Effects of endurance training under calorie restriction on energy substrate metabolism in mouse skeletal muscle and liver.

We investigated whether calorie restriction (CR) enhances metabolic adaptations to endurance training (ET). Ten-week-old male Institute of Cancer Research (ICR) mice were fed ad libitum or subjected to 30% CR. The mice were subdivided into sedentary and ET groups. The ET group performed treadmill running (20-25 m/min, 30 min, 5 days/week) for 5 weeks. We found that CR decreased glycolytic enzyme activity and monocarboxylate transporter (MCT) 4 protein content, while enhancing glucose transporter 4 protein content in the plantaris and soleus muscles. Although ET and CR individually increased citrate synthase activity in the plantaris muscle, the ET-induced increase in respiratory chain complex I protein content was counteracted by CR. In the soleus muscle, mitochondrial enzyme activity and protein levels were increased by ET, but decreased by CR. It has been suggested that CR partially interferes with skeletal muscle adaptation to ET.

[Jiaotaiwan improves brain glucose metabolism in a mouse model of Alzheimer's disease by activating the PI3K/AKT signaling pathway].

OBJECTIVE: To investigate the effect of Jiaotaiwan on brain insulin-PI3K/AKT pathway in a mouse model of Alzheimer's disease (AD). METHODS: Fifty 3-month-old male APP/PS1 double transgenic mice were randomized into AD model group, low-, medium- and high-dose Jiaotaiwan treatment groups, and donepezil treatment group. Cognitive functions of the mice were assessed using water maze and open field tests, and neuronal pathologies were observed with HE staining and Nissl staining; immunohistochemistry was used to detect amyloid Aß deposition in the brain. Fasting serum insulin levels of the mice were measured, and the expressions of Aß42, insulin-PI3K/AKT pathway components and downstream glucose transporters in the brain tissue were detected with RT-qPCR and Western blotting. RESULTS: The AD mouse models exhibited obvious impairment of learning and memory abilities, significantly reduced hippocampal neurons, and obvious Aß amyloid plaques in the brain tissue with increased Aß42 protein expression (P < 0.05) and insulin resistance index, decreased hippocampal PI3K expressions, lowered expressions of AKT and InR, reduced expressions of GLUT1, GLUT3, and GLUT4, and increased expression of GSK3ß in both the hippocampus and cortex. Treatment with Jiaotaiwan and donepezil both effectively improved memory ability of the mouse models, increased the number of hippocampal neurons, reduced Aß amyloid plaques and increased the expressions of PI3K, AKT, InR, GLUT1, GLUT3 and GLUT4 in the hippocampus and cortex. CONCLUSION: Jiaotaiwan improves learning and memory abilities of APP/PS1 double transgenic mice and delay the development of AD by activating the PI3K/AKT pathway and regulating the expression levels of its downstream GLUTs in the brain.

Metformin exposure during pregnancy and lactation affects offspring's long-term body weight and adipose tissue mass independent of the maternal metabolic state.

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.

Protein kinase-D1 and downstream signaling mechanisms involved in GLUT4 translocation in cardiac muscle.

The Ser/Thr kinase protein kinase-D1 (PKD1) is involved in induction of various cell physiological processes in the heart such as myocellular hypertrophy and inflammation, which may turn maladaptive during long-term stimulation. Of special interest is a key role of PKD1 in the regulation of cardiac substrate metabolism. Glucose and fatty acids are the most important substrates for cardiac energy provision, and the ratio at which they are utilized determines the health status of the heart. Cardiac glucose uptake is mainly regulated by translocation of the glucose transporter GLUT4 from intracellular stores (endosomes) to the sarcolemma, and fatty acid uptake via a parallel translocation of fatty acid transporter CD36 from endosomes to the sarcolemma. PKD1 is involved in the regulation of GLUT4 translocation, but not CD36 translocation, giving it the ability to modulate glucose uptake without affecting fatty acid uptake, thereby altering the cardiac substrate balance. PKD1 would therefore serve as an attractive target to combat cardiac metabolic diseases with a tilted substrate balance, such as diabetic cardiomyopathy. However, PKD1 activation also elicits cardiac hypertrophy and inflammation. Therefore, identification of the events upstream and downstream of PKD1 may provide superior therapeutic targets to alter the cardiac substrate balance. Recent studies have identified the lipid kinase phosphatidylinositol 4-kinase IIIß (PI4KIIIß) as signaling hub downstream of PKD1 to selectively stimulate GLUT4-mediated myocardial glucose uptake without inducing hypertrophy. Taken together, the PKD1 signaling pathway serves a pivotal role in cardiac glucose metabolism and is a promising target to selectively modulate glucose uptake in cardiac disease.

