Glucose Transporter Protein Research Articles

Adipocyte traits limiting cellular insulin responsiveness and glucose transport.

Adipocyte dysfunction is a hallmark of systemic insulin resistance. Insulin-responsive glucose transporter 4 (GLUT4) is downregulated in the insulin resistant state, and cellular insulin responsiveness varies depending on fat depot origin and degree of adipose expansion. Here, we have resolved factors limiting cellular insulin responsiveness, by examining adipocyte function and traits related to glucose transport at the cellular level. Subcutaneous (inguinal) and visceral (epididymal) adipocytes were isolated from C57BL/6J mice fed either chow or high-fat diet. Cell size was determined using coulter counter method, glucose uptake and cytosolic volume were assessed using glucose tracer assays. Total and GLUT4 protein content expressions were determined by Western blot. We found that basal glucose uptake per cell was preserved independent of diet or fat depot origin. Insulin-stimulated glucose uptake per cell was sustained in visceral adipocytes but decreased with adipose expansion in subcutaneous adipocytes. In parallel, the cytosolic space and total protein increased proportionally to total cellular volumetric expansion in visceral, but not in subcutaneous, adipocytes, whereas GLUT4 content decreased exclusively in expanding subcutaneous adipocytes. Together, these data support the existence of distinct phenotypic adipocyte traits that could limit cellular insulin responsiveness. Potentially, these characteristics account for fat depot-specific differences related to glucose transport capacity.NEW & NOTEWORTHY This work illustrates that adipocyte characteristics related to fat depot origin rather than adipocyte size per se limit cellular insulin responsiveness and glucose uptake in male C57BL/6J mice. These findings contribute to the overall understanding of factors limiting adipocyte function and how adipose progression affects insulin response and glucose transport capacity differently in diverse fat depots. Future studies examining whether the proposed characteristics hold true in adipocytes derived from female mice or human origin are needed.

Association between higher duodenal levels of the fructose carrier glucose transporter-5 and nonalcoholic fatty liver disease and liver fibrosis.

An increased dietary fructose intake has been shown to exert several detrimental metabolic effects and contribute to the pathogenesis of nonalcoholic fatty liver disease (NAFLD). An augmented intestinal abundance of the fructose carriers glucose transporter-5 (GLUT-5) and glucose transporter-2 (GLUT-2) has been found in subjects with obesity and type 2 diabetes. Herein, we investigated whether elevated intestinal levels of GLUT-5 and GLUT-2, resulting in a higher dietary fructose uptake, are associated with NAFLD and its severity. GLUT-5 and GLUT-2 protein levels were assessed on duodenal mucosa biopsies of 31 subjects divided into 2 groups based on ultrasound-defined NAFLD presence who underwent an upper gastrointestinal endoscopy. Individuals with NAFLD exhibited increased duodenal GLUT-5 protein levels in comparison to those without NAFLD, independently of demographic and anthropometric confounders. Conversely, no difference in duodenal GLUT-2 abundance was observed amongst the two groups. Univariate correlation analyses showed that GLUT-5 protein levels were positively related with body mass index, waist circumference, fasting and 2h post-load insulin concentrations, and insulin resistance (IR) degree estimated by homeostatic model assessment of IR (r=0.44; p=0.02) and liver IR (r=0.46; p=0.03) indexes. Furthermore, a positive relationship was observed between duodenal GLUT-5 abundance and serum uric acid concentrations (r=0.40; p=0.05), a product of fructose metabolism implicated in NAFLD progression. Importantly, duodenal levels of GLUT-5 were positively associated with liver fibrosis risk estimated by NAFLD fibrosis score. Increased duodenal GLUT-5 levels are associated with NAFLD and liver fibrosis. Inhibition of intestinal GLUT-5-mediated fructose uptake may represent a strategy for prevention and treatment of NAFLD.

Ginsenoside F4 Alleviates Skeletal Muscle Insulin Resistance by Regulating PTP1B in Type II Diabetes Mellitus.

