Generation of antibodies to an extracellular region of the transporters Glut1/Glut4 by immunization with a designed antigen.

Monoclonal antibodies are one of the fastest growing class of drugs. Nevertheless, relatively few biologics target multispanning membrane proteins because of technical challenges. To target relatively small extracellular regions of multiple membrane-spanning proteins, synthetic peptides, which are composed of amino acids corresponding to an extracellular region of a membrane protein, are often utilized in antibody discovery. However, antibodies to these peptides often do not recognize parental membrane proteins. In this study, we designed fusion proteins in which an extracellular helix of the membrane protein glucose transporter 1 (Glut1) was grafted onto the scaffold protein Adhiron. In the initial design, the grafted fragment did not form a helical conformation. Molecular dynamics simulations of full-length Glut1 suggested the importance of intramolecular interactions formed by surrounding residues in the formation of the helical conformation. A fusion protein designed to maintain such intramolecular interactions did form the desired helical conformation in the grafted region. We then immunized an alpaca with the designed fusion protein and obtained VHH (variable region of heavy-chain antibodies) using the phage display method. The binding of these VHH antibodies to the recombinant Glut1 protein was evaluated by surface plasmon resonance, and their binding to Glut1 on the cell membrane was further validated by flow cytometry. Furthermore, we also succeeded in the generation of a VHH against another integral membrane protein, glucose transporter 4 (Glut4) with the same strategy. These illustrates that our combined biochemical and computational approach can be applied to designing other novel fusion proteins for generating site-specific antibodies.

Two Prenylated Chalcones, 4-Hydroxyderricin, and Xanthoangelol Prevent Postprandial Hyperglycemia by Promoting GLUT4 Translocation via the LKB1/AMPK Signaling Pathway in Skeletal Muscle Cells.

SCOPE: Stimulation of glucose uptake in the skeletal muscle is crucial for the prevention of postprandial hyperglycemia. Insulin and certain polyphenols enhance glucose uptake through the translocation of glucose transporter 4 (GLUT4) in the skeletal muscle. The previous study reports that prenylated chalcones, 4-hydroxyderricin (4-HD), and xanthoangelol (XAG) promote glucose uptake and GLUT4 translocation in L6 myotubes, but their underlying molecular mechanism remains unclear. This study investigates the mechanism in L6 myotubes and confirms antihyperglycemia by 4-HD and XAG. METHODS AND RESULTS: In L6 myotubes, 4-HD and XAG promote glucose uptake and GLUT4 translocation through the activation of adenosine monophosphate-activated protein kinase (AMPK) and liver kinase B1 (LKB1) signaling pathway without activating phosphoinositide 3-kinase (PI3K)/protein kinase B (Akt) and Janus kinases (JAKs)/signal transducers and activators of transcriptions (STATs) pathways. Moreover, Compound C, an AMPK-specific inhibitor, as well as siRNA targeting AMPK and LKB1 completely canceled 4-HD and XAG-increased glucose uptake. Consistently, oral administration of 4-HD and XAG to male ICR mice suppresses acute hyperglycemia in an oral glucose tolerance test. CONCLUSION: In conclusion, LKB1/AMPK pathway and subsequent GLUT4 translocation in skeletal muscle cells are involved in Ashitaba chalcone-suppressed acute hyperglycemia.

Akt may associate with insulin-responsive vesicles via interaction with sortilin.

Insulin-responsive vesicles (IRVs) deliver the glucose transporter Glut4 to the plasma membrane in response to activation of the insulin signaling cascade: insulin receptor-IRS-PI3 kinase-Akt-TBC1D4-Rab10. Previous studies have shown that Akt, TBC1D4, and Rab10 are compartmentalized on the IRVs. Although functionally significant, the mechanism of Akt association with the IRVs remains unknown. Using pull-down assays, immunofluorescence microscopy, and cross-linking, we have found that Akt may be recruited to the IRVs via the interaction with the juxtamembrane domain of the cytoplasmic C terminus of sortilin, a major IRV protein. Overexpression of full-length sortilin increases insulin-stimulated phosphorylation of TBC1D4 and glucose uptake in adipocytes, while overexpression of the cytoplasmic tail of sortilin has the opposite effect. Our findings demonstrate that the IRVs represent both a scaffold and a target of insulin signaling.

