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Cellular heterogeneity is a well-accepted feature of tissues, and both transcriptional and metabolic diversity have been revealed by numerous approaches, including optical imaging. However, the high magnification objective lenses needed for high-resolution imaging provides information from only small layers of tissue, which can result in poor cell statistics. There is therefore an unmet need for an imaging modality that can provide detailed molecular and cellular insight within intact tissue samples in 3D. Using GFP-tagged GLUT4 as proof of concept, we present here a novel optical mesoscopy approach that allows precise measurement of the spatial location of GLUT4 within specific anatomical structures across the myocardium in ultrathick sections (5 mm×5 mm×3 mm) of intact mouse heart. We reveal distinct GLUT4 distribution patterns across cardiac walls and highlight specific changes in GLUT4 expression levels in response to high fat diet-feeding, and we identify sex-dependent differences in expression patterns. This method is applicable to any target that can be labelled for light microscopy, and to other complex tissues when organ structure needs to be considered simultaneously with cellular detail., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2024. Published by The Company of Biologists Ltd.)

Objective: Picalm (phosphatidylinositol-binding clathrin assembly protein), a ubiquitously expressed clathrin-adapter protein, is a well-known susceptibility gene for Alzheimer's disease, but its role in white adipose tissue (WAT) function has not yet been studied. Transcriptome analysis revealed differential expression of Picalm in WAT of diabetes-prone and diabetes-resistant mice, hence we aimed to investigate the potential link between Picalm expression and glucose homeostasis, obesity-related metabolic phenotypes, and its specific role in insulin-regulated GLUT4 trafficking in adipocytes., Methods: Picalm expression and epigenetic regulation by microRNAs (miRNAs) and DNA methylation were analyzed in WAT of diabetes-resistant (DR) and diabetes-prone (DP) female New Zealand Obese (NZO) mice and in male NZO after time-restricted feeding (TRF) and alternate-day fasting (ADF). PICALM expression in human WAT was evaluated in a cross-sectional cohort and assessed before and after weight loss induced by bariatric surgery. siRNA-mediated knockdown of Picalm in 3T3-L1-cells was performed to elucidate functional outcomes on GLUT4-translocation as well as insulin signaling and adipogenesis., Results: Picalm expression in WAT was significantly lower in DR compared to DP female mice, as well as in insulin-sensitive vs. resistant NZO males, and was also reduced in NZO males following TRF and ADF. Four miRNAs (let-7c, miR-30c, miR-335, miR-344) were identified as potential mediators of diabetes susceptibility-related differences in Picalm expression, while 11 miRNAs (including miR-23a, miR-29b, and miR-101a) were implicated in TRF and ADF effects. Human PICALM expression in adipose tissue was lower in individuals without obesity vs. with obesity and associated with weight-loss outcomes post-bariatric surgery. siRNA-mediated knockdown of Picalm in mature 3T3-L1-adipocytes resulted in amplified insulin-stimulated translocation of the endogenous glucose transporter GLUT4 to the plasma membrane and increased phosphorylation of Akt and Tbc1d4. Moreover, depleting Picalm before and during 3T3-L1 differentiation significantly suppressed adipogenesis, suggesting that Picalm may have distinct roles in the biology of pre- and mature adipocytes., Conclusions: Picalm is a novel regulator of GLUT4-translocation in WAT, with its expression modulated by both genetic predisposition to diabetes and dietary interventions. These findings suggest a potential role for Picalm in improving glucose homeostasis and highlight its relevance as a therapeutic target for metabolic disorders., Competing Interests: Declaration of competing interest MB received honoraria as a consultant and speaker from Amgen, AstraZeneca, Bayer, Boehringer-Ingelheim, Lilly, Novo Nordisk, Novartis, and Sanofi. All other authors declare no conflicts of interest. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript; or in the decision to publish the results. All other authors declare that there are no conflicts of interests., (Copyright © 2024 The Author(s). Published by Elsevier GmbH.. All rights reserved.)

Mitoxantrone (MTX) is a therapeutic agent used in the treatment of solid tumors and multiple sclerosis, recognized for its cardiotoxicity, with underlying molecular mechanisms not fully disclosed. The cardiotoxicity is influenced by risk factors, including age. Our study intended to assess the molecular effect of MTX on the cardiac muscle of old male CD-1 mice. Mice aged 19 months received a total cumulative dose of 4.5 mg/kg of MTX (MTX group) or saline solution (CTRL group). Two months post treatment, blood was collected, animals sacrificed, and the heart removed. MTX caused structural cardiac changes, which were accompanied by extracellular matrix remodeling, as indicated by the increased ratio between matrix metallopeptidase 2 and metalloproteinase inhibitor 2. At the metabolic level, decreased glycerol levels were found, together with a trend towards increased content of the electron transfer flavoprotein dehydrogenase. In contrast, lower glycolysis, given by the decreased content of glucose transporter GLUT4 and phosphofructokinase, seemed to occur. The findings suggest higher reliance on fatty acids oxidation, despite no major remodeling occurring at the mitochondrial level. Furthermore, the levels of glutamine and other amino acids (although to a lesser extent) were decreased, which aligns with decreased content of the E3 ubiquitin-protein ligase Atrogin-1, suggesting a decrease in proteolysis. As far as we know, this was the first study made in old mice with a clinically relevant dose of MTX, evaluating its long-term cardiac effects. Even two months after MTX exposure, changes in metabolic fingerprint occurred, highlighting enduring cardiac effects that may require clinical vigilance., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Our previous study proved that epicatechin (EC) and β-glucan (BG) from whole-grain highland barley synergistically modulate glucose metabolism in insulin-resistant HepG2 cells. However, the main target and the mechanism underlying the modulation of glucose metabolism in vivo remain largely unknown. In this study, cell transfection assay and microscale thermophoresis analysis revealed that EC and BG could directly bind to the insulin receptor (IR) and mammalian receptor for rapamycin (mTOR), respectively. Molecular dynamic analysis indicated that the key amino acids of binding sites were Asp, Met, Val, Lys, Ser, and Tys. EC supplementation upregulated the IRS-1/PI3K/Akt pathway, while BG upregulated the mTOR/Akt pathway. Notably, supplementation with EC + BG significantly increased Akt and glucose transporter type 4 (GLUT4) protein expressions, while decreasing glycogen synthase kinase 3β (GSK-3β) expression in liver cells as compared to the individual effects of EC and BG, indicating their synergistic effect on improving hepatic glucose uptake and glycogen synthesis. Consistently, supplementation with EC + BG significantly decreased blood glucose levels and improved oral glucose tolerance compared to EC and BG. Therefore, combined supplementation with EC and BG may bind to corresponding receptors, targeting synergistic activation of Akt expression, leading to the improvement of hepatic glucose metabolism and thereby ameliorating hyperglycemia in vivo.

Background: In China, the environmental concern of Dibutyl Phthalate (DBP) exposure significantly endangers human health by inducing insulin resistance (IR). Skeletal muscle tissue plays a critical role in this process. However, the precise molecular mechanisms through which DBP interferes with the insulin signaling pathway remain to be fully elucidated. This study aims to explore the molecular mechanisms by which DBP induces IR in skeletal muscle, focusing on the phosphatidylinositol 3-kinase (PI3K)-serine/threonine kinase (AKT)-glucose transporter 4 (GLUT4) signaling pathway., Methods: To investigate the molecular mechanisms underlying DBP-induced IR, an experimental study was established on a human skeletal muscle cell line (HSkMC). Expression levels of mRNA and proteins associated with key signaling genes within the insulin receptor (INSR)-insulin receptor substrate (IRS)-PI3K-AKT-GLUT4 pathway were assessed using quantitative real-time polymerase chain reaction (qRT-PCR) and Western blot techniques. Additionally, this study explored the effects of DBP alone and in combination with a PI3K inhibitor (BKM120) or phosphatase and tensin homolog (PTEN) overexpression lentivirus on these signaling components., Results: Results from this study demonstrated that DBP exposure significantly decreased mRNA levels of INSR , IRS1 , PI3K , AKT2 , and GLUT4 in HSkMC cells compared to untreated control cells. This reduction was exacerbated when DBP was combined with BKM120 or PTEN overexpression lentivirus, suggesting a synergistic effect. Furthermore, DBP treatment reduced the expression and phosphorylation of AKT2, indicating a disruption in the insulin signaling pathway., Conclusions: This study elucidates a molecular mechanism by which DBP induces IR in skeletal muscle cells, primarily through the deregulation of the PI3K-dependent insulin signaling pathway. These insights enhance comprehension of the pathophysiological changes associated with IR caused by environmental pollutants like DBP, potentially guiding future strategies for prevention and intervention.