Electrochemiluminescent evaluation of GLUT4 expression in rat adipocytes induced by Ganoderma lucidum polysaccharides.

Glucose transporter 4 (GLUT4) directly facilitates cellular uptake of glucose and plays a crucial role in mammalian adipose tissue glucose metabolism. In this work, we constructed a cytosensor for sensitive electrochemiluminescence (ECL) detection of GLUT4 in rat adipocytes (RA cells). A carbon nanotube sponge (CNTSP) was selected to fabricate a permeable electrode to overcome the steric hindrance of cells on the electrode. The expression of GLUT4 after treatment with Ganoderma lucidum polysaccharide (GLP) was assessed by analyzing the luminescence emitted from cell-surface ECL probes. Our preliminary results suggest that GLP promote the expression of GLUT4, thereby enhancing the uptake of the fluorescent glucose 2-NBDG. Treatment with GLP affected GLUT4 expression in RA cells in a dose-dependent manner. Additionally, the ECL cytosensor contributes to the development of ECL imaging of receptors on the cell surface for clinical drug evaluation.

Acetoacetate and d- and l-ß-hydroxybutyrate have distinct effects on basal and insulin-stimulated glucose uptake in L6 skeletal muscle cells.

Ketone bodies (acetoacetate and ß-hydroxybutyrate) are oxidized in skeletal muscle mainly during fasting as an alternative source of energy to glucose. Previous studies suggest that there is a negative relationship between increased muscle ketolysis and muscle glucose metabolism in mice with obesity and/or type 2 diabetes. Therefore, we investigated the connection between increased ketone body exposure and muscle glucose metabolism by measuring the effect of a 3-h exposure to ketone bodies on glucose uptake in differentiated L6 myotubes. We showed that exposure to acetoacetate at a typical concentration (0.2 mM) resulted in increased basal glucose uptake in L6 myotubes, which was dependent on increased membrane glucose transporter type 4 (GLUT4) translocation. Basal and insulin-stimulated glucose uptake was also increased with a concentration of acetoacetate reflective of diabetic ketoacidosis or a ketogenic diet (1 mM). We found that ß-hydroxybutyrate had a variable effect on basal glucose uptake: a racemic mixture of the two ß-hydroxybutyrate enantiomers (d and l) appeared to decrease basal glucose uptake, while 3 mM d-ß-hydroxybutyrate alone increased basal glucose uptake. However, the effects of the ketone bodies individually were not observed when acetoacetate was present in combination with ß-hydroxybutyrate. These results provide insight that will help elucidate the effect of ketone bodies in the context of specific metabolic diseases and nutritional states (e.g., type 2 diabetes and ketogenic diets).NEW & NOTEWORTHY A limited number of studies investigate the effect of ketone bodies at concentrations reflective of both typical fasting and ketoacidosis. We tested a mix of physiologically relevant concentrations of ketone bodies, which allowed us to highlight the differential effects of d- and l-ß-hydroxybutyrate and acetoacetate on skeletal muscle cell glucose uptake. Our findings will assist in better understanding the mechanisms that contribute to muscle insulin resistance and provide guidance on recommendations regarding ketogenic diets.

Preparation of a Novel Oat ß-Glucan-Chromium(III) Complex and Its Hypoglycemic Effect and Mechanism.

