Insulin-mediated translocation of GLUT-4-containing vesicles is preserved in denervated muscles

Skeletal muscle denervation decreases insulin-sensitive glucose uptake into this tissue as a result of marked GLUT-4 protein downregulation ( approximately 20% of controls). The process of insulin-stimulated glucose transport in muscle requires the movement or translocation of intracellular GLUT-4-rich vesicles to the cell surface, and it is accompanied by the translocation of several additional vesicular cargo proteins. Thus examining GLUT-4 translocation in muscles from denervated animals allows us to determine whether the loss of a major cargo protein, GLUT-4, affects the insulin-dependent behavior of the remaining cargo proteins. We find no difference, control vs. denervated, in the insulin-dependent translocation of the insulin-responsive aminopeptidase (IRAP) and the receptors for transferrin and insulin-like growth factor II/mannose 6-phosphate, proteins that completely (IRAP) or partially co-localize with GLUT-4. We conclude that 1) denervation of skeletal muscle does not block the specific branch of insulin signaling pathway that connects receptor proximal events to intracellular GLUT-4-vesicles, and 2) normal levels of GLUT-4 protein are not necessary for the structural organization and insulin-sensitive translocation of its cognate intracellular compartment. Muscle denervation also causes a twofold increase in GLUT-1. In normal muscle, all GLUT-1 is present at the cell surface, but in denervated muscle a significant fraction (25.1 +/- 6.1%) of this transporter is found in intracellular vesicles that have the same sedimentation coefficient as GLUT-4-containing vesicles but can be separated from the latter by immunoadsorption. These GLUT-1-containing vesicles respond to insulin and translocate to the cell surface. Thus the formation of insulin-sensitive GLUT-1-containing vesicles in denervated muscle may be a compensatory mechanism for the decreased level of GLUT-4.     

Mechanisms underlying eating-drinking transitions in rats

McFarland (1969) suggested two mechanisms whereby a switch could occur from one activity to another: ‘competition’, which he depicted as a gradual increase in the causal-factor strength (CFS) of activity 2, and ‘disinhibition’, which he depticted as a sudden decrease in the CFS of activity 1. We postulate two further mechanisms: ‘satiation’, depicted as a gradual decrease in the CFS of activity 1, and ‘inhibition’, depicted as a sudden increase in the CFS of activity 2. We suggest that disinhibition may be a less common mechanism of behavioural switching than is usually supposed, and describe three experiments suggesting that eat/drink and drink/eat switches in the rat occur by competition and/or satiation rather than by disinhibition and/or inhibition. In experiment 1, varying degrees of water deprivation were found to affect the timing of eat/drink switches in food-deprived rats; in experiment 2, varying rates of food availability were found to affect the timing of drink/eat switches in water-deprived rats; in experiment 3, drink/eat switches were delayed by allowing rats to ‘drink’ air instead of water.     

Mechanisms underlying stage-1 TRPL channel translocation in Drosophila photoreceptors

Background

TRP channels function as key mediators of sensory transduction and other cellular signaling pathways. In Drosophila, TRP and TRPL are the light-activated channels in photoreceptors. While TRP is statically localized in the signaling compartment of the cell (the rhabdomere), TRPL localization is regulated by light. TRPL channels translocate out of the rhabdomere in two distinct stages, returning to the rhabdomere with dark-incubation. Translocation of TRPL channels regulates their availability, and thereby the gain of the signal. Little, however, is known about the mechanisms underlying this trafficking of TRPL channels.

Methodology/Principal Findings

We first examine the involvement of de novo protein synthesis in TRPL translocation. We feed flies cycloheximide, verify inhibition of protein synthesis, and test for TRPL translocation in photoreceptors. We find that protein synthesis is not involved in either stage of TRPL translocation out of the rhabdomere, but that re-localization to the rhabdomere from stage-1, but not stage-2, depends on protein synthesis. We also characterize an ex vivo eye preparation that is amenable to biochemical and genetic manipulation. We use this preparation to examine mechanisms of stage-1 TRPL translocation. We find that stage-1 translocation is: induced with ATP depletion, unaltered with perturbation of the actin cytoskeleton or inhibition of endocytosis, and slowed with increased membrane sterol content.