AS160 expression, but not AS160 Serine-588, Threonine-642, and Serine-704 phosphorylation, is essential for elevated insulin-stimulated glucose uptake by skeletal muscle from female rats after acute exercise.

One exercise session can increase subsequent insulin-stimulated glucose uptake (ISGU) by skeletal muscle in both sexes. We recently found that muscle expression and phosphorylation of key sites of Akt substrate of 160 kDa (AS160; also called TBC1D4) are essential for the full-exercise effect on postexercise-ISGU (PEX-ISGU) in male rats. In striking contrast, AS160's role in increased PEX-ISGU has not been rigorously tested in females. Our rationale was to address this major knowledge gap. Wild-type (WT) and AS160-knockout (KO) rats were either sedentary or acutely exercised. Adeno-associated virus (AAV) vectors were engineered to express either WT-AS160 or AS160 mutated on key serine and threonine residues (Ser588, Thr642, and Ser704) to alanine to prevent their phosphorylation. AAV vectors were delivered to the muscle of AS160-KO rats to determine if WT-AS160 or phosphorylation-inactivated AS160 would influence PEX-ISGU. AS160-KO rats have lower skeletal muscle abundance of the GLUT4 glucose transporter protein. This GLUT4 deficit was rescued using AAV delivery of GLUT4 to determine if eliminating muscle GLUT4 deficiency would normalize PEX-ISGU. The novel results were as follows: (1) AS160 expression was required for greater PEX-ISGU; (2) rescuing muscle AS160 expression in AS160-KO rats restored elevated PEX-ISGU; (3) AS160's essential role for the postexercise increase in ISGU was not attributable to reduced muscle GLUT4 content; and (4) AS160 phosphorylation on Ser588, Thr642, and Ser704 was not essential for greater PEX-ISGU. In conclusion, these novel findings revealed that three phosphosites widely proposed to influence PEX-ISGU are not required for this important outcome in female rats.

Orange Peel Extract and Physical Exercise Synergistically Ameliorate Type 2 Diabetes Mellitus-Induced Dysmetabolism by Upregulating GLUT4 Concentration in Male Wistar Rats.

Diabetes mellitus (DM) is a chronic disease and one of the oldest known disorders. It is characterized by dysglycemia, dyslipidemia, insulin resistance (IR), and pancreatic cell dysfunction. Although different drugs, metformin (MET), glipizide, glimepiride, etc., have been introduced to treat type 2 DM (T2DM), these drugs are not without side effects. Scientists are now seeking natural treatments such as lifestyle modification and organic products known with limited side effects. Thirty-six male Wistar rats were randomized into six groups (n = 6 per group): control, DM untreated rats, DM+orange peel extract (OPE), DM+exercise (EX), DM+OPE +EX, and DM+MET. The administration was once daily through the oral route and lasted for 28 days. EX and OPE synergistically ameliorated the diabetic-induced increase in fasting blood sugar, homeostatic model assessment for insulin resistance (HOMA IR), total cholesterol (TC) and triglyceride (TG), TC/high-density lipoprotein (HDL), TG/HDL, triglyceride glucose (TyG) index, and hepatic lactate dehydrogenase, alanine transaminase, malondialdehyde, c-reactive protein, and tumour necrosis factor α when compared with the diabetic untreated group. Also, EX+OPE blunted DM-induced decrease in serum insulin, homeostasis model assessment of ß-cell function (HOMA-B), homeostasis model assessment of insulin sensitivity (HOMA S), quantitative insulin-sensitivity check index (QUICK 1), HDL, total antioxidant capacity, superoxide dismutase, and hepatic glycogen. Furthermore, EX+OPE ameliorated the observed DM-induced decrease in glucose transporter type 4 (GLUT 4), expression. This study showed that OPE and EX synergistically ameliorate T2DM-induced dysglycaemia, dyslipidaemia, and down-regulation of GLUT4 expression.