This study synthesized a novel oat ß-glucan (OBG)-Cr(III) complex (OBG-Cr(III)) and explored its structure, inhibitory effects on α-amylase and α-glucosidase, and hypoglycemic activities and mechanism in vitro using an insulin-resistant HepG2 (IR-HepG2) cell model. The Cr(III) content in the complex was found to be 10.87%. The molecular weight of OBG-Cr(III) was determined to be 7.736 × 104 Da with chromium ions binding to the hydroxyl groups of OBG. This binding resulted in the increased asymmetry and altered spatial conformation of the complex along with significant changes in morphology and crystallinity. Our findings demonstrated that OBG-Cr(III) exhibited inhibitory effects on α-amylase and α-glucosidase. Furthermore, OBG-Cr(III) enhanced the insulin sensitivity of IR-HepG2 cells, promoting glucose uptake and metabolism more efficiently than OBG alone. The underlying mechanism of its hypoglycemic effect involved the modulation of the c-Cbl/PI3K/AKT/GLUT4 signaling pathway, as revealed by Western blot analysis. This research not only broadened the applications of OBG but also positioned OBG-Cr(III) as a promising Cr(III) supplement with enhanced hypoglycemic benefits.

IGF1 promotes human myotube differentiation toward a mature metabolic and contractile phenotype.

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.

GPR30 selective agonist G-1 induced insulin resistance in ovariectomized mice on high fat diet and its mechanism.

BACKGROUND: Previous in vivo and in vitro studies have demonstrated that estrogen receptor agonist G-1 regulates glucose and lipid metabolism. This study focused on the effects of G-1 on cardiometabolic syndrome and anti-obesity under a high fat diet (HFD). METHODS: Bilateral ovariectomized female mice were fed an HFD for 6 weeks, and treated them with G-1. A cardiomyocyte insulin resistance model was used to simulate the in vivo environment. The main outcome measures were blood glucose, body weight, and serum insulin levels to assess insulin resistance, while cardiac function and degree of fibrosis were assessed by cardiac ultrasound and pathological observations. We also examined the expression of p-AMPK, p-AKT, and GLUT4 in mice hearts and in vitro models to explore the mechanism by which G-1 regulates insulin signaling. RESULTS: G-1 reduced body weight in mice on an HFD, but simultaneously increased blood glucose and promoted insulin resistance, resulting in myocardial damage. This damage included disordered cardiomyocytes, massive accumulation of glycogen, extensive fibrosis of the heart, and thickening of the front and rear walls of the left ventricle. At the molecular level, G-1 enhances gluconeogenesis and promotes glucose production by increasing the activity of pyruvate carboxylase (PC) while inhibiting GLUT4 translocation via the AMPK/TBC1D1 pathway, thereby limiting glucose uptake. CONCLUSION: Despite G-1's the potential efficacy in weight reduction, the concomitant induction of insulin resistance and cardiac impairment in conjunction with an HFD raises significant concerns. Therefore, comprehensive studies of its safety profile and effects under specific conditions are essential prior to clinical use.

Anti-Obesity and Anti-Diabetic Effects of Ostericum koreanum (Ganghwal) Extract.

"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.

Yishen Huatan Huoxue decoction and quercetin ameliorate decidualization dysfunction in polycystic ovary syndrome: A comprehensive investigation combining clinical trial and experimental studies.

Polycystic ovary syndrome (PCOS) is a common gynecological endocrine disorder characterized by a complex pathogenesis and limited treatment options. Yishen Huatan and Huoxue decoction (YHHD), as a traditional Chinese Medicine formula, has shown effectiveness in treating PCOS. However, the specific mechanisms by which YHHD exerts its therapeutic effects remain unclear. In this study, we performed to investigate the therapeutic effects of YHHD and quercetin on dehydroepiandrosterone-induced PCOS mice, and examine the effect of quercetin on the decidualization of T-HESCs under hyperinsulinemic conditions. The results showed that YHHD could reduce early miscarriage rates in PCOS patients and significantly improved glucose metabolism disorders, sex hormone levels, and the estrous cycles in PCOS mice. Quercetin could alleviate effect of high insulin levels and restore the low expression of insulin receptor substrate1/2 (IRS1/2) and glucose transporte 4 (GLUT4) in T-HESCs, demonstrating its potential to mitigate hyperinsulin-induced decidualization dysfunction via the GLUT4 signaling pathway mediated by IRS1/2. This study provides valuable molecular insights of YHHD and highlight the therapeutic potential of quercetin in treating decidualization dysfunction in PCOS.

