Insulin-dependent Glucose Transporter GLUT4 Research Articles

The SGLT2 inhibitor ertugliflozin causes a switch of cardiac substrate utilization leading to reduced cardiac mTOR-signaling, unfolded protein response and apoptosis

Abstract Introduction SGLT2 inhibitors reduce hospitalization for heart failure in patients with and without diabetes. The underlying mechanisms remain incompletely understood but might relate to the induction of a fasting like response with low blood glucose and insulin levels and increased ketone bodies. The study aimed to investigate underlying signaling pathways. Methods and results Cardiac hypertrophy was induced by transverse aortic constriction (TAC) surgery in 20-week-old C57Bl/6J mice. Mice were treated with the SGLT2 inhibitor ertugliflozin (225 mg/kg chow diet) or vehicle for a period of 10 weeks. Ertugliflozin significantly improved left ventricular systolic and diastolic function (dp/dtmax: TAC ctrl.: 8900±3362 mmHg/s vs. TAC ertu.: 12051±1604 mmHg/s (p<0.001) and dp/dtmin: TAC ctrl.: −7653±2770 mmHg/s vs. TAC ertu.: −10199±2463 mmHg/s (p<0.01); by millar catheter with dobutamine stress) and reduced myocardial fibrosis (p=0.17) and hypertrophy (p=0.09). This was paralleled by the expected fasting like response with lower glucose and insulin levels (HOMA-IR p<0.05) and increased ketone body concentrations (p<0.05). As a consequence cardiac insulin signaling (AKT-phosphorylation at Thr(308), 0.39-fold (p<0.01)) was reduced by ertugliflozin with less insulin-dependent glucose transporter GLUT4 expression (0.64-fold (p<0.05)) while fatty acid transporter CD36 (2.12-fold (p<0.001)) and the ketone body catabolizing key enzyme beta-hydroxybutyrate dehydrogenase BDH-1 were increased (1.59-fold (p<0.01)) in addition to AMPK-signaling (AMPK-phosphorylation at Thr(172), 1.62-fold (p<0.01)). This led to downstream inhibition of the mTOR pathway with reduced phosphorylation of p70S6K, 4E-BP1 and ULK1 (p70S6K-phosphorylation at Thr(389) (0.57-fold (p<0.05)), 4E-BP1-phosphorylation at Ser(65) (0.74-fold (p<0.05)) and ULK1-phosphorylation at Ser(757) (0.56-fold (p<0.01))). MTOR signaling critically mediates cardiac hypertrophy, endoplasmic reticulum stress, unfolded protein response (UPR) and adverse cardiac remodeling. Consistently, we found ertugliflozin to reduce ATF6 (0.69-fold (p<0.05)) and elf2α-phosphorylation at Ser(51) (0.66-fold (p=0.0611)) as well as downstream signaling (ATF4 0.58-fold (p<0.01); CHOP 0.36-fold (p<0.001)). This let to reduced caspase 3 (0.74-fold (p<0.05)), collagen I (0.60-fold (p<0.01)) and IL-1β (0.46-fold (p<0.01)) expression indicating less apoptosis, fibrosis and left ventricular remodeling with consequential reduction of BNP expression (0.60-fold (p<0.001)) in response to SGLT2 inhibition. Conclusion The SGLT2 inhibitor ertugliflozin improves left ventricular function in a murine model of cardiac hypertrophy. Mechanistically, this was associated with a metabolic switch of cardiac substrate utilization with reduced cardiac insulin- and increased cardiac AMPK-signaling leading to reduced cardiac mTOR-signaling, unfolded protein response and apoptosis. Funding Acknowledgement Type of funding sources: Private company. Main funding source(s): MSD