Mung bean peptides promote glucose uptake via Jak2 activation in L6 myotubes.

Mung beans are among the important edible legumes cultivated in Asia, Southern Europe, and Northern America. Mung beans contain 20-30% proteins with high digestibility and possess biological activities, but detailed health beneficial functions are not fully understood yet. In this study, we report the isolation and identification of active peptides from mung beans which promote glucose uptake and elucidate their mechanism in L6 myotubes. HTL, FLSSTEAQQSY, and TLVNPDGRDSY were isolated and identified as active peptides. These peptides promoted the translocation of glucose transporter 4 (GLUT4) to the plasma membrane. The tripeptide HTL promoted glucose uptake through the activation of adenosine monophosphate-activated protein kinase, while the oligopeptides FLSSTEAQQSY and TLVNPDGRDSY through the activation of the PI3K/Akt pathway. Furthermore, these peptides promoted the phosphorylation of Jak2 via interaction with the leptin receptor. Thus, mung bean is a promising functional food for the prevention of hyperglycemia and type 2 diabetes through promoting glucose uptake accompanied by JAK2 activation in the muscle cells.

Anemoside B4 Exerts Hypoglycemic Effect by Regulating the Expression of GLUT4 in HFD/STZ Rats.

Anemoside B4 (B4) is a saponin that is extracted from Pulsatilla chinensis (Bge.), and Regel exhibited anti-inflammatory, antioxidant, antiviral, and immunomodulatory activities. However, its hypoglycemic activity in diabetes mellitus has not been evaluated. Here, we explored the effect of B4 on hyperglycemia and studied its underlying mechanism of lowering blood glucose based on hyperglycemic rats in vivo and L6 skeletal muscle cells (L6) in vitro. The rats were fed a high-fat diet (HFD) for one month, combined with an intraperitoneal injection of 60 mg/kg streptozotocin (STZ) to construct the animal model, and the drug was administrated for two weeks. Blood glucose was detected and the proteins and mRNA were expressed. Our study showed that B4 significantly diminished fasting blood glucose (FBG) and improved glucose metabolism. In addition, B4 facilitated glucose utilization in L6 cells. B4 could enhance the expression of glucose transporter 4 (GLUT4) in rat skeletal muscle and L6 cells. Mechanistically, B4 elevated the inhibition of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways. Furthermore, we confirmed the effect of B4 on glucose uptake involved in the enhancement of GLUT4 expression in part due to PI3K/AKT signaling by using a small molecule inhibitor assay and constructing a GLUT4 promoter plasmid. Taken together, our study found that B4 ameliorates hyperglycemia through the PI3K/AKT pathway and promotes GLUT4 initiation, showing a new perspective of B4 as a potential agent against diabetes.

Akt substrate of 160 kDa is essential for the calorie restriction-induced increase in insulin-stimulated glucose uptake by skeletal muscle of female rats.

We evaluated effects of calorie restriction (CR; consuming 65% of ad libitum (AL) intake) for 8 weeks on female wildtype (WT) and Akt substrate of 160 kDa knockout (AS160-KO) rats. Insulin-stimulated glucose uptake (ISGU) was determined in isolated epitrochlearis muscles incubated with 0, 50, 100, or 500 µU/mL insulin. Phosphorylation of key insulin signaling proteins that control ISGU (Akt and AS160) was assessed by immunoblotting (Akt phosphorylation on Threonine-308, pAktThr308 and Serine-473, pAktSer473; AS160 phosphorylation on Serine-588, pAS160Ser588, and Threonine-642, pAS160Thr642). Abundance of proteins that regulate ISGU (GLUT4 glucose transporter protein and hexokinase II) was also determined by immunoblotting. The major results were as follows: (i) WT-CR versus WT-AL rats had greater ISGU with 100 and 500 µU/mL insulin; (ii) CR versus WT-AL rats had greater GLUT4 protein abundance; (iii) WT-CR versus WT-AL rats had greater pAktThr308 with 500 µU/mL insulin; (iv) WT-CR versus WT-AL rats did not differ for pAktSer473, pAS160Ser588, or pAS160Thr642 at any insulin concentration; (v) AS160-KO versus WT rats with each diet had lower ISGU at each insulin concentration, but not lower pAkt on either phosphosite; (vi) AS160-KO versus WT rats had lower muscle GLUT4 abundance regardless of diet; and (vii) AS160-KO-CR versus AS160-KO-AL rats did not differ for ISGU, GLUT4 abundance, pAkt on either phosphosite, or pAS160 on either phosphosite. These novel results demonstrated that AS160 expression, but not greater pAS160 on key phosphosites, was essential for the CR-induced increases in muscle ISGU and GLUT4 abundance of female rats.

