细胞骨架与血糖调节

细胞骨架由微丝、微管和中间丝构成,参与血糖调节这一复杂的生理过程,在胰岛素分泌、胰岛素功能和糖代谢相关酶类的细胞内分布等方面具有重要的作用。本文将从以上三个方面,对细胞骨架与血糖调节的关系加以综述。     

Insulin signaling in microdomains of the plasma membrane

Although the effects of insulin on glucose and lipid metabolism are well documented, gaps remain in our understanding of the precise molecular mechanisms of signal transduction. Recent evidence suggests that compartmentalization of signaling molecules and metabolic enzymes may explain the unique cellular effects of the hormone. Signal initiation from the insulin receptor is restricted in part to caveolae microdomains of the plasma membrane. A fraction of the insulin receptor directly interacts with caveolin, thus directing the protein to caveolae. Following its activation by insulin, the receptor recruits a series of adapter proteins, resulting in the activation of the G protein TC10, which also resides in caveolae. TC10 can influence a number of cellular processes, including changes in the actin cytoskeleton, recruitment of effector including the adapter protein CIP4, and assembly of the exocyst complex. These events play crucial roles in the trafficking, docking and fusion of vesicles containing the insulin-responsive glucose transporter Glut4 at the plasma membrane.     

Glucose regulates diacylglycerol intracellular levels and protein kinase C activity by modulating diacylglycerol kinase subcellular localization

Although chronic hyperglycemia reduces insulin sensitivity and leads to impaired glucose utilization, short term exposure to high glucose causes cellular responses positively regulating its own metabolism. We show that exposure of L6 myotubes overexpressing human insulin receptors to 25 mm glucose for 5 min decreased the intracellular levels of diacylglycerol (DAG). This was paralleled by transient activation of diacylglycerol kinase (DGK) and of insulin receptor signaling. Following 30-min exposure, however, both DAG levels and DGK activity returned close to basal levels. Moreover, the acute effect of glucose on DAG removal was inhibited by >85% by the DGK inhibitor R59949. DGK inhibition was also accompanied by increased protein kinase C-alpha (PKCalpha) activity, reduced glucose-induced insulin receptor activation, and GLUT4 translocation. Glucose exposure transiently redistributed DGK isoforms alpha and delta, from the prevalent cytosolic localization to the plasma membrane fraction. However, antisense silencing of DGKdelta, but not of DGKalpha expression, was sufficient to prevent the effect of high glucose on PKCalpha activity, insulin receptor signaling, and glucose uptake. Thus, the short term exposure of skeletal muscle cells to glucose causes a rapid induction of DGK, followed by a reduction of PKCalpha activity and transactivation of the insulin receptor signaling. The latter may mediate, at least in part, glucose induction of its own metabolism.     

The role of small G-proteins in the regulation of glucose transport (review).

Insulin increases the rate of glucose transport into fat and muscle cells by stimulating the translocation of intracellular Glut 4-containing vesicles to the plasma membrane. This results in a marked increase in the amount of the facilitative glucose transporter Glut 4 at the cell surface, allowing for an enhanced glucose uptake. This process requires a continuous cycling through the early endosomes, a Glut 4 specific storage compartment and the plasma membrane. The main effect of insulin is to increase the rate of Glut 4 trafficking from its specific storage compartment to the plasma membrane. The whole phenomenon involves signal transduction from the insulin receptor, vesicle trafficking (sorting and fusion processes) and actin cytoskeleton modifications, which are all supposed to require small GTPases. This review describes the potential role of the various members of the Ras, Rad, Rho, Arf and Rab families in the traffic of the Glut 4-containing vesicles.     