SHORT-TERM CRIZOTINIB TREATMENT REVERSES GLUT4 GLUCOSE TRANSPORTER DOWNREGULATION IN DIABETIC RAT HEART

Objective: Crizotinib is a multitarget ALK/c-MET/ROS1/RON inhibitor that can potentially inhibit non-intended receptor tyrosine kinases (RTKs) which play a role in cardiac development, function and/or metabolism. Tyrosine kinase inhibitors (TKIs) studies in diabetic patients showed antidiabetic effects of some TKIs and several potential mechanisms were suggested including mechanisms via RTKs. We examined RTKs after short-term crizotinib treatment in diabetic heart. Design and method: To induce diabetes, male Wistar rats were injected with streptozotocin (STZ; 80 mg/kg, i.p.), controls received vehicle (CON). After 3 days, diabetic rats were treated with crizotinib in a dose 25 mg/kg/day p.o. (STZ+CRI) or vehicle p.o. for 7 days (STZ). mRNA expression of selected RTKs were assessed by RT-qPCR method in rat left ventricle samples. Blood glucose, c-peptide and glucagon in plasma samples and expression of cardiac glucose transporters Glut1 and Glut4 were assessed to determine impact on glucose homeostasis. Results: Intriguingly, crizotinib had a hypoglycaemic effect in diabetic animals (plasma glucose in STZ: 19.2 mmol/L vs. STZ+CRI: 14.5 mmol/L., P<0.05). STZ animals had lower c-peptide and unaltered glucagon plasma levels. However, plasma levels of c-peptide and glucagon were not changed after crizotinib treatment suggesting altered glucose utilization in tissues. In STZ rats, we detected downregulation of insulin dependent glucose transporter Glut4 (by 40% as compared to CON, P < 0.05). Interestingly, crizotinib prevented this downregulation. Insulin non-dependent Glut1 glucose transporter remained stable across all groups. Diabetes upregulated cardiac insulin receptor and Igf-1 receptor without significant crizotinib impact. Regarding RTKs, we observed unaltered c-Met, Flt1 and Erbb2 expressions in diabetic hearts without influence of crizotinib. Conclusions: Crizotinib had a hypoglycaemic effect in diabetic animals after short-term treatment. This was independent of insulin and glucagon levels. Crizotinib prevented cardiac insulin-dependent Glut4 decrease. Downregulation of cardiac insulin receptor family RTKs expression remains stable under crizotinib suggesting modulation of alternative pathways responsible for crizotinib action in diabetic rat heart.

The tyrosine kinase inhibitor crizotinib influences blood glucose and mRNA expression of GLUT4 and PPARs in the heart of rats with experimental diabetes.

Tyrosine kinases inhibitors (TKIs) may alter glycaemia and may be cardiotoxic with importance in the diabetic heart. We investigated the effect of multi-TKI crizotinib after short-term administration on metabolic modulators of the heart of diabetic rats. Experimental diabetes mellitus (DM) was induced by streptozotocin (STZ; 80mg·kg-1, i.p.), and controls (C) received vehicle. Three days after STZ, crizotinib (STZ+CRI; 25mg·kg-1 per day p.o.) or vehicle was administered for 7days. Blood glucose, C-peptide, and glucagon were assessed in plasma samples. Receptor tyrosine kinases (RTKs), cardiac glucose transporters, and peroxisome proliferator-activated receptors (PPARs) were determined in rat left ventricle by RT-qPCR method. Crizotinib moderately reduced blood glucose (by 25%, P< 0.05) when compared to STZ rats. The drug did not affect levels of C-peptide, an indicator of insulin secretion, suggesting altered tissue glucose utilization. Crizotinib had no impact on cardiac RTKs. However, an mRNA downregulation of insulin-dependent glucose transporter Glut4 in the hearts of STZ rats was attenuated after crizotinib treatment. Moreover, crizotinib normalized Ppard and reduced Pparg mRNA expression in diabetic hearts. Crizotinib decreased blood glucose independently of insulin and glucagon. This could be related to changes in regulators of cardiac metabolism such as GLUT4 and PPARs.