Potentilla fulgens upregulate GLUT4, AMPK, AKT and insulin in alloxan-induced diabetic mice: an in vivo and in silico study.

OBJECTIVE: This study was designed to investigate whether the glucose lowering effects of Potentilla fulgens acts by modulating GLUT4, AKT2 and AMPK expression in the skeletal muscle and liver tissues. METHODOLOGY: Alloxan-induced diabetic mice treated with Potentilla fulgens was assessed for their blood glucose and insulin level, mRNA and protein expression using distinguished methods. Additionally, GLUT4, AKT2 and AMPK were docked with catechin, epicatechin, kaempferol, metformin, quercetin and ursolic acid reportedly present in Potentilla fulgens. RESULTS: Potentilla fulgens ameliorates hyperglycaemia and insulin sensitivity via activation of AKT2 and AMPK, increases the expression of GLUT4, AKT2, AMPKα1 and AMPKα2 whose levels are reduced under diabetic condition. Molecular docking revealed interacting residues and their binding affinities (-4.56 to -8.95 Kcal/mol). CONCLUSIONS: These findings provide more clarity vis-avis the mechanism of action of the phytoceuticals present in Potentilla fulgens extract which function through their action on GLUT4, PKB and AMPK.

Quercetin ameliorated insulin resistance via regulating METTL3-mediated N6-methyladenosine modification of PRKD2 mRNA in skeletal muscle and C2C12 myocyte cell line.

BACKGROUND AND AIMS: N6-Methyladenosine (m6A) modification is involved in many pathological processes, including insulin resistance (IR). Quercetin (Que), a bioactive compound with strong antioxidant activity, has potential therapeutic effects on IR-related metabolic diseases. The aim of this study is to investigate the roles of m6A and Que in hyperinsulinemia. METHODS AND RESULTS: Male C57Bl/6 mice received a high-fat diet (HFD) for 8 weeks to establish an IR model. Que treatment reduced the body weight, blood glucose, plasma triglycerides (TG) and serum insulin, ameliorated IR, and decreased oxidative stress in HFD-fed mice. Cellular IR model was established in C2C12 cells by palmitic acid (PA) stimulation, and a noncytotoxic dose of Que was found to promote glucose uptake and inhibit oxidative stress. Moreover, methyltransferase-like 3 (METTL3) and serine-threonine kinase protein kinase D2 (PRKD2) was downregulated in skeletal muscle of HFD-fed mouse and in PA-induced C2C12 cells. The online bioinformatic tool SRAMP revealed that there were multiple m6A modification sites in the PRKD2 mRNA sequence. Downregulation of METTL3 enhanced PRKD2 expression by reducing m6A level and promoting mRNA stability in PRKD2 mRNA transcript. Que decreased m6A, METTL3, and phosphorylated insulin receptor substrate 1 (p-IRS1) levels, increased the protein expression of PRKD2, glucose transporter type 4 (GLUT4) and p-AKT, promoted glucose uptake, and reduced oxidative stress in PA-induced C2C12 cells. Moreover, METTL3 overexpression or PRKD2 silence reversed the inhibitory effects of Que on the levels of MDA and p-IRS1 and the promotive effects on glucose uptake, superoxide dismutase (SOD), GSH and GLUT4 and p-AKT levels. CONCLUSION: Que promoted glucose uptake, repressed oxidative stress and improved IR through METTL3-mediated m6A of PRKD2 mRNA.