During insulin resistance, the heart undergoes a metabolic shift in which fatty acids (FA) account for roughly about 99% of the ATP production. This metabolic shift is indicative of impaired glucose metabolism. A shift in FA metabolism with impaired glucose tolerance can increase reactive oxygen species (ROS), lipotoxicity, and mitochondrial dysfunction, ultimately leading to cardiomyopathy. Thyroid hormones (TH) may improve the glucose intolerance by increasing glucose reabsorption and metabolism in peripheral tissues, but little is known on its effects on cardiac tissue during insulin resistance. In the present study, insulin resistant Otsuka Long Evans Tokushima Fatty (OLETF) rats were used to assess the effects of exogenous thyroxine (T4) on glucose metabolism in cardiac tissue. Rats were assigned to four groups: (1) lean, Long Evans Tokushima Otsuka (LETO; n=6), (2) LETO + T4 (8 μg/100 g BM/d × 5 wks; n = 7), (3) untreated OLETF (n = 6), and (4) OLETF + T4 (8 μg/100 g BM/d × 5 wks; n = 7). T4 increased GLUT4 gene expression by 85% in OLETF and increased GLUT4 protein translocation to the membrane by 294%. Additionally, T4 increased p-AS160 by 285%, phosphofructokinase-1 (PFK-1) mRNA, the rate limiting step in glycolysis, by 98% and hexokinase II by 64% in OLETF. T4 decreased both CPT2 mRNA and protein expression in OLETF. The results suggest that exogenous T4 has the potential to increase glucose uptake and metabolism while simultaneously reducing fatty acid transport in the heart of insulin resistant rats. Thus, L-thyroxine may have therapeutic value to help correct the impaired substrate metabolism associated with diabetic cardiomyopathy., Competing Interests: Declaration of competing interest None., (Published by Elsevier B.V.)

Our recent studies have identified p21-activated kinase 4 (PAK4) as a key regulator of lipid catabolism in the liver and adipose tissue, but its role in glucose homeostasis in skeletal muscle remains to be explored. In this study, we find that PAK4 levels are highly upregulated in the skeletal muscles of diabetic humans and mice. Skeletal muscle-specific Pak4 ablation or administering the PAK4 inhibitor in diet-induced obese mice retains insulin sensitivity, accompanied by AMPK activation and GLUT4 upregulation. We demonstrate that PAK4 promotes insulin resistance by phosphorylating AMPKα2 at Ser491, thereby inhibiting AMPK activity. We additionally show that skeletal muscle-specific expression of a phospho-mimetic mutant AMPKα2 S491D impairs glucose tolerance, while the phospho-inactive mutant AMPKα2 S491A improves it. In summary, our findings suggest that targeting skeletal muscle PAK4 may offer a therapeutic avenue for type 2 diabetes., (© 2024. The Author(s).)

The increasing prevalence of obesity, type 2 diabetes mellitus (T2DM), and gestational diabetes (GDM) among pregnant women has risen dramatically worldwide. The antihyperglycemic drug metformin is the most common drug for T2DM treatment in non-pregnant individuals; nevertheless, it is increasingly being used for diabetes-complicated pregnancies. Studies on the long-term metabolic effects of this drug in offspring remain scarce. This work aimed to determine the effect of metformin exposure during pregnancy and lactation on the offspring of a model of diet-induced maternal hyperglycemia. Cohorts of pregnant mice were fed a 46% fat diet (HFD) or a control standard diet (SD). A group of dams were exposed to metformin during pregnancy and lactation. After weaning, the offspring were fed SD for 8 weeks and then challenged with a 46% HFD after puberty for 12 weeks. Irrespective of the maternal diet, offspring of metformin-exposed mothers had a lower body weight and reduced inguinal white adipose tissue (iWAT) mass after HFD challenge. This was associated with increased expression of Pparg, Fabp4, Glut4, Srebp1, and Fasn in the iWAT during adulthood in the metabolically impaired dams exposed to metformin, suggesting increased adipogenesis and de novo lipogenesis. Increased expression of Fasn associated with decreased methylation levels at its promoter and proximal coding region in the iWAT was found. These results suggest that metformin modulates gene expression levels by epigenetic mechanisms in maternal metabolic-impaired conditions., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024. Published by Elsevier B.V.)

Energy metabolism is essential for insect development, reproduction and detoxification. Insects often reallocate energy and resources to manage external stress, balancing the demands of detoxification and reproduction. Glucose transport 4 (Glut4), a glucose transporter, is involved in glucose and lipid metabolism. However, the specific molecular mechanism of Glut4 in insect reproduction, and its role in the response to insecticide-induced oxidative stress remain unclear. In this study, LmGlut4 was identified and analyzed in Locusta migratoria. Silencing of LmGlut4 significantly reduced vitellogenin (Vg) biosynthesis in the fat body and Vg absorption by oocytes, ultimately hindering ovarian development and oocyte maturation. Knockdown of LmGlut4 also inhibited the biosynthesis of key insect hormones, such as juvenile hormone (JH), 20-hydroxyecdysone (20E) and insulin. Furthermore, LmGlut4 knockdown led to reduced triglyceride (TG) and glycogen content in the fat body and ovary, as well as decreased capacity for trehalose biosynthesis in adipocytes. Additionally, dsLmGlut4-treated locusts showed heightened sensitivity to deltamethrin, leading to increased triglyceride depletion during detoxification. This study sheds light on the biological function of LmGlut4 in the ovary and provides potential target genes for exploring biological pest management strategies., Competing Interests: Declaration of competing interest The authors declare that they have no competing financial interests or personal relationships., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Context: Obesity is related to insulin resistance, and adipose tissue-secreted TNF- α may play a role in inducing obesity. TNF- α activates inflammatory protein kinase and impairs insulin signalling., Objectives: We investigated the effect of betulinic acid on insulin resistance caused by TNF- α treatment in 3T3-L1 adipocytes., Material and Methods: 3T3-L1 was exposed to TNF- α in the presence and absence of betulinic acid. Various parameters such as glucose uptake assay, cell viability, expression of proteins involved in insulin resistance were studied., Results: Betulinic acid increased glucose uptake in TNF- α pre-treated cells and inhibited the activation of PTP1B and JNK and reduced IκB α degradation. Tyrosine phosphorylation was increased, and serine phosphorylation was decreased in IRS-1., Discussion: Betulinic acid restored TNF- α impaired insulin signalling and increased PI3K activation and phosphorylation of Akt and increased plasma membrane expression of GLUT 4, which stimulated glucose uptake concentration-dependently., Conclusion: These results suggest that betulinic acid is effective at improving TNF- α -induced insulin resistance in adipocytes via inhibiting the activation of negative regulator of insulin signalling and inflammation-activated protein kinase and may potentially improve insulin resistance.