Conclusions/Significance

Our results indicate that translocation of TRPL out of the rhabdomere is likely due to protein transport, and not degradation/re-synthesis. Re-localization from each stage to the rhabdomere likely involves different strategies. Since TRPL channels can translocate to stage-1 in the absence of ATP, with no major requirement of the cytoskeleton, we suggest that stage-1 translocation involves simple diffusion through the apical membrane, which may be regulated by release of a light-dependent anchor in the rhabdomere.     

Mechanisms of impaired calcium handling underlying subclinical diastolic dysfunction in diabetes

Isolated diastolic dysfunction is found in almost half of asymptomatic patients with well-controlled diabetes and may precede diastolic heart failure. However, mechanisms that underlie diastolic dysfunction during diabetes are not well understood. We tested the hypothesis that isolated diastolic dysfunction is associated with impaired myocardial Ca(2+) handling during type 1 diabetes. Streptozotocin-induced diabetic rats were compared with age-matched placebo-treated rats. Global left ventricular myocardial performance and systolic function were preserved in diabetic animals. Diabetes-induced diastolic dysfunction was evident on Doppler flow imaging, based on the altered patterns of mitral inflow and pulmonary venous flows. In isolated ventricular myocytes, diabetes resulted in significant prolongation of action potential duration compared with controls, with afterdepolarizations occurring in diabetic myocytes (P < 0.05). Sustained outward K(+) current and peak outward component of the inward rectifier were reduced in diabetic myocytes, while transient outward current was increased. There was no significant change in L-type Ca(2+) current; however, Ca(2+) transient amplitude was reduced and transient decay was prolonged by 38% in diabetic compared with control myocytes (P < 0.05). Sarcoplasmic reticulum Ca(2+) load (estimated by measuring the integral of caffeine-evoked Na(+)-Ca(2+) exchanger current and Ca(2+) transient amplitudes) was reduced by approximately 50% in diabetic myocytes (P < 0.05). In permeabilized myocytes, Ca(2+) spark amplitude and frequency were reduced by 34 and 20%, respectively, in diabetic compared with control myocytes (P < 0.05). Sarco(endo)plasmic reticulum Ca(2+)-ATPase-2a protein levels were decreased during diabetes. These data suggest that in vitro impairment of Ca(2+) reuptake during myocyte relaxation contributes to in vivo diastolic dysfunction, with preserved global systolic function, during diabetes.     

Mechanisms underlying the chronic pioglitazone treatment-induced improvement in the impaired endothelium-dependent relaxation seen in aortas from diabetic rats

The objectives of this study were to determine the effects of chronic treatment with pioglitazone, a peroxisome proliferator-activated receptor gamma agonist, on the impaired endothelium-dependent relaxation seen in aortas from established streptozotocin (STZ)-induced diabetic rats, and to identify some of the molecular mechanisms involved. Starting at 8 weeks of diabetes, pioglitazone (10 mg/kg) was administered to STZ-induced diabetic rats for 4 weeks. In untreated STZ rats (vs age-matched control rats): (1) ACh-induced relaxation, cGMP accumulation, phosphorylation of the cGMP-dependent protein kinase substrate vasodilator-stimulated phosphoprotein at Ser-239 [an established biochemical end-point of nitric oxide (NO)/cGMP signaling], and Cu/Zn-superoxide dismutase (SOD) expression and SOD activity were all reduced; (2) aortic superoxide generation, nitrotyrosine expression, and NAD(P)H oxidase activity were increased; (3) plasma endothelin-1 (ET-1) and aortic c-Jun (AP-1 component) protein expressions were increased. Pioglitazone treatment markedly corrected the above abnormalities. Collectively, these results suggest that pioglitazone treatment improves endothelium-dependent relaxation by reducing oxidative stress via increased SOD activity, decreased NAD(P)H oxidase activity, and a decreased ET-1 level, and that this decreased ET-1 level may be attributable to an inhibition of the AP-1 signaling pathway.     