Autocrine FGF1 signaling promotes glucose uptake in adipocytes.

Fibroblast growth factor 1 (FGF1) is an autocrine growth factor released from adipose tissue during over-nutrition or fasting to feeding transition. While local actions underlie the majority of FGF1's anti-diabetic functions, the molecular mechanisms downstream of adipose FGF receptor signaling are unclear. We investigated the effects of FGF1 on glucose uptake and its underlying mechanism in murine 3T3-L1 adipocytes and in ex vivo adipose explants from mice. FGF1 increased glucose uptake in 3T3-L1 adipocytes and epididymal WAT (eWAT) and inguinal WAT (iWAT). Conversely, glucose uptake was reduced in eWAT and iWAT of FGF1 knockout mice. We show that FGF1 acutely increased adipocyte glucose uptake via activation of the insulin-sensitive glucose transporter GLUT4, involving dynamic crosstalk between the MEK1/2 and Akt signaling proteins. Prolonged exposure to FGF1 stimulated adipocyte glucose uptake by MEK1/2-dependent transcription of the basal glucose transporter GLUT1. We have thus identified an alternative pathway to stimulate glucose uptake in adipocytes, independent from insulin, which could open new avenues for treating patients with type 2 diabetes.

A popular fermented soybean food of Northeast India exerted promising antihyperglycemic potential via stimulating PI3K/AKT/AMPK/GLUT4 signaling pathways and regulating muscle glucose metabolism in type 2 diabetes.

This study examined the antidiabetic efficacy of popular fermented soybean foods (FSF) of Northeast (NE) India. Results showed that among different FSF, aqueous extract of Hawaijar (AEH), a traditional FSF of Manipur, NE India, significantly augmented glucose utilization in cultured myotubes treated with high glucose (HG, 25 mM). Furthermore, AEH also upregulated glucose uptake, glucose-6-phosphate level, and phopho-PI3K/phospho-AKT/phospho-AMPK/GLUT4 protein expression in HG-treated myotubes. In vivo studies demonstrated that AEH supplementation (50, 100, or 200 mg/kg body weight/day, oral gavaging, 16 weeks) reduced body weight, fasting blood glucose, glycated hemoglobin, insulin resistance, and glucose intolerance in rats fed with high-fat diet (HFD). AEH supplementation stimulated phopho-PI3K/phospho-AKT/phospho-AMPK/GLUT4 signaling cascades involved in glucose metabolism of muscle tissues in diabetic rats. Chemical profiling of AEH (SDS-PAGE, immunoblotting, and HRMS) suggests the possible role of bioactive proteins/peptides and isoflavones underlying the antihyperglycemic potential AEH. Results from this study will be helpful for developing food-based prophylactics/therapeutics in managing hyperglycemia. PRACTICAL APPLICATIONS: Fermented soybean foods are gaining acceptance due to multiple health benefits. This study for the first time reports the antidiabetic potential of Hawaijar, an indigenous fermented soybean food of North-East India. Higher abundance of bioactive compounds (isoflavones and proteins/peptides) in Hawaijar may be responsible for the alleviation of impaired glucose metabolism associated with diabetes. The findings may be helpful for the development of a novel therapeutic to achieve better control of hyperglycemia and improve the lives of the patient population with diabetes.

Role of metformin in functional endometrial hyperplasia and polycystic ovary syndrome involves the regulation of MEG3/miR­223/GLUT4 and SNHG20/miR­4486/GLUT4 signaling.