Type 2 diabetes mellitus (T2DM) is a chronic metabolic disease with increasing morbidity. Protein tyrosine phosphatase 1B (PTP1B) is a major negative regulator of the insulin signaling cascade and has attracted intensive investigation in the T2DM study. Ginseng is widely used to treat metabolic diseases, while the effects of ginsenoside F4 (F4) on T2DM have remained unknown. Here, we identify F4 as an inhibitor of skeletal muscle insulin resistance. The results showed that F4 significantly improved the hyperglycemic state of db/db mice, alleviated dyslipidemia, and promoted skeletal muscle glucose uptake. This phenomenon was closely related to the inhibition of the PTP1B activity. On the one hand, the inhibition of PTP1B activity by F4 resulted in increased insulin receptor (INSR) and insulin receptor substrate 1 tyrosine phosphorylation and enhanced insulin sensitivity. On the other hand, F4 as a PTP1B inhibitor inhibited the inositol-requiring enzyme 1 (IRE-1)/recombinant TNF receptor associated factor 2 (TRAF2)/c-Jun N-terminal kinase signaling pathway and alleviated skeletal muscle endoplasmic reticulum (ER) stress, thereby reducing IRS-1 serine phosphorylation. Both finally activated the PI3K/AKT signaling pathway and promoted glucose transporter protein 4 translocation to the cell membrane for glucose uptake. Taken together, our experiments demonstrate that F4 activates the insulin signaling pathway by inhibiting the activity of PTP1B while inhibiting the IRE-1/TRAF2/JNK signaling pathway, enhancing insulin sensitivity, and alleviating ER stress in the skeletal muscle of db/db mice. Our results indicate that F4 can be used as a PTP1B inhibitor for the treatment of T2DM.

Peanut skin procyanidins reduce intestinal glucose transport protein expression, regulate serum metabolites and ameliorate hyperglycemia in diabetic mice

One of diabetic characteristics is the postprandial hyperglycemia. Inhibiting glucose uptake may be beneficial for controlling postprandial blood glucose levels and regulating the glucose metabolism Peanut skin procyanidins (PSP) have shown a potential for lowering blood glucose; however, the underlying mechanism through which PSP regulate glucose metabolism remains unknown. In the current study, we investigated the effect of PSP on intestinal glucose transporters and serum metabolites using a mouse model of diabetic mice. Results showed that PSP improved glucose tolerance and systemic insulin sensitivity, which coincided with decreased expression of sodium-glucose cotransporter 1 and glucose transporter 2 in the intestinal epithelium induced by an activation of the phospholipase C β2/protein kinase C signaling pathway. Moreover, untargeted metabolomic analysis of serum samples revealed that PSP altered arachidonic acid, sphingolipid, glycerophospholipid, bile acids, and arginine metabolic pathways. The study provides new insight into the anti-diabetic mechanism of PSP and a basis for further research.

Alcohol blunts pregnancy-mediated insulin resistance and reduces fetal brain glucose despite elevated fetal gluconeogenesis, and these changes associate with fetal weight outcomes.

Prenatal alcohol exposure (PAE) impairs fetal growth and neurodevelopment. Although alcohol is well known to alter metabolism, its impact on these processes during pregnancy is largely unexplored. Here, we investigate how alcohol affects maternal-fetal glucose metabolism using our established mouse binge model of PAE. In the dam, alcohol reduces the hepatic abundance of glucose and glycolytic intermediates, and the gluconeogenic enzymes glucose-6-phosphtase and phosphoenolpyruvate carboxykinase. Fasting blood glucose is also reduced. In a healthy pregnancy, elevated maternal gluconeogenesis and insulin resistance ensures glucose availability for the fetus. Glucose and insulin tolerance tests reveal that alcohol impairs the dam's ability to acquire insulin resistance. Alcohol-exposed dams have enhanced glucose clearance (p < .05) in early gestation, after just two days of alcohol, and this persists through late term when fetal glucose needs are maximal. However, maternal plasma insulin levels, hepatic insulin signaling, and the abundance of glucose transporter proteins remain unchanged. In the PAE fetus, the expression of hepatic gluconeogenic genes is elevated, and there is a trend for elevated blood and liver glucose levels. In contrast, fetal brain and placental glucose levels remain low. This reduced maternal fasting glucose, reduced hepatic glucose, and elevated glucose clearance inversely correlated with fetal body and brain weight. Taken together, these data suggest that alcohol blunts the adaptive changes in maternal glucose metabolism that otherwise enhance fetal glucose availability. Compensatory attempts by the fetus to increase glucose pools via gluconeogenesis do not normalize brain glucose. These metabolic changes may contribute to the impaired fetal growth and brain development that typifies PAE.