Insulin stimulation of GLUT-4 translocation: a model for regulated recycling

Insulin stimulates glucose transport in muscle and fat cells by causing the redistribution of a facilitative glucose transporter, GLUT-4, from an intracellular compartment to the cell surface. But what is this intracellular GLUT-4 compartment? It may be a specialized compartment, perhaps analogous to synaptic vesicles, or may simply be part of the endosomal system. Other constituents of this compartment might be regulators of GLUT-4 movement to the cell surface, and their identification should make it possible to find the link between the insulin signal transduction pathway and GLUT-4 translocation.     

Short-term exercise enhances insulin-stimulated GLUT-4 translocation and glucose transport in adipose cells

This investigation examined the effectsof short-term exercise training on insulin-stimulated GLUT-4 glucosetransporter translocation and glucose transport activity in rat adiposecells. Male Wistar rats were randomly assigned to a sedentary (Sed) orswim training group (Sw, 4 days; final 3 days: 2 × 3 h/day). Adipose cell size decreased significantly but minimally(~20%), whereas total GLUT-4 increased by 30% in Sw vs. Sed rats.Basal3-O-methyl-D-[¹⁴C]glucosetransport was reduced by 62%, whereas maximally insulin-stimulated (MIS) glucose transport was increased by 36% in Sw vs. Sed rats. MIScell surface GLUT-4 photolabeling was 44% higher in the Sw vs. Sedanimals, similar to the increases observed in MIS glucose transportactivity and total GLUT-4. These results suggest that increases intotal GLUT-4 and GLUT-4 translocation to the cell surface contribute tothe increase in MIS glucose transport with short-term exercisetraining. In addition, the results suggest that the exercisetraining-induced adaptations in glucose transport occur more rapidlythan previously thought and with minimal changes in adipose cell size.

     

Mechanisms underlying gastric antiulcerative activity of nitroxides in rats

Reactive oxygen-derived species and redox-active metals are implicated in mediation of the pathogenesis of gastric mucosal damage and ulceration. Therefore, common strategies of intervention employ metal chelators, antioxidative enzymes, and low-molecular-weight antioxidants (LMWA). The aim of the present study was to elaborate the mechanism(s) responsible for the protection provided by nitroxide radicals in the experimental model of gastric ulceration.

Fasted male rats were treated ig with 1 ml 96% ethanol, with or without ig pretreatment with nitroxide or hydroxylamine. In several experiments, rats were injected ip or iv with iron(III) or iron(II) prior to ethanol administration. Rats were sacrificed 10 min after ethanol administration, the stomach was removed, washed and lesion area measured. Pretreatment with iron(III) complexed to nitrilotriacetate or citrate, aggravated the extent of the gastric injury. Conversely, iron(III) inhibited the formation of lesions. The nitroxides were rapidly reduced to their respective hydroxylamines and demonstrated antiulcerative activity for rats treated with iron. However, injecting the hydroxylamine resulted in a similar tissue distribution of nitroxide/hydroxylamine but did not provide protection.

The results show that: (a) the nitroxide radicals, rather than their respective non-radical reduced form, are the active species responsible for protection; (b) nitroxides protect by dismutating O^·-₂ and possibly indirectly increasing the NO level; (c) unlike classical LMWA which are reducing agents, nitroxides inhibit gastric damage by acting as mild oxidants, oxidizing reduced metals and pre-empting the Fenton reaction; and (d) the nitroxides act catalytically as recycling antioxidants.     

Mechanisms underlying gastric antiulcerative activity of nitroxides in rats

Reactive oxygen-derived species and redox-active metals are implicated in mediation of the pathogenesis of gastric mucosal damage and ulceration. Therefore, common strategies of intervention employ metal chelators, antioxidative enzymes, and low-molecular-weight antioxidants (LMWA). The aim of the present study was to elaborate the mechanism(s) responsible for the protection provided by nitroxide radicals in the experimental model of gastric ulceration. Fasted male rats were treated ig with 1 ml 96% ethanol, with or without ig pretreatment with nitroxide or hydroxylamine. In several experiments, rats were injected ip or iv with iron(III) or iron(II) prior to ethanol administration. Rats were sacrificed 10 min after ethanol administration, the stomach was removed, washed and lesion area measured. Pretreatment with iron(III) complexed to nitrilotriacetate or citrate, aggravated the extent of the gastric injury. Conversely, iron(II) inhibited the formation of lesions. The nitroxides were rapidly reduced to their respective hydroxylamines and demonstrated antiulcerative activity for rats treated with iron. However, injecting the hydroxylamine resulted in a similar tissue distribution of nitroxide/hydroxylamnine but did not provide protection. The results show that: (a) the nitroxide radicals, rather than their respective non-radical reduced form, are the active species responsible for protection; (b) nitroxides protect by dismutating O2*- and possibly indirectly increasing the NO level; (c) unlike classical LMWA which are reducing agents, nitroxides inhibit gastric damage by acting as mild oxidants, oxidizing reduced metals and pre-empting the Fenton reaction; and (d) the nitroxides act catalytically as recycling antioxidants.     