Metformin (MET) can effectively treat endometrial hyperplasia (EH), and the expression of glucose transporter type 4 insulin­responsive (GLUT4) is closely associated with the development of EH. The present study aimed to verify the effect of MET in functional EH and polycystic ovary syndrome (PCOS). H&E staining was performed to analyze the severity of EH, and immunohistochemistry was performed to evaluate the expression of GLUT4 in the endometrium of PCOS rats. Reverse transcription­quantitative PCR was used to calculate the expression of long non­coding (lnc)RNA­maternally expressed gene 3 (MEG3), lncRNA­small nucleolar RNA host gene 20 (SNHG20), GLUT4 mRNA, microRNA (miR)­223 and miR­4486. Sequence analysis and luciferase assays were performed to explore the regulatory relationship among certain lncRNAs, miRNAs and target genes. EH in PCOS rats was efficiently inhibited by MET administration. The increased expression of GLUT4 in PCOS rats was attenuated by MET treatment. Moreover, the expression levels of lncRNA­MEG3 and lncRNA­SNHG20 were significantly inhibited in the endometrium of PCOS rats. MET treatment also showed remarkable efficiency in restoring the expression of lncRNA­MEG3 and lncRNA­SNHG20. Meanwhile, the expression levels of miR­223 and miR­4486 were notably elevated in the endometrium of PCOS rats, while MET treatment reduced the expression of miR­223 and miR­4486 in PCOS rats. Furthermore, a luciferase assay confirmed the inhibitory relationship between miR­223 and lncRNA­MEG3/GLUT4 expression, as well as between miR­4486 and lncRNA­SNHG20/GLUT4 expression. GLUT4 knockdown restored the decreased viability of HCC­94 cells induced by overexpression of lncRNA­MEG3. To conclude, MET exhibited a therapeutic effect in the treatment of EH by modulating the lncRNA­MEG3/miR­223/GLUT4 and lncRNA­SNHG20/miR­4486/GLUT4 signaling pathways. This work provides mechanistic insight into the development of EH.

Trafficking regulator of GLUT4-1 (TRARG1) is a GSK3 substrate.

Trafficking regulator of GLUT4-1, TRARG1, positively regulates insulin-stimulated GLUT4 trafficking and insulin sensitivity. However, the mechanism(s) by which this occurs remain(s) unclear. Using biochemical and mass spectrometry analyses we found that TRARG1 is dephosphorylated in response to insulin in a PI3K/Akt-dependent manner and is a novel substrate for GSK3. Priming phosphorylation of murine TRARG1 at serine 84 allows for GSK3-directed phosphorylation at serines 72, 76 and 80. A similar pattern of phosphorylation was observed in human TRARG1, suggesting that our findings are translatable to human TRARG1. Pharmacological inhibition of GSK3 increased cell surface GLUT4 in cells stimulated with a submaximal insulin dose, and this was impaired following Trarg1 knockdown, suggesting that TRARG1 acts as a GSK3-mediated regulator in GLUT4 trafficking. These data place TRARG1 within the insulin signaling network and provide insights into how GSK3 regulates GLUT4 trafficking in adipocytes.

Structure and activity study of tripeptide IRW in TNF-α induced insulin resistant skeletal muscle cells.

Egg white protein ovotransferrin derived peptide IRW (Ile-Arg-Trp) was found to improve tumor necrosis factor alpha (TNF-α) or angiotensin II induced insulin resistance in L6 cells. Our recent study further showed that this peptide can improve glucose tolerance in high fat diet fed C57BL/6 mice. However, the structural requirements of IRW, especially the significance of each amino acid residue of IRW, is unknown. The study was aimed to investigate the structure and activity relationships of IRW in TNF-α induced insulin resistance L6 cells. The peptides were designed to determine the significance of individual amino acids in IRW using alanine scanning (replacing one amino acid at one time), the order of the peptide sequence and the constituting elements of IRW. Among the tested peptides and amino acids, only IRA and IR showed the same effects as that of IRW: enhanced glucose uptake, improvement in the impaired insulin signaling pathway and increased glucose transporter protein 4 (GLUT4) translocation in TNF-α treated L6 myotubes. This study demonstrated that C-terminal W is not essential to the activity of IRW. Further study is necessary to establish if IR and IRA show similar effects to that of IRW in vivo.

Lowering of Blood Glucose Levels with the Peptide Mixture Deglusterol in In Vitro and In Vivo Models.

