome proliferator-activated receptor gamma coactivator-1 alpha (PGC-1α), cellular-myelocytomatosis viral oncogene (c-Myc), hypoxia inducible factor-1α (HIF-1α), glucose transporter 4 (GLUT4) or pyruvate dehydrogenase kinase 1 (PDHK1), could abolish ISL-induced inhibition of cell viability in GC cells. ConclusionThese findings implicated that ISL inhibits GC growth by decreasing GLUT4 mediated glucose uptake and inducing PDHK1/PGC-1α-mediated energy metabolic collapse through depressing protein expression of c-Myc and HIF-1α in GC, suggesting its potential application for GC treatment.

O304 ameliorates hyperglycemia in mice by dually promoting muscle glucose effectiveness and preserving β-cell function

Although insulin mediated glucose uptake in skeletal muscle is a major mechanism ensuring glucose disposal in humans, glucose effectiveness, i.e., the ability of glucose itself to stimulate its own uptake independent of insulin, accounts for roughly half of the glucose disposed during an oral glucose tolerance test. Both insulin dependent and insulin independent skeletal muscle glucose uptake are however reduced in individuals with diabetes. We here show that AMPK activator O304 stimulates insulin independent glucose uptake and utilization in skeletal muscle and heart in vivo, while preventing glycogen accumulation. Combined glucose uptake and utilization requires an increased metabolic demand and we show that O304 acts as a mitochondrial uncoupler, i.e., generates a metabolic demand. O304 averts gene expression changes associated with metabolic inflexibility in skeletal muscle and heart of diabetic mice and reverts diabetic cardiomyopathy. In Type 2 diabetes, insulin resistance elicits compensatory insulin hypersecretion, provoking β-cell stress and eventually compensatory failure. In db/db mice O304 preserves β-cell function by preventing decline in insulin secretion, β-cell mass, and pancreatic insulin content. Thus, as a dual AMPK activator and mitochondrial uncoupler O304 mitigates two central defects of T2D; impaired glucose uptake/utilization and β-cell failure, which today lack effective treatment.

Comparison of a modified 2-step insulin response test performed with porcine zinc insulin and an oral glucose test to detect hyperinsulinemic Icelandic horses

Both, oral and intravenous (IV) testing protocols, are recommended and still used to detect insulin dysregulation (ID) in equids. However, IV tests mainly focus on peripheral insulin resistance (IR), while oral tests assess hyperinsulinemia (HI), which are different aspects of ID. The objective of this study was to describe if horses with HI also demonstrate IR and consequently can be detected by a modified 2-step insulin response test (2-step IRT) performed with a veterinary approved porcine zinc insulin (PZI). Twelve Icelandic horses were subjected to an OGT and 2-step IRT in a crossover study. Serum insulin concentrations during the OGT revealed that six horses were hyperinsulinemic (HI) while six were not (NON-HI). To describe the glucose response to IV injected PZI, the decline of plasma glucose concentration within the first 30 min was analyzed. Glucose reduction was similar in horses with and without HI during the 2-step IRT over time. Additionally, none of the horses reached a glucose reduction of ≥ 50% at 30 min. The results of the present study indicated that a comparable insulin mediated glucose uptake may be observed in horses with and without HI during a modified 2-step IRT. While six out of twelve horses were identified as HI by the OGT, all twelve horses were identified as IR by the modified 2-step IRT performed with PZI underlining the importance, but difficulty in choosing the right diagnostic tool in clinical settings to assess ID.

Characterizing Adrenergic Regulation of Glucose Transporter 4-Mediated Glucose Uptake and Metabolism in the Heart

Whereas adrenergic stimulation promotes cardiac function that demands more fuel and energy, how this receptor controls cardiac glucose metabolism is not defined. This study shows that the cardiac β2 adrenoreceptor (β2AR) is required to increase glucose transporter 4 (GLUT4)-mediated glucose uptake in myocytes and glucose oxidation in working hearts via activating the cardiac β2AR and promotes the G inhibitory-phosphoinositide 3-kinase-protein kinase B cascade to increase phosphorylation of TBC1D4 (aka AS160), a Rab guanosine triphosphatase-activating protein, which is a key enzyme to mobilize GLUT4. Furthermore, deleting G-protein receptor kinase phosphorylation sites of β2AR blocked adrenergic stimulation of GLUT4-mediated glucose uptake in myocytes and hearts. This study defines a molecular pathway that controls cardiac GLUT4-mediated glucose uptake and metabolism under adrenergic stimulation.

