Paneth cell α-defensins HD-5 and HD-6 display differential degradation into active antimicrobial fragments.

Antimicrobial peptides, in particular α-defensins expressed by Paneth cells, control microbiota composition and play a key role in intestinal barrier function and homeostasis. Dynamic conditions in the local microenvironment, such as pH and redox potential, significantly affect the antimicrobial spectrum. In contrast to oxidized peptides, some reduced defensins exhibit increased vulnerability to proteolytic degradation. In this report, we investigated the susceptibility of Paneth-cell-specific human α-defensin 5 (HD-5) and -6 (HD-6) to intestinal proteases using natural human duodenal fluid. We systematically assessed proteolytic degradation using liquid chromatography-mass spectrometry and identified several active defensin fragments capable of impacting bacterial growth of both commensal and pathogenic origins. Of note, incubation of mucus with HD-5 resulted in 255-8,000 new antimicrobial combinations. In contrast, HD-6 remained stable with consistent preserved nanonet formation. In vivo studies demonstrated proof of concept that a HD-5 fragment shifted microbiota composition (e.g., increases of Akkermansia sp.) without decreasing diversity. Our data support the concept that secretion of host peptides results in an environmentally dependent increase of antimicrobial defense by clustering in active peptide fragments. This complex clustering mechanism dramatically increases the host's ability to control pathogens and commensals. These findings broaden our understanding of host modulation of the microbiome as well as the complexity of human mucosal defense mechanisms, thus providing promising avenues to explore for drug development.

Distinct patterns of tissue-specific lipid accumulation during the induction of insulin resistance in mice by high-fat feeding.

AIMS/HYPOTHESIS: While it is well known that diet-induced obesity causes insulin resistance, the precise mechanisms underpinning the initiation of insulin resistance are unclear. To determine factors that may cause insulin resistance, we have performed a detailed time-course study in mice fed a high-fat diet (HFD). METHODS: C57Bl/6 mice were fed chow or an HFD from 3 days to 16 weeks and glucose tolerance and tissue-specific insulin action were determined. Tissue lipid profiles were analysed by mass spectrometry and inflammatory markers were measured in adipose tissue, liver and skeletal muscle. RESULTS: Glucose intolerance developed within 3 days of the HFD and did not deteriorate further in the period to 12 weeks. Whole-body insulin resistance, measured by hyperinsulinaemic-euglycaemic clamp, was detected after 1 week of HFD and was due to hepatic insulin resistance. Adipose tissue was insulin resistant after 1 week, while skeletal muscle displayed insulin resistance at 3 weeks, coinciding with a defect in glucose disposal. Interestingly, no further deterioration in insulin sensitivity was observed in any tissue after this initial defect. Diacylglycerol content was increased in liver and muscle when insulin resistance first developed, while the onset of insulin resistance in adipose tissue was associated with increases in ceramide and sphingomyelin. Adipose tissue inflammation was only detected at 16 weeks of HFD and did not correlate with the induction of insulin resistance. CONCLUSIONS/INTERPRETATION: HFD-induced whole-body insulin resistance is initiated by impaired hepatic insulin action and exacerbated by skeletal muscle insulin resistance and is associated with the accumulation of specific bioactive lipid species.

Targeted disruption of inducible nitric oxide synthase protects against obesity-linked insulin resistance in muscle.

Inducible nitric oxide synthase (iNOS) is induced by inflammatory cytokines in skeletal muscle and fat. It has been proposed that chronic iNOS induction may cause muscle insulin resistance. Here we show that iNOS expression is increased in muscle and fat of genetic and dietary models of obesity. Moreover, mice in which the gene encoding iNOS was disrupted (Nos2-/- mice) are protected from high-fat-induced insulin resistance. Whereas both wild-type and Nos2-/- mice developed obesity on the high-fat diet, obese Nos2-/- mice exhibited improved glucose tolerance, normal insulin sensitivity in vivo and normal insulin-stimulated glucose uptake in muscles. iNOS induction in obese wild-type mice was associated with impairments in phosphatidylinositol 3-kinase and Akt activation by insulin in muscle. These defects were fully prevented in obese Nos2-/- mice. These findings provide genetic evidence that iNOS is involved in the development of muscle insulin resistance in diet-induced obesity.