An 8-Week study on the effects of high and Moderate-Intensity interval exercises on mitochondrial MOTS-C changes and their relation to metabolic markers in male diabetic sand rats.

Mitochondrial dysfunction is a significant feature of type 2 diabetes. MOTS-C, a peptide derived from mitochondria, has positive effects on metabolism and exercise capacity. This study explored the impact of high and moderate-intensity interval exercises on mitochondrial MOTS-C alterations and their correlation with metabolic markers in male diabetic sand rats. Thirty male sand rats were divided into six groups: control, MIIT, DM + HIIT, DM + MIIT, DM, and HIIT (5 rats each). Diabetes was induced using a high-fat diet (HFD) combined with streptozotocin (STZ). The Wistar sand rats in exercise groups underwent 8 weeks of interval training of varying intensities. Post sample collection, protein expressions of PCG-1a, AMPK, and GLUT4 were assessed through Western blot analysis, while MOTS-C protein expression was determined using ELISA. Both exercise intensity and diabetes significantly affected the levels of PCG-1a, MOTS-C, GLUT4 proteins, and insulin resistance (p < 0.001). The combined effect of diabetes status and exercise intensity on these levels was also significant (p < 0.001). However, the diabetes effect varied when comparing high-intensity to moderate-intensity exercise. The moderate-intensity exercise group with diabetes showed higher levels of PCG-1a, MOTS-C, and GLUT4 proteins and reduced insulin resistance levels (p < 0.001). Exercise intensity (p = 0.022) and diabetes (p = 0.008) significantly influenced AMPK protein levels. The interplay between diabetes status and exercise intensity on AMPK protein levels was noteworthy, with the moderate-intensity diabetes group exhibiting higher AMPK levels than the high-intensity diabetes group (p < 0.001). In conclusion, exercise elevates the levels of PCG-1a, MOTS-C, GLUT4, and AMPK proteins, regulating insulin resistance in diabetic sand rats. Given the AMPK-MOTS-C mitochondrial pathway's mechanisms, interval exercises might enhance the metabolic rates and general health of diabetic rodents.

Matrix mechanics regulates muscle regeneration by modulating kinesin-1 activity.

Sarcopenia, a prevalent muscle disease characterized by muscle mass and strength reduction, is associated with impaired skeletal muscle regeneration. However, the influence of the biomechanical properties of sarcopenic skeletal muscle on the efficiency of the myogenic program remains unclear. Herein, we established a mouse model of sarcopenia and observed a reduction in stiffness within the sarcopenic skeletal muscle in vivo. To investigate whether the biomechanical properties of skeletal muscle directly impact the myogenic program, we established an in vitro system to explore the intrinsic mechanism involving matrix stiffness control of myogenic differentiation. Our findings identify the microtubule motor protein, kinesin-1, as a mechano-transduction hub that senses and responds to matrix stiffness, crucial for myogenic differentiation and muscle regeneration. Specifically, kinesin-1 activity is positively regulated by stiff matrices, facilitating its role in transporting mitochondria and enhancing translocation of the glucose transporter GLUT4 to the cell surface for glucose uptake. Conversely, the softer matrices significantly suppress kinesin-1 activity, leading to the accumulation of mitochondria around nuclei and hindering glucose uptake by inhibiting GLUT4 membrane translocation, consequently impairing myogenic differentiation. The insights gained from the in-vitro system highlight the mechano-transduction significance of kinesin-1 motor proteins in myogenic differentiation. Furthermore, our study confirms that enhancing kinesin-1 activity in the sarcopenic mouse model restores satellite cell expansion, myogenic differentiation, and muscle regeneration. Taken together, our findings provide a potential target for improving muscle regeneration in sarcopenia.