This study aimed to investigate the blood glucose-lowering effect of the peptide complex Deglusterol, which was isolated from corn extract, in insulin-resistance models. It was found to inhibit insulin receptor substrate (IRS) Ser302 phosphorylation, known as the insulin resistance mechanism, through the inhibition of tumor necrosis factor-α (TNF-α) signaling and the induction of AMP-activated protein kinase phosphorylation. Furthermore, the phosphorylation of IRS Tyr632, phosphoinositide 3-kinase (PI3K), and AKT that is involved in the insulin action mechanism was decreased by TNF-α, whereas Deglusterol increased their phosphorylations, leading to an increase of glucose uptake rate by 190% through glucose transporter type 4 (GLUT4) compared with TNF-α-treated group in C2C12 cells. In addition to insulin signaling activation, Deglusterol treatment resulted in significantly greater mRNA expressions of IRS (190%) and GLUT4 (140%) as well as that of leptin (260%) and adiponectin (140%), which are indicators of insulin sensitivity. In animal models with type 2 diabetes, the blood glucose concentrations in the Deglusterol-administered group were significantly reduced by 50% compared with the control group. Deglusterol suppressed insulin resistance and restored insulin sensitivity, which contributed to lowering blood glucose concentrations in the insulin-resistant models, suggesting its potential as a blood glucose-lowering agent for people at high risk of type 2 diabetes or prediabetes.

Diane-35 and Metformin Induce Autophagy and Apoptosis in Polycystic Ovary Syndrome Women with Early-Stage Endometrial Carcinoma.

OBJECTIVE: Women with polycystic ovary syndrome (PCOS) are at increased risk ofendometrial carcinoma (EC). Previous studies indicated that the combined therapy of Diane-35 and metformin significantly suppresses disease progression in PCOS patients with early EC; however, the mechanisms remain unclear. METHODS: An established murine model of PCOS with early EC, clinical specimens, and human EC cells was used in this study. The levels of protein and mRNA were measured with Western blotting and RT-PCR, respectively. Cell proliferation was determined with MTT, colony formation, and flow cytometry. Proteins were analyzed with immunofluorescence and immunohistochemistry. RESULTS: Diane-35 and metformin significantly inhibited proliferative activity and promoted apoptosis in EC cells. Additionally, cell autophagy was induced by the combined therapy. Quantitive PCR revealed that Diane-35 and metformin decreased androgen receptor (AR) expression but elevated GLUT4 expression. AR was found to repress GLUT4 expression by binding to the promoter of GLUT4. Moreover, the combined treatment mediated the onset of cellular autophagy by regulating the mTORC pathway via the suppression of IGF-1 and inhibited the development of EC by the activation of the PI3K/mTORC pathway. CONCLUSION: The results and previous clinical evidence support the use of Diane-35 and metformin combination therapy for patients with PCOS and early EC.

GLUT3 inhibitor discovery through in silico ligand screening and in vivo validation in eukaryotic expression systems.

The passive transport of glucose and related hexoses in human cells is facilitated by members of the glucose transporter family (GLUT, SLC2 gene family). GLUT3 is a high-affinity glucose transporter primarily responsible for glucose entry in neurons. Changes in its expression have been implicated in neurodegenerative diseases and cancer. GLUT3 inhibitors can provide new ways to probe the pathophysiological role of GLUT3 and tackle GLUT3-dependent cancers. Through in silico screening of an ~ 8 million compounds library against the inward- and outward-facing models of GLUT3, we selected ~ 200 ligand candidates. These were tested for in vivo inhibition of GLUT3 expressed in hexose transporter-deficient yeast cells, resulting in six new GLUT3 inhibitors. Examining their specificity for GLUT1-5 revealed that the most potent GLUT3 inhibitor (G3iA, IC50 ~ 7 µM) was most selective for GLUT3, inhibiting less strongly only GLUT2 (IC50 ~ 29 µM). None of the GLUT3 inhibitors affected GLUT5, three inhibited GLUT1 with equal or twofold lower potency, and four showed comparable or two- to fivefold better inhibition of GLUT4. G3iD was a pan-Class 1 GLUT inhibitor with the highest preference for GLUT4 (IC50 ~ 3.9 µM). Given the prevalence of GLUT1 and GLUT3 overexpression in many cancers and multiple myeloma's reliance on GLUT4, these GLUT3 inhibitors may discriminately hinder glucose entry into various cancer cells, promising novel therapeutic avenues in oncology.