운동과 산화질소의 기능

There is increasing evidence that nitric oxide (NO) is an important hemodynamic and metabolic regulator during the performance of physical activity. Futhermore, there are adaptations in this system as a result of exercise training that likely contribute to increased functional capacity and the cardioprotective effects associated with higher fitness levels. Exercise has particular efficacy in restoring dysfunction of the vascular endotherial NO system, which is becoming established as a precursor to the atherosclerotic process.&#x0D; The production of NO from L-arginine is catalysed by the nitric oxide synthase (NOS), which involves NOS Ⅰ, NOS Ⅱ, and NOS Ⅲ. NO has been implicated in such diverse processes as vasodilation, inhibition of platelet aggregation, immune function, cell growth, neurotransmission, metabolic regulation and excitation-contraction coupling. The vast animal literature, together with more recent human studies, indicates that endurance exercise training for a period ranging from days to several weeks enhances basal release of NO from aorta, active and inactive muscle and coronary arteries. This adaptation may contribute to the reduction in resting blood pressure and increase in blood flow to skeletal muscle. Also, NO system is modified by training in the setting of cardiovascular disease in humans and that these effects may contribute to increased functional capacity. Recent animal study data also suggest that this benefit may extend to skeletal muscle NO, which appears, among other functions, to mediate glucose uptake during exercise. However, the role of NO in the coronary circulation and skeletal muscle, particularly with regard to glucose uptake, is yet to be elucidated.

Fluoxetine increases astrocytic glucose uptake and glycolysis in corticosterone-induced depression through restricting GR-TXNIP-GLUT1 Pathway.

Antidepressant fluoxetine can affect cerebral glucose metabolism in clinic, but the underlying molecular mechanism remains poorly understood. Here, we examined the effect of fluoxetine on brain regional glucose metabolism in a rat model of depression induced by repeated corticosterone injection, and explored the molecular mechanism. Fluoxetine was found to recover the decrease of 18F-fluorodeoxyglucose (18F-FDG) signal in prefrontal cortex (PFC), and increased 2-[N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-D-glucose (2-NBDG, a fluorescent glucose analog) uptake in an astrocyte-specific manner in ex vivo cultured PFC slices from corticosterone-induced depressive rats, which were consistent with its improvement of animal depressive behaviors. Furthermore, fluoxetine restricted nuclear translocation of glucocorticoid receptor (GR) to suppress the transcription of thioredoxin interacting protein (TXNIP). Subsequently, it promoted glucose transporter 1 (GLUT1)-mediated glucose uptake and glycolysis of PFC astrocytes through suppressing TXNIP expression under corticosterone-induced depressive state. More importantly, fluoxetine could improve glucose metabolism of corticosterone-stimulated astrocytes via TXNIP-GLUT1 pathway. These results demonstrated that fluoxetine increased astrocytic glucose uptake and glycolysis in corticosterone-induced depression via restricting GR-TXNIP-GLUT1 pathway. The modulation of astrocytic glucose metabolism by fluoxetine was suggested as a novel mechanism of its antidepressant action.

637 Non-redundant roles for GLUT1 and GLUT3 in melanoma and primary melanocytes

The GLUT1 transporter mediates glucose uptake in most tissues and is upregulated in both inflammatory and malignant skin diseases. Most melanomas overexpress both GLUT1 and GLUT3, but it is unclear whether the closely related transporters are functionally redundant. Consistent with its function in keratinocytes, GLUT1 knockdown in melanoma cells (UACC-62 and Mel-juso) inhibited glucose uptake and cell proliferation. Notably, GLUT3 knockdown did not impair glucose transport yet showed even more dramatic effects on cell proliferation and morphology. Fractionation revealed the preferential localization of GLUT1 to the plasma membrane while GLUT3 preferentially localized to endosomes. RNA-sequencing, Western blotting, and immunofluorescence revealed an essential role for GLUT3 in signal transducer and activator of transcription 3 (STAT3) activation, which has been implicated in melanoma growth and metastasis. Deletion of GLUT1 or GLUT3 in primary mouse melanocytes through Tyrosinase-cre expression confirmed a critical role for GLUT3 in STAT3 activation and non-redundant roles for GLUT1 and GLUT3 in pigmentation and melanocyte proliferation ex vivo. These studies identify unique functions for GLUT1 and GLUT3 in melanoma and melanocytes and highlight the GLUT3-STAT3 signaling axis as a potential therapeutic target in melanoma.