Purpose: Garlic extract (GA) is purported to enhance antioxidant and anti-inflammatory activity and glucose regulation in humans. The present study investigated the effects of post-exercise GA supplementation on GLUT4 expression, glycogen replenishment, and the transcript factors involved with mitochondrial biosynthesis in exercised human skeletal muscle., Methods: The single-blinded crossover counterbalanced study was completed by 12 participants. Participants were randomly divided into either GA (2000 mg of GA) or placebo trials immediately after completing a single bout of cycling exercise at 75% Maximal oxygen uptake (VO 2max ) for 60 minutes. Participants consumed either GA (2000 mg) or placebo capsules with a high glycemic index carbohydrate meal (2 g carb/body weight) immediately after exercise. Muscle samples were collected at 0-h and 3-h post-exercise. Muscle samples were used to measure glycogen levels, GLUT4 protein expression, as well as transcription factors for glucose uptake, and mitochondria biogenesis. Plasma glucose, insulin, glycerol, non-esterified fatty acid (NEFA) concentrations, and respiratory exchange ratio (RER) were also analyzed during the post-exercise recovery periods., Results: Skeletal muscle glycogen replenishment was significantly elevated during the 3-h recovery period for GA concurrent with no difference in GLUT4 protein expression between the garlic and placebo trials. PGC1-α gene expression was up-regulated for both GA and placebo after exercise ( p  < 0.05). Transcript factors corresponding to muscle mitochondrial biosynthesis were significantly enhanced under acute garlic supplementation as demonstrated by TFAM and FIS1. However, the gene expression of SIRT1, ERRα, NFR1, NFR2, MFN1, MFN2, OPA1, Beclin-1, DRP1 were not enhanced, nor were there any improvements in GLUT4 expression, following post-exercise garlic supplementation., Conclusion: Acute post-exercise garlic supplementation may improve the replenishment of muscle glycogen, but this appears to be unrelated to the gene expression for glucose uptake and mitochondrial biosynthesis in exercised human skeletal muscle.

Skeletal muscle is one of the predominant sites involved in glucose disposal, accounting for ∼80% of postprandial glucose uptake, and plays a critical role in maintaining glycemic homeostasis. Dysregulation of energy metabolism in skeletal muscle is involved in developing insulin resistance and type 2 diabetes (T2D). Transcriptomic responses of skeletal muscle to exercise found that the expression of Klf3 was increased in T2D Goto-Kakizaki (GK) rats and decreased after exercise with improved hyperglycemia and insulin resistance, implying that Klf3 might be associated with insulin sensitivity and glucose metabolism. We also found that knockdown of Klf3 promoted basal and insulin-stimulated glucose uptake in L6 myotubes, whereas overexpression of Klf3 resulted in the opposite. Through pairwise comparisons of L6 myotubes transcriptome, we identified 2,256 and 1,988 differentially expressed genes in Klf3 knockdown and overexpression groups, respectively. In insulin signaling, the expression of Slc2a4 , Akt2 , Insr , and Sorbs1 was significantly increased by Klf3 knockdown and decreased with Klf3 overexpression; Ptprf and Fasn were markedly downregulated in Klf3 reduced group and upregulated in Klf3 overexpressed group. Moreover, downregulation of Klf3 promoted the expression of glucose transporter 4 (GLUT4) and protein kinase B (AKT) proteins, as well as the translocation of GLUT4 to the cell membrane in the basal situation, and enhanced insulin sensitivity, characterized by increased insulin-stimulated GLUT4 translocation and AKT, TBC1 domain family member 1 (TBC1D1) and TBC1 domain family member 4 (TBC1D4) phosphorylation, whereas overexpression of Klf3 showed contrary results. These results suggest that Klf3 affects glucose uptake and insulin sensitivity via insulin signal transduction and intracellular metabolism, offering a novel potential treatment strategy for T2D. NEW & NOTEWORTHY The knockdown of Klf3 increased glucose uptake and improved insulin sensitivity in L6 myotubes, whereas its overexpression had the opposite effect. To explore the underlying mechanisms, we evaluated the transcriptional profiles of L6 myotubes after Klf3 knockdown and overexpression and revealed that metabolism and insulin-related pathways were significantly impacted. Klf3 also influenced the expression or modification of glucose transporter 4 (GLUT4), protein kinase B (AKT), TBC1 domain family member 1 (TBC1D1), and TBC1 domain family member 4 (TBC1D4) in the insulin signaling pathway, affecting insulin sensitivity and glucose uptake.

Advanced glycated end products (AGEs) are cytotoxic compounds that are mainly increased in diabetes mellitus (DM), kidney failure, inflammation, and in response to the ingestion of AGE-rich diets. AGEs can also impair glycemic homeostasis by decreasing the expression of the Slc2a4 (solute carrier family 2 member 4) gene and its GLUT4 (solute carrier family 2, facilitated glucose transporter member 4) protein in muscle. However, the mechanisms underlying AGE's effect on adipocytes have not been demonstrated yet. This study investigated the effects of AGEs upon Slc2a4 /GLUT4 expression in 3T3-L1 adipocytes, as well as the potential role of NFKB (nuclear factor NF-kappa-B) activity in the effects observed. Adipocytes were cultured in the presence of control albumin (CA) or advanced glycated albumin (GA) at concentrations of 0.4, 3.6, and 5.4 mg/mL for 24 h or 72 h. Slc2a4 , Rela, and Nfkb1 mRNAs were measured by RT-qPCR, GLUT4, IKKA/B, and p50/p65 NFKB subunits using Western blotting, and p50/p65 binding into the Slc2a4 promoter was analyzed by chromatin immunoprecipitation (ChIP) assay. GA at 0.4 mg/mL increased Slc2a4 /GLUT4 expression after 24 h and 72 h (from 50% to 100%), but at 5.4 mg/mL, Slc2a4 /GLUT4 expression decreased at 72 h (by 50%). Rela and Nfkb1 expression increased after 24 h at all concentrations, but this effect was not observed at 72 h. Furthermore, 5.4 mg/mL of GA increased the p50/p65 nuclear content and binding into Slc2a4 at 72 h. In summary, this study reveals AGE-induced and NFKB-mediated repression of Slc2a4 /GLUT4 expression. This can compromise the adipocyte glucose utilization, contributing not only to the worsening of glycemic control in DM subjects but also the impairment of glycemic homeostasis in non-DM subjects under the high intake of AGE-rich foods.

Chronic lipid overconsumption, associated with the Western diet, causes excessive cardiac lipid accumulation, insulin resistance, and contractile dysfunction, altogether termed lipotoxic cardiomyopathy (LCM). Existing treatments for LCM are limited. Traditional Chinese Medicine (TCM) has been shown as beneficial in diabetes and its complications. The following compounds-Resveratrol, Quercetin, Berberine, Baicalein, and Isorhamnetin-derived from TCM and often used to treat type 2 diabetes. However, virtually nothing is known about their effects in the lipid-overexposed heart. Lipid-induced insulin resistance was generated in HL-1 cardiomyocytes and adult rat cardiomyocytes by 24 h exposure to high palmitate. Upon simultaneous treatment with each of the TCM compounds, we measured myocellular lipid accumulation, insulin-stimulated fatty acid and glucose uptake, phosphorylation levels of AKT and ERK1/2, plasma membrane appearance of GLUT4 and CD36, and expression of oxidative stress-/inflammation-related genes and contractility. In lipid-overloaded cardiomyocytes, all the selected TCM compounds prevented lipid accumulation. These compounds also preserved insulin-stimulated CD36 and GLUT4 translocation and insulin-stimulated glucose uptake in an Akt-independent manner. Moreover, all the TCM compounds prevented and restored lipid-induced contractile dysfunction. Finally, some (not all) of the TCM compounds inhibited oxidative stress-related SIRT3 expression, and others reduced inflammatory TNFα expression. Their ability to restore CD36 trafficking makes all these TCM compounds attractive natural supplements for LCM treatment.