The role of small G-proteins in the regulation of glucose transport

Insulin increases the rate of glucose transport into fat and muscle cells by stimulating the translocation of intracellular Glut 4-containing vesicles to the plasma membrane. This results in a marked increase in the amount of the facilitative glucose transporter Glut 4 at the cell surface, allowing for an enhanced glucose uptake. This process requires a continuous cycling through the early endosomes, a Glut 4 specific storage compartment and the plasma membrane. The main effect of insulin is to increase the rate of Glut 4 trafficking from its specific storage compartment to the plasma membrane. The whole phenomenon involves signal transduction from the insulin receptor, vesicle trafficking (sorting and fusion processes) and actin cytoskeleton modifications, which are all supposed to require small GTPases. This review describes the potential role of the various members of the Ras, Rad, Rho, Arf and Rab families in the traffic of the Glut 4-containing vesicles.     

Sedimentation characteristics of subcellular vesicles associated with internalized insulin and those bound with intracellular glucose transport activity

Comparative studies were made on the sedimentation characteristics of microsomal vesicles associated with internalized [¹²⁵I]iodoinsulin and those bound with intracellular glucose transport activity. Upon linear sucrose density gradient centrifugation, the internalized hormone formed a peak slightly, but significantly, on the higher density side of the peak of intracellular glucose transport activity. After a long centrifugation, the peak of ¹²⁵I activity became lower and broader than that of glucose transport activity. Internalized ¹²⁵I activity was also found in the medium-density microsomal fraction, which had little glucose transport activity. Accumulation of ¹²⁵I activity in the medium-density fraction and that in the low-density fraction were both completed in approximately 10 min. Under basal conditions, little, if any, insulin binding activity was detectable in either the medium- or low-density microsomal fractions; in contrast, some glucose transport activity was always present in the low-density fraction. These results indicate that the subcellular distribution of internalized insulin and of intracellular glucose transport activity are different, suggesting that the pathways of intracellular processing of the insulin receptor and the glucose transport mechanism are different.     

Regulation of glucose transport in skeletal muscle.

The entry of glucose into muscle cells is achieved primarily via a carrier-mediated system consisting of protein transport molecules. GLUT-1 transporter isoform is normally found in the sarcolemmal (SL) membrane and is thought to be involved in glucose transport under basal conditions. With insulin stimulation, glucose transport is accelerated by translocating GLUT-4 transporters from an intracellular pool out to the T-tubule and SL membranes. Activation of transporters to increase the turnover number may also be involved, but the evidence is far from conclusive. When insulin binds to its receptor, it autophosphorylates tyrosine and serine residues on the beta-subunit of the receptor. The tyrosine residues are thought to activate tyrosine kinases, which in turn phosphorylate/activate as yet unknown second messengers. Insulin receptor antibodies, however, have been reported to increase glucose transport without increasing kinase activity. Insulin resistance in skeletal muscle is a major characteristic of obesity and diabetes mellitus, especially NIDDM. A decrease in the number of insulin receptors and the ability of insulin to activate receptor tyrosine kinase has been documented in muscle from NIDDM patients. Most studies report no change in the intracellular pool of GLUT-4 transporters available for translocation to the SL. Both the quality and quantity of food consumed can regulate insulin sensitivity. A high-fat, refined sugar diet, similar to the typical U.S. diet, causes insulin resistance when compared with a low-fat, complex-carbohydrate diet. On the other hand, exercise increases insulin sensitivity. After an acute bout of exercise, glucose transport in muscle increases to the same level as with maximum insulin stimulation. Although the number of GLUT-4 transporters in the sarcolemma increases with exercise, neither insulin or its receptor is involved. After an initial acute phase, which may involve calcium as the activator, a secondary phase of increased insulin sensitivity can last for up to a day after exercise. The mechanism responsible for the increased insulin sensitivity with exercise is unknown. Regular exercise training also increases insulin sensitivity, which can be documented several days after the final bout of exercise, and again the mechanism is unknown. An increase in the muscle content of GLUT-4 transporters with training has recently been reported. Even though significant progress has been made in the past few years in understanding glucose transport in skeletal muscle, the mechanisms involved in regulating transport are far from being understood.     