223-LB: Interactive Effects of Dietary Iron and Chronic Hypoxia on Glucose Homeostasis in a T2D Mouse Model

The micronutrient iron is a risk factor for type II diabetes (T2D) . The effects of iron are, in part, mediated by the iron-sensing hypoxia inducible factor (HIF) pathway. The interactions of iron and hypoxia in the progression of T2D are not fully understood. Mice were fed a fast-food (FF) diet supplemented with either normal iron (35 mg/kg) or high iron (2000 mg/kg) under normoxia and hypoxia. Hypoxic mice had a significant reduction in weight compared to normoxic mice (by 16% for 35mg-Fe diet and 14% for 2000mg-Fe diet, p&amp;lt;0.001) . There was an interactive effect of iron and hypoxia on fasting glucose and glucose tolerance (p=0.016, p=0.41 respectively) in which the combination of hypoxia and normal iron yielded the best improvement. For insulin-mediated glucose uptake mechanisms, insulin sensitivity was observed with high iron and also under hypoxia when using the homeostatic model assessment (HOMA-IR) . Direct measurement using the insulin tolerance test showed no additional sensitivity was observed with hypoxia, suggesting non-insulin mediated glucose uptake (NIMGU) mechanisms. Indirect calorimetry demonstrates that hypoxia and iron interact to increase carbohydrate utilization and energy expenditure (p&amp;lt;0.001 and p=0.005, respectively) . Measured eWAT tissue mass showed significant reduction in tissue mass for hypoxic mice. Transcriptional profile of mouse eWAT shows significant upregulation of classic HIF target genes involved in glycolysis, mitochondrial metabolism, and intracellular iron homeostasis in hypoxic mice and interestingly also in normoxic high iron mice. Lastly, protein expression of HIF regulators FIH1 and PHD2 were significantly reduced (25-40% reduction, p&amp;lt;0.05 for FIH and p&amp;lt;0.01 for PHD2) under hypoxia. We conclude that the observed effects of dietary iron are dependent on oxygen levels, with the best protection against T2D offered by normal iron and hypoxia. Enhanced glucose disposal is due to NIMGU mechanisms, with the two HIF regulatory arms, PHD2 and FIH1, also mediating effects. Results from this study support novel targeted therapies and dietary changes to treat T2D. Disclosure A. V. Harrison: None. D. A. Mcclain: None. S. T. Sink: None. F. Lorenzo: None. Funding NIDDK Research Service of the Veterans Administration

Kutkin, iridoid glycosides enriched fraction of Picrorrhiza kurroa promotes insulin sensitivity and enhances glucose uptake by activating PI3K/Akt signaling in 3T3-L1 adipocytes