Exercise increases MEF2A abundance in rat cardiac muscle by downregulating microRNA-223-5p

Exercise plays an important role in cardiac health and enhances the transport of glucose in cardiac muscle by increasing the glucose transporter-4 (GLUT4) content at the cell membrane. The GLUT4 gene is a target of myocyte enhancer transcription factor 2A (MEF2A). Several transcription factors are regulated by microRNAs (miRs), small non-coding RNAs that control gene expression at the posttranscriptional level. In this study we tested the hypothesis that exercise regulates the expression of miR-223 and that MEF2A is a direct target of miR-223. Quantitative reverse transcriptase polymerase chain reaction (qRT-PCR) and western blot experiments showed that GLUT4 gene expression and protein abundance increased by 30 and 23%, respectively, in the microsomal fraction immediately after exercise, and had returned to control levels after 18 h. In contrast, the increase in GLUT4 in the membrane fraction was delayed. Exercise also increased the protein abundance of transcription factors involved in GLUT4 expression. Immediately after exercise, the protein abundance of MEF2A, nuclear respiratory factor 1 (NRF1), and forkhead box O1 (FOXO1) increased by 18, 30, and 40%, respectively. qRT-PCR experiments showed that miR-223-3p and miR-223-5p expression decreased immediately after exercise by 60 and 30%, respectively, and luciferase assays indicated that MEF2A is a target of the 5p strand of miR-223. Overexpression of miR-223-5p in H9c2 cells decreased the protein abundance of MEF2A. Our results suggest that the exercise-induced increase in GLUT4 content in cardiac muscle is partly due to the posttranscriptional increase in MEF2A protein abundance caused by the decrease in miR-223-5p expression. The exercise-induced decrease in miR-223-3p expression likely contributes to the increases in NRF1 and FOXO1 abundance and GLUT4 content.

Low-temperature photothermal-induced alkyl radical release facilitates dihydroartemisinin-triggered “valve-off” starvation therapy

The high nutrient and energy demand of tumor cells compared to normal cells to sustain rapid proliferation offer a potentially auspicious avenue for implementing starvation therapy. However, conventional starvation therapy, such as glucose exhaustion and vascular thrombosis, can lead to systemic toxicity and exacerbate tumor hypoxia. Herein, we developed a new “valve-off” starvation tactic, which was accomplished by closing the valve of glucose transporter protein 1 (GLUT1). Specifically, dihydroartemisinin (DHA), 2,20-azobis [2-(2-imidazolin-2-yl) propane] dihydrochloride (AI), and Ink were co-encapsulated in a sodium alginate (ALG) hydrogel. Upon irradiation with the 1064 nm laser, AI rapidly disintegrated into alkyl radicals (R•), which exacerbated the DHA-induced mitochondrial damage through the generation of reactive oxygen species and further reduced the synthesis of adenosine triphosphate (ATP). Simultaneously, the production of R• facilitated DHA-induced starvation therapy by suppressing GLUT1, which in turn reduced glucose uptake. Systematic in vivo and in vitro results suggested that this radical-enhanced “valve-off” strategy for inducing tumor cell starvation was effective in reducing glucose uptake and ATP levels. This integrated strategy induces tumor starvation with efficient tumor suppression, creating a new avenue for controlled, precise, and concerted tumor therapy.

Upregulated miR‑378a‑3p expression suppresses energy metabolism and promotes apoptosis by targeting a GLUT‑1/ALDOA/PKM2 axis in esophageal carcinoma.

Esophageal squamous cell carcinoma (ESCC) is an aggressive malignancy of the digestive system with increasing incidence and mortality rates. The biological roles of microRNA (miR)-378a-3p in tumor cells remain contested, and the mechanisms underlying the functions, energy metabolism, and cell survival mechanisms in ESCC cells are yet to be fully elucidated. In the present study, miR-378a-3p overexpression and negative control plasmids were transfected into ECA-109 cells using electroporation. Western blotting was used to detect the relative expression of proteins, and flow cytometry was used to detect cell apoptosis. Subsequently, ELISA assays were performed to determine enzyme activity, and an ATP detection kit was used to measure ATP content. Dual-luciferase reporter assays were performed to identify the target genes of miR-378a-3p. The results of the present study demonstrated that miR-378a-3p inhibited the gene expression and enzyme activities of glucose transporter protein 1 (GLUT-1), Aldolase A (ALDOA), and pyruvate kinase M2 (PKM2), all of which are involved in the glycolytic pathway of cells. Energy metabolism was suppressed by miR-378a-3p by reducing ATP content, and this downregulated the expression of Bcl-2 and Survivin. Moreover, increased miR-378a-3p expression promoted cell apoptosis in the early stages by increasing the expression levels and the activity of Bad and Caspase-3, while inhibiting the expression levels of Bcl-2 and Survivin. The results of the present study also demonstrated that GLUT-1/ALDOA/PKM2 were target genes of miR-378a-3p. Notably, miR-378a-3p blocked energy production and promoted the apoptosis of tumor cells via the downregulation of glycolytic enzyme expression and by reducing the mitochondrial membrane potential in ESCC. Bad, Caspase-3, Survivin, and Bcl-2 may be associated with blocking energy production and promoting apoptosis via miR-378a-3p in ESCC cells.