Spheroidization of ultrasonic degraded corn silk polysaccharide to enhance bioactivity by the anti-solvent precipitation method.

BACKGROUND: Corn silk is a very important by-product of corn production with medicinal value. Corn silk polysaccharide (CSP) is the main active ingredient. In the present study, ultrasound and spheroidization by anti-solvent were applied to improve the biological activity of CSP. RESULTS: The results showed that ultrasonic degradation improved the α-glucosidase inhibitory activity of CSP by changing its physicochemical characteristics. As the anti-solvent ratio increased, the particle size of the nanoparticles (NPs) from the spheroidization of ultrasonic-degraded corn silk polysaccharide (UCSP) gradually increased, and NP-1 exhibited the highest inhibitory effect of α-glucosidase. Isothermal titration calorimetry (ITC) results indicated that the enhanced activity might be due to more α-glucosidase binding sites with NP-1 compared with no spheroidization. Western blotting results showed that NP-1 could improve the 2-[N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl) amino]-2-deoxy-d-glucose (2-NBDG) uptake in the L6 cells by regulating the phosphatidylinositol 3-kinase (PI3K)/Akt signal pathway and the translocation of glucose transporter 4 (GLUT4). NP-1 also exhibited excellent stability in different environments. CONCLUSION: The study revealed that ultrasonic treatment and spheroidization processing showed potential applications for improving the biological activity of polysaccharides. © 2021 Society of Chemical Industry.

Antidiabetic with antilipidemic and antioxidant effects of flindersine by enhanced glucose uptake through GLUT4 translocation and PPARγ agonism in type 2 diabetic rats.

ETHNOPHARMACOLOGICAL RELEVANCE: Medicinal plants have been used by the people of developing countries to treat various diseases. WHO also recommends the use of medicines from plants source. In that, diabetes also one of the diseases that have been treated traditionally by several people all over the world. In India, Toddalia asiatica (L.) Lam. (Rutaceae) is also a medicinal plant used traditionally for the treatment of diabetes in Ayurveda. Moreover, T. asiatica is also used in a polyherbal formulation to treat diabetes. AIM OF THE STUDY: This study examined the antidiabetic with antilipidemic and antioxidant effects of flindersine isolated from T. asiatica leaves. MATERIALS AND METHODS: Diabetes was induced in Wistar rats by feeding a high-fat diet (HFD) for 15 days and injecting a single dose of 40 mg/kg b. wt. of Streptozotocin (STZ). Five days post-injection, the grouped diabetic rats were treated with 20 and 40 mg/kg of flindersine. RESULTS: Flindersine resulted in a clear decline of blood glucose levels during 28 days of treatment in two different doses. Flindersine also significantly (P ≤ 0.05; P ≤ 0.005) reduced the body weight gain, plasma insulin concentration, urea, creatinine, total cholesterol (TC), triglycerides (TG) and free fatty acids (FFA) levels and significantly increased (P ≤ 0.05; P ≤ 0.005) the total protein level, superoxide dismutase (SOD), catalase (CAT) and glutathione peroxidase (GPx) activities compared to the standard drug, pioglitazone. Additionally, flindersine restored the glucose transporter protein 4 (GLUT4), adenosine monophosphate protein kinase (AMPK) and peroxisome proliferator-activated receptor γ (PPARγ) expressions in adipose tissues and skeletal muscles. CONCLUSION: It has been found that flindersine has potent antilipidemic and antidiabetic activities by improving insulin sensitivity by enhancing the phosphorylation of AMPK, GLUT4 translocation, and PPARγ agonism on adipose tissue and skeletal muscles of diabetic rats.