Regulated transport of the glucose transporter GLUT4

In muscle and fat cells, insulin stimulates the delivery of the glucose transporter GLUT4 from an intracellular location to the cell surface, where it facilitates the reduction of plasma glucose levels. Understanding the molecular mechanisms that mediate this translocation event involves integrating our knowledge of two fundamental processes--the signal transduction pathways that are triggered when insulin binds to its receptor and the membrane transport events that need to be modified to divert GLUT4 from intracellular storage to an active plasma membrane shuttle service.     

Osmotic water transport with glucose in GLUT2 and SGLT

Carrier-mediated water cotransport is currently a favored explanation for water movement against an osmotic gradient. The vestibule within the central pore of Na⁺-dependent cotransporters or GLUT2 provides the necessary precondition for an osmotic mechanism, explaining this phenomenon without carriers. Simulating equilibrative glucose inflow via the narrow external orifice of GLUT2 raises vestibular tonicity relative to the external solution. Vestibular hypertonicity causes osmotic water inflow, which raises vestibular hydrostatic pressure and forces water, salt, and glucose into the outer cytosolic layer via its wide endofacial exit. Glucose uptake via GLUT2 also raises oocyte tonicity. Glucose exit from preloaded cells depletes the vestibule of glucose, making it hypotonic and thereby inducing water efflux. Inhibiting glucose exit with phloretin reestablishes vestibular hypertonicity, as it reequilibrates with the cytosolic glucose and net water inflow recommences. Simulated Na⁺-glucose cotransport demonstrates that active glucose accumulation within the vestibule generates water flows simultaneously with the onset of glucose flow and before any flow external to the transporter caused by hypertonicity in the outer cytosolic layers. The molar ratio of water/glucose flow is seen now to relate to the ratio of hydraulic and glucose permeability rather than to water storage capacity of putative water carriers.     

Changes in calmodulin immunocytochemical localization associated with capacitation and acrosomal exocytosis of ram spermatozoa

The aim of this study was to determine the localization of calmodulin (CaM) in ram sperm and the possible changes during in vitro capacitation (CA) and the ionophore-induced acrosome reaction (AR). Likewise, changes in intracellular calcium levels ([Ca²⁺]_i) were also analysed by using flow cytometry. CA was induced in vitro in a medium containing BSA, CaCl₂, NaHCO₃, and AR by the addition of the calcium ionophore A23187. The acrosomal status was assessed by the chlortetracycline-fluorescence (CTC) assay. Flow cytometry (FC) analyses were performed by loading samples with Fluo-3 AM, that emits fluorescence at a high [Ca²⁺]_i, combined with propidium iodide (PI) that allowed us to discriminate sperm with/without an integral plasma membrane both with high/low [Ca²⁺]_i. Immunocytochemistry localized CaM to the flagellum, and some sperm also contained CaM in the head (equatorial and post-acrosomal regions). CA and AR resulted in a slight increase in the post-acrosomal labelling. The treatment of sperm with increasing concentrations of two CaM antagonists, W7 and calmidazolium (CZ), accounted for an increase in capacitated and acrosome-reacted CTC-sperm patterns. CZ induced a significant reduction in the content of three protein tyrosine-phosphorylated bands of approximately of 30, 40 and 45 kDa. However, W7 showed no significant effect at any of the studied concentrations. Neither of them significantly influenced protein serine and threonine phosphorylation. FC analysis revealed that the main subpopulation in the control samples contained 70% of the total sperm with integral plasma membrane and a medium [Ca²⁺]_i. After CA, 67.1% of the sperm preserved an integral membrane with a higher [Ca²⁺]_i. After AR, only 7.2% of the total sperm preserved intact membranes with a very high [Ca²⁺]_i. These results imply that CaM appears to be involved in ram sperm capacitation, and both treatments increased its localization in the post-acrosomal region.     