METABOLIC CONTROL OF HIGH-FREQUENCY GAMMA OSCILLATIONS IN THE BRAIN

A high-frequency electrical activity across the range of 30–100 Hz, known as gamma rhythms, is observed in many regions of the brain. This phenomenon serves to synchronize the activity of various neural networks intended to process, transmit, store and receive information. Gamma rhythms play a key role in such processes of higher nervous activity as attention, sensory perception and memory formation. Impairment of gamma rhythms is a common symptom of diseases associated with cognitive impairment, including Alzheimer’s disease, epilepsy and schizophrenia. Recent studies have shown that a particular population of GABAergic-inhibiting neurons, i.e. parvalbumin-positive (PV+) interneurons, is the source of high-frequency oscillations. Maintenance of gamma rhythms is an extremely energy-intensive process that relies on a high rate of oxidative phosphorylation in the mitochondria of neurons and is limited by the presence of glucose. Insulin may be involved in the metabolic control of gamma oscillations, since PV+ interneurons selectively express the insulin-dependent glucose transporter GLUT4, which can provide an additional glucose influx under near-limit functioning conditions as in the case of high-frequency gamma oscillations. This review generalized available literature data on the relationship between metabolism and a high-frequency electrical brain activity, with an emphasis on the possible contribution of central insulin resistance to disturbances of gamma rhythms in the brain.

Metformin augments the levels of molecules that regulate the expression of the insulin-dependent glucose transporter GLUT4 in the endometria of hyperinsulinemic PCOS patients

Does treatment with the insulin sensitizer metformin modify the levels and activation of proteins related to the expression of the insulin-dependent glucose transporter (GLUT4), such as adenosine monophosphate-activated protein kinase (AMPK) and myocyte enhancer factor 2A (MEF2A), in endometria from hyperinsulinemic hyperandrogenemic polycystic ovary syndrome (PCOS h-Ins) patients? In PCOS h-Ins patients, metformin increases endometrial levels of GLUT4 mRNA and protein levels by normalizing the quantity and activation of molecules that regulate GLUT4 expression to healthy values. These changes could improve endometrial metabolic function. PCOS is an endocrine-metabolic disorders closely associated with insulin resistance. In particular, the insulin signaling pathway is impaired in endometria from these patients and the concentration of GLUT4, as well as the molecules involved in its translocation to the cell surface, is decreased. However, there are limited data about the mechanisms that regulate the GLUT4 expression in the endometria and the effect of metformin on them. This is a case-control study in the setting of a research unit, approved by the Ethical Committees of our institution. The groups whose endometria were studied were PCOS h-Ins (n = 8); PCOS patients with hyperandrogenemia hyperinsulinemia taking only metformin for at least 3 months (PCOS-MTF, n = 8) and healthy fertile women at the time of hysterectomy because of benign pathology as controls (CE, n = 8). Steroids and sex hormone-binding globulin were measured and glucose and insulin levels were evaluated during an oral glucose tolerance test. Protein levels for αAMPK (catalytic subunit of AMPK), phosphorylated (p)-AMPKαThr(172) (activating phosphorylation site), MEF2A, p-MEF2AThr312 (activating phosphorylation site) and GLUT4 were assessed by western blot and immunohistochemistry. In addition, GLUT4 gene expression was evaluated by RT-PCR. We found significantly lower levels of MEF2A and p-MEF2AThr312 in PCOS h-Ins compared with CE endometria (P < 0.05). Also, we detected lower levels of p-AMPKαThr(172) in PCOS h-Ins endometria compared with the PCOS-MTF group (P < 0.05). The ratios of phospho-AMPK/total AMPK and phospho-MEF2A/total MEF2A were significantly increased in the PCOS-MTF compared with the PCOS h-Ins group (P < 0.05). The RT-PCR experiments showed lower levels of GLUT4 mRNA transcripts in PCOS h-Ins compared with PCOS-MTF-treated group (P < 0.05), the protein levels of GLUT4 were decreased in a similar way. The limited number of patients included in this study who presented large clinical variability. Therefore, it would be necessary to recruit a greater number of patients to minimize our data dispersion in order to prove the clinical benefits of metformin described by others. Since the insulin sensitizer metformin increases the expression of the GLUT4, it may improve endometrial physiology in PCOS patients and, therefore, promote better reproductive outcomes. These results suggest that in PCOS patients, metformin may act directly at the endometrial level and decrease insulin resistance condition by increasing the expression of GLUT4 and, in this way, indirectly restore endometrial function. This work was supported by Fondo Nacional de Desarrollo Científico y Tecnológico (grant number 1095127 to M.V.). None of the authors has any conflict of interest to declare.

