Glucose Transport In Response Research Articles

Utilizing the apical-out enteroids in vitro model to investigate intestinal glucose transport, barrier function, oxidative stress, and inflammatory responses in broiler chickens

IntroductionConventional 2D intestinal epithelial cell lines have been widely used in investigating intestinal functions, yet with limitations in recapitulating the in vivo gut physiology of chickens. A recently established chicken enteroid model with apical-out nature and the presence of leukocyte components represents intestinal mucosal functions. The objectives of this study were to 1) evaluate basic gut nutrient transport and barrier functions in this model and 2) identify the model’s effectiveness in studying inflammation and oxidative stress responses.MethodsEnteroids were generated from individual villus units isolated from the small intestine of Cobb500 broiler embryos. Enteroid viability, morphology, and epithelial cell markers were monitored; barrier function was evaluated based on the permeability to fluorescein isothiocyanate–dextran (FD4) with or without EDTA and lipopolysaccharide (LPS) challenges; nutrient transport was evaluated by fluorescence-labeled glucose (2NBD-G) with or without transporter blockade; the oxidative status was indicated by reactive oxygen species (ROS). Inflammatory and oxidative challenges were induced by LPS and menadione treatment, respectively. Selected marker gene expressions, including tight junction proteins (CLDN-1, CLDN-2, ZO-1, and OCCL), epithelial cell markers (Lgr-5, LYZ, and MUC-2), cytokines (IL-1β, IL-6, IL-8, IL-10, TNF-α, and INF-γ), and antioxidant enzymes (Nrf-2, catalase, and SOD), were determined by using RT-qPCR. Data were analyzed by one-way ANOVA among treatment groups.ResultsEnteroid cell activity was stable from day (d) 2 to d 6 and declined at d 7. Epithelial cell marker and cytokine expressions were stable from d 4 to d 6. FD4 permeability was increased after the EDTA treatment (P ≤ 0.05). Transporter-mediated 2NBD-G absorption was observed, which was reduced with glucose transporter blockade (P ≤ 0.05). Enteroids showed classic responses to LPS challenges, including upregulated gene expressions of IL-1β and IL-6, downregulated gene expressions of ZO-1 and OCCL, and increased FD4 permeability (P ≤ 0.05). Enteroids showed increased ROS generation (P ≤ 0.05) in response to oxidative stress.DiscussionIn conclusion, this apical-out enteroid model is a stable alternative in vitro model that exhibits intestinal barrier, nutrient transport, oxidation, and inflammation functions. With this enteroid model, we developed two challenge protocols for evaluating intestinal functions under oxidative stress and inflammation conditions.

Smoothelin-Like Protein 1 Regulates Development and Metabolic Transformation of Skeletal Muscle in Hyperthyroidism.

Hyperthyroidism triggers a glycolytic shift in skeletal muscle (SKM) by altering the expression of metabolic proteins, which is often accompanied by peripheral insulin resistance. Our previous results show that smoothelin-like protein 1 (SMTNL1), a transcriptional co-regulator, promotes insulin sensitivity in SKM. Our aim was to elucidate the role of SMTNL1 in SKM under physiological and pathological 3,3′,5-Triiodo-L-thyronine (T3) concentrations. Human hyper- and euthyroid SKM biopsies were used for microarray analysis and proteome profiler arrays. Expression of genes related to energy production, nucleic acid- and lipid metabolism was changed significantly in hyperthyroid samples. The phosphorylation levels and activity of AMPKα2 and JNK were increased by 15% and 23%, respectively, in the hyperthyroid samples compared to control. Moreover, SMTNL1 expression showed a 6-fold decrease in the hyperthyroid samples and in T3-treated C2C12 cells. Physiological and supraphysiological concentrations of T3 were applied on differentiated C2C12 cells upon SMTNL1 overexpression to assess the activity and expression level of the elements of thyroid hormone signaling, insulin signaling and glucose metabolism. Our results demonstrate that SMTNL1 selectively regulated TRα expression. Overexpression of SMTNL1 induced insulin sensitivity through the inhibition of JNK activity by 40% and hampered the non-genomic effects of T3 by decreasing the activity of ERK1/2 through PKCδ. SMTNL1 overexpression reduced IRS1 Ser307 and Ser612 phosphorylation by 52% and 53%, respectively, in hyperthyroid model to restore the normal responsiveness of glucose transport to insulin. SMTNL1 regulated glucose phosphorylation and balances glycolysis and glycogen synthesis via the downregulation of hexokinase II by 1.3-fold. Additionally, mitochondrial respiration and glycolysis were measured by SeaHorse analysis to determine cellular metabolic function/phenotype of our model system in real-time. T3 overload strongly increased the rate of acidification and a shift to glycolysis, while SMTNL1 overexpression antagonizes the T3 effects. These lines of evidence suggest that SMTNL1 potentially prevents hyperthyroidism-induced changes in SKM, and it holds great promise as a novel therapeutic target in insulin resistance.