Host-Microbe Interplay in the Cardiometabolic Benefits of Dietary Polyphenols.

Polyphenols are nonessential phytonutrients abundantly found in fruits and vegetables. A wealth of data from preclinical models and clinical trials consistently supports cardiometabolic benefits associated with dietary polyphenols in murine models and humans. Furthermore, a growing number of studies have shown that specific classes of polyphenols, such as proanthocyanidins (PACs) and ellagitannins, as well as the stilbenoid resveratrol, can alleviate several features of the metabolic syndrome. Moreover, mounting evidence points to the gut microbiota as a key mediator of the health benefits of polyphenols. In this review we summarize recent findings supporting the beneficial potential of polyphenols against cardiometabolic diseases, with a focus on the role of host-microbe interactions.

Yogurt, diet quality and lifestyle factors.

Yogurt consumption has been associated with healthy dietary patterns and lifestyles, better diet quality and healthier metabolic profiles. Studies have shown that frequent yogurt consumers do not only have higher nutrient intakes, but also an improved diet quality, which includes higher consumption of fruits and vegetables, whole grains, and dairy compared with low or non-consumers indicating better compliance with dietary guidelines. Recent epidemiological and clinical evidence suggests that yogurt contributes to better metabolic health because of its effects on the control of body weight, energy homeostasis and glycemic control. Furthermore, yogurt consumers have been shown to be more physically active (⩾ 2 h/week), smoke less, have higher education and knowledge of nutrition compared with non-consumers. Thus, yogurt consumption may be considered a signature of a healthy diet through its nutritional content, impact on metabolic health including the control of energy balance, body weight and glycemia and its relationships with healthier behaviors and lifestyle factors.

Sustained activation of insulin receptors internalized in GLUT4 vesicles of insulin-stimulated skeletal muscle.

Exposure of target cells to insulin results in the formation of ligand receptor complexes on the cell surface and their subsequent internalization into the endosomal apparatus. A current view is that endocytosis of the insulin receptor (IR) kinase results in its rapid deactivation and sorting of the IR back to the cell surface or to late endocytic compartments. We report herein that, in skeletal muscle, in vivo stimulation with insulin induced a rapid internalization of the IR to an insulin-sensitive GLUT4-enriched intracellular membrane fraction. After 30 min of stimulation, IR content and tyrosine phosphorylation were increased by three and nine times in that fraction, respectively, compared with unstimulated muscles. In vitro autophosphorylation assays revealed that the kinase activity of internalized IRs was markedly augmented (eight to nine times) by insulin. In marked contrast with hepatic endosomes or adipocyte low-density microsomes, no IR tyrosine dephosphorylation activity was observed in GLUT4-enriched vesicles isolated from skeletal muscle. The activated IR was recovered in immunopurified GLUT4 vesicles after insulin stimulation. Insulin also increased tyrosine-phosphorylated insulin receptor substrate 1 and phosphatidylinositol 3-kinase adapter (p85) subunit contents in the intracellular membrane fraction, but these signaling molecules were not directly associated with GLUT4 vesicles. These results show that, in skeletal muscle, the activated IR reaches a GLUT4-enriched compartment where its activity is apparently sustained. We propose that compartmentalization of activated IRs to GLUT4 vesicles may play a role in sustaining insulin signaling at this locus in skeletal muscle.

Selective impairment in GLUT4 translocation to transverse tubules in skeletal muscle of streptozotocin-induced diabetic rats.