C2C12 myocytes lack an insulin-responsive vesicular compartment despite dexamethasone-induced GLUT4 expression

Insulin regulates the uptake of glucose into skeletal muscle and adipocytes by redistributing the tissue-specific glucose transporter GLUT4 from intracellular vesicles to the cell surface. To date, GLUT4 is the only protein involved in insulin-regulated vesicular traffic that has this tissue distribution, thus raising the possibility that its expression alone may allow formation of an insulin-responsive vesicular compartment. We show here that treatment of differentiating C2C12 myoblasts with dexamethasone, acting via the glucocorticoid receptor, causes a >or=10-fold increase in GLUT4 expression but results in no significant change in insulin-stimulated glucose transport. Signaling from the insulin receptor to its target, Akt2, and expression of the soluble N-ethylmaleimide-sensitive factor-attachment protein receptor, or SNARE, proteins syntaxin 4 and vesicle-associated membrane protein are normal in dexamethasone-treated C2C12 cells. However, these cells show no insulin-dependent trafficking of the insulin-responsive aminopeptidase or the transferrin receptor, respective markers for intracellular GLUT4-rich compartments and endosomes that are insulin responsive in mature muscle and adipose cells. Therefore, these data support the hypothesis that GLUT4 expression by itself is insufficient to establish an insulin-sensitive vesicular compartment.     

An analysis of the relationship between the cellular distribution and the rate of turnover for the separate classes of unoccupied, noncovalently occupied, and covalently occupied insulin receptor

To further investigate insulin's role in regulating the turnover of insulin receptor during down-regulation in 3T3-L1 adipocytes, the relationship between the cellular distribution and turnover of unoccupied, noncovalently occupied, and covalently occupied receptor was examined. At steady-state 12% of the unoccupied receptors and 46% of covalently occupied receptors are intracellular. The apparent first-order rate constant (Kapp) for turnover of the total pool of covalently occupied receptors (0.16 h-1) is 3.8-fold higher than that for unoccupied receptors (0.042 h-1). When unlabeled insulin is added, identical values for both Kapp (0.10 h-1) and distribution (26% internal) are measured for noncovalently and covalently occupied receptors. The rate constant (Kdeg), describing the relative sensitivity of internalized receptor to degradation, is identical (0.36-0.41 h-1) for unoccupied, noncovalently occupied, and permanently occupied pools of internal receptor. Mechanisms for down-regulation postulating: (a) an occupancy-dependent alteration in the conformation of internal receptor increasing receptor sensitivity to internal proteases, (b) a preferential sorting of internal occupied receptor to degradative pathways, or (c) induction of intracellular proteases by insulin, would all reflect a substantial change in Kdeg for occupied receptor and thus are unlikely mechanisms by which insulin increases the rate of receptor turnover. The turnover of insulin receptor in 3T3-L1 adipocytes is regulated primarily by its intracellular concentration and not by the state of occupancy of internalized receptor.     

GLUT4 is retained by an intracellular cycle of vesicle formation and fusion with endosomes

The intracellularly stored GLUT4 glucose transporter is rapidly translocated to the cell surface upon insulin stimulation. Regulation of GLUT4 distribution is key for the maintenance of whole body glucose homeostasis. We find that GLUT4 is excluded from the plasma membrane of adipocytes by a dynamic retention/retrieval mechanism. Our kinetic studies indicate that GLUT4-containing vesicles continually bud and fuse with endosomes in the absence of insulin and that these GLUT4 vesicles are 5 times as likely to fuse with an endosome as with the plasma membrane. We hypothesize that this intracellular cycle of vesicle budding and fusion is an element of the active mechanism by which GLUT4 is retained. The GLUT4 trafficking pathway does not extensively overlap with that of furin, indicating that the trans-Golgi network, a compartment in which furin accumulates, is not a significant storage reservoir of GLUT4. An intact microtubule cytoskeleton is required for insulin-stimulated recruitment to the cell surface, although it is not required for the basal budding/fusion cycle. Nocodazole disruption of the microtubule cytoskeleton reduces the insulin-stimulated exocytosis of GLUT4, accounting for the reduced insulin-stimulated translocation of GLUT4 to the cell surface.     