Resveratrol improves palmitic acid‑induced insulin resistance via the DDIT4/mTOR pathway in C2C12 cells.

The present study aimed to establish a model of palmitic acid (PA)‑induced insulin resistance (IR) in C2C12 cells and to determine the mechanism underlying how resveratrol (RSV) improves IR. C2C12 cells were divided into the control (CON), PA, PA + RSV, PA + RSV + DNA damage‑inducible transcript 4 (DDIT4)‑small interfering (si)RNA and PA + RSV + MHY1485 (mTOR agonist) groups. Glucose contents in culture medium and triglyceride contents in cells were determined. Oil red O staining was performed to observe the pathological changes in the cells. Reverse transcription‑quantitative PCR and western blotting were conducted to evaluate the mRNA and protein expression levels, respectively, of DDIT4, mTOR, p70 ribosomal protein S6 kinase (p70S6K), insulin receptor substrate (IRS)‑1, PI3K, AKT and glucose transporter 4 (GLUT4). Compared with in the CON group, glucose uptake was decreased, cellular lipid deposition was increased, phosphorylated (p)‑IRS‑1, p‑mTOR and p‑p70S6K protein expression levels were increased, and p‑PI3K, p‑AKT, GLUT4 and DDIT4 protein expression levels were decreased in the PA group. By contrast, compared with in the PA group, culture medium glucose content and cellular lipid deposition were decreased, p‑PI3K, p‑AKT, GLUT4 and DDIT4 protein expression levels were increased, p‑IRS‑1 protein expression levels were decreased, and mTOR and p70S6K mRNA and protein expression levels were decreased in the PA + RSV group. Compared with in the PA + RSV group, DDIT4 protein and mRNA expression levels were reduced in the PA + RSV + DDIT4‑siRNA group, but showed no change in the PA + RSV + MHY1485 group. Following transfection with DDIT4‑siRNA or treatment with MHY1485, the effects of RSV on improving IR and lipid metabolism were weakened, mTOR and p70S6K protein expression levels were upregulated, p‑PI3K, p‑AKT and GLUT4 protein expression levels were down‑regulated, p‑IRS‑1 protein expression levels were upregulated, and culture medium glucose content and cellular lipid deposition were increased. In conclusion, RSV may improve PA‑induced IR in C2C12 cells through the DDIT4/mTOR/IRS‑1/PI3K/AKT/GLUT4 signaling pathway, as well as via improvements in glucose and lipid metabolism.

Prognostic impact of the combination of HIF‑1α and GLUT1 in patients with oesophageal squamous cell carcinoma.

