Studies on insulin/IGF-1 hybrid and IGF-1 growth-promoting functional region

Single-chain insulin/IGF-1 hybrid-[Ins/IGF-1(C)], single-chain porcine insulin precursor-(PIP), and B10Asp PIP were prepared by protein engineering. Their growth-promoting activities in mouse breast cancer cell line GR2H6 are 10, 0.2, and 2 times that of insulin, respectively, and 29%, 0.6%, and 6% of that of IGF-1, indicating that the C domain and 9Glu of IGF-1 are important for its growth-promoting activity. Given these results and previous reports, we propose that the C domain, 9Glu, and 23Phe-26Asn beta bend are involved in the growth-promoting functional region of IGF-1.     

Glucose deprivation and hypoxia increase the expression of the GLUT1 glucose transporter via a specific mRNA cis-acting regulatory element

The expression of the glucose transporter type-1 (GLUT1) gene is up-regulated in hypoxia and glucose deprivation. A 10 nucleotide (nt) cis-acting regulatory element (CAE), which is located within nt 2181-2190 of the GLUT1 3'-untranslated region (CAE2181-2190), increases the expression of a GLUT1-luciferase reporter gene and decreases its mRNA decay. The present study investigated the role of the GLUT1 CAE2181-2190 in glucose deprivation and hypoxia using stable transfectants. Glucose and O2 deprivation produced a marked increase in the expression of the GLUT1 reporter gene carrying the CAE2181-2190, and this effect was additive. Glucose deprivation and/or hypoxia induced no significant changes in the expression of the reporter gene wherein the GLUT1 CAE2181-2190 was site-directed deleted. Data presented here suggest that the GLUT1 CAE2181-2190 participates in the increase of GLUT1 gene expression in glucose deprivation and hypoxia.     

Reduced insulin/IGF-1 signalling and human longevity

Evidence is accumulating that aging is hormonally regulated by an evolutionarily conserved insulin/IGF-1 signalling (IIS) pathway. Mutations in IIS components affect lifespan in Caenorhabditis elegans, Drosophila melanogaster and mice. Most long-lived IIS mutants also show increased resistance to oxidative stress. In D. melanogaster and mice, the long-lived phenotype of several IIS mutants is restricted to females. Here, we analysed the relationship between IIS signalling, body height and longevity in humans in a prospective follow-up study. Based on the expected effects (increased or decreased signalling) of the selected variants in IIS pathway components (GHRHR, GH1, IGF1, INS, IRS1), we calculated composite IIS scores to estimate IIS pathway activity. In addition, we analysed the relative impact on lifespan and body size of the separate variants in multivariate models. In women, lower IIS scores are significantly associated with lower body height and improved old age survival. Multivariate analyses showed that these results were most pronounced for the GH1 SNP, IGF1 CA repeat and IRS1 SNP. In females, for variant allele carriers of the GH1 SNP, body height was 2 cm lower (P = 0.007) and mortality 0.80-fold reduced (P = 0.019) when compared with wild-type allele carriers. Thus, in females, genetic variation causing reduced IIS activation is beneficial for old age survival. This effect was stronger for the GH1 SNP than for variation in the conserved IIS genes that were found to affect longevity in model organisms.     

Development of insulin sensitivity in rat skeletal muscle. Studies of glucose transporter and insulin receptor mRNA levels.

Expression of GLUT-4 and insulin receptor mRNAs was investigated in rat skeletal muscle by Northern hybridization. GLUT-4 mRNA was barely detectable in foetal muscle, was expressed at low levels by 1-8 days and at 2-3-fold higher levels during and after weaning (18-40 days). In contrast there was little change in insulin receptor mRNA levels prior to weaning and a reduction in mRNA abundance between 18 and 40 days. Weaning rats on to a diet rich in fat prevented the increase in GLUT-4 abundance seen between 15 and 29 days in animals weaned on a high-carbohydrate diet.     

Activation of the glucose transporter GLUT4 by insulin.

The transport of glucose into cells and tissues is a highly regulated process, mediated by a family of facilitative glucose transporters (GLUTs). Insulin-stimulated glucose uptake is primarily mediated by the transporter isoform GLUT4, which is predominantly expressed in mature skeletal muscle and fat tissues. Our recent work suggests that two separate pathways are initiated in response to insulin: (i) to recruit transporters to the cell surface from intracellular pools and (ii) to increase the intrinsic activity of the transporters. These pathways are differentially inhibited by wortmannin, demonstrating that the two pathways do not operate in series. Conversely, inhibitors of p38 mitogen-activated protein kinase (MAPK) imply that p38 MAPK is involved only in the regulation of the pathway leading to the insulin-stimulated activation of GLUT4. This review discusses the evidence for the divergence of GLUT4 translocation and activity and proposed mechanisms for the regulation of GLUT4.     