Exogenous thyroxine increases cardiac GLUT4 translocation in insulin resistant OLETF rats.

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.

GLUT4 localisation with the plasma membrane is unaffected by an increase in plasma free fatty acid availability.

BACKGROUND: Insulin-stimulated glucose uptake into skeletal muscle occurs via translocation of GLUT4 from intracellular storage vesicles to the plasma membrane. Elevated free fatty acid (FFA) availability via a lipid infusion reduces glucose disposal, but this occurs in the absence of impaired proximal insulin signalling. Whether GLUT4 localisation to the plasma membrane is subsequently affected by elevated FFA availability is not known. METHODS: Trained (n = 11) and sedentary (n = 10) individuals, matched for age, sex and body mass index, received either a 6 h lipid or glycerol infusion in the setting of a concurrent hyperinsulinaemic-euglycaemic clamp. Sequential muscle biopsies (0, 2 and 6 h) were analysed for GLUT4 membrane localisation and microvesicle size and distribution using immunofluorescence microscopy. RESULTS: At baseline, trained individuals had more small GLUT4 spots at the plasma membrane, whereas sedentary individuals had larger GLUT4 spots. GLUT4 localisation with the plasma membrane increased at 2 h (P = 0.04) of the hyperinsulinemic-euglycemic clamp, and remained elevated until 6 h, with no differences between groups or infusion type. The number of GLUT4 spots was unchanged at 2 h of infusion. However, from 2 to 6 h there was a decrease in the number of small GLUT4 spots at the plasma membrane (P = 0.047), with no differences between groups or infusion type. CONCLUSION: GLUT4 localisation with the plasma membrane increases during a hyperinsulinemic-euglycemic clamp, but this is not altered by elevated FFA availability. GLUT4 appears to disperse from small GLUT4 clusters located at the plasma membrane to support glucose uptake during a hyperinsulinaemic-euglycaemic clamp.

A Metabolomics Study of the Effects of Eleutheroside B on Glucose and Lipid Metabolism in a Zebrafish Diabetes Model.

(1) Background: Diabetes is a common metabolic disease that seriously endangers human health. In the present study, we investigated the therapeutic effects of the active ingredient Eleutheroside B (EB) from the traditional Chinese medicine Eleutheroside on diabetes mellitus in a zebrafish model. Concomitant hepatic injury was also analysed, along with the study of possible molecular mechanisms using metabolomics technology. This work should provide some theoretical references for future experimental studies. (2) Methods: A zebrafish diabetes model was constructed by soaking in a 1.75% glucose solution and feeding a high-fat diet. The intervention drug groups were metformin (100 µg∙mL-1) and EB (50, 100, and 150 µg∙mL-1) via water-soluble exposure for 30 days. Glucose, TG, TC, LDL-C, and HDL-C were evaluated in different treatment groups. GLUT4 protein expression was also evaluated in each group, and liver injury was observed by HE staining. Metabolomics techniques were used to investigate the mechanism by which EB regulates endogenous markers and metabolic pathways during the development of diabetes. (3) Results: All EB treatment groups in diabetic zebrafish showed significantly reduced body mass index (BMI) and improved blood glucose and lipid profiles. EB was found to upregulate GLUT4 protein expression and ameliorate the liver injury caused by diabetes. Metabolomics studies showed that EB causes changes in the metabolic profile of diabetic zebrafish. These were related to the regulation of purine metabolism, cytochrome P450, caffeine metabolism, arginine and proline metabolism, the mTOR signalling pathway, insulin resistance, and glycerophospholipid metabolism. (4) Conclusions: EB has a hypoglycaemic effect in diabetic zebrafish as well as significantly improving disorders of glycolipid metabolism. The mechanism of action of EB may involve regulation of the mTOR signalling pathway, purine metabolism, caffeine metabolism, and glycerophospholipid metabolism.