Increased GLUT-4 translocation mediates enhanced insulin sensitivity of muscle glucose transport after exercise

The purpose of this study was to determinewhether the increase in insulin sensitivity of skeletal muscle glucosetransport induced by a single bout of exercise is mediated by enhancedtranslocation of the GLUT-4 glucose transporter to the cell surface.The rate of3-O-[³H]methyl-D-glucosetransport stimulated by a submaximally effective concentration ofinsulin (30 µU/ml) was approximately twofold greater in the musclesstudied 3.5 h after exercise than in those of the sedentary controls(0.89 ± 0.10 vs. 0.43 ± 0.05 µmol · ml¹ · 10 min¹; means ± SE forn = 6/group). GLUT-4 translocation wasassessed by using theATB-[2-³H]BMPAexofacial photolabeling technique. Prior exercise resulted in greatercell surface GLUT-4 labeling in response to submaximal insulintreatment (5.36 ± 0.45 dpm × 10³/g in exercised vs. 3.00 ± 0.38 dpm × 10³/g insedentary group; n = 10/group) thatclosely mirrored the increase in glucose transport activity. The signalgenerated by the insulin receptor, as reflected in the extent ofinsulin receptor substrate-1 tyrosine phosphorylation, was unchangedafter the exercise. We conclude that the increase in muscle insulinsensitivity of glucose transport after exercise is due to translocationof more GLUT-4 to the cell surface and that this effect is not due topotentiation of insulin-stimulated tyrosine phosphorylation.

     

Mechanisms underlying the supra-maximal thermogenesis elicited by aminophylline in rats

Previous studies showed that acute treatment with aminophylline (AMPY) significantly elevated maximum thermogenesis and improved cold tolerance in rats and man in severe cold. However, the exact mechanism by which AMPY enhances thermogenesis was unknown. Rats receiving enprofylline (ENPRO) (1.5 and 15 mg/kg, i.p.), a selective phosphodiesterase inhibitor, failed to show enhanced thermogenesis. In contrast, treatment with a selective adenosine receptor antagonist, 8-phenyltheophylline(8-PT; 2.5 to 10 mg/kg, i.p.), significantly increased (p less than 0.05) thermogenesis and cold tolerance. However, the maximal thermogenic effect by optimal dose of 8-PT (5 mg/kg) was significantly lower than that with optimal dose of AMPY (18.7 mg/kg, i.p.); the deficit could be eradicated by combining optimal 8-PT dose with a low dose of AMPY (1.25 mg/kg), but not with ENPRO. These results indicate that the thermogenic effect of AMPY is not by inhibition of phosphodiesterase but at least partially by antagonism of adenosine receptors. It is also apparent that older mechanisms in addition to adenosine antagonism are also involved in AMPY's thermogenic action.     