Identification of a novel association for the WWOX/HIF1A axis with gestational diabetes mellitus (GDM).

BackgroundAlthough the WW-domain-containing oxidoreductase (WWOX)/Hypoxia-inducible factor 1 (HIF1) pathway is a well-known regulator of cellular glucose and energy metabolism in pathophysiological processes, its role in gestational diabetes mellitus (GDM), remains elusive. We undertook this study to determine the effect of WWOX/HIF1A signaling on the expression of glucose metabolism genes in GDM patients.MethodsLeukocytes were obtained from 135 pregnant women with (n = 98) or without (n = 37) GDM and, in turn, 3 months (n = 8) and 1 year (n = 12) postpartum. Quantitative RT-PCR was performed to determine gene expression profiles of the WWOX/HIF1A-related genes, including those involved in glucose transport (SLC2A1, SLC2A4), glycolytic pathway (HK2, PKM2, PFK, LDHA), Wnt pathway (DVL2, CTNNB1), and inflammatory response (NFKB1).ResultsGDM patients displayed a significant downregulation of WWOX with simultaneous upregulation of HIF1A which resulted in approximately six times reduction in WWOX/HIF1A ratio. As a consequence, HIF1A induced genes (SLC2A1, HK2, PFK, PKM) were found to be overexpressed in GDM compared to normal pregnancy and negative correlate with WWOX/HIF1A ratio. The postpartum WWOX expression was higher than during GDM, but its level was comparable to that observed in normal pregnancy.ConclusionsThe obtained results suggest a significant contribution of the WWOX gene to glucose metabolism in patients with gestational diabetes. Decreased WWOX expression in GDM compared to normal pregnancy, and in particular reduction of WWOX/HIF1A ratio, indicate that WWOX modulates HIF1α activity in normal tissues as described in the tumor. The effect of HIF1α excessive activation is to increase the expression of genes encoding proteins directly involved in the glycolysis which may lead to pathological changes in glucose metabolism observed in gestational diabetes.

The p21-activated kinase 2 (PAK2), but not PAK1, regulates contraction-stimulated skeletal muscle glucose transport.

AimMuscle contraction stimulates skeletal muscle glucose transport. Since it occurs independently of insulin, it is an important alternative pathway to increase glucose transport in insulin‐resistant states, but the intracellular signaling mechanisms are not fully understood. Muscle contraction activates group I p21‐activated kinases (PAKs) in mouse and human skeletal muscle. PAK1 and PAK2 are downstream targets of Rac1, which is a key regulator of contraction‐stimulated glucose transport. Thus, PAK1 and PAK2 could be downstream effectors of Rac1 in contraction‐stimulated glucose transport. The current study aimed to test the hypothesis that PAK1 and/or PAK2 regulate contraction‐induced glucose transport.MethodsGlucose transport was measured in isolated soleus and extensor digitorum longus (EDL) mouse skeletal muscle incubated either in the presence or absence of a pharmacological inhibitor (IPA‐3) of group I PAKs or originating from whole‐body PAK1 knockout, muscle‐specific PAK2 knockout or double whole‐body PAK1 and muscle‐specific PAK2 knockout mice.ResultsIPA‐3 attenuated (−22%) the increase in glucose transport in response to electrically stimulated contractions in soleus and EDL muscle. PAK1 was dispensable for contraction‐stimulated glucose transport in both soleus and EDL muscle. Lack of PAK2, either alone (−13%) or in combination with PAK1 (−14%), partly reduced contraction‐stimulated glucose transport compared to control littermates in EDL, but not soleus muscle.ConclusionContraction‐stimulated glucose transport in isolated glycolytic mouse EDL muscle is partly dependent on PAK2, but not PAK1.