Isolation and characterization of a macromolecular complex associated with the outer acrosomal membrane of bovine spermatozoa

The acrosomal membrane of mammalian spermatozoa is segregated into domains of different structure and function. The outer acrosomal membrane of the apical and principal segments is the only domain to participate in the membrane fusion events of the acrosome reaction, but the molecular basis for this function is not resolved. In previous studies of bovine spermatozoa, we noted that a unique structural feature of the outer acrosomal membrane was an adherent layer of electron-dense material on its luminal surface (ES Surface, Branton et al., 1975). In this study, we report the isolation of this material and we describe both its structural and biochemical characteristics. Cauda epididymal spermatozoa were extracted with 1% Triton X-100 to solubilize cytoplasmic and membrane components; detergent treatment solubilized the outer acrosomal membrane but not its adherent electron-dense complex. Homogenization released this complex from the spermatozoa and it was then resolved into a homogeneous fraction by centrifugation on Percoll density gradients. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of this fraction revealed a spectrum of polypeptides including components of 290 kDa, 280 kDa, 260 kDa, 115 kDa, 81 kDa, 58 kDa, and 46 kDa and a family of interrelated components in the 34-12 kDa range. This complex possesses protein kinase activity that phosphorylates specific endogeneous polypeptides in a cAMP-independent manner. In addition, several polypeptides of the 34-12 kDa family specifically bind 125I-calmodulin. One consistent structural response of the isolated complex was that its edges wound into a spiral configuration. We speculate that this membrane-associated assembly plays a functional role in the membrane fusion events of the acrosome reaction.     

Berberine acutely activates the glucose transport activity of GLUT1

Berberine, which has a long history of use in Chinese medicine, has recently been shown to have efficacy in the treatment of diabetes. While the hypoglycemic effect of berberine has been clearly documented in animal and cell line models, such as 3T3-L1 adipocytes and L6 myotube cells, the mechanism of action appears complex with data implicating activation of the insulin signaling pathway as well as activation of the exercise or AMP kinase-mediated pathway. There have been no reports of the acute affects of berberine on the transport activity of the insulin-insensitive glucose transporter, GLUT1. Therefore, we examined the acute effects of berberine on glucose uptake in L929 fibroblast cells, a cell line that express only GLUT1. Berberine- activated glucose uptake reaching maximum stimulation of five-fold at >40 μM. Significant activation (P < 0.05) was measured within 5 min reaching a maximum by 30 min. The berberine effect was not additive to the maximal stimulation by other known stimulants, azide, methylene blue or glucose deprivation, suggesting shared steps between berberine and these stimulants. Berberine significantly reduced the K_m of glucose uptake from 6.7 ± 1.9 mM to 0.55 ± 0.08 mM, but had no effect on the V_max of uptake. Compound C, an inhibitor of AMP kinase, did not affect berberine-stimulated glucose uptake, but inhibitors of downstream kinases partially blocked berberine stimulation. SB203580 (inhibitor of p38 MAP kinase) did not affect submaximal berberine activation, but did lower maximal berberine stimulation by 26%, while PD98059 (inhibitor of ERK kinase) completely blocked submaximal berberine activation and decreased the maximal stimulation by 55%. It appears from this study that a portion of the hypoglycemic effects of berberine can be attributed to its acute activation of the transport activity of GLUT1.     

A plasma membrane-associated hyaluronidase is localized to the posterior acrosomal region of stallion sperm and is associated with spermatozoal function.

Sperm hyaluronidase has been implicated in sperm penetration of the extracellular matrix of the cumulus oophorus and may play a crucial role in gamete interaction and fertility in mammals. The objectives of this study were to characterize the enzyme activity of equine sperm hyaluronidase and to investigate its cellular distribution. Zymography of stallion sperm plasma membrane extracts was used to identify hyaluronidase activity in protein bands. Affinity-purified polyclonal IgG raised against equine sperm hyaluronidase was used to label fresh and capacitated stallion sperm, followed by indirect immunofluorescence. Equine sperm plasma membrane extracts displayed 3 major protein bands with potent hyaluronidase activity of approximately 54, 59, and 83 kDa. Under reducing conditions, a single protein band was observed at 62 kDa, although the reduced sample exhibited no enzyme activity. The polyclonal IgG labeled the postacrosomal region of stallion sperm and was redistributed over the acrosomal region during in vitro capacitation in a significant percentage of sperm cells. These studies suggest that a specific protein localized to the equine sperm head displays hyaluronidase activity, gets redistributed over the acrosomal region during capacitation, and may be important in fertility in this species.     

The role of N-glycosylation of GLUT1 for glucose transport activity.