Oesophageal squamous cell carcinoma (ESCC) is a common type of carcinoma. Hypoxia is associated with chemo- and radio-resistance, which may lead to a poor prognosis. Hypoxia-inducible factor-1α (HIF-1α) is the main transcriptional regulator of the cellular response to low oxygen levels. Moreover, it can trigger the expression of critical genes, including glucose transporter protein type 1 (GLUT1). The aim of the present study was to evaluate the roles of HIF-1α and GLUT1 in ESCC and their usefulness as prognostic markers. HIF-1α and GLUT1 were measured in four ESCC cell lines, namely Eca109, KYSE150, TE-1 and TE-10, by western blotting following culture under normoxic and hypoxic conditions. In addition, xenograft tumors were established in mice using normoxic and hypoxic Eca109 cells and the chemosensitivity of the xenografts to 5-fluorouracil (5-FU) was evaluated. Furthermore, HIF-1α and GLUT1 were analysed by immunochemistry in the tumor tissues of patients with ESCC and the associations of their expression levels with clinicopathological parameters were investigated. The results revealed that HIF-1α and GLUT1 protein expression was weak in all four cell lines under a normoxic atmosphere but increased following culture in a hypoxic environment. In vivo, 5-FU inhibited tumor growth more strongly in normoxic Eca109 ×enografts than hypoxic Eca109 ×enografts. Higher levels of apoptosis were also detected in the normoxic Eca109 ×enografts via western blotting and TUNEL analysis. In patients with ESCC, HIF-1α expression was associated with advanced ESCC while GLUT1 expression was associated with the sex of the patients. Multivariate analysis demonstrated that HIF-1α and GLUT1 were negatively associated with progression-free survival (PFS) and overall survival (OS). Additionally, a combination of HIF-1α and GLUT1 expression was a predictor of RFS and OS in patients with ESCC without lymph node metastasis but not those with lymph node metastasis. The study demonstrated that HIF-1α and GLUT1 were strongly expressed in vitro and in xenograft models when cells were exposed to hypoxia. The simultaneous high expression of HIF-1α and GLUT1 was associated with poorer survival, and may play an important role in ESCC chemoresistance and the prognosis of ESCC.

Determinants of sugar-induced influx in the mammalian fructose transporter GLUT5

In mammals, glucose transporters (GLUT) control organism-wide blood-glucose homeostasis. In human, this is accomplished by 14 different GLUT isoforms, that transport glucose and other monosaccharides with varying substrate preferences and kinetics. Nevertheless, there is little difference between the sugar-coordinating residues in the GLUT proteins and even the malarial Plasmodium falciparum transporter PfHT1, which is uniquely able to transport a wide range of different sugars. PfHT1 was captured in an intermediate ‘occluded’ state, revealing how the extracellular gating helix TM7b has moved to break and occlude the sugar-binding site. Sequence difference and kinetics indicated that the TM7b gating helix dynamics and interactions likely evolved to enable substrate promiscuity in PfHT1, rather than the sugar-binding site itself. It was unclear, however, if the TM7b structural transitions observed in PfHT1 would be similar in the other GLUT proteins. Here, using enhanced sampling molecular dynamics simulations, we show that the fructose transporter GLUT5 spontaneously transitions through an occluded state that closely resembles PfHT1. The coordination of D-fructose lowers the energetic barriers between the outward- and inward-facing states, and the observed binding mode for D-fructose is consistent with biochemical analysis. Rather than a substrate-binding site that achieves strict specificity by having a high affinity for the substrate, we conclude GLUT proteins have allosterically coupled sugar binding with an extracellular gate that forms the high-affinity transition-state instead. This substrate-coupling pathway presumably enables the catalysis of fast sugar flux at physiological relevant blood-glucose concentrations.

The Effect of GLUT1 and HIF-1α Expressions on Glucose Uptake and Patient Survival in Non-Small-Cell Lung Carcinoma

Lung cancer is the second-most-common cancer while being the leading cause of cancer deaths worldwide. It has been found that glucose transporter 1 (GLUT1) and hypoxia-inducible factor 1α (HIF-1α) are overexpressed in various malignancies and that they correlate with the maximum standard uptake values (SUVmax) on 18F-fluorodeoxyglucose-positron emission tomography/computed tomography (18F-FDG PET/CT) and poor prognosis. In this study, we aim to evaluate the relationship between the SUVmax, GLUT1, and HIF-1α expression with primary tumor size, histological type, lymph node metastases, and patient survival. Of the 48 patients with non-small-cell lung cancer, those with squamous cell carcinomas (SCCs) had significantly higher GLUT1 and HIF-1α immunohistochemical expressions in comparison to adenocarcinomas (ACs), while there was no statistically significant difference in FDG accumulation between them. No significant correlation was noted between either GLUT1 or HIF-1α protein expression and FDG uptake and overall survival. However, an analysis of tumor transcriptomics showed a significant difference in overall survival depending on mRNA expression; patients with SCC and high HIF-1α levels survived longer compared to those with low HIF-1α levels, while patients with AC and low GLUT1 levels had a higher average survival time than those with high GLUT1 levels. Further studies are needed to determine the prognostic value of the expression of these factors depending on the histologic type.