From the fruits of Cordia dichotoma , 11 new phenolic compounds, dichotomins A-K, were isolated, together with 19 known compounds. Through the analysis of detailed NMR data and HRESIMS data, the planar structures of all compounds were confirmed. Using NMR calculations, the absolute configuration of dichotomins A-K was elucidated by comparing their observed and computed electronic circular dichroism (ECD) spectra. Dichotomin H ( 8 ) and dichotomin I ( 9 ) were determined as two pairs of enantiomers. The enantiomers of compounds 8 and 9 were separated using chiral-phase high-performance liquid chromatography (HPLC), and the stereostructure of each enantiomer was determined by similarly calculating the ECD. Compounds 3 , 5, 7 , 17 , 18 , 23 - 25 , and 27 - 30 increased glucose uptake by 1.04- to 2.85-folds at concentrations of 30 μg/mL. Further studies revealed that compounds 3 and 5 had a moderate effect on glucose transporter 4 (GLUT4) translocation activity in L6 cells. At 30 μg/mL, compound 3 significantly enhanced AMPK phosphorylation and GLUT4 expression. As a whole, compound 3 has the potential to be a drug candidate for the treatment of type 2 diabetes mellitus (T2DM).

Insulin resistance, the hallmark of type 2 diabetes mellitus (T2DM), has emerged as a pathological feature in Alzheimer's disease (AD). Given the shared role of insulin resistance in T2DM and AD, repurposing peripheral insulin sensitizers is a promising strategy to preserve neuronal insulin sensitivity and prevent AD. 1-Deoxynojirimycin (DNJ), a bioactive iminosugar, exhibited insulin-sensitizing effects in metabolic tissues and was detected in brain tissue post-oral intake. However, its impact on brain and neuronal insulin signaling has not been described. Here, we investigated the effect of DNJ treatment on insulin signaling and AD markers in insulin-resistant human SK-N-SH neuroblastoma, a cellular model of neuronal insulin resistance. Our findings show that DNJ increased the expression of insulin signaling genes and the phosphorylation status of key molecules implicated in insulin resistance (Y1146-pIRβ, S473-pAKT, S9-GSK3B) while also elevating the expression of glucose transporters Glut3 and Glut4, resulting in higher glucose uptake upon insulin stimuli. DNJ appeared to mitigate the insulin resistance-driven increase in phosphorylated tau and Aβ 1-42 levels by promoting insulin-induced phosphorylation of GSK3B (a major tau kinase) and enhancing mRNA expression of the insulin-degrading enzyme (IDE) pivotal for insulin and Aβ clearance. Overall, our study unveils probable mechanisms underlying the potential benefits of DNJ for AD, wherein DNJ attenuates tau and amyloid pathologies by reversing neuronal insulin resistance. This provides a scientific basis for expanding the use of DNJ-containing products for neuroprotective purposes and prompts further research into compounds with similar mechanisms of action., (© 2024 The Author(s). The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

Alzheimer's disease (AD) is a neurological condition that is connected with a decline in a person's memory as well as their cognitive ability. One of the key topics of AD research has been the exploration of metabolic causes. We investigated the effects of treadmill exercise and intranasal insulin on learning and memory impairment and the expression of IGF1, BDNF, and GLUT4 in hypothalamus. The animals were put into 9 groups at random. In this study, we examined the impact of insulin on spatial memory in male Wistar rats and analyzed the effects of a 4-week pretreatment of moderate treadmill exercise and insulin on the mechanisms of improved hypothalamic glucose metabolism through changes in gene and protein expression of IGF1, BDNF, and GLUT4. We discovered that rat given Aβ 25-35 had impaired spatial learning and memory, which was accompanied by higher levels of Aβ plaque burden in the hippocampus and lower levels of IGF1, BDNF, and GLUT4 mRNA and protein expression in the hypothalamus. Additionally, the administration of exercise training and intranasal insulin results in the enhancement of spatial learning and memory impairments, the reduction of plaque burden in the hippocampus, and the enhancement of the expression of IGF1, BDNF, and GLUT4 in the hypothalamus of rats that were treated with Aβ 25-35 . Our results show that the improvement of learning and spatial memory due to the improvement of metabolism and upregulation of the IGF1, BDNF, and GLUT4 pathways can be affected by pretreatment exercise and intranasal insulin., (© 2024. The Author(s).)

Background: Alterations in the placental expression of glucose transporters (GLUTs), the crucial maternal-fetal nutrient transporters, have been found in women with hyperglycemia in pregnancy (HIP). However, there is still uncertainty about the underlying effect of the high-glucose environment on placental GLUTs expression in HIP., Methods: We quantitatively evaluated the activity of mammalian target of rapamycin (mTOR) and expression of GLUTs (GLUT1, GLUT3, and GLUT4) in the placenta of women with normal pregnancies (CTRL, n = 12) and pregnant women complicated with poorly controlled type 2 diabetes mellitus (T2DM, n = 12) by immunohistochemistry. In addition, BeWo cells were treated with different glucose concentrations to verify the regulation of hyperglycemia. Then, changes in the expression of GLUTs following the activation or suppression of the mTOR pathway were also assessed using MHY1485/rapamycin (RAPA) treatment or small interfering RNA (siRNA)-mediated silencing approaches. Moreover, we further explored the alteration and potential upstream regulatory role of methyltransferase-like 3 (METTL3) when exposed to hyperglycemia., Results: mTOR, phosphorylated mTOR (p-mTOR), and GLUT1 protein levels were upregulated in the placenta of women with T2DM compared with those CTRL. In BeWo cells, mTOR activity increased with increasing glucose concentration, and the expression of GLUT1, GLUT3, and GLUT4 as well as GLUT1 cell membrane translocation were upregulated by hyperglycemia to varying degrees. Both the drug-mediated and genetic depletion of mTOR signaling in BeWo cells suppressed GLUTs expression, whereas MHY1485-induced mTOR activation upregulated GLUTs expression. Additionally, high glucose levels upregulated METTL3 expression and nuclear translocation, and decreasing METTL3 levels suppressed GLUTs expression and mTOR activity and vice versa . Furthermore, in METTL3 knockdown BeWo cells, the inhibitory effect on GLUTs expression was eliminated by activating the mTOR signaling pathway using MHY1485., Conclusion: High-glucose environment-induced upregulation of METTL3 in trophoblasts regulates the expression of GLUTs through mTOR signaling, contributing to disordered nutrient transport in women with HIP., (Copyright © 2024 The Chinese Medical Association, produced by Wolters Kluwer, Inc. under the CC-BY-NC-ND license.)

Type 2 diabetes mellitus (T2DM) remains a global health concern despite current treatment options. This study investigated the potential of Tapinanthus cordifolius (TC) leaf extract as a therapeutic agent for T2DM. T2DM was induced in rats using a high-fat diet and streptozotocin. Diabetic rats received daily oral administration of TC extract (200, 400, or 800 mg/kg) and metformin (400 mg/kg) or remained untreated for 21 days. Blood glucose levels, body weight, diabetic symptoms, oxidative stress markers, and gene expression of metabolic regulators were assessed. TC treatment significantly reduced blood glucose levels and restored body weight in diabetic rats, comparable to the effects of metformin. TC also increased antioxidant enzyme activities (SOD, GST, and CAT) and decreased lipid peroxidation in various tissues. Furthermore, TC upregulated gene expression of glucose transporter type 4 (GLUT-4) and adiponectin receptor 2 (ADIPOR-2) while downregulating pro-inflammatory cytokines TNF-α and IL-6. This study provides the first in vivo evidence supporting TC leaf extract's anti-diabetic and antioxidant efficacy. The findings suggest that TC holds promise as a natural therapeutic agent for managing T2DM through multiple mechanisms, including improved glycemic control, enhanced insulin sensitivity, and protection against oxidative stress and tissue damage. In conclusion, this study validates the ethnobotanical use of TC as an anti-diabetic agent. Further research is warranted to isolate the bioactive compounds responsible for these effects., Competing Interests: Declaration of Competing Interest This is to declare that the authors have no conflicts of interest to disclose related to the study and contents of the above manuscript submitted for publication in Biomedicine & Pharmacotherapy. The study was conceived and designed by the authors. The experiments, data analysis and interpretation were performed by the authors. The manuscript was written by the authors. The collection and use of plant materials complied with institutional guidelines. The animal study protocols were approved by the institutional research ethics committee. The authors did not receive any specific funding or grant for this research work. The laboratory equipment used was provided by the affiliated institution of the authors. The authors have no financial, consultant, institutional or other relationships that might lead to bias or conflict of interest regarding the results, discussion and conclusions presented in the manuscript. The authors declare that the contents of this manuscript are original and that any liability for claims related to the content of this manuscript shall be borne by the authors. The study results have not been previously published elsewhere nor submitted for publication to any other journal. The authors adhered to ethical guidelines and legislation in the course of the research., (Copyright © 2024 The Authors. Published by Elsevier Masson SAS.. All rights reserved.)