Inhibition of glucose uptake and suppression of glucose transporter 1 mRNA expression in L929 cells by tumour necrosis factor-alpha

Recombinant human tumour necrosis factor-alpha (rhTNF-alpha) arrested the growth and suppressed glucose uptake of mouse fibrosarcoma L929 cells in vitro. When the cells were treated with rhTNF-alpha for 24 hours, the mRNA level of glucose transporter 1 (GLUT 1), which is the only GLUT found to be present in L929 cells in our study, was suppressed in a dose-dependent manner. Since the growth of tumour cells depends mainly on glucose catabolism, our findings may indicate that rhTNF-alpha inhibits L929 cells growth by lowering the glucose transport through suppression of GLUT 1 mRNA expression in the cells.     

3T3-L1 adipocyte glucose transporter (HepG2 class): sequence and regulation of protein and mRNA expression by insulin, differentiation, and glucose starvation

A glucose transporter cDNA (GLUT) clone was isolated from mouse 3T3-L1 adipocytes and sequenced. The nucleotide and deduced amino acid sequences were, respectively, 95 and 99% homologous to those of the rat brain transporter. The mouse cDNA and a polyclonal antibody recognizing the corresponding in vitro translation product were used to compare changes in transporter mRNA and protein levels during differentiation, glucose starvation, and chronic insulin exposure of 3T3-L1 preadipocytes. The respective cellular content of transporter mRNA and protein were increased 6.6- and 7.8-fold during differentiation, and 3.8- and 2.5-fold from chronic insulin exposure of differentiated adipocytes. Glucose starvation increased transporter mRNA and protein levels 2.2- and 3.5-fold in undifferentiated preadipocytes and 1.8- and 3.1-fold in differentiated adipocytes. Starvation of undifferentiated cells completely converted the native transporter to an incompletely glycosylated form, while increasing basal transport rates 4.5-fold. Either full glycosylation is not required to produce a functionally active transporter, or starvation causes a unique predifferentiation induction of the normally absent "responsive" transporter. The changes in transporter protein expression elicited by differentiation were attributed primarily to increased rates of transporter synthesis, while the disproportionate changes in mRNA and protein expression from chronic insulin treatment and starvation suggested these conditions increase synthesis and decrease turnover rates in regulating transporter protein expression. Although chronic insulin exposure and glucose starvation each raised the expression of transporter protein greater than 3-fold and basal transport rates 2.5- to 4.5-fold, no significant increase in the insulin responsiveness of 3T3-L1 preadipocytes or differentiated adipocytes was observed. Thus, the changes in the transporter mRNA and protein expression observed in this study were most consistent with their being associated with the regulated expression of a basal or low level insulin-responsive transporter.     

Short-term insulin treatment and aortic expressions of IGF-1 receptor and VEGF mRNA in diabetic rats

We investigated the relationship between the changes in vascular responsiveness and growth factor mRNA expressions induced by 1-wk treatment with high-dose insulin in control and established streptozotocin (STZ)-induced diabetes. Aortas from diabetic rats, but not those from insulin-treated diabetic rats, showed impaired endothelium-dependent relaxation in response to ACh (vs. untreated controls). The ACh-induced nitrite plus nitrate (NOx) level showed no significant difference between controls and diabetics. Insulin treatment increased NOx only in diabetics. In diabetics, insulin treatment significantly increased the aortic expressions of endothelial nitric oxide synthase (eNOS) mRNA and VEGF mRNA. The expression of IGF-1 mRNA was unaffected by diabetes or by insulin treatment. In contrast, the mRNA for the aortic IGF-1 receptor was increased in diabetics and further increased in insulin-treated diabetics. In aortic strips from age-matched control rats, IGF-1 caused a concentration-dependent relaxation. This relaxation was significantly stronger in strips from STZ-induced diabetic rats. These results suggest that in STZ-diabetic rats, short-term insulin treatment can ameliorate endothelial dysfunction by inducing overexpression of eNOS and/or VEGF mRNAs possibly via IGF-1 receptors. These receptors were increased in diabetes, perhaps as result of insulin deficiency.     