Signalling through the insulin receptor

The insulin receptor substrates function at the heart of the insulin signalling network. It has recently become apparent that the intracellular localisation of these molecules is regulated in a precise manner that is critical for both the generation and the termination of the insulin signal. Some insulin receptor substrate isoforms appear to be associated with an insoluble matrix that resembles the cytoskeleton. When inappropriately dissociated from this matrix the signalling network collapses concomitant with loss of insulin sensitivity.     

Deteriorating insulin resistance due to WL15 peptide from cysteine and glycine-rich protein 2 in high glucose-induced rat skeletal muscle L6 cells

This study investigates the antioxidant and antidiabetic activity of the WL15 peptide derived from Channa striatus on regulating the antioxidant property in the rat skeletal muscle cell line (L6) and enhancing glucose uptake via glucose metabolism. Increased oxidative stress plays a major role in the development of diabetes and its complications. Strategies are needed to mitigate the oxidative stress that can reduce these pathogenic processes. Our results showed that with treatment with WL15 peptide, the reactive oxygen species significantly decreased in L6 myotubes in a dose-dependent manner, and increased antioxidant enzymes help to prevent the formation of lipid peroxidation in L6 myotubes. The cytotoxicity of WL15 is evaluated in the L6 cells and found to be non-cytotoxic at the tested concentration. Also, for the analysis of glucose uptake activity in L6 cells, the 2-(N-[7-nitrobenz-2-oxa-1,3-diazol-4-yl]amino)-2-deoxy- d -glucose assay was performed in the presence of wortmannin and genistein inhibitors. WL15 demonstrated antidiabetic activities through a dose-dependent increase in glucose uptake (64%) and glycogen storage (7.8 mM). The optimal concentration for the maximum activity was found to be 50 µM. In addition, studies of gene expression in L6 myotubes demonstrated upregulation of antioxidant genes and genes involved in the pathway of insulin signaling. In cell-based assays, WL15 peptide decreased intracellular reactive oxygen species levels and demonstrated insulin mimic activity by enhancing the primary genes involved in the insulin signaling pathway by increased glucose uptake indicating that glucose transporter type 4 (GLUT4) is regulated from the intracellular pool to the plasma membrane.     

Changes in insulin receptor,hexokinase and NADPH producing enzymes in Choroid plexus during experimental diabetes

The activities of insulin receptor and the enzymes hexokinase (EC 2.7.1.1) and NADP-dependent malic enzyme (EC1.1.1.40), glucose 6-phosphate dehydrogenase (EC 1.1.1.49) and isocitrate dehydrogenase (EC 1.1.1.42) were measured in rat choroid plexus in alloxan induced diabetes. A significant decrease was observed in the activities of all the enzymes except isocitrate dehydrogenase and also the choroid plexus insulin receptor activity was decreased. A reversal of the efect was observed with insulin administration to diabetic rats. It may be concluded that the enzymes of choroid plexus together with insulin receptor are directly controlled by-the concentration of insulin.     

Regulation of glycogen synthase activity in the isolated mouse soleus muscle effect of insulin,epinephrine, glucose and anti-insulin receptor antibodies