We previously reported that insulin induces the translocation of GLUT4 to both the plasma membrane and the transverse tubules (T-tubules) in rat skeletal muscle (Am J Physiol 270:E667-E676, 1996). The aim of the present study was to investigate whether the insulin-resistant glucose utilization of skeletal muscle from streptozotocin (STZ)-induced diabetic rats is linked to an impaired translocation of GLUT4 to the plasma membrane, the T-tubules, or both surface compartments. Whole-body insulin-mediated glucose disposal, assessed during a hyperinsulinemic-euglycemic clamp, was reduced by 48% (P < 0.01) in diabetic rats as compared with controls. Subcellular membrane fractions enriched with plasma membranes, T-tubules, or GLUT4-enriched intracellular membranes were isolated from hindlimb muscles of control and insulin-stimulated rats, and GLUT4 content was measured by Western blot analysis. In the absence of insulin (unstimulated), GLUT4 content in muscle of diabetic rats was markedly lower (by approximately 40%) in both the T-tubules and the intracellular membrane fraction as compared with controls. In contrast, the transporter protein levels were similar in the plasma membrane fraction. In skeletal muscle of control animals, the hyperinsulinemic clamp induced GLUT4 translocation from the intracellular membrane pool to both the plasma membrane and the T-tubule-enriched fractions (approximately 2.2-fold to approximately 2.5-fold). Surprisingly, insulin increased plasma membrane GLUT4 content to comparable levels in control and diabetic rat skeletal muscle. However, insulin-mediated GLUT4 translocation to the T-tubules was significantly reduced in the same muscle. Whole-body insulin action was significantly correlated with GLUT4 protein levels in the T-tubules, but not with the transporter content in either plasma membranes or intracellular membranes. These results strongly suggest that peripheral resistance to insulin action on glucose disposal in STZ-induced diabetic rats is caused by a selective impairment of GLUT4 translocation to skeletal muscle T-tubules.

Defective insulin-induced GLUT4 translocation in skeletal muscle of high fat-fed rats is associated with alterations in both Akt/protein kinase B and atypical protein kinase C (zeta/lambda) activities.

The cellular mechanism by which high-fat feeding induces skeletal muscle insulin resistance was investigated in the present study. Insulin-stimulated glucose transport was impaired ( approximately 40-60%) in muscles of high fat-fed rats. Muscle GLUT4 expression was significantly lower in these animals ( approximately 40%, P < 0.05) but only in type IIa-enriched muscle. Insulin stimulated the translocation of GLUT4 to both the plasma membrane and the transverse (T)-tubules in chow-fed rats. In marked contrast, GLUT4 translocation was completely abrogated in the muscle of insulin-stimulated high fat-fed rats. High-fat feeding markedly decreased insulin receptor substrate (IRS)-1-associated phosphatidylinositol (PI) 3-kinase activity but not insulin-induced tyrosine phosphorylation of the insulin receptor and IRS proteins in muscle. Impairment of PI 3-kinase function was associated with defective Akt/protein kinase B kinase activity (-40%, P < 0.01) in insulin-stimulated muscle of high fat-fed rats, despite unaltered phosphorylation (Ser473/Thr308) of the enzyme. Interestingly, basal activity of atypical protein kinase C (aPKC) was elevated in muscle of high fat-fed rats compared with chow-fed controls. Whereas insulin induced a twofold increase in aPKC kinase activity in the muscle of chow-fed rats, the hormone failed to further increase the kinase activity in high fat-fed rat muscle. In conclusion, it was found that GLUT4 translocation to both the plasma membrane and the T-tubules is impaired in the muscle of high fat-fed rats. We identified PI 3-kinase as the first step of the insulin signaling pathway to be impaired by high-fat feeding, and this was associated with alterations in both Akt and aPKC kinase activities.

Expression of nitric oxide synthase in skeletal muscle: a novel role for nitric oxide as a modulator of insulin action.