GLUT-4 translocation in skeletal muscle studied with a cell-free assay: involvement of phospholipase D

GLUT-4-containing membranes immunoprecipitated from insulin-stimulated rat skeletal muscle produce the phospholipase D (PLD) product phosphatidic acid. In vitro stimulation of PLD in crude membrane with ammonium sulfate (5 mM) resulted in transfer of GLUT-4 (3.0-fold vs. control) as well as transferrin receptor proteins from large to small membrane structures. The in vitro GLUT-4 transfer could be blocked by neomycin (a PLD inhibitor), and neomycin also reduced insulin-stimulated glucose transport in intact incubated soleus muscles. Furthermore, protein kinase B(beta) (PKB(beta)) was found to associate with the GLUT-4 protein and was transferred to small vesicles in response to ammonium sulfate in vitro. Finally, addition of cytosolic proteins, prepared from basal skeletal muscle, and GTP nucleotides to an enriched GLUT-4 membrane fraction resulted in in vitro transfer of GLUT-4 to small membranes (6.8-fold vs. unstimulated control). The cytosol and nucleotide-induced GLUT-4 transfer could be blocked by neomycin and N-ethylmaleimide. In conclusion, we have developed a cell-free assay that demonstrates in vitro GLUT-4 transfer. This transfer may suggest release of GLUT-4-containing vesicles from donor GLUT-4 membranes involving PLD activity and binding of PKB(beta) to GLUT-4.     

Mechanisms underlying intracisternal TRH-induced stimulation of gastric acid secretion in rats

The neurohumoral pathways mediating intracisternal TRH-induced stimulation of gastric acid secretion were investigated. In urethane-anesthetized rats, with gastric and intrajugular cannulas, TRH or the analog [N-Val2]-TRH (1 microgram) injected intracisternally increased gastric acid output for 90 min. Serum gastrin levels were not elevated significantly. Under these conditions the TRH analog, unlike TRH, was devoid of thyrotropin-releasing activity as measured by serum TSH levels. In pylorus-ligated rats, gastrin values were not modified 2 h after peptide injection whereas gastric acid output was enhanced. TRH (0.1-1 micrograms) stimulated vagal efferent discharge, recorded from a multifiber preparation of the cervical vagus in urethane-anesthetized rats and the response was dose-dependent. The time course of vagal activation was well correlated with the time profile of gastric stimulation measured every 2 min. These results demonstrated that gastric acid secretory stimulation elicited by intracisternal TRH is not related to changes in circulating levels of gastrin or TSH but is mediated by the activation of efferent vagal pathways that stimulated parietal cell secretion.     

Increased expression of GLUT-4 and hexokinase in rat epitrochlearis muscles exposed to AICAR in vitro.

Exercise acutely stimulates muscle glucose transport and also brings about an adaptive increase in the capacity of muscle for glucose uptake by inducing increases in GLUT-4 and hexokinase.(1) Recent studies have provided evidence that activation of AMP protein kinase (AMPK) is involved in the stimulation of glucose transport by exercise. The purpose of this study was to determine whether activation of AMPK is also involved in mediating the adaptive increases in GLUT-4 and hexokinase. To this end, we examined the effect of incubating rat epitrochlearis muscles in culture medium for 18 h in the presence or absence of 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), which enters cells and is converted to the AMP analog ZMP, thus activating AMPK. Exposure of muscles to 0.5 mM AICAR in vitro for 18 h resulted in an approximately 50% increase in GLUT-4 protein and an approximately 80% increase in hexokinase. This finding provides strong evidence in support of the hypothesis that the activation of AMPK that occurs in muscle during exercise is involved in mediating the adaptive increases in GLUT-4 and hexokinase.     

Mechanisms underlying the cardiovascular responses to intrathecal vasopressin administration in rats

Vasopressinergic pathways within the spinal cord have been implicated in the control of cardiovascular function. This study was undertaken to determine the mechanisms whereby intrathecally administered arginine vasopressin (AVP) increases blood pressure and heart rate in anesthetized rats. The cardiovascular responses to intrathecal AVP administration were significantly attenuated after intravenous administration of the ganglionic blocking agent, chlorisondamine chloride, as were the pressor responses following alpha-adrenergic receptor blockade with phentolamine and the heart rate responses following beta-receptor blockade with propranolol. Intrathecal administration of the V1 vasopressin receptor antagonist d(CH2)5Tyr(Me)AVP completely blocked the cardiovascular responses to intrathecal AVP injections, but did not significantly alter the responses to intrathecal substance P injections. There was no evidence for the involvement of the renin-angiotensin system in the pressor responses to intrathecal AVP, as (i) an angiotensin II receptor blocking agent, [Sar1, Val5, Ala8]angiotensin, failed to significantly alter the responses to intrathecal AVP, and (ii) plasma renin levels did not change following administration of the peptide. Intrathecal injections of [3H]AVP suggest that only small amounts of the peptide may cross into the plasma during the time in which the cardiovascular variables are changing. These data provide evidence that intrathecally administered AVP discretely activates the sympathetic outflow to the heart and vasculature, and confirm the neurally mediated nature of the response.     