Cloning of a rabbit brain glucose transporter cDNA and alteration of glucose transporter mRNA during tissue development

A full-length cDNA clone that codes for glucose transporter protein was isolated from a rabbit brain cDNA library by using synthetic oligonucleotide probe derived from the sequence of human glucose transporter cDNA. The coding region shared 93.2% nucleotide and 97.0% amino-acid similarities with those of human glucose transporter and 89.4% nucleotide and 97.4% amino-acid similarities with those of rat transporter. Northern blot analysis revealed that glucose transporter mRNA is most abundant in the placenta and that it is also abundant in the brain. The fat tissue, heart, liver, and skeletal muscle of adult rats contained a very small amount of mRNA, while heart, liver, skeletal muscle and kidney of fetal rats contained a very high amount of glucose transporter mRNA. These results suggest that this type of glucose transporter might be closely related with cell proliferation and tissue development.     

补偿生长对异育银鲫IGF-1、IGFBP-1水平及IGF-1、IGF-1RmRNA表达的影响

该实验分析饥饿和恢复投喂对异育银鲫血液IGF-1和IGFBP-1水平和肝脏IGF-1、白肌IGF-1RmRNA表达量的影响。结果显示:饥饿期(14d)血液中IGF-1和IGFBP-1水平逐渐下降,在饥饿第14天均出现显著性降低(P<0.05);恢复投喂后第1天IGF-1迅速恢复到对照组水平,而IGFBP-1水平仍显著低于对照组(P<0.05),随后逐渐升高,直至于恢复投喂第14天后显著高于对照组水平(P<0.05);饥饿期肝脏IGF-1mRNA表达量呈下降趋势,但与对照组无显著性差异(P>0.05);恢复投喂初期(第1、3天),IGF-1mRNA表达量仍继续下降(P<0.05),对营养条件的变化反应滞后,至第7天,表达水平恢复到对照组水平。白肌IGF-1RmRNA表达水平在饥饿第3天出现显著性下降(P<0.05),继续饥饿其水平出现补偿性升高;恢复投喂后第14天IGF-1RmRNA表达量显著高于对照组水平(P<0.05)。该结果揭示恢复投喂期高水平的IGFBP-1含量和IGF-1RmRNA表达量可能通过提高IGF-1的促生长作用参与异育银鲫的补偿生长调节。     

Acute and chronic regulation of phosphoenolpyruvate carboxykinase mRNA by insulin and glucose

Using the well differentiated rat hepatoma Fao we have studied the regulation of phosphoenolpyruvate carboxykinase (PEPCK) mRNA by insulin and glucose and compared these results to glucose production as estimated by glucose release into the medium. Fao cells possess an active gluconeogenic pathway and, when grown in glucose-free medium, release glucose for over 8 h. Addition of the cAMP analog, 8-(4-chlorophenyl-thio) cAMP (8-CTP-cAMP) or increasing the concentration of dihydroxyacetone and oxaloacetate results in an increase in glucose release which can be suppressed by insulin at concentrations between 1 and 100 nM. These effect of cAMP and insulin are associated with parallel changes in the level of mRNAPEPCK. Insulin treatment reduces mRNAPEPCK levels in these cells by 80%; this effect is transient reaching a maximum at 2-4 h. Addition of glucose to cells grown in glucose-free (G-) medium produces a decrease in mRNAPEPCK which is similar in magnitude and kinetics to that produced by insulin. Conversely, when cells grown in normal medium are placed in G- medium mRNAPEPCK levels triple over a period of 8 h, then return toward the basal value. Cells grown in G- medium or in G- medium plus 10nM insulin for 1 yr exhibit only slightly increased levels of mRNAPEPCK and respond to both 8-CTP-cAMP, and insulin, although the response to 8-CTP-cAMP is slightly blunted. These data indicate that glucose and insulin can play independent roles in regulation of PEPCK gene expression, and that these regulatory effects are usually transient.     

Up-regulation of Na-coupled glucose transporter SGLT1 by caveolin-1

The Na⁺-coupled glucose transporter SGLT1 (SLC5A1) accomplishes concentrative cellular glucose uptake even at low extracellular glucose concentrations. The carrier is expressed in renal proximal tubules, small intestine and a variety of nonpolarized cells including several tumor cells. The present study explored whether SGLT1 activity is regulated by caveolin-1, which is known to regulate the insertion of several ion channels and carriers in the cell membrane. To this end, SGLT1 was expressed in Xenopus oocytes with or without additional expression of caveolin-1 and electrogenic glucose transport determined by dual electrode voltage clamp experiments. In SGLT1-expressing oocytes, but not in oocytes injected with water or caveolin-1 alone, the addition of glucose to the extracellular bath generated an inward current (I_g), which was increased following coexpression of caveolin-1. Kinetic analysis revealed that caveolin-1 increased maximal I_g without significantly modifying the glucose concentration required to trigger half maximal I_g (K_M). According to chemiluminescence and confocal microscopy, caveolin-1 increased SGLT1 protein abundance in the cell membrane. Inhibition of SGLT1 insertion by brefeldin A (5 μM) resulted in a decline of I_g, which was similar in the absence and presence of caveolin-1. In conclusion, caveolin-1 up-regulates SGLT1 activity by increasing carrier protein abundance in the cell membrane, an effect presumably due to stimulation of carrier protein insertion into the cell membrane.     