Previous studies have shown that nitric oxide synthase (NOS), the enzyme that catalyzes the formation of nitric oxide (NO), is expressed in skeletal muscle. The aim of the present study was to test the hypothesis that NO can modulate glucose metabolism in slow- and fast-twitch skeletal muscles. Calcium-dependent NOS was detected in skeletal muscle, and the enzyme activity was greater in fast-type extensor digitorum longus (EDL) muscles than in slow-type soleus muscles. Both the neuronal-type (nNOS) and endothelial-type (eNOS) enzymes are expressed in resting skeletal muscles. However, nNOS protein was only detected in EDL muscles, whereas eNOS protein contents were comparable in soleus and EDL muscles. NOS expression in muscle cryosections (diaphorase histochemistry) was located in vascular endothelium and in muscle fibers, and the staining was greater in type IIb than in type I and IIa fibers. The macrophage-type inducible NOS (iNOS) was not detected in resting muscle, but endotoxin treatment induced its expression, concomitant with elevated NO production. iNOS induction was associated with impaired insulin-stimulated glucose uptake in isolated rat muscles. In vitro, NOS blockade with specific inhibitors did not affect basal or insulin-stimulated glucose transport in EDL or soleus muscles. In contrast, the NO donors GEA 5024 and sodium nitroprusside induced dose-dependent inhibition (up to 50%) of maximal insulin-stimulated glucose transport in both muscles with minor effects on basal uptake values. GEA 5024 also blunted insulin-stimulated glucose transport and amino acid uptake in cultured L6 muscle cells without affecting insulin binding to its receptor. On the other hand, the permeable cGMP analogue dibutyryl cGMP did not affect muscle glucose transport. These results strongly suggest that NO modulates insulin action in both slow- and fast-type skeletal muscles. This novel autocrine action of NO in muscle appears to be mediated by cGMP-independent pathways.

Insulin induces the translocation of GLUT4 from a unique intracellular organelle to transverse tubules in rat skeletal muscle.

Skeletal muscle surface membrane is constituted by the PM domain and its specialized deep invaginations known as TTs. We have shown previously that insulin induces a rapid translocation of GLUT4s from an IM pool to the PM in rat skeletal muscle (6). In this study, we have investigated the possibility that insulin also stimulates the translocation of GLUT4 proteins to TTs, which constitute the largest area of the cell surface envelope. PM, TTs, and IM components of control and insulinized skeletal muscle were isolated by subcellular fractionation. The TTs then were purified further by removing vesicles of SR origin by using a Ca-loading procedure. Ca-loading resulted in a five- to sevenfold increase in the purification of TTs in the unloaded fraction relative to the loaded fraction, assessed by immunoblotting with an anti-DHP-receptor monoclonal antibody. In contrast, estimation of the content of Ca(2+)-ATPase protein (a marker of SR) with a specific polyclonal antibody revealed that most, if not all, SR vesicles were recovered in the Ca-loaded fraction. Western blotting with an anti-COOH-terminal GLUT4 protein polyclonal antibody revealed that acute insulin injection in vivo (30 min) increased the content of GLUT4 (by 90%) in isolated PMs and markedly enhanced (by 180%) GLUT4 content in purified TTs. Importantly, these insulin-dependent changes in GLUT4 content of PM and purified TTs were seen in the absence of changes in the alpha 1-subunit of the Na(+)-K(+)-ATPase, a surface membrane marker.(ABSTRACT TRUNCATED AT 250 WORDS)

The transferrin receptor defines two distinct contraction-responsive GLUT4 vesicle populations in skeletal muscle.