Oral post-exercise garlic extract supplementation enhances glycogen replenishment but does not up-regulate mitochondria biogenesis mRNA expression in human-exercised skeletal muscle.

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 (VO2max) 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.

Maladaptive response following glucose overload in GLUT4-overexpressing H9C2 cardiomyoblasts.

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.

Flow cytometry protocol for GLUT4-myc detection on cell surfaces.

Insulin and muscle contraction trigger GLUT4 translocation to the plasma membrane, which increases glucose uptake by muscle cells. Insulin resistance and Type 2 diabetes are the result of impaired GLUT4 translocation. Quantifying GLUT4 translocation is essential for comprehending the intricacies of both physiological and pathophysiological processes involved in glucose metabolism. The most commonly used methods for measuring GLUT4 translocation are the ELISA-type assay and the immunofluorescence assay. While some reports suggest that flow cytometry could be useful in quantifying GLUT4 translocation, this technique is not frequently used. Much of our current understanding of the regulation of GLUT4 has been based on experiments using the rat myoblast cell line (L6 cell) which expresses GLUT4 with a myc epitope on the exofacial loop. In the present study, we use the L6-GLUT4myc cell line to develop a flow cytometry-based approach to detect GLUT4 translocation. Flow cytometry offers the advantages of both immunofluorescence and ELISA-based assays. It allows easy identification of separate cell populations in the sample, similar to immunofluorescence, while providing results based on a population-level analysis of multiple individual cells, like an ELISA-based assay. Our results demonstrate a 0.6-fold increase with insulin stimulation compared with basal conditions. Finally, flow cytometry consistently yielded results across different experiments and exhibited sensitivity under the tested conditions.

Abrus precatorius Leaf Extract Stimulates Insulin-mediated Muscle Glucose Uptake: In vitro Studies and Phytochemical Analysis.

Diabetes mellitus, linked with insulin resistance and hyperglycaemia, is a leading cause of mortality. Glucose uptake through glucose transporter type 4, especially in skeletal muscle, is crucial for maintaining euglycaemia and is a key pathway targeted by antidiabetic medication. Abrus precatorius is a medicinal plant with demonstrated antihyperglycaemic activity in animal models, but its mechanisms are unclear.This study evaluated the effect of a 50% ethanolic (v/v) A. precatorius leaf extract on (1) insulin-stimulated glucose uptake and (2) related gene expression in differentiated C2C12 myotubes using rosiglitazone as a positive control, and (3) generated a comprehensive phytochemical profile of A. precatorius leaf extract using liquid chromatography-high resolution mass spectrometry to elucidate its antidiabetic compounds. A. precatorius leaf extract significantly increased insulin-stimulated glucose uptake, and insulin receptor substrate 1 and Akt substrate of 160 kDa gene expression; however, it had no effect on glucose transporter type 4 gene expression. At 250 µg/mL A. precatorius leaf extract, the increase in glucose uptake was significantly higher than 1 µM rosiglitazone. Fifty-five phytochemicals (primarily polyphenols, triterpenoids, saponins, and alkaloids) were putatively identified, including 24 that have not previously been reported from A. precatorius leaves. Abrusin, precatorin I, glycyrrhizin, hemiphloin, isohemiphloin, hispidulin 4'-O-ß-D-glucopyranoside, homoplantaginin, and cirsimaritin were putatively identified as known major compounds previously reported from A. precatorius leaf extract. A. precatorius leaves contain antidiabetic phytochemicals and enhance insulin-stimulated glucose uptake in myotubes via the protein kinase B/phosphoinositide 3-kinase pathway by regulating insulin receptor substrate 1 and Akt substrate of 160 kDa gene expression. Therefore, A. precatorius leaves may improve skeletal muscle insulin sensitivity and hyperglycaemia. Additionally, it is a valuable source of bioactive phytochemicals with potential therapeutic use for diabetes.