Mechanisms underlying increased release of endothelin-1 from aorta in diabetic rats

To clarify the mechanism underlying increased endothelin-1 release in diabetic rats, we examined its release from thoracic aortas obtained from streptozotocin-induced diabetic rats. The methoxamine-induced contraction was significantly inhibited by BQ-123 plus BQ-788 (specific antagonists for ET(A) and ET(B) receptors) in diabetic, but not control rats. Preincubation with phosphoramidon also inhibited the methoxamine-induced contraction in diabetic but not control rats. The expression of prepro endothelin-1 mRNA was significantly enhanced in aortas from streptozotocin-induced diabetic rats. These results suggest that the increases in the basal and alpha-agonist-induced release of endothelin-1 in the diabetic state may be due to an overexpression of the mRNA for prepro endothelin-1.     

Inhibition of calpain results in impaired contraction-stimulated GLUT4 translocation in skeletal muscle

It was previously found that transgenic mice that overexpress the calpain inhibitor calpastatin (CsTg) have an approximately 3-fold increase in GLUT4 protein in their skeletal muscles. Despite the increase in GLUT4, which appears to be due to inhibition of its proteolysis by calpain, insulin-stimulated glucose transport is not increased in CsTg muscles. PKB (Akt) protein level is reduced approximately 60% in CsTg muscles, suggesting a possible mechanism for the relative insulin resistance. Muscle contractions stimulate glucose transport by a mechanism that is independent of insulin signaling. The purpose of this study was to test the hypothesis that the threefold increase in GLUT4 in CsTg would result in a large increase in contraction-stimulated glucose transport. CAMKII and AMPK mediate steps in the contraction-stimulated pathway. The protein levels of AMPK and CAMKII were increased three- to fourfold in CsTg muscles, suggesting that these proteins are also calpain substrates. Despite the large increases in GLUT4, AMPK, and CAMKII, contraction-stimulated GLUT4 translocation and glucose transport were not increased above wild-type values. These findings suggest that inhibition of calpain results in impairment of a step in the GLUT4 translocation process downstream of the insulin- and contraction-signaling pathways. They also provide evidence that CAMKII and AMPK are calpain substrates.     

Role of PDE3B in insulin-induced glucose uptake, GLUT-4 translocation and lipogenesis in primary rat adipocytes

In adipocytes, phosphorylation and activation of PDE3B is a key event in the antilipolytic action of insulin. The role of PDE4, another PDE present in adipocytes, is not yet known. In this work we investigate the role of PDE3B and PDE4 in insulin-induced glucose uptake, GLUT-4 translocation and lipogenesis. Inhibition of PDE3 (OPC3911, milrinone) but not PDE4 (RO 20-1724) lowered insulin-induced glucose uptake and lipogenesis, especially in the presence of isoproterenol (a general beta-adrenergic agonist), CL316243, a selective beta3-adrenergic agonist, and pituitary adenylate cyclase-activating peptide. The inhibitory effect of OPC3911 was associated with reduced translocation of GLUT-4 from the cytosol to the plasma membrane. Both OPC3911 and RO 20-1724 increased protein kinase A (PKA) activity and lipolysis. H89, a PKA inhibitor, did not affect OPC3911-mediated inhibition of insulin-induced glucose uptake and lipogenesis, whereas 8-pCPT-2'-O-Me-cAMP, an Epac agonist which mediates PKA independent cAMP signaling events, mimicked all the effects of OPC3911. Insulin-mediated activation of protein kinase B, a kinase involved in insulin-induced glucose uptake, was apparently not altered by OPC3911. In summary, our data suggest that PDE3B, but not PDE4, contributes to the regulation of insulin-induced glucose uptake, GLUT-4 translocation, and lipogenesis presumably by regulation of a cAMP/Epac signalling mechanisms.