The effects of insulin, epinephrine, glucose and anti-insulin receptor antibodies on enzymes involved in the regulation of glycogen synthesis were investigared in the isolated mouse soleus muscle. Insulin maximally increased the percentage of glycogen synthase active form after 15 min in the absence of glucose in the extracellular medium; half-maximal and maximal effects were obtained with 1.5 and 33 nM insulin, respectively. The basal percentage of glycogen phosphorylase active form was not altered by insulin. Antibodies to the insulin receptor had similar effects to those of insulin on both enzymes. The percentage of glycogen synthase active form was maximally decreased and that of phosphorylase maximally increased after a 2 min exposure to epinephrine in the absence of extracellular glucose. Glucose alone had no effect on muscle glycogen synthase. When muscles were incubated with insulin (33 nM) plus glucose (20 mM) for 5–10 min, the increase in the percentage of glycogen synthase active form was greater than with insulin alone. This enhancing effect of glucose on insulin activation of glycogen synthase disappeared after 20 min. The results suggest the existence of two mechanisms whereby insulin activates muscle glycogen synthase. The main effect is operative in the absence of extracellular glucose and occurs at insulin concentrations close to the physiological range. The other effect requires glucose and may result from the stimulation by insulin of glucose transport and/or metabolism.     

Effects of insulin and histamine in themselves and in combination on the glucose metabolism of Tetrahymena

The glucose metabolism and the related intracellular processes of Tetrahymena pyriformis GL cells are measurably influenced by several hormones of higher organisms, among others by the endocytosis stimulating hormone histamine, and the glucose metabolism regulating hormone insulin. Histamine does not interfere with the glucose metabolizing action of insulin, but markedly enhances the utilization of glucose, to judge from a significant decrease in the PAS-positive (hexose) component of histamine-exposed Tetrahymena pyriformis GL cells.     

Regulation of glycogen synthase activity in the isolated mouse soleus muscle. Effect of insulin, epinephrine, glucose and anti-insulin receptor antibodies

The effects of insulin, epinephrine, glucose and anti-insulin receptor antibodies on enzymes involved in the regulation of glycogen synthesis were investigated in the isolated mouse soleus muscle. Insulin maximally increased the percentage of glycogen synthase active form after 15 min in the absence of glucose in the extracellular medium; half-maximal and maximal effects were obtained with 1.5 and 33 mM insulin, respectively. The basal percentage of glycogen phosphorylase active form was not altered by insulin. Antibodies to the insulin receptor had similar effects to those of insulin on both enzymes. The percentage of glycogen synthase active form was maximally decreased and that of phosphorylase maximally increased after a 2 min exposure to epinephrine in the absence of extracellular glucose. Glucose alone had no effect on muscle glycogen synthase. When muscles were incubated with insulin (33 nM) plus glucose (20 mM) for 5-10 min, the increase in the percentage of glycogen synthase active form was greater than with insulin alone. This enhancing effect of glucose on insulin activation of glycogen synthase disappeared after 20 min. The results suggest the existence of two mechanisms whereby insulin activates muscle glycogen synthase. The main effect is operative in the absence of extracellular glucose and occurs at insulin concentrations close to the physiological range. The other effect requires glucose and may result from the stimulation by insulin of glucose transport and/or metabolism.     

Proteomic Analysis of GLUT4 Storage Vesicles Reveals Tumor Suppressor Candidate 5 (TUSC5) as a Novel Regulator of Insulin Action in Adipocytes

Insulin signaling augments glucose transport by regulating glucose transporter 4 (GLUT4) trafficking from specialized intracellular compartments, termed GLUT4 storage vesicles (GSVs), to the plasma membrane. Proteomic analysis of GSVs by mass spectrometry revealed enrichment of 59 proteins in these vesicles. We measured reduced abundance of 23 of these proteins following insulin stimulation and assigned these as high confidence GSV proteins. These included established GSV proteins such as GLUT4 and insulin-responsive aminopeptidase, as well as six proteins not previously reported to be localized to GSVs. Tumor suppressor candidate 5 (TUSC5) was shown to be a novel GSV protein that underwent a 3.7-fold increase in abundance at the plasma membrane in response to insulin. siRNA-mediated knockdown of TUSC5 decreased insulin-stimulated glucose uptake, although overexpression of TUSC5 had the opposite effect, implicating TUSC5 as a positive regulator of insulin-stimulated glucose transport in adipocytes. Incubation of adipocytes with TNFα caused insulin resistance and a concomitant reduction in TUSC5. Consistent with previous studies, peroxisome proliferator-activated receptor (PPAR) γ agonism reversed TNFα-induced insulin resistance. TUSC5 expression was necessary but insufficient for PPARγ-mediated reversal of insulin resistance. These findings functionally link TUSC5 to GLUT4 trafficking, insulin action, insulin resistance, and PPARγ action in the adipocyte. Further studies are required to establish the exact role of TUSC5 in adipocytes.     