Human hippocampal energy metabolism is impaired during cognitive activity in a lipid infusion model of insulin resistance.

Neuronal glucose uptake was thought to be independent of insulin, being facilitated by glucose transporters GLUT1 and GLUT3, which do not require insulin signaling. However, it is now known that components of the insulin-mediated glucose uptake pathway, including neuronal insulin synthesis and the insulin-dependent glucose transporter GLUT4, are present in brain tissue, particularly in the hippocampus. There is considerable recent evidence that insulin signaling is crucial to optimal hippocampal function. The physiological basis, however, is not clear. We propose that while noninsulin-dependent GLUT1 and GLUT3 transport is adequate for resting needs, the surge in energy use during sustained cognitive activity requires the additional induction of insulin-signaled GLUT4 transport. We studied hippocampal high-energy phosphate metabolism in eight healthy volunteers, using a lipid infusion protocol to inhibit insulin signaling. Contrary to conventional wisdom, it is now known that free fatty acids do cross the blood–brain barrier in significant amounts. Energy metabolism within the hippocampus was assessed during standardized cognitive activity. 31Phosphorus magnetic resonance spectroscopy was used to determine the phosphocreatine (PCr)-to-adenosine triphosphate (ATP) ratio. This ratio reflects cellular energy production in relation to concurrent cellular energy expenditure. With lipid infusion, the ratio was significantly reduced during cognitive activity (PCr/ATP 1.0 ± 0.4 compared with 1.4 ± 0.4 before infusion, P = 0.01). Without lipid infusion, there was no reduction in the ratio during cognitive activity (PCr/ATP 1.5 ± 0.3 compared with 1.4 ± 0.4, P = 0.57). This provides supporting evidence for a physiological role for insulin signaling in facilitating increased neuronal glucose uptake during sustained cognitive activity. Loss of this response, as may occur in type 2 diabetes, would lead to insufficient neuronal energy availability during cognitive activity.

Advanced glycation endproducts in horses with insulin-induced laminitis

Advanced glycation endproducts (AGEs) have been implicated in the pathogenesis of cancer, inflammatory conditions and diabetic complications. An interaction of AGEs with their receptor (RAGE) results in increased release of pro-inflammatory cytokines and reactive oxygen species (ROS), causing damage to susceptible tissues. Laminitis, a debilitating foot condition of horses, occurs in association with endocrine dysfunction and the potential involvement of AGE and RAGE in the pathogenesis of the disease has not been previously investigated. Glucose transport in lamellar tissue is thought to be largely insulin-independent (GLUT-1), which may make the lamellae susceptible to protein glycosylation and oxidative stress during periods of increased glucose metabolism. Archived lamellar tissue from horses with insulin-induced laminitis (n=4), normal control horses (n=4) and horses in the developmental stages (6h, 12h and 24h) of the disease (n=12) was assessed for AGE accumulation and the presence of oxidative protein damage and cellular lipid peroxidation. The equine-specific RAGE gene was identified in lamellar tissue, sequenced and is now available on GenBank. Lamellar glucose transporter (GLUT-1 and GLUT-4) gene expression was assessed quantitatively with qRT-PCR in laminitic and control horses and horses in the mid-developmental time-point (24h) of the disease. Significant AGE accumulation had occurred by the onset of insulin-induced laminitis (48h) but not at earlier time-points, or in control horses. Evidence of oxidative stress was not found in any group. The equine-specific RAGE gene was not expressed differently in treated and control animals, nor was the insulin-dependent glucose transporter GLUT-4. However, the glucose transporter GLUT-1 was increased in lamellar tissue in the developmental stages of insulin-induced laminitis compared to control horses and the insulin-independent nature of the lamellae may facilitate AGE formation. However, due to the lack of AGE accumulation during disease development and a failure to detect an increase in ROS or upregulation of RAGE, it appears unlikely that oxidative stress and protein glycosylation play a central role in the pathogenesis of acute, insulin-induced laminitis.