Background: Glucose overload drives diabetic cardiomyopathy by affecting the tricarboxylic acid pathway. However, it is still unknown how cells could overcome massive chronic glucose influx on cellular and structural level., Methods/materials: Expression profiles of hyperglycemic, glucose transporter-4 (GLUT4) overexpressing H9C2 (KE2) cardiomyoblasts loaded with 30 mM glucose (KE230L) and wild type (WT) cardiomyoblasts loaded with 30 mM glucose (WT30L) were compared using proteomics, real-time polymerase quantitative chain reaction analysis, or Western blotting, and immunocytochemistry., Results: The findings suggest that hyperglycemic insulin-sensitive cells at the onset of diabetic cardiomyopathy present complex changes in levels of structural cell-related proteins like tissue inhibitor of metalloproteases-1 (1.3 fold), intercellular adhesion molecule 1 (1.8 fold), type-IV-collagen (3.2 fold), chaperones (Glucose-Regulated Protein 78: 1.8 fold), autophagy (Autophagosome Proteins LC3A, LC3B: 1.3 fold), and in unfolded protein response (UPR; activating transcription factor 6α expression: 2.3 fold and processing: 2.4 fold). Increased f-actin levels were detectable with glucose overload by immnocytochemistry. Effects on energy balance (1.6 fold), sirtuin expression profile (Sirtuin 1: 0.7 fold, sirtuin 3: 1.9 fold, and sirtuin 6: 4.2 fold), and antioxidant enzymes (Catalase: 0.8 fold and Superoxide dismutase 2: 1.5 fold) were detected., Conclusion: In conclusion, these findings implicate induction of chronic cell distress by sustained glucose accumulation with a non-compensatory repair reaction not preventing final cell death. This might explain the chronic long lasting pathogenesis observed in developing heart failure in diabetes mellitus., (© 2024 The Authors. Diabetes, Obesity and Metabolism published by John Wiley & Sons Ltd.)

Management of chronic obesity-associated metabolic disorders is a key challenge for biomedical researchers. During chronic obesity, visceral adipose tissue (VAT) undergoes substantial transformation characterized by a unique lipid-rich hypoxic AT microenvironment which plays a crucial role in VAT dysfunction, leading to insulin resistance (IR) and type 2 diabetes. Here, we demonstrate that obese AT microenvironment triggers the release of miR-210-3p microRNA-loaded extracellular vesicles from adipose tissue macrophages, which disseminate miR-210-3p to neighboring adipocytes, skeletal muscle cells, and hepatocytes through paracrine and endocrine actions, thereby influencing insulin sensitivity. Moreover, EVs collected from Dicer-silenced miR-210-3p-overexpressed bone marrow-derived macrophages induce glucose intolerance and IR in lean mice. Mechanistically, miR-210-3p interacts with the 3'-UTR of GLUT4 mRNA and silences its expression, compromising cellular glucose uptake and insulin sensitivity. Therapeutic inhibition of miR-210-3p in VAT notably rescues high-fat diet-fed mice from obesity-induced systemic glucose intolerance. Thus, targeting adipose tissue macrophage-specific miR-210-3p during obesity could be a promising strategy for managing IR and type 2 diabetes., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Skeletal muscle mediates the beneficial effects of exercise, thereby improving insulin sensitivity and reducing the risk for type 2 diabetes. Current human skeletal muscle models in vitro are incapable of fully recapitulating its physiological functions especially muscle contractility. By supplementation of insulin-like growth factor 1 (IGF1), a growth factor secreted by myofibers in vivo, we aimed to overcome these limitations. We monitored the differentiation process starting from primary human CD56-positive myoblasts in the presence/absence of IGF1 in serum-free medium in daily collected samples for 10 days. IGF1-supported differentiation formed thicker multinucleated myotubes showing physiological contraction upon electrical pulse stimulation (EPS) following day 6 . Myotubes without IGF1 were almost incapable of contraction. IGF1 treatment shifted the proteome toward skeletal muscle-specific proteins that contribute to myofibril and sarcomere assembly, striated muscle contraction, and ATP production. Elevated PPARGC1A, MYH7, and reduced MYH1/2 suggest a more oxidative phenotype further demonstrated by higher abundance of proteins of the respiratory chain and elevated mitochondrial respiration. IGF1-treatment also upregulated glucose transporter (GLUT)4 and increased insulin-dependent glucose uptake compared with myotubes differentiated without IGF1. To conclude, addition of IGF1 to serum-free medium significantly improves the differentiation of human myotubes that showed enhanced myofibril formation, response to electrical pulse stimulation, oxidative respiratory capacity, and glucose metabolism overcoming limitations of previous standards. This novel protocol enables investigation of muscular exercise on a molecular level. NEW & NOTEWORTHY Human skeletal muscle models are highly valuable to study how exercise prevents type 2 diabetes without invasive biopsies. Current models did not fully recapitulate the function of skeletal muscle especially during exercise. By supplementing insulin-like growth factor 1 (IGF1), the authors developed a functional human skeletal muscle model characterized by inducible contractility and increased oxidative and insulin-sensitive metabolism. The novel protocol overcomes the limitations of previous standards and enables investigation of exercise on a molecular level.

" Ganghwal " is a widely used herbal medicine in Republic of Korea, but it has not been reported as a treatment strategy for obesity and diabetes within adipocytes. In this study, we determined that Ostericum koreanum extract (OKE) exerts an anti-obesity effect by inhibiting adipogenesis and an anti-diabetic effect by increasing the expression of genes related to glucose uptake in adipocytes and inhibiting α-glucosidase activity. 3T3-L1 preadipocytes were differentiated for 8 days in methylisobutylxanthine, dexamethasone, and insulin medium, and the effect of OKE was confirmed by the addition of 50 and 100 µg/mL of OKE during the differentiation process. This resulted in a reduction in lipid accumulation and the expression of PPARγ (Peroxisome proliferator-activated receptor γ) and C/EBPα (CCAAT enhancer binding protein α). Significant activation of AMPK (AMP-activated protein kinase), increased expression of GLUT4 (Glucose Transporter Type 4), and inhibition of α-glucosidase activity were also observed. These findings provide the basis for the anti-obesity and anti-diabetic effects of OKE. In addition, OKE has a significant antioxidant effect. This study presents OKE as a potential natural product-derived material for the treatment of patients with metabolic diseases such as obesity- and obesity-induced diabetes., Competing Interests: The authors declare no conflicts of interest.

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., (© 2024 Wiley‐VCH GmbH.)

Skeletal muscle is the largest metabolic tissue responsible for systemic glucose handling. Glucose uptake into skeletal tissue is highly dynamic and delicately regulated, in part through the controlled expression and subcellular trafficking of multiple types of glucose transporters. Although the roles of GLUT4 in skeletal muscle metabolism are well established, the physiological significance of other, seemingly redundant, glucose transporters remain incompletely understood. Nonetheless, recent studies have shed light on the roles of several glucose transporters, such as GLUT1 and GLUT10, in skeletal muscle. Mice experiments suggest that GLUT10 could be a novel player in skeletal muscle metabolism in the context of mechanical overload, which is in line with the meta-analytical results of gene expression changes after resistance exercise in humans. Herein we discuss the knowns, unknowns, and implications of these recent findings., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., (© 2023 Federation of European Biochemical Societies.)