Insulin and contraction increase glucose transport in an additive fashion in skeletal muscle. However, it is still unclear whether they do so by inducing the recruitment of GLUT4 transporters from the same or distinct intracellular compartments to the plasma membrane and the T-tubules. Using the transferrin receptor as a recognized marker of recycling endosomes, we have examined whether insulin and/or contraction recruit GLUT4 from this pool to either the plasma membranes or T-tubules, isolated by subcellular fractionation of perfused hindlimb muscles. Either stimulus independently increased GLUT4 translocation from an intracellular fraction to both the plasma membrane and T-tubules. The combination of insulin and contraction induced a marked (approximately threefold) and almost fully additive increase in GLUT4 content, but only in the plasma membrane. Insulin did not stimulate transferrin receptor recruitment from the GLUT4-containing intracellular fraction to either the plasma membrane or the T-tubules. In contrast, contraction stimulated the recruitment of the transferrin receptor from the same GLUT4-containing intracellular fraction to the plasma membrane but not to the T-tubules. Contraction-induced recruitment of the transferrin receptor was also observed from immunopurified GLUT4 vesicles. It is concluded that muscle contraction stimulates translocation of GLUT4 from two distinct intracellular compartments: 1) a population of recycling endosomes that is selectively recruited to the plasma membrane and 2) from GLUT4 storage vesicles that are also insulin-responsive and recruited to both the plasma membrane and the T-tubules. The lack of additive translocation of GLUT4 to the T-tubules may be linked to the failure of GLUT4-containing recycling endosomes to be recruited to these structures.

Differential regulation of GLUT1 and GLUT4 glucose transporters in skeletal muscle of a new model of type II diabetes. The obese SHR/N-cp rat.

The obese diabetic SHR/N-cp rat is a newly developed strain that inherits obesity as an autosomal recessive trait. These rats display early-onset hyperinsulinemia and hyperglycemia, which are hallmarks of type II diabetes. This study was undertaken to determine the expression and the subcellular distribution of the GLUT1 and GLUT4 glucose transporters in skeletal muscle of obese diabetic SHR rats. D-glucose-protectable cytochalasin-B binding to subcellular membrane fractions of hindlimb muscles was used to determine glucose transporter number. GLUT1 and GLUT4 glucose transporter isotypes were detected using antibodies to the COOH-terminal region of the GLUT1 and GLUT4 proteins. Glucose transporter number was significantly lower (-40%) in crude unfractionated membranes of obese diabetic SHR than of lean SHR muscles. When crude membranes were fractionated to separate plasma membranes and the intracellular membranes containing glucose transporters, the number of cytochalasin-B binding sites was found to be markedly lower (-50%) in intracellular membranes and slightly but not significantly reduced (-20%) in plasma membranes of muscle from obese diabetic SHR compared with lean SHR rats. Western blot analysis revealed that a lower GLUT4 protein abundance (-40%) accounts for the reduced glucose transporter number in intracellular membranes of obese diabetic SHR compared with lean SHR muscles. GLUT4 protein content was also reduced by 50% in plasma membranes from obese SHR muscles relative to lean rat muscles.(ABSTRACT TRUNCATED AT 250 WORDS)

Opposite effects of hyperglycemia and insulin deficiency on liver glycogen synthase phosphatase activity in the diabetic rat.

The specific effect of hyperglycemia on the reported decrease in liver glycogen synthase phosphatase activity was studied in STZ-induced diabetic rats with normal fasting insulinemia. Four groups of animals were investigated: control (nondiabetic), diabetic hyperglycemic (STZ), diabetic normoglycemic (STZ followed by 3-day phloridzin treatment), and a diabetic normoglycemic group injected with glucose to reinstate hyperglycemia. None of the treatments significantly altered fasting plasma insulin and glucagon concentrations. We found that hepatic synthase phosphatase activity decreased in STZ-induced diabetic rats and was further markedly reduced when glycemia was normalized in the diabetic animals. This additional decrease in phosphatase activity was almost fully reversed when hyperglycemia was restored by acute glucose infusion of the normoglycemic diabetic rats. In parallel, the levels of liver G6P and F6P were markedly reduced in the diabetic normoglycemic rats and restored with reinstatement of hyperglycemia. In contrast, liver microsomal glucose-6-phosphatase activity was enhanced and glucokinase activity was lowered in all diabetic groups, regardless of glycemia. Our results indicate that hyperglycemia per se counteracts part of the loss of hepatic synthase phosphatase in diabetic animals and provokes the stable conversion of synthase phosphatase from a less active to a more active form.