To elucidate a functional role of N-glycosylation in glucose transporters, we introduced oligonucleotide-directed mutagenesis in GLUT1 cDNA to remove the possible site for N-linked glycosylation. The wild-type and the mutated GLUT1 cDNAs which induced a mutation of Asn at residue 45 to Asp, Tyr, or Gln were transfected and stably expressed into Chinese hamster ovary cells. The expressed wild-type and the mutated GLUT1 was demonstrated to be a broad band of a 45-60-kDa form and a sharp band of a 38-kDa form on Western blot analysis, respectively, indicating no glycosylation in the mutated GLUT1. Although the cell surface labeling of the glucose transporters demonstrated the presence of the glycosylation-defective glucose transporters on the cells surface, photoaffinity labeling of glycosylation-defective GLUT1 with [3H] cytochalasin B and a photoreactive mannose derivative, [3H]2-N-4-(1-azi-2,2,2,trifluoroethyl)benzoyl-1,3-bis(D-mannos+ ++-4-yloxy)-2- propylamine in the membranes was observed to be 40-70 and 15-30% of that of the wild-type GLUT1, respectively. The kinetic study of 2-deoxyglucose uptake revealed that the glycosylation-defective GLUT1 had a 2-2.5-fold greater Km value for 2-deoxyglucose uptake compared with the wild-type GLUT1. These observations strongly suggest that 1) N-glycosylation of GLUT1 glucose transporter is only on Asn 45 and 2) N-glycosylation plays an important role in maintaining a structure of glucose transporter with high affinity for glucose, thus, with high transport activity.     

GLUT4在胰岛素调控葡萄糖转运中作用

机体的血糖平衡调节主要依赖于胰岛素,其中一个重要的机制是胰岛素通过调控GLUT4的囊泡运转来调节脂肪细胞和肌细胞对葡萄糖的摄取。由胰岛素受体介导的一系列磷酸化过程能调节一些关键的GLUT4转运相关蛋白质的活性,这些蛋白质包括小GTP酶、拴系复合体和囊泡融合体。而这些蛋白质又反过来通过内膜系统调节GLUT4储存囊泡的生成、滞留,并调控这些囊泡的靶向出胞方式。了解这些过程有助于解释2型糖尿病中胰岛素耐受的机制,并可能为糖尿病提供新的靶向治疗方法。     

The murine heterochromatin protein M31 is associated with the chromocenter in round spermatids and Is a component of mature spermatozoa

In mature sperm the normal nucleosomal packaging of DNA found in somatic and meiotic cells is transformed into a highly condensed form of chromatin which consists mostly of nucleoprotamines. Although sperm DNA is highly condensed it is nevertheless packaged into a highly defined nuclear architecture which may be organized by the heterochromatic chromocenter. One major component of heterochromatin is the heterochromatin protein 1 which is involved in epigenetic gene silencing. In order to investigate the possible involvement of heterochromatin protein in higher order organization of sperm DNA we studied the localization of the murine homologue of heterochromatin protein 1, M31, during chromatin reorganization in male germ cell differentiation. Each cell type in the testis showed a unique distribution pattern of M31. Colocalization to the heterochromatic regions were found in Sertoli cells, in midstage pachytene spermatocytes, and in round spermatids in which M31 localizes to the centromeric chromocenter. M31 cannot be detected in elongated spermatids or mature spermatozoa immunocytologically, but could be detected in mature spermatozoa by Western blotting. We suggest that M31, a nuclear protein involved in the organization of chromatin architecture, is involved in higher order organization of sperm DNA.     

Effects of cinnamaldehyde on the glucose transport activity of GLUT1

There is accumulating evidence that cinnamon extracts contain components that enhance insulin action. However, little is know about the effects of cinnamon on non-insulin stimulated glucose uptake. Therefore, the effects of cinnamaldehyde on the glucose transport activity of GLUT1 in L929 fibroblast cells were examined under both basal conditions and conditions where glucose uptake is activated by glucose deprivation. The data reveal that cinnamaldehyde has a dual action on the glucose transport activity of GLUT1. Under basal conditions it stimulates glucose uptake and reaches a 3.5 fold maximum stimulation at 2.0 mM. However, cinnamaldehyde also inhibits the activation of glucose uptake by glucose deprivation in a dose dependent manner. Experiments with cinnamaldehyde analogs reveal that these activities are dependent on the α,β-unsaturated aldehyde structural motif in cinnamaldehyde. The inhibitory, but not the stimulatory activity of cinnamaldehyde was maintained after a wash-recovery period. Pretreatment of cinnamaldehyde with thiol-containing compounds, such as β-mercaptoethanol or cysteine, blocked the inhibitory activity of cinnamaldehyde. These results suggest that cinnamaldehyde inhibits the activation of GLUT1 by forming a covalent link to target cysteine residue/s. This dual activity of cinnamaldehyde on the transport activity of GLUT1 suggests that cinnamaldehyde is not a major contributor to the anti-diabetic properties of cinnamon.     