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., Competing Interests: Conflict of interest The authors declare that they have no conflicts of interest with the contents of this article., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Ethnopharmacological Relevance: Cola nitida (Vent.) Schott & Endl. are among the common medicinal plants employed in traditional medicine for treating diabetes and its complications., Aim of the Study: The present study investigated the effect of Cola nitida infusion on the expression of key genes involved in insulin signaling vis-à-vis Insulin receptor substrate 1 (IRS-1), tumor protein P53 gene, glucose transporter type 4 (GLUT4), phosphoinositide 3-kinases (PI3K) and B-cell lymphoma-2 (BCL2) in skeletal muscles of type 2 diabetic (T2D) rats., Methods: Type 2 diabetic rats were administered C. nitida infusion at low and high doses (150 and 300 mg/kg bodyweight, respectively), while a high dose of the infusion was also administered to a normal toxicological group. Metformin served as the standard antidiabetic drug. The rats were sacrificed at the end of the experimental period. Their psoas muscles were harvested and assayed for the expressions of IRS1, p53, GLUT4, PI3K and BCL2. The studied genes were further subjected to enrichment analysis using the ShinyGO 0.76 online software., Results: Induction of T2D upregulated the expressions of IRS-1, p53, PI3K and BCL2 in psoas muscles, while concomitantly downregulating GLUT4 expression. These expressions were significantly reversed in type 2 diabetic rats treated with C. nitida infusion, and the results were statistically significant compared to metformin. Gene enrichment analysis revealed that the genes were linked to intrinsic pathways and biological processes involved in insulin resistance. The infusion further improved muscle glucose uptake, ex vivo. Molecular docking and molecular dynamics stimulation of C. nitida infusion phytoconstituents, caffeine and theobromine with IRS-1, p53, GLUT4, PI3K and BCL2 revealed a strong binding affinity as evident by the RMSD and RMSF values., Conclusion: These results indicate the potentials of C. nitida infusion to improve glucose homeostasis in skeletal muscles of type 2 diabetic rats., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 The Authors. Published by Elsevier B.V. All rights reserved.)

Ethnopharmacological Relevance: Cortex fraxini (also known as qinpi)-the bark of Fraxinus rhynchophylla Hance (Oleaceae)-is widely used as a Chinese traditional medicinal for its anti-inflammatory and anti-hyperuricemic activities., Aim of the Study: Obesity-induced insulin resistance (IR) is driving the rising incidence of type 2 diabetes mellitus and is related to pathological adipose tissue remodeling. Esculin, a major active component of Cortex fraxini, has anti-diabetic effects. However, whether esculin improves obesity-induced IR by regulating adipose tissue remodeling is unclear. The aims of the present study were to assess the effects of esculin on obesity-induced IR and to explore the underlying mechanisms., Materials and Methods: Obese IR C57BL/6J mice were treated with esculin (40 or 80 mg/kg/day) for 4 weeks. Oral glucose tolerance tests were used to assess insulin sensitivity. Histological analyses were performed to analyze the number and size distribution of adipocytes. Glucose uptake was assessed using 2-NBDG., Results: Esculin had no effect on body weight gain but reduced fasting blood glucose, improved oral glucose tolerance, and increased insulin sensitivity. Esculin reduced adipocyte size and the expression levels of collagen 4A1 and tumor necrosis factor α and increased the number of adipocytes and the expression of vascular endothelial growth factor A. Esculin promoted the differentiation of 3T3-L1 cells and upregulated the mRNA expression of CCAAT/enhancer-binding protein α and peroxisome proliferator-activated receptor-γ, activated the insulin receptor substrate 1 (IRS1)/phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway, and enhanced the translocation of glucose transporter type 4 (GLUT4) and glucose uptake in adipocytes treated with palmitic acid., Conclusions: These data suggest that esculin increases insulin sensitivity by improving adipose tissue remodeling and activating the IRS1/PI3K/AKT/GLUT4 pathway., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Glucose transporter-4 (GLUT4) represents the major glucose transporter isoform responsible for glucose uptake into insulin-sensitive cells, primarily in skeletal muscle and adipose tissues. In insulin-resistant conditions, such as type 2 diabetes mellitus, GLUT4 expression and/or translocation to the cell plasma membrane is reduced, compromising cell energy metabolism. Therefore, the use of synthetic or naturally occurring molecules able to stimulate GLUT4 expression represents a good tool for alternative treatments of insulin resistance. The present study aimed to investigate the effects of essential oils (EOs) derived from Pinus spp. ( P. nigra and P. radiata ) and of their main terpenoid constituents (α- and β-pinene) on the expression/translocation of GLUT4 in myoblast C2C12 murine cells. For this purpose, the chemical profiles of the EOs were first analyzed through gas chromatography-mass spectrometry (GC-MS). Cell viability was assessed by MTT assay, and GLUT4 expression/translocation was evaluated through RT-qPCR and flow cytometry analyses. The results showed that only the P. nigra essential oil (PnEO) and α-pinene can increase the transcription of the Glut4/Scl2a4 gene, resulting in a subsequent increase in the amount of GLUT4 produced and its plasma membrane localization. Moreover, the PnEO or α-pinene can induce Glut4 expression both during myogenesis and in myotubes. In summary, the PnEO and α-pinene emulate insulin's effect on the GLUT4 transporter expression and its translocation to the muscle cell surface., Competing Interests: The authors declare no conflicts of interest.

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.

The ability of insulin to stimulate glucose transport in muscle and fat cells is mediated by the regulated delivery of intracellular vesicles containing glucose transporter-4 (GLUT4) to the plasma membrane, a process known to be defective in disease such as Type 2 diabetes. In the absence of insulin, GLUT4 is sequestered in tubules and vesicles within the cytosol, collectively known as the GLUT4 storage compartment. A subset of these vesicles, known as the 'insulin responsive vesicles' are selectively delivered to the cell surface in response to insulin. We have previously identified Syntaxin16 (Sx16) and its cognate Sec1/Munc18 protein family member mVps45 as key regulatory proteins involved in the delivery of GLUT4 into insulin responsive vesicles. Here we show that mutation of a key residue within the Sx16 N-terminus involved in mVps45 binding, and the mutation of the Sx16 binding site in mVps45 both perturb GLUT4 sorting, consistent with an important role of the interaction of these two proteins in GLUT4 trafficking. We identify Threonine-7 (T7) as a site of phosphorylation of Sx16 in vitro . Mutation of T7 to D impairs Sx16 binding to mVps45 in vitro and overexpression of T7D significantly impaired insulin-stimulated glucose transport in adipocytes. We show that both AMP-activated protein kinase (AMPK) and its relative SIK2 phosphorylate this site. Our data suggest that Sx16 T7 is a potentially important regulatory site for GLUT4 trafficking in adipocytes., Competing Interests: Gwyn W Gould is an Academic Editor for PeerJ., (©2023 Bremner et al.)