Loss of insulin signaling in hepatocytes leads to severe insulin resistance and progressive hepatic dysfunction

The liver plays a central role in the control of glucose homeostasis and is subject to complex regulation by substrates, insulin, and other hormones. To investigate the effect of the loss of direct insulin action in liver, we have used the Cre-loxP system to inactivate the insulin receptor gene in hepatocytes. Liver-specific insulin receptor knockout (LIRKO) mice exhibit dramatic insulin resistance, severe glucose intolerance, and a failure of insulin to suppress hepatic glucose production and to regulate hepatic gene expression. These alterations are paralleled by marked hyperinsulinemia due to a combination of increased insulin secretion and decreased insulin clearance. With aging, the LIRKO liver exhibits morphological and functional changes, and the metabolic phenotype becomes less severe. Thus, insulin signaling in liver is critical in regulating glucose homeostasis and maintaining normal hepatic function.     

Insulin receptor substrate 1-induced inhibition of endoplasmic reticulum Ca2+ uptake in beta-cells. Autocrine regulation of intracellular ca2+ homeostasis and insulin secretion.

To understand the role of the insulin receptor pathway in beta-cell function, we have generated stable beta-cells (betaIRS1-A) that overexpress by 2-fold the insulin receptor substrate-1 (IRS-1) and compared them to vector-expressing controls. IRS-1 overexpression dramatically increased basal cytosolic Ca2+ levels from 81 to 278 nM, but it did not affect Ca2+ response to glucose. Overexpression of the insulin receptor also caused an increase in cytosolic Ca2+. Increased cytosolic Ca2+ was due to inhibition of Ca2+ uptake by the endoplasmic reticulum, because endoplasmic reticulum Ca2+ uptake and content were reduced in betaIRS1-A cells. Fractional insulin secretion was significantly increased 2-fold, and there was a decrease in betaIRS1-A insulin content and insulin biosynthesis. Steady-state insulin mRNA levels and glucose-stimulated ATP were unchanged. High IRS-1 levels also reduced beta-cell proliferation. These data demonstrate a direct link between the insulin receptor signaling pathway and the Ca2+-dependent pathways regulating insulin secretion of beta-cells. We postulate that during regulated insulin secretion, released insulin binds the beta-cell insulin receptor and activates IRS-1, thus further increasing cytosolic Ca2+ by reducing Ca2+ uptake. We suggest the existence of a novel pathway of autocrine regulation of intracellular Ca2+ homeostasis and insulin secretion in the beta-cell of the endocrine pancreas.     

Evidence for the involvement of vicinal sulfhydryl groups in the insulin stimulation of intracellular glucose metabolism in Zajdela Hepatoma cells

Phenylarsine oxide (PhAsO), a dithiol reagent that blocks insulin stimulation of glucose transport in 3T3 L1 cells, also altered insulin stimulation of intracellular glucose metabolism in Zajdela Hepatoma cultured cells. PhAsO (2 M) similarly inhibited the insulin-induced glycogen and lipid syntheses without modifying the basal level of these processes, cell viability or the ATP content. Prior incubation of the cells with PhAsO did not prevent insulin binding to the cells, or activation of the receptor tyrosine kinase, while it minimally (16%) altered receptor internalization. These results indicate that cellular dithiols located at a post-receptor step are involved in the transduction of the insulin signal to intracellular glucose metabolism.