Postprandial Changes in Plasma Free Amino Acids in Weanling Horses

s / Journal of Equine Veterinary Science 31 (2011) 230-356 328 (PROC MIXED) of SAS (SAS Institute Inc.) with horse as random. Differences were considered at P .05. Results and Discussion: Compared to control horses, mRNA abundance of IR and GLUT4 was lower (P .01) in the SIM of PPID horses. For GLUT1, GLUT2, GLUT5 and SGLT1, mRNA abundance did not differ between PPID and control horses. For SGLT1, GLUT2, GLUT1 and GLUT5 and, mRNA abundance did not differ between PPID and control horses. Protein expression of GLUT-4 was confirmed in all horses tested by visualization of a single band on Western blot, changing in relative abundance with the amount of protein loaded. Immunohistochemical staining revealed abundant labeling of GLUT4 in the epithelium and the muscularis mucosa and was confirmed in muscle tissue. Others have also reported GLUT4 transcript, albeit in low abundance, in small intestinal mucosa of the pig [5]. Results indicate that PPID down-regulates the expression of insulin-dependent glucose transporter GLUT4 and IR at the transcription level in the proximal jejunum of the horse. Thus far, dysfunctional glucose uptake into muscle and fat cells has been shown to contribute to the onset of type II diabetes, but little attention has been given to the role of the intestine in the pathology of the condition. In equine species, it is possible that intestinal GLUT4 plays a critical in controlling post-prandial blood glucose clearance. Conclusion: Horses with PPID had lower mRNA abundance of genes encoding for insulin-dependent glucose transporter GLUT4 and IR compared to non-PPID horses. These results shed light on the physiological mechanisms for increased susceptibility to nutritionally induced laminitis in PPID-afflicted horses.

Translocation of the insulin-regulated aminopeptidase to the cell surface: detection by radioligand binding.

Insulin-regulated aminopeptidase (IRAP) and the insulin-dependent glucose transporter GLUT4 colocalize in specific intracellular vesicles (that is, GLUT4 vesicles). These vesicles move slowly to the cell surface, but their translocation is markedly enhanced by insulin, resulting in higher glucose uptake. Previous studies of the insulin-mediated translocation of IRAP to the cell surface have been hampered by the laborious detection of IRAP at the cell surface. We aimed to develop a more direct and faster method to detect IRAP. To this end, we used model systems with well-characterized IRAP: CHO-K1 cells expressing endogenous IRAP and recombinant HEK293 cells expressing human IRAP. A more widespread application of the method was demonstrated by the use of 3T3-L1 adipocytes. After stimulation of the cells with insulin, internalization of IRAP was inhibited by the addition of phenyl arsine oxide (PAO). Then, cell-surface IRAP was detected by the high-affinity binding of radiolabelled angiotensin (Ang) IV (either 125I or 3H). We monitored the time- and concentration dependence of insulin-mediated translocation of IRAP in both cell lines and 3T3-L1 adipocytes. A plateau was reached between 6 and 8 min, and 10(-7) M insulin led to the highest amount of IRAP at the cell surface. Based on the capacity of the IRAP apoenzyme to display high affinity for radiolabelled Ang IV and on the ability of PAO to inhibit IRAP internalization, we developed a more direct and faster method to measure insulin-mediated translocation of IRAP to the cell surface.

The trans-10, cis-12 isomer of conjugated linoleic acid decreases adiponectin assembly by PPARγ-dependent and PPARγ-independent mechanisms