Objectives: Adipogenesis is the fat cell formation process regulated by peroxisome proliferator-activated receptors ( PPARγ ). The insulin-responsive glucose transporter 4 ( GLUT4 ) has a major role in glucose uptake and metabolism in insulin target tissues (i.e., adipose and muscle cells). The interplay between PPARγ and GLUT4 is essential for proper glucose homeostasis. This study aimed to isolate, elucidate, and investigate the effect of an isolated compound from Penicillium citrinum XT6 on adipogenesis, PPARγ , and GLUT4 expression in 3T3-L1 adipocytes., Methods: The isolated compound was determined by analyzing spectroscopic data (LC-MS, FT-IR, Spectrophotometry UV-Vis, and NMR). The adipogenesis activity of the isolated compound in 3T3-L1 cells was determined by the Oil Red O staining method. RT-PCR was used to analyze the gene expression of PPARγ and GLUT4., Results: Di-(2-ethylhexyl)-phthalate (DEHP) was the isolated compound from P.citrinum XT6. The results revealed adipogenesis stimulation and inhibition, as well as PPARγ and GLUT4 expressions ., Conclusions: DEHP showed a non-monotonic dose-response (NMDR) effect on adipogenesis and PPARγ and GLUT4 expression. It is the first study that reveals DEHP's NMDR effects on lipid and glucose metabolism in adipocytes., (© 2023 Walter de Gruyter GmbH, Berlin/Boston.)

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 β-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.

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., (© 2023 The Authors. The FASEB Journal published by Wiley Periodicals LLC on behalf of Federation of American Societies for Experimental Biology.)

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.

Background: Hyperglycemia-induced oxidative stress accelerates the process of apoptosis in tissues. Dilleniaindica (DI) is a medicinal plant, and its fruit contains many therapeutic properties. The therapeutic activity of the Methanolic Fruit Extract (MFE) of DI in attenuating oxidative stress and apoptosis in the liver and kidney tissues of alloxan-induced diabetic mice was analyzed in the present study., Methods: High-Performance Thin Layer Chromatography (HPTLC) profiling of MFE was conducted. GLUT4 protein expression analysis and lipid peroxidation assays were conducted to check for MFE effect by administering in diabetic mice. An ultrastructural study was conducted for both the tissues. In apoptotic studies, the TUNEL assay and apoptotic protein expression analysis was conducted., Results: High-Performance Thin Layer Chromatography (HPTLC) profiling of MFE showed the presence of two crucial antioxidants, ascorbic acid, and naringenin. In GLUT-4 protein expression analysis, MFE suppresses hyperglycemia by upregulating GLUT4 protein expression. Lipid peroxidation assay showed a decrease in malondialdehyde (MDA) upon MFE administration in diabetic mice. An ultrastructural study was conducted, and MFE was found to restore cellular alterations in diabetic tissues. In apoptotic studies, the TUNEL assay shows that MFE treatment showed fewer apoptotic cells than the diabetic group. The study also observed decreased caspase 3 protein expression and increased Bcl-2 protein expression., Conclusions: Therefore, it is inferred from the study that MFE can exert a protective effect by suppressing hyperglycemia and modulating oxidative stress and apoptosis in alloxan-administered diabetic mice., Competing Interests: The authors declare no conflict of interest., (© 2023 The Author(s). Published by IMR Press.)

Insulin resistance (IR) is one of the hallmarks of heart failure (HF). Abnormalities in skeletal muscle (SM) metabolism have been identified in patients with HF. However, the underlying mechanisms of IR development in SM in HF are poorly understood. Herein, we hypothesize that HF upregulates miR-133b in SM and in turn alters glucose metabolism and the propensity toward IR. Mitochondria isolated from SM of mice with HF induced by transverse aortic constriction (TAC) showed lower respiration and downregulation of muscle-specific components of the tricarboxylic acid (TCA) cycle, AMP deaminase 1 (AMPD1), and fumarate compared with those from control animals. RNA-Seq and subsequent qPCR validation confirmed upregulation of SM-specific microRNA (miRNA), miR-133b, in TAC versus sham animals. miR-133b overexpression alone resulted in significantly lower mitochondrial respiration, cellular glucose uptake, and glycolysis along with lower ATP production and cellular energy reserve compared with the scramble (Scr) in C2C12 cells. miR-133b binds to the 3'-untranslated region (UTR) of KLF15 , the transcription factor for the insulin-sensitive glucose transporter, GLUT4. Overexpression of miR-133b lowers GLUT4 and lowers pAkt in presence of insulin in C2C12 cells. Finally, lowering miR-133b in primary skeletal myocytes isolated from TAC mice using antagomir-133b reversed the changes in KLF15, GLUT4, and AMPD1 compared with the scramble-transfected myocytes. Taken together, these data demonstrate a role for SM miR-133b in altered glucose metabolism in HF and suggest the therapeutic potential in HF to improve glucose uptake and glycolysis by restoring GLUT4 abundance. The data uncover a novel mechanism for IR and ultimately SM metabolic abnormalities in patients with HF. NEW & NOTEWORTHY Heart failure is associated with systemic insulin resistance and abnormalities in glucose metabolism but the underlying mechanisms are poorly understood. In the skeletal muscle, the major peripheral site of glucose utilization, we observe an increase in miR-133b in heart failure mice, which reduces the insulin-sensitive glucose transporter (GLUT4), glucose uptake, and metabolism in C2C12 and in myocytes. The antagomir for miR-133b restores GLUT4 protein and markers of metabolism in skeletal myocytes from heart failure mice demonstrating that miR-133b is an exciting target for systemic insulin resistance in heart failure and an important player in the cross talk between the heart and the periphery in the heart failure syndrome.

De novo lipogenesis (DNL) in visceral adipose tissue (VAT) is associated with systemic insulin sensitivity. DNL in VAT is regulated through ChREBP activity and glucose uptake through Glut4 (encoded by Slc2a4 ). Slc2a4 expression, ChREBP activity, and DNL are decreased in obesity, the underlying cause however remains unidentified. We hypothesize that increased DNA methylation in an enhancer region of Slc2a4 decreases Slc2a4 expression in obesity and insulin resistance. We found that SLC2A4 expression in VAT of morbidly obese subjects with high HbA1c (>6.5%, n = 35) is decreased, whereas DNA methylation is concomitantly increased compared to morbidly obese subjects with low HbA1c (≤6.5%, n = 65). In diet-induced obese (DIO) mice, DNA methylation of Slc2a4 persistently increases with the onset of obesity and insulin resistance, while gene expression progressively decreases. The regulatory impact of DNA methylation in the investigated enhancer region on SLC2A4 gene expression was validated with a reporter gene assay. Additionally, treatment of 3T3 pre-adipocytes with palmitate/oleate during differentiation decreased DNA methylation and increased Slc2a4 expression. These findings highlight a potential regulation of Slc2a4 by DNA methylation in VAT, which is induced by fatty acids and may play a role in the progression of obesity and insulin resistance in humans.

The decreased expression and dysfunction of glucose transporter 4 (GLUT4), the insulin-responsive glucose transporter, are closely related to the occurrence of insulin resistance (IR). To improve the expression of GLUT4 may represent a promising strategy to prevent and treat IR and type 2 diabetes (T2DM). Here, we demonstrate that the natural compound tectorigenin (TG) enhances GLUT4 expression, glucose uptake and insulin responsiveness via activating AMP-activated protein kinase (AMPK)/myocyte enhancer factor 2 (MEF2) signaling in both normal and IR skeletal muscle cells and tissues. Accordingly, prophylactic and therapeutic uses of TG can significantly ameliorate IR and hyperglycemia in T2DM mice. Mechanistically, we identify protein kinase A catalytic subunit α (PKACα) as the target of TG to increase GLUT4 expression and TG-PKACα binding promotes the dissociation of PKACα from the regulatory subunits, leading to the activation of PKA/AMPK signaling. PKACα knockdown in local quadriceps muscles almost completely abolished the therapeutic effects of TG on IR and T2DM, as well as the enhancement on AMPK signaling and GLUT4 expression in skeletal muscle. This study supports TG as a new drug candidate to treat IR and its related diseases, but also enriches our knowledge of PKA signaling in glucose metabolism in skeletal muscle., Competing Interests: Competing Interests: The authors have declared that no competing interest exists., (© The author(s).)