PERIOPERATIVE DIABETIC KETOACIDOSIS ASSOCIATED WITH SODIUM-GLUCOSE CO-TRANSPORTER-2 INHIBITORS : A SYSTEMATIC REVIEW

DKA or diabetic ketoacidosis is an emergency condition caused by hyperglycemia in which excessive acid is produced in the blood. Patients with type 1 and type 2 diabetes who undergo surgery, have an infection, or are under extreme stress can also develop DKA. The body generates the hormone adrenaline to combat infection and stress, but this can have a negative effect on blood glucose levels (adrenaline is counterinsulin). This can worsen if the patient refuses to take diabetes medication or inject insulin during stressful or infectious situations. The fundamental components of the pathophysiological mechanisms at play in diabetic ketoacidosis (DKA) include shifts in hormone levels and the ensuing inflammatory response. Alterations in the concentration of hormones lead to shifts in the production and consumption of glucose, as well as an increase in lipolysis and the formation of ketone bodies. Canagliflozin, dapagliflozin, empagliflozin, and ertugliflozin are agents approved by the Food and Drug Administration (FDA) for the treatment of type 2 diabetes mellitus (DM) in adults. Acting on proteins, the four agents are SGLT2 inhibitor class agents. SGLT-2 is expressed in the kidney's proximal tubule. This may result in a decrease in filtered glucose reabsorption, a decrease in the renal threshold for glucose (RTG), and an increase in glucose excretion via urine. Diabetes patients treated with sodium–glucose transport protein 2 (SGLT2) inhibitors have also been reported to experience DKA. The results of a study indicate that DKA is uncommon among T2DM patients treated with SGLT2 inhibitors.

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.

ERK/PKM2 Is Mediated in the Warburg Effect and Cell Proliferation in Arsenic-Induced Human L-02 Hepatocytes.

This study aimed to investigate the potential role of pyruvate kinase M2 (PKM2) and extracellular regulated protein kinase (ERK) in arsenic-induced cell proliferation. L-02 cells were treated with 0.2 and 0.4 μmol/L As3+, glycolysis inhibitor (2-deoxy-D-glucose,2-DG), ERK inhibitor [1,4-diamino-2,3-dicyano-1,4-bis(2-aminophenylthio)-butadiene, U0126] or transfected with PKM2 plasmid. Cell viability, proliferation, lactate acid production, and glucose intake capacity were determined by CCK-8 assay, EdU assay, lactic acid kit and 2-deoxy-2-[(7-nitro-2,1,3-benzoxadiazol-4-yl) amino]-D-glucose (2-NBDG) uptake kit, respectively. Also, levels of PKM2, phospho-PKM2S37, glucose transporter protein 1 (GLUT1), lactate dehydrogenase A (LDHA), ERK, and phospho-ERK were detected using Western blot and the subcellular localization of PKM2 in L-02 cells was detected by immunocytochemistry (ICC). Treatment with 0.2 and 0.4 μmol/L As3+ for 48 h increased the viability and proliferation of L-02 cells, the proportion of 2-NBDG+ cell and lactic acid in the culture medium, and GLUT1, LDHA, PKM2, phospho-PKM2S37, and phospho-ERK levels and PKM2 in nucleus. Compared with the 0.2 μmol/L As3+ treatment group, the lactic acid in the culture medium, cell proliferation and cell viability, and the expression of GLUT1 and LDHA were reduced in the group co-treated with siRNA-PKM2 and arsenic or in the group co-treated with U0126. Moreover, the arsenic-increased phospho-PKM2S37/PKM2 was decreased by U0126. Therefore, ERK/PKM2 plays a key role in the Warburg effect and proliferation of L-02 cells induced by arsenic, and also might be involved in arsenic-induced upregulation of GLUT1 and LDHA. This study provides a theoretical basis for further elucidating the carcinogenic mechanism of arsenic.

Bone marrow mesenchymal stem cell-derived exosomes reduce insulin resistance and obesity in mice via the PI3K/AKT signaling pathway.