BackgroundTherapeutic failure and drug resistance are common sequelae to insulin resistance associated with type 2 diabetes mellitus (T2DM). Consequently, there is an unmet need of alternative strategies to overcome insulin resistance associated complications. PurposeTo demonstrate whether Kutkin (KT), iridoid glycoside enriched fraction of Picrorhiza kurroa extract (PKE) has potential to increase the insulin sensitivity vis à vis glucose uptake in differentiated adipocytes. MethodsMolecular interaction of KT phytoconstituents, picroside-I (P-I) & picroside- II (P-II) with peroxisome proliferator-activated receptor gamma (PPARγ), phosphatidylinositol 3-kinase (PI3K) and protein kinase B (Akt) were analyzed in silico. Cellular viability and adipogenesis were determined by following 3-(4, 5-Dimethylthiazol-2-Yl)-2, 5-Diphenyltetrazolium bromide (MTT) assay and Oil Red-O staining. Further, ELISA kit based triglycerides and diacylglycerol-O-Acyltransferase-1 (DGAT1) were assessed in differentiated adipocytes. ELISA based determination were performed to check the levels of adiponectin and tumor necrosis factor alpha (TNF-α). However, Flow cytometry and immunofluorescence based assays were employed to measure the glucose uptake and glucose transporter 4 (glut4) expression in differentiated adipocytes, respectively. Further to explore the targeted signaling axis, mRNA expression levels of PPARγ, CCAAT/enhancer binding protein α (CEBPα), and glut4 were determined using qRT-PCR and insulin receptor substrate-1 (IRS-1), Insulin receptor substrate-2 (IRS-2), PI3K/Akt, AS160, glut4 followed by protein validation using immunoblotting in differentiated adipocytes. ResultsIn silico analysis revealed the binding affinities of major constituents of KT (P-I& P-II) with PPARγ/PI3K/Akt. The enhanced intracellular accumulation of triglycerides with concomitant activation of PPARγ and C/EBPα in KT treated differentiated adipocytes indicates augmentation of adipogenesis in a concentration-dependent manner. Additionally, at cellular level, KT upregulated the expression of DAGT1, and decreases fatty acid synthase (FAS), and lipoprotein lipase (LPL), further affirmed improvement in lipid milieu. It was also observed that KT upregulated the levels of adiponectin and reduced TNFα expression, thus improving the secretory functions of adipocytes along with enhanced insulin sensitivity. Furthermore, KT significantly promoted insulin mediated glucose uptake by increasing glut4 translocation to the membrane via PI3/Akt signaling cascade. The results were further validated using PI3K specific inhibitor, wortmannin and findings revealed that KT treatment significantly enhanced the expression and activation of p-PI3K/PI3K and p-Akt/Akt even in case of treatment with PI3K inhibitor wortmannin alone and co-treatment with KT in differentiated adipocytes and affirmed that KT as activator of PI3K/Akt axis in the presence of inhibitor as well. ConclusionCollectively, KT fraction of PKE showed anti-diabetic effects by enhancing glucose uptake in differentiated adipocytes via activation of PI3K/Akt signaling cascade. Therefore, KT may be used as a promising novel natural therapeutic agent for managing T2DMand to the best of our knowledge, this is the first report, showing the efficacy and potential molecular mechanism of KT in enhancing insulin sensitivity and glucose uptake in differentiated adipocytes.

An Exercise‐Driven, Insulin‐Independent Glucose Uptake Pathway in Contracting Skeletal Muscle

Glucose uptake by skeletal muscles, which import and store up to 80% of circulating glucose, plays a major role in glucose homeostasis. Prior research has focused largely on the insulin regulated GLUT4 transporter, which mediates glucose uptake in quiescent (non‐contracting) muscles using a canonical insulin‐dependent translocation mechanism. An ongoing paradox in the field is that actively contracting muscles take up glucose at 50‐100x greater rates than quiescent muscles, despite declining insulin levels. For this reason, exercise is a potent treatment for disorders of insulin insufficiency and insulin resistance. However, despite more than 30 years of study, the mechanism by which contracting muscles take up glucose without a requirement for insulin remains incompletely understood. It is proposed that GLUT4‐translocation occurs using signaling pathways that bypass the insulin receptor. However, no GLUT4 models have accounted for all the glucose taken up by contracting muscles or the great discrepancy between measured GLUT4 translocation (2‐fold increase in membrane density) and glucose uptake rate (50‐100‐fold increase), suggesting the existence of additional mechanisms. The goals of this study were to:i) develop a technology based on Inductively Coupled Plasma‐Mass Spectroscopy (ICP‐MS) that allows simultaneous detection of multiple transported species, including 13C‐labeled hexoses; and ii) test the hypothesis that contraction‐stimulated glucose uptake may be linked to Na,K‐ATPase (NKA) transport, which is also dramatically stimulated during muscle contraction. We measured uptake of Rb (a congener for K+), and 13C‐[6C]‐D‐glucose or 13C‐[6C]‐2‐deoxy glucose in isolated mouse EDL muscles stimulated to contract ex vivo. Muscles were obtained from WT mice. Glucose uptake was measured for 5 min at 32 C in a physiological solution containing 200 mM tracer RbCl and 11mM 13C glucose. Following uptake, the Rb and 13C content of the muscle were measured by ICP‐MS. We achieved a Limit of Detection of 54uM for 13C glucose in mouse muscle. This detection power for 13C by ICP‐MS is entirely new and was achieved by combining technical approaches: i) introduction of 13C‐based hexose substrates; ii) modifications to sample preparation that reduce background carbon; iii) ratiometric monitoring of the 13C/12C signal; and iv) 13C‐optimized instrument conditions. Contracting skeletal muscle took up 13C‐[6C]‐labeled‐D‐glucose at a rate of 10.8 mM/g‐min, and Rb at a rate of 1.2 mM/g‐min. The ability to detect glucose and other carbon‐based organic compounds by ICP‐MS is expected to advance studies of coupled transport and other biological processes that require multi‐species detection.