RabGAPs in skeletal muscle function and exercise.

The two closely related RabGAPs TBC1D1 and TBC1D4 are key signaling factors of skeletal muscle substrate utilization. In mice, deficiency in both RabGAPs leads to reduced skeletal muscle glucose transport in response to insulin and lower GLUT4 abundance. Conversely, Tbc1d1 and Tbc1d4 deficiency results in enhanced lipid use as fuel in skeletal muscle, through yet unknown mechanisms. In humans, variants in TBC1D1 and TBC1D4 are linked to obesity, insulin resistance and type 2 diabetes. While the specific function in metabolism of each of the two RabGAPs remains to be determined, TBC1D1 emerges to be controlling exercise endurance and physical capacity, whereas TBC1D4 may rather be responsible for maintaining muscle insulin sensitivity, muscle contraction, and exercise. There is growing evidence that TBC1D1 also plays an important role in skeletal muscle development, since it has been found to be associated to meat production traits in several livestock species. In addition, TBC1D1 protein abundance in skeletal muscle is regulated by both, insulin receptor and insulin-like growth factor-1 (IGF-1) receptor signaling. This review focuses on the specific roles of the two key signaling factors TBC1D1 and TBC1D4 in skeletal muscle metabolism, development and exercise physiology.

Hyperbaric oxygen toxicity in brain: A case of hyperoxia induced hypoglycemic brain syndrome

Hyperbaric oxygen exposure is a recent hazzard for higher animals that originated as humans began underwater construction, exploration, and sports. Exposure can lead to abnormal brain EEG, convulsions, and death, the time to onset of each stage of pathology decreasing with increase in oxygen pressure. We provide evidence that hyperoxia, through oxidative phosphorylation, increases the energy state ([ATP]/[ADP][Pi]) of cells critical to providing glucose to cells behind the blood brain barrier (BBB). Brain cells without an absolute dependence on glucose metabolism; i.e. those having sufficient ATP synthesis using lactate and glutamate as oxidizable substrates, are not themselves very adversely affected by hyperoxia. The increased energy state and decrease in free [AMP], however, suppress glucose transport through the blood brain barrier (BBB) and into cells behind the BBB. Glucose has to pass in sequence through three steps of transport by facilitated diffusion and transporter activity for each step is regulated in part by AMP dependent protein kinase. The physiological role of this regulation is to increase glucose transport in response to hypoxia and/or systemic hypoglycemia. Hyperoxia, however, through unphysiological decrease in free [AMP] suppresses 1) glucose transport through the BBB (endothelial GLUT1 transporters) into cerebrospinal fluid (CSF); 2) glucose transport from CSF into cells behind the BBB (GLUT3 transporters) and (GLUT4 transporters). Cumulative suppression of glucose transport results in local regions of hypoglycemia and induces hypoglycemic failure. It is suggested that failure is initiated at axons and synapses with insufficient mitochondria to meet their energy requirements.

Chemical denervation using botulinum toxin increases Akt expression and reduces submaximal insulin-stimulated glucose transport in mouse muscle

Botulinum toxin A (botox) is a toxin used for spasticity treatment and cosmetic purposes. Botox blocks the excitation of skeletal muscle fibers by preventing the release of acetylcholine from motor nerves, a process termed chemical denervation. Surgical denervation is associated with increased expression of the canonical insulin-activated kinase Akt, lower expression of glucose handling proteins GLUT4 and hexokinase II (HKII) and insulin resistant glucose uptake, but it is not known if botox has a similar effect. To test this, we performed a time-course study using supra-maximal insulin-stimulation in mouse soleus ex vivo. No effect was observed in the glucose transport responsiveness at day 1, 7 and 21 after intramuscular botox injection, despite lower expression of GLUT4, HKII and expression and phosphorylation of TBC1D4. Akt protein expression and phosphorylation of the upstream kinase Akt were increased by botox treatment at day 21. In a follow-up study, botox decreased submaximal insulin-stimulated glucose transport. The marked alterations of insulin signaling, GLUT4 and HKII and submaximal insulin-stimulated glucose transport are a potential concern with botox treatment which merit further investigation in human muscle. Furthermore, the botox-induced chemical denervation model may be a less invasive alternative to surgical denervation.