The concerted effect of alpha-chlorohydrin and glucose on the ATP concentration in spermatozoa is associated with the accumulation of glycolytic intermediates

In the absence of a glycolysable sugar the effect of 1 mM-RS-alpha-chlorohydrin on the ATP concentration in ram or boar spermatozoa was relatively small but the addition of 0.10 or 0.03 mM-glucose initiated a rapid loss of ATP. When the spermatozoa were incubated with 0.05 mM-RS-alpha-chlorohydrin, the addition of 1.0 mM (ram) or 0.06 mM (boar)-glucose was required to produce ATP dissipation. In ram spermatozoa treated with 0.05 or 1.00 mM-RS-alpha-chlorohydrin, ATP loss was caused by 10 mM-fructose or 10 mM-mannose but not by 10 mM-glycerol or 10 mM-inositol. In boar spermatozoa incubated with 1 mM-RS-alpha-chlorohydrin the addition of 10 mM-L-lactate plus 1.0 mM-pyruvate protected the spermatozoa against the ability of 1.0 mM-glucose to produce a decline in ATP concentration. Every combination of treatments capable of inducing a marked decline in ATP concentration also caused a dramatic (20-100-fold) increase in the concentration of fructose 1,6-bisphosphate. An increase in fructose 1,6-bisphosphate concentration was never observed when the ATP concentration was unaffected. We conclude that it is very probable that the concerted effect of alpha-chlorohydrin and glycolysable sugar is responsible for the contraceptive action of alpha-chlorohydrin in vivo and that fructose 1,6-bisphosphate is implicated in its mechanism of action.     

Localization of a syntaxin isoform, syntaxin 2, to the acrosomal region of rodent spermatozoa

The acrosome reaction includes a membrane fusion event that is a prerequisite for sperm penetration through the zona pellucida and subsequent fertilization. Since SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) proteins have been shown to be key players in membrane fusion during regulated exocytosis in nerve terminals and secretory cells, and since the acrosome reaction has some features in common with regulated exocytosis, we hypothesized that SNARE proteins might also regulate acrosomal exocytosis. RT-PCR analysis demonstrated the expression of SNARE proteins, three isoforms of syntaxin 2 (2A, 2B, and 2C) and syntaxin 4A, in rat testes. Immunoblot analysis with anti-syntaxin 2 antibody showed that the protein was expressed in rodent spermatozoa, and that it was associated with membrane components of spermatozoa prepared by sucrose density gradient centrifugation. Confocal laser scanning microscopy with double immunolabeling revealed that syntaxin 2 was colocalized with acrin 1, a 90 kDa acrosomal protein, over the acrosomal region of spermatozoa but was not associated with the posterior half of head or tail. Localization of syntaxin 2 over the acrosomal region was supported by the finding that it was shed from sperm heads during an acrosome reaction induced by calcium ionophore A23187 in vitro. In view of the putative role of syntaxin proteins in other membrane fusion systems, these data suggest that syntaxin 2 may be involved in regulating the acrosomal reaction in rodent spermatozoa.     

Distinct regulation of glucose transport and GLUT1/GLUT3 transporters by glucose deprivation and IGF-I in chromaffin cells

Effects of prolonged metabolic (glucose deprivation) and hormonal [insulin-like growth factor I (IGF-I)] challenge on regulation of glucose transporter (GLUT) expression, glucose transport rate and possible signaling pathways involved were studied in the neuroendocrine chromaffin cell. The results show that bovine chromaffin cells express both GLUT1 and GLUT3. Glucose deprivation and IGF-I activation led to an elevation of GLUT1 and GLUT3 mRNA, the strongest effect being that of IGF-I on GLUT3 mRNA. Both types of stimulus increased the GLUT1 protein content in a cycloheximide (CHX)-sensitive manner, and the glucose transport rate was elevated by 3- to 4-fold after 48 h under both experimental conditions. IGF-I-induced glucose uptake was totally suppressed by CHX. In contrast, only approximately 50% of transport activation in glucose-deprived cells was sensitive to the protein synthesis inhibitor. Specific inhibitors of mTOR/FRAP and p38 MAPK each partially blocked IGF-I-stimulated glucose transport, but had no effect on transport rate in glucose-deprived cells. The results are consistent with IGF-I-activated transport being completely dependent on new GLUT protein synthesis while the enhanced transport in glucose-deprived cells was partially achieved independent of new synthesis of proteins, suggesting a mechanism relying on preexisting transporters.     