Statins Aggravate the Risk of Insulin Resistance in Human Muscle.

Beside their beneficial effects on cardiovascular events, statins are thought to contribute to insulin resistance and type-2 diabetes. It is not known whether these effects are long-term events from statin-treatment or already triggered with the first statin-intake. Skeletal muscle is considered the main site for insulin-stimulated glucose uptake and therefore, a primary target for insulin resistance in the human body. We analyzed localization and expression of proteins related to GLUT4 mediated glucose uptake via AMPKα or AKT in human skeletal muscle tissue from patients with statin-intake >6 months and in primary human myotubes after 96 h statin treatment. The ratio for AMPKα activity significantly increased in human skeletal muscle cells treated with statins for long- and short-term. Furthermore, the insulin-stimulated counterpart, AKT, significantly decreased in activity and protein level, while GSK3ß and mTOR protein expression reduced in statin-treated primary human myotubes, only. However, GLUT4 was normally distributed whereas CAV3 was internalized from plasma membrane around the nucleus in statin-treated primary human myotubes. Statin-treatment activates AMPKα-dependent glucose uptake and remains active after long-term statin treatment. Permanent blocking of its insulin-dependent counterpart AKT activation may lead to metabolic inflexibility and insulin resistance in the long run and may be a direct consequence of statin-treatment.

Capsaicin and Zinc Promote Glucose Uptake in C2C12 Skeletal Muscle Cells through a Common Calcium Signalling Pathway.

Capsaicin and zinc have recently been highlighted as potential treatments for glucose metabolism disorders; however, the effect of these two natural compounds on signalling pathways involved in glucose metabolism is still uncertain. In this study, we assessed the capsaicin- or zinc- induced activation of signalling molecules including calcium/calmodulin-dependent protein kinase 2 (CAMKK2), cAMP-response element-binding protein (CREB), and target of rapamycin kinase complex 1 (TORC1). Moreover, the expression status of genes associated with the control of glucose metabolism was measured in treated cells. The activation of cell signalling proteins was then evaluated in capsaicin- or zinc treated cells in the presence or absence of cell-permeant calcium chelator (BAPTA-AM) and the CAMKK inhibitor (STO-609). Finally, capsaicin- and zinc-induced glucose uptake was measured in the cells pre-treated with or without BAPTA-AM. Our results indicate that calcium flux induced by capsaicin or zinc led to activation of calcium signalling molecules and promoting glucose uptake in skeletal muscle cells. Pharmacological inhibition of CAMKK diminished activation of signalling molecules. Moreover, we observed an increase in intracellular cAMP levels in the cells after treatment with capsaicin and zinc. Our data show that capsaicin and zinc mediate glucose uptake in C2C12 skeletal muscle cells through the activation of calcium signalling.

GLUT-2 mediated glucose uptake analysis of Duranta repens: In-silico and In-vitro approach.