Combined Hyperglycemia- and Hyperinsulinemia-Induced Insulin Resistance in Adipocytes Is Associated With Dual Signaling Defects Mediated by PKC-ζ.

A hyperglycemic and hyperinsulinemic environment characteristic of type 2 diabetes causes insulin resistance. In adipocytes, defects in both insulin sensitivity and maximum response of glucose transport have been demonstrated. To investigate the molecular mechanisms, freshly isolated rat adipocytes were incubated in control (5.6 mM glucose, no insulin) and high glucose (20 mM)/high insulin (100 nM) (HG/HI) for 18 hours to induce insulin resistance. Insulin-resistant adipocytes manifested decreased sensitivity of glucose uptake associated with defects in insulin receptor substrate (IRS)-1 Tyr phosphorylation, association of p85 subunit of phosphatidylinositol-3-kinase, Akt Ser473 and Thr308 phosphorylation, accompanied by impaired glucose transporter 4 translocation. In contrast, protein kinase C (PKC)-ζ activity was augmented by chronic HG/HI. Inhibition of PKC-ζ with a specific cell-permeable peptide reversed the signaling defects and insulin sensitivity of glucose uptake. Transfection of dominant-negative, kinase-inactive PKC-ζ blocked insulin resistance, whereas constitutively active PKC-ζ recapitulated the defects. The HG/HI incubation was associated with stimulation of IRS-1 Ser318 and Akt Thr34 phosphorylation, targets of PKC-ζ. Transfection of IRS-1 S318A and Akt T34A each partially corrected insulin signaling, whereas combined transfection of both completely normalized insulin signaling. In vivo hyperglycemia/hyperinsulinemia in rats for 48 hours similarly resulted in activation of PKC-ζ and increased phosphorylation of IRS-1 Ser318 and Akt Thr34. These data indicate that impairment of insulin signaling by chronic HG/HI is mediated by dual defects at IRS-1 and Akt mediated by PKC-ζ.

Hypothalamic Glucose Transport in Humans During Experimentally Induced Hypoglycemia-Associated Autonomic Failure.

Upregulated brain glucose transport in response to recurrent hypoglycemia may contribute to the development of hypoglycemia-associated autonomic failure (HAAF) and impaired awareness of hypoglycemia. Whether recurrent hypoglycemia alters glucose transport in the hypothalamus is unknown. To test the hypothesis that hypothalamic glucose transport will increase in healthy volunteers preconditioned with recurrent hypoglycemia to induce HAAF. University medical center. Thirteen healthy subjects underwent paired euglycemic and hypoglycemic preconditioning studies separated by at least 1 month. Following preconditioning, hypothalamic glucose transport was measured by magnetic resonance spectroscopy (MRS) in the afternoon on day 2 of each preconditioning protocol. The ratio of maximal transport rate to cerebral metabolic rate of glucose (Tmax/CMRglc), obtained from MRS-measured glucose in the hypothalamus as a function of plasma glucose. HAAF was successfully induced based on lower epinephrine, glucagon, and cortisol during the third vs first hypoglycemic preconditioning clamp (P ≤ 0.01). Hypothalamic glucose transport was not different following recurrent euglycemia vs hypoglycemia (Tmax/CMRglc 1.62 ± 0.09 after euglycemia preconditioning and 1.75 ± 0.14 after hypoglycemia preconditioning; P was not significant). Hypothalamic glucose concentrations measured by MRS were not different following the two preconditioning protocols. Glucose transport kinetics in the hypothalamus of healthy humans with experimentally induced HAAF were not different from those measured without HAAF. Future studies of patients with diabetes and impaired awareness of hypoglycemia will be necessary to determine if the existence of the diabetes state is required for this adaptation to hypoglycemia to occur.

A Role for Focal Adhesion Kinase in the Stimulation of Glucose Transport in Cardiomyocytes.