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, pAkt Thr308 and Serine-473, pAkt Ser473 ; AS160 phosphorylation on Serine-588, pAS160 Ser588 , and Threonine-642, pAS160 Thr642 ). 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 pAkt Thr308 with 500 µU/mL insulin; ( iv ) WT-CR versus WT-AL rats did not differ for pAkt Ser473 , pAS160 Ser588 , or pAS160 Thr642 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.

Background: Type 2 Diabetes mellitus (T2DM) is a major risk factor for cardiovascular disease and associated with poor outcome after myocardial infarction (MI). In T2DM, cardiac metabolic flexibility, i.e. the switch between carbohydrates and lipids as energy source, is disturbed. The RabGTPase-activating protein TBC1D4 represents a crucial regulator of insulin-stimulated glucose uptake in skeletal muscle by controlling glucose transporter GLUT4 translocation. A human loss-of-function mutation in TBC1D4 is associated with impaired glycemic control and elevated T2DM risk. The study's aim was to investigate TBC1D4 function in cardiac substrate metabolism and adaptation to MI., Methods: Cardiac glucose metabolism of male Tbc1d4-deficient (D4KO) and wild type (WT) mice was characterized using in vivo [ 18 F]-FDG PET imaging after glucose injection and ex vivo basal/insulin-stimulated [ 3 H]-2-deoxyglucose uptake in left ventricular (LV) papillary muscle. Mice were subjected to cardiac ischemia/reperfusion (I/R). Heart structure and function were analyzed until 3 weeks post-MI using echocardiography, morphometric and ultrastructural analysis of heart sections, complemented by whole heart transcriptome and protein measurements., Results: Tbc1d4-knockout abolished insulin-stimulated glucose uptake in ex vivo LV papillary muscle and in vivo cardiac glucose uptake after glucose injection, accompanied by a marked reduction of GLUT4. Basal cardiac glucose uptake and GLUT1 abundance were not changed compared to WT controls. D4KO mice showed mild impairments in glycemia but normal cardiac function. However, after I/R D4KO mice showed progressively increased LV endsystolic volume and substantially increased infarction area compared to WT controls. Cardiac transcriptome analysis revealed upregulation of the unfolded protein response via ATF4/eIF2α in D4KO mice at baseline. Transmission electron microscopy revealed largely increased extracellular matrix (ECM) area, in line with decreased cardiac expression of matrix metalloproteinases of D4KO mice., Conclusions: TBC1D4 is essential for insulin-stimulated cardiac glucose uptake and metabolic flexibility. Tbc1d4-deficiency results in elevated cardiac endoplasmic reticulum (ER)-stress response, increased deposition of ECM and aggravated cardiac damage following MI. Hence, impaired TBC1D4 signaling contributes to poor outcome after MI., (© 2023. The Author(s).)

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.

We observed that exercise and calorie restriction reduced the body weight and blood glucose levels, concurrently improving insulin resistance and glucose tolerance in obese/diabetic model KKAy mice. Analysis of gene expression in the skeletal muscle showed enhanced mRNA levels of GLUT4 (glucose uptake), ATGL (lipolytic enzyme), and slow-twitch myosin heavy chain, which may contribute to the antiobesity and antidiabetic effects., (© The Author(s) 2022. Published by Oxford University Press on behalf of Japan Society for Bioscience, Biotechnology, and Agrochemistry.)

Adipocytes are key to metabolic regulation, exhibiting insulin-stimulated glucose transport that is underpinned by the insulin-stimulated delivery of glucose transporter type 4 (SLC2A4, also known and hereafter referred to as GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, and increase cell surface GLUT4 levels. Adipocytokines, such as adiponectin, are secreted via a similar mechanism. We used genome editing to knock out syntaxin-4, a protein reported to mediate fusion between GLUT4-containing vesicles and the plasma membrane in 3T3-L1 adipocytes. Syntaxin-4 knockout reduced insulin-stimulated glucose transport and adiponectin secretion by ∼50% and reduced GLUT4 levels. Ectopic expression of haemagglutinin (HA)-tagged GLUT4 conjugated to GFP showed that syntaxin-4-knockout cells retain significant GLUT4 translocation capacity, demonstrating that syntaxin-4 is dispensable for insulin-stimulated GLUT4 translocation. Analysis of recycling kinetics revealed only a modest reduction in the exocytic rate of GLUT4 in knockout cells, and little effect on endocytosis. These analyses demonstrate that syntaxin-4 is not always rate limiting for GLUT4 delivery to the cell surface. In sum, we show that syntaxin-4 knockout results in reduced insulin-stimulated glucose transport, depletion of cellular GLUT4 levels and inhibition of adiponectin secretion but has only modest effects on the translocation capacity of the cells. This article has an associated First Person interview with Hannah L. Black and Rachel Livingstone, joint first authors of the paper., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2022. Published by The Company of Biologists Ltd.)

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., (© 2022 Wiley Periodicals LLC.)

Glucose transporter 1 (GLUT1) is believed to solely mediate basal (insulin-independent) glucose uptake in skeletal muscle; yet recent work has demonstrated that mechanical overload, a model of resistance exercise training, increases muscle GLUT1 levels. The primary objective of this study was to determine if GLUT1 is necessary for basal or overload-stimulated muscle glucose uptake. Muscle-specific GLUT1 knockout (mGLUT1KO) mice were generated and examined for changes in body weight, body composition, metabolism, systemic glucose regulation, muscle glucose transporters, and muscle [ 3 H]-2-deoxyglucose uptake ± the GLUT1 inhibitor BAY-876. [ 3 H]-hexose uptake ± BAY-876 was also examined in HEK293 cells-expressing GLUT1-6 or GLUT10. mGLUT1KO mice exhibited no impairments in body weight, lean mass, whole body metabolism, glucose tolerance, basal or overload-stimulated muscle glucose uptake. There was no compensation by the insulin-responsive GLUT4. In mGLUT1KO mouse muscles, overload stimulated higher expression of mechanosensitive GLUT6, but not GLUT3 or GLUT10. In control and mGLUT1KO mouse muscles, 0.05 µM BAY-876 impaired overload-stimulated, but not basal glucose uptake. In the GLUT-HEK293 cells, BAY-876 inhibited glucose uptake via GLUT1, GLUT3, GLUT4, GLUT6, and GLUT10. Collectively, these findings demonstrate that GLUT1 does not mediate basal muscle glucose uptake and suggest that a novel glucose transport mechanism mediates overload-stimulated glucose uptake.

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., Competing Interests: Declaration of competing interest The authors declare that they have no competing interests., (Copyright © 2022 The Italian Diabetes Society, the Italian Society for the Study of Atherosclerosis, the Italian Society of Human Nutrition and the Department of Clinical Medicine and Surgery, Federico II University. Published by Elsevier B.V. All rights reserved.)

Insulin-induced GLUT4 translocation to the plasma membrane in muscle and adipocytes is crucial for whole-body glucose homeostasis. Currently, GLUT4 trafficking assays rely on overexpression of tagged GLUT4. Here we describe a high-content imaging platform for studying endogenous GLUT4 translocation in intact adipocytes. This method enables high fidelity analysis of GLUT4 responses to specific perturbations, multiplexing of other trafficking proteins and other features including lipid droplet morphology. Using this multiplexed approach we showed that Vps45 and Rab14 are selective regulators of GLUT4, but Trarg1 , Stx6 , Stx16 , Tbc1d4 and Rab10 knockdown affected both GLUT4 and TfR translocation. Thus, GLUT4 and TfR translocation machinery likely have some overlap upon insulin-stimulation. In addition, we identified Kif13A, a Rab10 binding molecular motor, as a novel regulator of GLUT4 traffic. Finally, comparison of endogenous to overexpressed GLUT4 highlights that the endogenous GLUT4 methodology has an enhanced sensitivity to genetic perturbations and emphasises the advantage of studying endogenous protein trafficking for drug discovery and genetic analysis of insulin action in relevant cell types., (© 2022 Diaz-Vegas et al.)

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.