Type-2 diabetes mellitus is a common metabolic disorder characterized by insulin resistance, a relative impairment in insulin secretion, and a certain degree of genetic predisposition. The rapid rise in the prevalence of diabetes mellitus around the world has assisted in the development of new pharmacologically active compounds. The current study was aimed to investigate and validate the anti-diabetic activity of wild-grown plant Duranta repens L. In-silico molecular docking via AutoDock tools 4.2 and in-vitro glucose uptake assay using yeast cells was performed to investigate the anti-diabetic property of plant Duranta repens. Further, mRNA-based gene ontology enrichment analysis was performed to predict the imitated ontology by the bio-actives from Duranta repens. The in-silico study results reveal that among the 9 active phytoconstituents docked against GLUT-2 protein, α-onocerin possessed the highest binding affinity of -10.23kcal/mol with no predicted adverse effects and also complies with Lipinski's rule of five. Also,in-vitro studies reflected in a 5mM glucose solution, hydro-alcoholic extract of Duranta repens at different concentrations enhanced glucose uptake in yeast cells. Duranta repens extractenhanced theglucose uptake in yeast cells which may be due to the presence of α-onocerin; possessed the better interaction. Also, no adverse effects were predicted for α-onocerin. Thus, it can be speculated that Duranta repens maypossess anti-diabetic activity which may be due to α-onocerin and other related bioactives; needs to be further confirmed vi aexperimental studies.

Drosophila Solute Carrier 5A5 Regulates Systemic Glucose Homeostasis by Mediating Glucose Absorption in the Midgut.

The small intestine is the initial site of glucose absorption and thus represents the first of a continuum of events that modulate normal systemic glucose homeostasis. A better understanding of the regulation of intestinal glucose transporters is therefore pertinent to our efforts in curbing metabolic disorders. Using molecular genetic approaches, we investigated the role of Drosophila Solute Carrier 5A5 (dSLC5A5) in regulating glucose homeostasis by mediating glucose uptake in the fly midgut. By genetically knocking down dSLC5A5 in flies, we found that systemic and circulating glucose and trehalose levels are significantly decreased, which correlates with an attenuation in glucose uptake in the enterocytes. Reciprocally, overexpression of dSLC5A5 significantly increases systemic and circulating glucose and trehalose levels and promotes glucose uptake in the enterocytes. We showed that dSLC5A5 undergoes apical endocytosis in a dynamin-dependent manner, which is essential for glucose uptake in the enterocytes. Furthermore, we showed that the dSLC5A5 level in the midgut is upregulated by glucose and that dSLC5A5 critically directs systemic glucose homeostasis on a high-sugar diet. Together, our studies have uncovered the first Drosophila glucose transporter in the midgut and revealed new mechanisms that regulate glucose transporter levels and activity in the enterocyte apical membrane.

The “Levine effect” and the father of modern diabetes research

The “Levine effect” and the father of modern diabetes research

Inositol supplementation and body mass index: A systematic review and meta-analysis of randomized clinical trials.

BackgroundInositol is a sugar‐alcohol and recognized as a key component of cell membrane phospholipids. It has crucial role in the cell signaling pathways and contribute to improving glycemic responses. Although some earlier studies have revealed the effect of inositol mediating glucose uptake by improving insulin sensitivity, the benefit of inositol supplementation in patients with overweight and obesity is not completely understood. This study aimed to assess the impact of inositol supplementation on body mass index (BMI) through a systematic review and meta‐analysis of controlled clinical trials.MethodsA systematic search was performed to August 2021 in the following databases: PubMed‐Medline, Embase, Web of Science and Scopus. Fifteen controlled clinical trials investigating the effect of inositol on adult's BMI were finally included in the study. A random‐effects model was employed to estimate the effect size. Subgroup analysis was performed by dose, duration, age, type of inositol. Meta‐regression was used to investigate presence of any linear relationship. Begg's and Egger's tests were carried out to detect small study effect.ResultsThe results of pooled analysis showed that inositol supplementation significantly decreased BMI scores (WMD = −0.41 kg/m2; 95% CI: −0.78, −0.04; p = 0.028). Subgroup analysis was performed to identify the source of heterogeneity among studies (I 2 = 73.9%, p < 0.001), demonstrating supplementation duration, baseline BMI, mean age of participants, type of inositol and dosage were potential sources of heterogeneity. The effect of intervention was more clinically significant in participants with polycystic ovary syndrome (PCOS) and overweight/obesity. Inositol in the form of myo‐inositol (MI) had stronger effect on BMI reduction.ConclusionThe meta‐analysis suggests that oral inositol supplementation has positive effect on BMI reduction. Inositol supplementation could be considered as an adjunct treatment to improve body mass index.