Stimulation of glucose transport is markedly impaired in cardiomyocytes exposed to free fatty acids (FFA), despite relative preservation of canonical insulin- or metabolic stress signaling. We determined whether Focal Adhesion Kinase (FAK) activity is required for stimulation of glucose transport in cardiomyocytes, and whether FAK downregulation participates in FFA-induced impairment of glucose transport stimulation. Glucose transport, measured in isolated cultured cardiomyocytes, was acutely stimulated either by insulin treatment, or by metabolic inhibition with oligomycin resulting in AMP-activated kinase (AMPK) activation. FAK activity was inhibited pharmacologically by preincubation with PF-573,228 (PF). FAK activity was assessed from its autophosphorylation on residue Y397, and from the phosphorylation of its target paxillin on Y118. Y397 FAK phosphorylation was reduced in cultured cardiomyocytes chronically exposed to FFA. Preincubation with PF prior to determination of glucose transport resulted in a significant reduction of oligomycin-stimulated glucose transport, with a lesser reduction in insulin-stimulated glucose transport. Insulin and AMPK signaling was unaffected by PF preincubation. siRNA-mediated FAK knockdown also resulted in reduced oligomycin-stimulated glucose transport. Chronic treatment of FFA-exposed cardiomyocytes with phenylephrine or a phorbol ester restored FAK activity and improved glucose transport. In conclusion, stimulation of glucose transport in cardiomyocytes requires FAK activity prior to stimulation. The chronic reduction of FAK activity in cardiomyocytes exposed to FFA contributes to the loss of glucose transport responsiveness to insulin or metabolic inhibition. J. Cell. Biochem. 118: 670-677, 2017. © 2016 Wiley Periodicals, Inc.

Experimental type II diabetes and related models of impaired glucose metabolism differentially regulate glucose transporters at the proximal tubule brush border membrane.

What is the central question of this study? Although SGLT2 inhibitors represent a promising treatment for patients suffering from diabetic nephropathy, the influence of metabolic disruption on the expression and function of glucose transporters is largely unknown. What is the main finding and its importance? In vivo models of metabolic disruption (Goto-Kakizaki typeII diabetic rat and junk-food diet) demonstrate increased expression of SGLT1, SGLT2 and GLUT2 in the proximal tubule brush border. In the typeII diabetic model, this is accompanied by increased SGLT- and GLUT-mediated glucose uptake. A fasted model of metabolic disruption (high-fat diet) demonstrated increased GLUT2 expression only. The differential alterations of glucose transporters in response to varying metabolic stress offer insight into the therapeutic value of inhibitors. SGLT2 inhibitors are now in clinical use to reduce hyperglycaemia in typeII diabetes. However, renal glucose reabsorption across the brush border membrane (BBM) is not completely understood in diabetes. Increased consumption of a Western diet is strongly linked to typeII diabetes. This study aimed to investigate the adaptations that occur in renal glucose transporters in response to experimental models of diet-induced insulin resistance. The study used Goto-Kakizaki typeII diabetic rats and normal rats rendered insulin resistant using junk-food or high-fat diets. Levels of protein kinaseC-βI (PKC-βI), GLUT2, SGLT1 and SGLT2 were determined by Western blotting of purified renal BBM. GLUT- and SGLT-mediated d-[(3) H]glucose uptake by BBM vesicles was measured in the presence and absence of the SGLT inhibitor phlorizin. GLUT- and SGLT-mediated glucose transport was elevated in typeII diabetic rats, accompanied by increased expression of GLUT2, its upstream regulator PKC-βI and SGLT1 protein. Junk-food and high-fat diet feeding also caused higher membrane expression of GLUT2 and its upstream regulator PKC-βI. However, the junk-food diet also increased SGLT1 and SGLT2 levels at the proximal tubule BBM. Glucose reabsorption across the proximal tubule BBM, via GLUT2, SGLT1 and SGLT2, is not solely dependent on glycaemic status, but is also influenced by diet-induced changes in glucose metabolism. We conclude that different metabolic disturbances result in complex adaptations in renal glucose transporter protein levels and function.

The Akt switch model: Is location sufficient?