Activation of p38 mitogen-activated protein kinase alpha and beta by insulin and contraction in rat skeletal muscle: potential role in the stimulation of glucose transport.

The stress-activated p38 mitogen-activated protein kinase (MAPK) was recently shown to be activated by insulin in muscle and adipose cells in culture. Here, we explore whether such stimulation is observed in rat skeletal muscle and whether muscle contraction can also affect the enzyme. Insulin injection (2 U over 3.5 min) resulted in increases in p38 MAPK phosphorylation measured in soleus (3.2-fold) and quadriceps (2.2-fold) muscles. Increased phosphorylation (3.5-fold) of an endogenous substrate of p38 MAPK, cAMP response element binder (CREB), was also observed. After in vivo insulin treatment, p38 MAPKalpha and p38 MAPKbeta isoforms were found to be activated (2.1- and 2.4-fold, respectively), using an in vitro kinase assay, in immunoprecipitates from quadriceps muscle extracts. In vitro insulin treatment (1 nmol/l over 4 min) and electrically-induced contraction of isolated extensor digitorum longus (EDL) muscle also doubled the kinase activity of p38 MAPKalpha and p38 MAPKbeta. The activity of both isoforms was inhibited in vitro by 10 micromol/l SB203580 in all muscles. To explore the possible participation of p38 MAPK in the stimulation of glucose uptake, EDL and soleus muscles were exposed to increasing doses of SB203580 before and during stimulation by insulin or contraction. SB203580 caused a significant reduction in the insulin- or contraction-stimulated 2-deoxyglucose uptake. Maximal inhibition (50-60%) occurred with 10 micromol/l SB203580. These results show that p38 MAPKalpha and -beta isoforms are activated by insulin and contraction in skeletal muscle. The data further suggest that activation of p38 MAPK may participate in the stimulation of glucose uptake by both stimuli in rat skeletal muscle.

Effect of diabetes on glucoregulation. From glucose transporters to glucose metabolism in vivo.

Peripheral resistance to insulin is a prominent feature of both insulin-dependent and non-insulin-dependent diabetes. Skeletal muscle is the primary site responsible for decreased insulin-induced glucose utilization in diabetic subjects. Glucose transport is the rate-limiting step for glucose utilization in muscle, and that cellular process is defective in human and animal diabetes. The transport of glucose across the muscle cell plasma membrane is mediated by glucose transporter proteins, and two isoforms (GLUT1 and GLUT4) are expressed in muscle. Insulin acutely increases glucose transport in muscle by selectively stimulating the recruitment of the GLUT4 transporter (but not GLUT1) from an intracellular pool to the plasma membrane. In skeletal muscles of streptozocin-induced diabetic rats, there is a decreased GLUT4 protein content in intracellular and plasma membranes. In these rats, insulin induced the mobilization of GLUT4 from the internal pool, but the incorporation of the transporter protein into the plasma membrane is diminished. Conversely, the content of the GLUT1 transporter increases in the plasma membrane of these diabetic rats. Normalization of glycemia with phlorizin fully restores the amount of GLUT1 and GLUT4 proteins to normal levels in the plasma membrane without altering insulin levels. This suggests that glycemia regulates the number of glucose transporters at the cell surface, GLUT1 varying directly and GLUT4 inversely, to glycemia. The regulatory role of glycemia also can be seen in diabetic dogs in vivo, where correction of hyperglycemia with phlorizin restores, at least in part, the defective metabolic clearance rate of glucose seen in these animals. In addition to acutely stimulating glucose transport in muscle, insulin controls exercise- and possibly stress-mediated glucose uptake in vivo, by preventing hyperglycemia and by restraining the effects of catecholamines on lipolysis and/or muscle glycogenolysis. Finally, we postulated a neural pathway that requires the permissive effect of insulin to increase glucose uptake by the muscle. Thus, insulin, glucose, and neural pathways regulate muscle glucose utilization in vivo and are, therefore, important determinants of glucoregulation in diabetes.