The adipocyte-derived secretory protein adiponectin functions as an insulin-sensitizing agent. In plasma, adiponectin exists as low, medium, and high molecular weight oligomers. Treatment with trans-10, cis-12 conjugated linoleic acid (t-10, c-12 CLA) reduces levels of adiponectin as well as triglyceride (TG) in mice and adipocyte cell culture models. The aim of this study was to determine whether the effects of t-10, c-12 CLA on adiponectin and TG are mediated through modulation of the transcription factor peroxisome proliferator-activated receptor gamma (PPARgamma). 3T3-L1 cells were treated either during or after differentiation into adipocytes with 100 microM t-10, c-12 CLA with or without 10 microM troglitazone, a PPARgamma agonist, or 1 microM GW9662, a PPARgamma antagonist, and adiponectin and TG levels were analyzed. Treatment with t-10, c-12 CLA reduced TG as well as cellular and secreted adiponectin levels and impaired the assembly of adiponectin oligomers. These changes were accompanied by decreases in PPARgamma mass. Troglitazone was able to reverse the t-10, c-12 CLA-mediated decrease in TG levels and restore the assembly of adiponectin oligomers but was unable to restore adiponectin synthesis. Conversely, treatment with GW9662 decreased TG mass and impaired adiponectin oligomer assembly but did not decrease total adiponectin mass. In a reporter assay, t-10, c-12 CLA appeared to be a partial PPARgamma agonist and prevented the stimulation of reporter activity by troglitazone. Therefore, the t-10, c-12 CLA isomer appears to alter adipocyte adiponectin metabolism through PPARgamma-dependent and PPARgamma-independent mechanisms.

Muscle GLUT4 in cirrhosis

The insulin-dependent glucose transporter GLUT4 mediates 50-80% of whole body glucose uptake, but its relation to the frequent glucose intolerance in patients with liver cirrhosis is unknown. Thirty patients and seven healthy controls underwent a 2-h oral glucose tolerance test and later a muscle biopsy. Levels of GLUT4 total protein and mRNA content were determined in muscle biopsies by polyclonal antibody labelling and RT-PCR, respectively. GLUT4 protein content in the cirrhosis group was not different from that of the controls, but at variance with the control subjects it correlated closely with measures of glucose tolerance (R(2)=0.45; p=0.003). GLUT4 mRNA of the patients with cirrhosis was reduced to 56% of control value (95% ci: 27-86%; p=0.015) and was inversely related to the level of basal hyper-insulinemia (R(2)=0.39; p=0.004). In cirrhosis GLUT4 protein content was quantitatively intact, while limiting glucose tolerance. This indicates loss of redundancy of the major glucose transport system, possibly related to the markedly decreased expression of its gene. Hyper-insulinemia may be a primary event. Our findings implicate the muscular GLUT4 system in the glucose intolerance of liver cirrhosis by a mechanism different from that in diabetes.

PPARgamma induces the insulin-dependent glucose transporter GLUT4 in the absence of C/EBPalpha during the conversion of 3T3 fibroblasts into adipocytes.

To define the molecular mechanisms that control GLUT4 expression during adipogenesis, NIH-3T3 fibroblasts ectopically expressing different adipogenic transcription factors (C/EBPbeta, C/EBPdelta, C/EBPalpha, and PPARgamma) under the control of a tetracycline-responsive inducible (C/EBPs) or a constitutive retroviral (PPARgamma) expression system were used. Enhanced production of C/EBPbeta (beta2 cell line), C/EBPbeta together with C/EBPdelta (beta/delta39 cell line), C/EBPalpha (alpha1 cell line), or PPARgamma (Pgamma2 cell line) in cells exposed to dexamethasone and the PPARgamma ligand ciglitazone (a thiazolidinedione) resulted in expression of GLUT4 mRNA as well as other members of the adipogenic gene program, including aP2 and adipsin. Focusing our studies on the beta/delta39 cells, we have demonstrated that C/EBPbeta along with C/EBPdelta in the presence of dexamethasone induces PPARgamma, adipsin, and aP2 mRNA production; however, GLUT4 mRNA is only expressed in cells exposed to ciglitazone. In addition, enhanced expression of a ligand-activated form of PPARgamma in the beta/delta39 fibroblasts stimulates synthesis of GLUT4 protein and gives rise to a population of adipocytic cells that take up glucose in direct response to insulin. C/EBPalpha is not expressed in the beta/delta39 cells under conditions that stimulate the adipogenic program. This observation suggests that PPARgamma alone or in combination with C/EBPbeta and C/EBPdelta is capable of activating GLUT4 gene expression.