Akt/PKB is a biochemical regulator that functions as an important cross-talk node between several signalling pathways in the mammalian cell. In particular, Akt is a key mediator of glucose transport in response to insulin. The phosphorylation (activation) of only a small percentage of the Akt pool of insulin-sensitive cells results in maximal translocation of glucose transporter 4 (GLUT4) to the plasma membrane (PM). This enables the diffusion of glucose into the cell. The dysregulation of Akt signalling is associated with the development of diabetes, cancer and cardiovascular disease.Akt is synthesised in the cytoplasm in the inactive state. Under the influence of insulin, it moves to the PM, where it is phosphorylated to form pAkt. Although phosphorylation occurs only at the PM, pAkt is found in many cellular locations, including the PM, the cytoplasm, and the nucleus. Indeed, the spatial distribution of pAkt within the cell appears to be an important determinant of downstream regulation.Here we present a simple, linear, four-compartment ordinary differential equation (ODE) model of Akt activation that tracks both the biochemical state and the physical location of Akt. This model embodies the main features of the activation of this important cross-talk node and is consistent with the experimental data. In particular, it allows different downstream signalling motifs without invoking separate feedback pathways. Moreover, the model is computationally tractable, readily analysed, and elucidates some of the apparent anomalies in insulin signalling via Akt.

Rac1: an emerging player in stretch-stimulated glucose transport.

Rac1: an emerging player in stretch-stimulated glucose transport.

Stretch-stimulated glucose transport in skeletal muscle is regulated by Rac1.

Rac1 regulates stretch-stimulated (i.e. mechanical stress) glucose transport in muscle. Actin depolymerization decreases stretch-induced glucose transport in skeletal muscle. Rac1 is a required part of the mechanical stress-component of the contraction-stimulus to glucose transport in skeletal muscle. An alternative to the canonical insulin signalling pathway for glucose transport is muscle contraction/exercise. Mechanical stress is an integrated part of the muscle contraction/relaxation cycle, and passive stretch stimulates muscle glucose transport. However, the signalling mechanism regulating stretch-stimulated glucose transport is not well understood. We recently reported that the actin cytoskeleton regulating GTPase, Rac1, was activated in mouse muscle in response to stretching. Rac1 is a regulator of contraction- and insulin-stimulated glucose transport, however, its role in stretch-stimulated glucose transport and signalling is unknown. We therefore investigated whether stretch-induced glucose transport in skeletal muscle required Rac1 and the actin cytoskeleton. We used muscle-specific inducible Rac1 knockout mice as well as pharmacological inhibitors of Rac1 and the actin cytoskeleton in isolated soleus and extensor digitorum longus muscles. In addition, the role of Rac1 in contraction-stimulated glucose transport during conditions without mechanical load on the muscles was evaluated in loosely hanging muscles and muscles in which cross-bridge formation was blocked by the myosin ATPase inhibitors BTS and Blebbistatin. Knockout as well as pharmacological inhibition of Rac1 reduced stretch-stimulated glucose transport by 30-50% in soleus and extensor digitorum longus muscle. The actin depolymerizing agent latrunculin B similarly decreased glucose transport in response to stretching by 40-50%. Rac1 inhibition reduced contraction-stimulated glucose transport by 30-40% in tension developing muscle but did not affect contraction-stimulated glucose transport in muscles in which force development was prevented. Our findings suggest that Rac1 and the actin cytoskeleton regulate stretch-stimulated glucose transport and that Rac1 is a required part of the mechanical stress-component of the contraction-stimulus to glucose transport in skeletal muscle.

Roles of TBC1D1 and TBC1D4 in insulin- and exercise-stimulated glucose transport of skeletal muscle.