Insulin-like growth factor I circumvents defective insulin action in human myotonic dystrophy skeletal muscle cells.

Primary human skeletal muscle cell cultures derived from muscles of a myotonic dystrophy (DM) fetus provided a model in which both resistance to insulin action described in DM patient muscles and the potential ability of insulin-like growth factor I (IGF-I) to circumvent this defect could be investigated. Basal glucose uptake was the same in cultured DM cells as in normal myotubes. In DM cells, a dose of 10 nM insulin produced no stimulatory effect on glucose uptake, and at higher concentrations, stimulation of glucose uptake remained significantly lower than that in normal myotubes. In addition, basal and insulin-mediated protein synthesis were both significantly reduced compared with those in normal cells. In DM myotubes, insulin receptor messenger RNA expression and insulin receptor binding were significantly diminished, whereas the expression of GLUT1 and GLUT4 glucose transporters was not affected. These results indicate that impaired insulin action is retained in DM cultured myotubes. The action of recombinant human IGF-I (rhIGF-I) was evaluated in this cellular model. We showed that rhIGF-I is able to stimulate glucose uptake to a similar extent as in control cells and restore normal protein synthesis level in DM myotubes. Thus, rhIGF-I is able to bypass impaired insulin action in DM myotubes. This provides a solid foundation for the eventual use of rhIGF-I as an effective treatment of muscle weakness and wasting in DM.

Marked depletion of GLUT4 glucose transporters in transverse tubules of skeletal muscle from streptozotocin-induced diabetic rats.

The principal goal of the present study was to determine the subcellular content of GLUT4 in diabetic rat muscle, and to test the hypothesis that a reduced abundance of the transporter protein in transverse tubules is responsible for impaired glucose utilization in that tissue. GLUT4 protein levels were measured in hindlimb muscle homogenates as well as in subcellular membrane fractions enriched with either plasma membranes, transverse tubules, or GLUT4-containing intracellular membranes from control and diabetic (streptozotocin-induced) rats. GLUT4 protein contents in diabetic muscle homogenates was reduced by 30% as compared to control rats. Subcellular fractionation experiments revealed that GLUT4 contents in transverse tubules-enriched fractions was markedly decreased (by 55-60%) in skeletal muscle of diabetic animals whereas no significant reductions in GLUT4 abundance was observed in the plasma membrane fraction. Moreover, GLUT4 was markedly depleted (by 45%) in the GLUT4-enriched intracellular membrane fraction. These results indicate that GLUT4 is markedly depleted in both the intracellular pool and in the cell surface membranes in muscle of STZ-diabetic rats. Most strikingly, this study demonstrates that transverse tubules and not the plasma membrane are the main sites of cell surface GLUT4 depletion in diabetic muscle.

Expression of beta subunit isoforms of the Na+,K(+)-ATPase is muscle type-specific.