Role of SGK1 kinase in regulating glucose transport via glucose transporter GLUT4

Insulin stimulates glucose transport into muscle and fat cells by enhancing GLUT4 abundance in the plasma membrane through activation of phosphatidylinositol 3-kinase (PI3K). Protein kinase B (PKB) and PKCzeta are known PI3K downstream targets in the regulation of GLUT4. The serum- and glucocorticoid-inducible kinase SGK1 is similarly activated by insulin and capable to regulate cell surface expression of several metabolite transporters. In this study, we evaluated the putative role of SGK1 in the modulation of GLUT4. Coexpression of the kinase along with GLUT4 in Xenopus oocytes stimulated glucose transport. The enhanced GLUT4 activity was paralleled by increased transporter abundance in the plasma membrane. Disruption of the SGK1 phosphorylation site on GLUT4 ((S274A)GLUT4) abrogated the stimulating effect of SGK1. In summary, SGK1 promotes glucose transporter membrane abundance via GLUT4 phosphorylation at Ser274. Thus, SGK1 may contribute to the insulin and GLUT4-dependent regulation of cellular glucose uptake.     

Reduced insulin-stimulated glucose transport in denervated muscle is associated with impaired Akt-alpha activation

Insulin signaling was examined in muscle made insulin resistant by short-term (24-h) denervation. Insulin-stimulated glucose transport in vitro was reduced by 28% (P < 0.05) in denervated muscle (DEN). In control muscle (SHAM), insulin increased levels of surface-detectable GLUT-4 (i.e., translocated GLUT-4) 1.8-fold (P < 0.05), whereas DEN surface GLUT-4 was not increased by insulin (P > 0.05). Insulin treatment in vivo induced a rapid appearance of phospho[Ser(473)]Akt-alpha in SHAM 3 min after insulin injection. In DEN, phospho[Ser(473)]Akt-alpha also appeared at 3 min, but Ser(473)-phosphorylated Akt-alpha was 36% lower than in SHAM (P < 0. 05). In addition, total Akt-alpha protein in DEN was 37% lower than in SHAM (P < 0.05). Akt-alpha kinase activity was lower in DEN at two insulin levels tested: 0.1 U insulin/rat (-22%, P < 0.05) and 1 U insulin/rat (-26%, P < 0.01). These data indicate that short-term (24-h) denervation, which lowers insulin-stimulated glucose transport, is associated with decreased Akt-alpha activation and impaired insulin-stimulated GLUT-4 appearance at the muscle surface.     

Antiproliferative activity is predominantly associated with ellagitannins in raspberry extracts

Raspberry extracts enriched in polyphenols, but devoid of organic acids, sugars and vitamin C, were prepared by sorption to C18 solid phase extraction matrices and tested for their ability to inhibit the proliferation of human cervical cancer (HeLa) cells in vitro. The raspberry extract reduced proliferation in a dose-dependent manner whether this was judged by cell number or measurements of cell viability. However, measurements based on cell viability were more accurate and gave an EC(50) value of 17.5 microg/ml gallic acid equivalents (GAE) at day 4 of culture. Raspberry extracts were fractionated by sorption to Sephadex LH-20 into an unbound fraction, which was obviously enriched in anthocyanins, and a bound fraction. The unbound anthocyanin-enriched fraction was much less effective in reducing proliferation then the original extract and gave an EC(50) value estimated at 67 microg/ml. The LH-20 bound fraction was more effective than the original raspberry extract (EC(50)=13 microg/ml) suggesting that the main anti-proliferative agents were retained in the bound fraction. Analysis of the original extract, the unbound and the LH20 bound fractions by LC-MS confirmed that the unbound fraction was enriched in anthocyanins and the bound fraction primarily contained ellagitannins. The ellagitannin-rich bound fraction had the highest antioxidant capacity as measured by the ferric reducing antioxidant potential (FRAP) assay. The mechanism by which the ellagitannins inhibit proliferation of cancer cells is discussed.