This review focuses on two paralogue Rab GTPase activating proteins known as TBC1D1 Tre-2/BUB2/cdc 1 domain family (TBC1D) 1 and TBC1D4 (also called Akt Substrate of 160kDa, AS160) and their roles in controlling skeletal muscle glucose transport in response to the independent and combined effects of insulin and exercise. Convincing evidence implicates Akt2-dependent TBC1D4 phosphorylation on T642 as a key part of the mechanism for insulin-stimulated glucose uptake by skeletal muscle. TBC1D1 phosphorylation on several insulin-responsive sites (including T596, a site corresponding to T642 in TBC1D4) does not appear to be essential for in vivo insulin-stimulated glucose uptake by skeletal muscle. In vivo exercise or ex vivo contraction of muscle result in greater TBC1D1 phosphorylation on S237 that is likely to be secondary to increased AMP-activated protein kinase activity and potentially important for contraction-stimulated glucose uptake. Several studies that evaluated both normal and insulin-resistant skeletal muscle stimulated with a physiological insulin concentration after a single exercise session found that greater post-exercise insulin-stimulated glucose uptake was accompanied by greater TBC1D4 phosphorylation on several sites. In contrast, enhanced post-exercise insulin sensitivity was not accompanied by greater insulin-stimulated TBC1D1 phosphorylation. The mechanism for greater TBC1D4 phosphorylation in insulin-stimulated muscles after acute exercise is uncertain, and a causal link between enhanced TBC1D4 phosphorylation and increased post-exercise insulin sensitivity has yet to be established. In summary, TBC1D1 and TBC1D4 have important, but distinct roles in regulating muscle glucose transport in response to insulin and exercise.

Contraction-stimulated glucose transport in muscle is controlled by AMPK and mechanical stress but not sarcoplasmatic reticulum Ca(2+) release.

Understanding how muscle contraction orchestrates insulin-independent muscle glucose transport may enable development of hyperglycemia-treating drugs. The prevailing concept implicates Ca2+ as a key feed forward regulator of glucose transport with secondary fine-tuning by metabolic feedback signals through proteins such as AMPK. Here, we demonstrate in incubated mouse muscle that Ca2+ release is neither sufficient nor strictly necessary to increase glucose transport. Rather, the glucose transport response is associated with metabolic feedback signals through AMPK, and mechanical stress-activated signals. Furthermore, artificial stimulation of AMPK combined with passive stretch of muscle is additive and sufficient to elicit the full contraction glucose transport response. These results suggest that ATP-turnover and mechanical stress feedback are sufficient to fully increase glucose transport during muscle contraction, and call for a major reconsideration of the established Ca2+ centric paradigm.

0366: A role for focal adhesion kinase in the stimulation of glucose transport in cardiomyocytes

Stimulation of glucose transport in response to insulin or metabolic stress is an important determinant of cardiomyocytes function and survival, particularly during ischemia-reperfusion episodes. Stimulation of glucose transport is markedly impaired in cardiomyocytes exposed to free fatty acids (FA), despite relative preservation of insulin- or metabolic stress signaling. To determine whether Focal Adhesion Kinase (FAK) activity is required for stimulation of glucose transport in cardiomyocytes, and whether FAK downregulation participates in FA-induced impairment of glucose transport stimulation. Glucose transport, measured in isolated cardiomyocytes, was acutely stimulated either by insulin treatment, or by metabolic inhibition with oligomycin resulting in AMP-activated kinase (AMPK) activation. FAK activity was inhibited pharmacologically by preincubation with PF-573,228 (PF). FAK activity was assessed from its autophosphorylation on residue Y397, and from the phosphorylation of paxillin on Y118. Y397 FAK phosphorylation was reduced in cardiomyocytes chronically exposed to FA. Preincubation with PF prior to determination of glucose transport resulted in a significant reduction of oligomycin-stimulated glucose transport, with a modest reduction in insulin-stimulated glucose transport. Insulin and AMPK signaling was unaffected by PF preincubation. Intriguingly metabolic stress provoked Y397 FAK dephosphorylation and deactivation, as evidenced by a concomitant reduction in Y118 paxillin phosphorylation. stimulation of glucose transport by insulin or metabolic stress in cardiomyocytes requires FAK activity prior to stimulation; FAK activity is however acutely reduced during metabolic stress. The chronic reduction of FAK activity in cardiomyocytes exposed to FFA partially explains the loss of glucose transport responsiveness to insulin or metabolic inhibition.