Hindlimb skeletal muscles of the rat express two isoforms of the alpha (alpha 1 and alpha 2) and two isoforms of the beta (beta 1 and beta 2) subunits of the Na+,K(+)-ATPase. Because several muscles constitute the hindlimb, we investigated if specific isoforms are expressed in particular muscles. Northern blot analysis using isoform-specific cDNA probes demonstrated that soleus muscle expressed only the beta 1 transcript, whereas EDL or white gastrocnemius muscles expressed only the beta 2 transcript, and red gastrocnemius muscle expressed both mRNAs. All muscles tested expressed both alpha 1 and alpha 2 transcripts, albeit to various degrees: alpha 1 transcripts were present to about the same extent in all muscles but alpha 2 mRNA was 4-fold more abundant in soleus than in EDL for the same amount of total RNA. Beta subunit protein levels were investigated in purified plasma membrane fractions of pooled red (soleus + red gastrocnemius + red quadriceps) or white (white gastrocnemius + white quadriceps) muscles using isoform-specific antibodies. Red muscles expressed mostly the beta 1 protein while white muscles expressed mostly the beta 2 subunit. Both muscle groups had similar levels of alpha 1 or alpha 2 subunits, and crude membranes isolated from red muscles had 30% higher Na+,K(+)-ATPase activity than white muscle membranes. We conclude that oxidative muscles (slow and fast twitch) express beta 1 subunits, whereas glycolytic, fast twitch muscles express beta 2 subunits, and that both beta isoforms support the Na+,K(+)-ATPase activity of the alpha subunits.

The GLUT4 glucose transporter and the alpha 2 subunit of the Na+,K(+)-ATPase do not localize to the same intracellular vesicles in rat skeletal muscle.

The GLUT4 glucose transporter and the alpha 2 subunit of the Na+,K(+)-ATPase of rat skeletal muscle are two proteins which redistribute from intracellular membranes to plasma membranes following in vivo insulin stimulation. Here we show that although both proteins co-segregate after subcellular fractionation of unstimulated rat hindlimb muscles, they do not share the same intracellular residence inside the muscle fibre. By immunogold single- and double-labeling on ultrathin muscle cryosections with specific antibodies, the GLUT4 glucose transporter and the Na+,K(+)-ATPase alpha 2 subunit were observed on different vesicular structures within the cell. GLUT4 was detected on subsarcolemmal and perinuclear membranes, and at the junction between myofibrillar A and I bands where triads are localized. The alpha 2 subunit of the Na+,K(+)-ATPase was observed at the plasma membrane and in distinct subsarcolemmal vesicles and intermyofibrillar membranes. Quantitative analysis of double-labeling of GLUT4 and Na+,K(+)-ATPase alpha 2 subunit revealed that less than 6% of the two proteins co-localize in the same continuous vesicular structures. The differential intracellular localization of the two proteins was further confirmed by immunopurification of GLUT4-containing membranes from muscle homogenates, in which the alpha 2 subunit of the Na+,K(+)-ATPase was found only at the same extent as the alpha 1 subunit of the enzyme, a protein exclusively present at the plasma membrane.

Effects of insulin on regional blood flow and glucose uptake in Wistar and Sprague-Dawley rats.

The euglycemic-hyperinsulinemic clamp technique in conscious Sprague-Dawley and Wistar rats chronically instrumented with intravascular catheters and pulsed Doppler flow probes was used to examine insulin's actions on regional blood flow and glucose metabolism. The effect of insulin on in vivo and in vitro glucose utilization in individual muscles was estimated using [3H]-2-deoxy-D-glucose. We found that in both strains, insulin (4, 32, and 64 mU x kg(-1) x min(-1)) causes similar cardiovascular changes characterized by slight increases in blood pressure (at high dose), vasodilation in renal and hindquarter vascular beds, and vasoconstriction (at high dose) in the superior mesenteric vascular bed. However, at the lowest dose of insulin tested, we found a smaller insulin sensitivity index and a lower insulin-stimulated in vivo glucose uptake in extensor digitorum longus (EDL) muscles of Wistar versus Sprague-Dawley rats. Higher insulin-stimulated glucose transport activity was found in isolated soleus muscle, while greater basal glucose transport was noted in isolated EDL muscle from Sprague-Dawley versus Wistar rats. These results provide further evidence for an insulin blood flow-regulatory effect and suggest that strain characteristics (differences in muscle perfusion, hindquarter composition, or fiber insulin sensitivity) constitute a major determinant in the variation in whole-body insulin sensitivity.