Obesity is a common chronic metabolic disease that induces chronic systemic inflammation in the body, eventually leading to related complications such as insulin resistance (IR), type 2 diabetes mellitus, and metabolic syndromes such as cardiovascular disease. Exosomes transfer bioactive substances to neighboring or distal cells through autosomal, paracrine, or distant secretion, regulating the gene and protein expression levels of receptor cells. In this study, we investigated the effect of mouse bone marrow mesenchymal stem cell-derived exosomes (BMSC-Exos) on high-fat diet obese mice and mature 3T3-L1 adipocyte models of IR. BMSC-Exo treatment of obese mice promoted their metabolic homeostasis, including reduction of obesity, inhibition of M1-type proinflammatory factor expression, and improvement of insulin sensitivity. In vitro analysis revealed that BMSC-Exos improved IR and lipid droplet accumulation in mature 3T3-L1 adipocytes treated with palmitate (PA). Mechanistically, BMSC-Exos cause increased glucose uptake and improved IR in high-fat chow-fed mice and PA-acting 3T3-L1 adipocytes by activating the phosphoinositide 3-kinases/protein kinase B (PI3K/AKT) signaling pathway and upregulating glucose transporter protein 4 (GLUT4) expression. This study offers a new perspective for the development of treatments for IR in obese and diabetic patients.

Therapeutic exploration of polyherbal formulation against letrozole induced PCOS rats: A mechanistic approach

ObjectiveThis study aimed to develop an effective alternative medicine with multi potential herbs against polycystic ovarian syndrome (PCOS) in rats induced by letrozole treatment. Materials and methodPolyherbal syrup was prepared with a combination of S. asoca bark, G. sylvestre leaves, P. daemia aerial parts, C. zeylanium stem bark, C. bonduc seeds, and W. somnifera roots ethanolic extract. In vitro cell viability study, adenosine monophosphate-activated protein kinase (AMPK), and glucose transporter 4 (GLUT4) gene expression assay were carried out on the Chinese Hamster Ovarian (CHO) cell line. For the PCOS induction letrozole (1 mg/kg p. o.) was given for 21 consecutive days. The PCOS induction was confirmed by measuring estrus irregularity, insulin resistance by oral glucose tolerance test (OGTT), and hyperandrogenism by measuring serum total testosterone level 21 days after completion of letrozole treatment. After induction of PCOS, metformin (155 mg/kg p. o.), and polyherbal syrup (100 mg/kg, 200 mg/kg, and 400 mg/kg p. o.) were administered for further 28 days. The treatment efficacy was measured by measuring serum lipid profile, fasting insulin level, sex hormones level, ovarian steroidogenic enzymes, ovarian tissue insulin receptor, AMPK, and GLUT4 protein expression levels, and histomorphological studies. The post-treatment effect was confirmed by reproductive performance studies. ResultsLetrozole-induced PCOS rats showed significant estrus irregularity, abnormal sex hormones levels, and hyperandrogenism indicated by showing increased free androgenic index and decreased sex hormones binding globulin (SHBG) level. The insulin resistance in PCOS rats was indicated by increased fasting glucose levels with impaired glucose clearance in the OGT test. Homeostasis Model Assessment Index of Insulin Resistance (HOMA-IR) increased level, also decreases INSR, GLUT4, and AMPK mRNA expression in ovarian cells confirming the insulin resistance in PCOS rats. Ovarian histology in PCOS rats also showed many follicular cysts, atretic follicles, and the absence of corpus luteum. The administration of polyherbal syrup, in a dose-dependent manner, effectively restored these alterations. The treatment of polyherbal formulation 400 mg/kg possesses highly significant efficacy over the treatment of metformin in PCOS rats. It mainly acts by reducing peripheral and ovarian hyperandrogenism and improves insulin sensitivity via activating the insulin receptor and AMP-activated kinase-mediated transcription and translation of GLUT4 from the cytoplasm to the ovarian membrane improves glucose uptake and promotes the follicular development and ovulation. The higher fertility rate, delivery index, and survival of delivered pups confirm the broader and superior efficacy of PCOS. These beneficial actions are mainly attributable to the formulation's inclusion of the key secondary metabolites flavonoids and phytosterols. In conclusion, the prepared polyherbal syrup was found to be the safest and most effective alternative medicine for both endocrinal and metabolic complications of PCOS women.

Expression of Glucose Transporters 1 and 3 in the Placenta of Pregnant Women with Gestational Diabetes Mellitus.