Chapter Nine - Estrogen and Mitochondria Function in Cardiorenal Metabolic Syndrome

The cardiorenal metabolic syndrome (CRS) consists of a constellation of cardiac, renal, and metabolic disorders including insulin resistance (IR), obesity, metabolic dyslipidemia, high-blood pressure, and evidence of early cardiac and renal disease. Mitochondria dysfunction often occurs in the CRS, and this dysfunction is promoted by excess reactive oxygen species, genetic factors, IR, aging, and altered mitochondrial biogenesis. Recently, it has been shown that there are important sex-related differences in mitochondria function and metabolic, cardiovascular, and renal components. Sex differences in the CRS have mainly been attributed to the estrogen's effects that are mainly mediated by estrogen receptor (ER) α, ERβ, and G-protein coupled receptor 30. In this review, we discuss the effects of estrogen on the mitochondrial function, insulin metabolic signaling, glucose transport, lipid metabolism, and inflammatory responses from liver, pancreatic β cells, adipocytes, skeletal muscle, and cardiovascular tissue.

Soybean Toxin (SBTX) Impairs Fungal Growth by Interfering with Molecular Transport, Carbohydrate/Amino Acid Metabolism and Drug/Stress Responses

Soybean toxin (SBTX) is an antifungal protein from soybeans with broad inhibitory activity against the growth and filamentation of many fungi, including human and plant pathogenic species such as Candida albicans, Candida parapsilosis, Aspergillus niger, Penicillium herquei, Cercospora sojina and Cercospora kikuchii. Understanding the mechanism by which SBTX acts on fungi and yeasts may contribute to the design of novel antifungal drugs and/or the development of transgenic plants resistant to pathogens. To this end, the polymorphic yeast C. albicans was chosen as a model organism and changes in the gene expression profile of strain SC5314 upon exposure to SBTX were examined. Genes that were differentially regulated in the presence of SBTX were involved in glucose transport and starvation-associated stress responses as well as in the control of both the induction and repression of C. albicans hyphal formation. Transmission electron microscopy showed that C. albicans cells exposed to SBTX displayed severe signs of starvation and were heavily granulated. Our data were indicative of C. albicans cell starvation despite sufficient nutrient availability in the medium; therefore, it can be speculated that SBTX blocks nutrient uptake systems. Because neither the starvation signal nor the alkaline response pathway lead to the induction of hyphae, we hypothesise that conflicting signals are transmitted to the complex regulatory network controlling morphogenesis, eventually preventing the filamentation signal from reaching a significant threshold.

Dual effect of the heart-targeting cytokine cardiotrophin-1 on glucose transport in cardiomyocytes

Cardiotrophin-1 (CT-1) is a heart-targeting cytokine that is increased in the metabolic syndrome due to overexpression in the adipocytes. The effects of CT-1 on cardiomyocyte substrate metabolism remain unknown. We therefore determined the effects of CT-1 on basal and stimulated glucose transport in cardiomyocytes exposed to a low dose (1nM) or a high dose (10nM). Dose–response curves for insulin showed that 1nM CT-1 reduced insulin responsiveness, while 10nM CT-1 increased insulin responsiveness. In either condition insulin sensitivity was unaffected. Similarly 1nM CT-1 reduced the stimulation of glucose transport in response to metabolic stress, induced by the mitochondrial poison oligomycin, while 10nM CT-1 increased this response. Reduction of stimulated glucose transport by 1nM CT-1 was associated with overexpression of SOCS-3, a protein known to hinder proximal insulin signaling, and increased phosphorylation of STAT5. In cardiomyocytes exposed to 1nM CT-1 there was also reduced phosphorylation of Akt and AS160 in response to insulin, and of AMPK in response to oligomycin. Insulin-stimulated glucose transport and signaling were restored by inhibition of STAT5 activity. On the other hand in cardiomyocytes exposed to 10nM CT-1 there was increased phosphorylation of the AS160 and Akt in response to insulin. Most importantly, basal and oligomycin-stimulated phosphorylation of AMPK was markedly increased in cardiomyocytes exposed to 10nM CT-1. The enhancement of basal and stimulated-glucose transport was abolished in cardiomyocytes treated with the calmodulin-dependent kinase II (CaMKII) inhibitor KN93, and so was AMPK phosphorylation. This suggests that activation of CaMKII mediates activation of AMPK by a high dose of CT-1 independently of metabolic stress. Our results point to a role for CT-1 in the regulation of myocardial glucose metabolism and implicate entirely separate mechanisms in the inhibitory or stimulatory effects of CT-1 on glucose transport at low or high concentrations respectively.