The annual prevalence of gestational diabetes mellitus-characterized by an increase in blood glucose in pregnant women-has been increasing worldwide. The goal of this study was to evaluate the expression of glucose transporter 1 (GLUT1) and glucose transporter 3 (GLUT3) in the placenta of women with gestational diabetes mellitus. Sixty-five placentas from women admitted to the King Saud University Medical City, Riyadh, Saudi Arabia, were analyzed; 34 and 31 placentas were from healthy pregnant women and women with gestational diabetes, respectively. The expressions of GLUT1 and GLUT3 were assessed using RT-PCR, Western blotting, and immunohistochemical methods. The degree of apoptosis in the placental villi was estimated via a TUNEL assay. The results of the protein expression assays and immunohistochemical staining showed that the levels of GLUT1 and GLUT3 were significantly higher in the placentas of pregnant women with gestational diabetes than those in the placentas of healthy pregnant women. In addition, the findings showed an increase in apoptosis in the placenta of pregnant women with gestational diabetes compared to that in the placenta of healthy pregnant women. However, the results of gene expression assays showed no significant difference between the two groups. Based on these results, we conclude that gestational diabetes mellitus leads to an increased incidence of apoptosis in the placental villi and alters the level of GLUT1 and GLUT3 protein expressions in the placenta of women with gestational diabetes. Understanding the conditions in which the fetus develops in the womb of a pregnant woman with gestational diabetes may help researchers understand the underlying causes of the development of chronic diseases later in life.

Autosomal Recessively Inherited Glucose Transporter-1 Deficiency Syndrome with Acanthocytosis: A Case Report

AbstractGlucose transporter-1 deficiency syndrome (GLUT-1DS) is a rare, autosomal dominantly inherited disorder due to a heterozygous mutation in the gene solute channel 2A1 (SLC2A1).This gene encodes the glucose transporter protein-1. Autosomal recessive inheritance is extremely rare. Similarly, only very few reports are found in the literature of hematological manifestations in this syndrome. We report an autosomal recessively inherited GLUT-1DS, due to a de novo mutation, with the classical infantile presentation associated with concomitant acanthocytosis. In this case report, the second child born to consanguineous parents with initial refractory neonatal seizures and subsequent poorly controlled epilepsy and developmental regression is discussed. The most notable investigation findings supportive of his underlying diagnosis were very low cerebrospinal fluid (CSF) glucose and CSF lactate levels. His CSF:plasma glucose ratio was 1:7.6. He was anemic with a hemoglobin of 8.8 g/dL with his blood film showing marked acanthocytosis. His elder brother who also had refractory epilepsy and developmental regression had similar hypoglycorrhachia, low CSF:plasma glucose ratio, and mild anemia with acanthocytosis, and he died before establishment of a diagnosis. Our patient was diagnosed to have a novel mutation SLC2A1 c.184A &gt; G p.(Thr62Ala), for which both parents were heterozygous, confirming autosomal recessive inheritance. Commencement of a ketogenic diet resulted in improvement of his seizures and slow gain in development. It also resulted in gradual disappearance of acanthocytes from his peripheral blood. This case describes a rare case of classical GLUT-1DS, autosomal recessively inherited, due to a novel mutation. The acanthocytosis in his blood smear is another rare association minimally reported in GLUT-1DS. The cause of his abnormal red blood cell morphology is unclear. It is possibly related to cation leakage reported in some rare mutations of the SLC2A1 gene needs reference.

Anti-diabetic potential of apigenin, luteolin, and baicalein via partially activating PI3K/Akt/Glut-4 signaling pathways in insulin-resistant HepG2 cells

Dietary flavonoids are abundant in natural plants and possess multiple pharmacological and nutritional activities. In this study, apigenin, luteolin, and baicalein were chosen to evaluate their anti-diabetic effect in high-glucose and dexamethasone induced insulin-resistant (IR) HepG2 cells. All flavonoids improves the glucose consumption and glycogen synthesis abilities in IR-HepG2 cells via activating glucose transporter protein 4 (GLUT4) and phosphor-glycogen synthase kinase (GSK-3β). These flavonoids significantly inhibited the production of reactive oxygen species (ROS) and advanced glycation end-products (AGEs), which were closely related to the suppression of the phosphorylation form of NF-κB and P65. The expression levels of insulin receptor substrate-1 (IRS-1), insulin receptor substrate-2 (IRS-2) and phosphatidylinositol 3-kinase (PI3K)/protein kinase B (Akt) pathway in IR-HepG2 cells were all partially activated by the flavonoids, with variable effects. Furthermore, the intracellular metabolic conditions of the flavonoids were also evaluated.