Vascular endothelial insulin/IGF-1 signaling controls skin wound vascularization

Type 2 diabetes mellitus affects 6% of western populations and represents a major risk factor for the development of skin complications, of which impaired wound healing, manifested in e.g. "diabetic foot ulcer", is most prominent. Impaired angiogenesis is considered a major contributing factor to these non-healing wounds. At present it is still unclear whether diabetes-associated wound healing and skin vascular dysfunction are direct consequences of impaired insulin/IGF-1 signaling, or secondary due to e.g. hyperglycemia. To directly test the role of vascular endothelial insulin signaling in the development of diabetes-associated skin complications and vascular function, we inactivated the insulin receptor and its highly related receptor, the IGF-1 receptor, specifically in the endothelial compartment of postnatal mice, using the inducible Tie-2CreERT (DKO(IVE)) deleter. Impaired endothelial insulin/IGF-1 signaling did not have a significant impact on endothelial homeostasis in the skin, as judged by number of vessels, vessel basement membrane staining intensity and barrier function. In contrast, challenging the skin through wounding strongly reduced neo-angiogenesis in DKO(IVE) mice, accompanied by reduced granulation tissue formation reduced. These results show that endothelial insulin/IGF signaling is essential for neo-angiogenesis upon wounding, and imply that reduced endothelial insulin/IGF signaling directly contributes to diabetes-associated impaired healing.     

Overexpression of glucose transporter modulates insulin biosynthesis in insulin producing cell line

Glucose transporter (GT) has been suggested to be involved in the insulin biosynthesis. However, the functional relationship between GT and insulin biosynthesis is not well understood. In this report, we have generated rat pancreatic B cell lines (RINr) that stably overexpress a cDNA encoding the brain type GT. These cell lines showed 3- to 4-fold increase in insulin mRNA and protein. These results suggest that GT might have some relationship to the insulin biosynthesis in the pancreatic B cells.     

Modulation of basal glucose transporter Km in the adipocyte by insulin and other factors

We have previously described experimental conditions where basal methylglucose transport in adipocytes exhibited an apparent Km of approximately 35 mM. Under those conditions insulin stimulated transport predominantly by decreasing the transport Km (Whitesell, R. R., and Abumrad, N. A. (1985) J. Biol. Chem. 260, 2894-2899). Our findings were in contrast with earlier reports that the Km of basal glucose transport was low (3-5 mM) and similar to that of transport in insulin-treated cells. In this study we have investigated the effect of different experimental conditions on the kinetics of basal glucose transport in adipocytes. When transport was assayed at 37 degrees C, cell agitation for 10 min prior to the transport assay decreased the basal Km from 35 to 12 mM. Deprivation of metabolic substrate produced a further reduction down to 2 mM. Refeeding starved cells with 1 mM glucose returned the Km back up to 12 mM in agitated cells and to 40 mM in stabilized cells. The effects of agitation to lower and of glucose to raise the basal Km were prevented by preincubating cells with dinitrophenol. Cell agitation or substrate lack did not alter the Vmax of basal transport and were without effect on both Km and Vmax in insulin-treated cells. The temperature dependencies of the kinetics of basal and stimulated transport were studied. A decrease in the assay temperature from 37 to 23 degrees C caused both basal Km and Vmax to drop proportionately from 25 to 5 mM, and 13 to 3.6 nmol/(microliter X min), respectively. In insulin-stimulated cells, only the Vmax was decreased (Km went from 3.5 to 3 mM, Vmax from 45 to 17 nmol/(microliter X min]. The results support the concept that experimental conditions can produce large changes in the Km of basal glucose transporters. Furthermore they explain why, under certain assay conditions (with temperatures around 23 degrees C or with deprivation of metabolic substrate), the effect of insulin on transport Km is not observed. Our data also suggest that basal transport characteristics do not persist in insulin-treated cells. We would propose that one of the actions of insulin (in addition to raising Vmax) is to change the characteristics of basal transporters by overriding metabolic factors which keep the Km high. Alternatively, insulin could cause the disappearance of basal transporters as new and different ones are recruited from intracellular stores.     

Release of PAI-1 by human preadipocytes and adipocytes independent of insulin and IGF-1

The effect of insulin and insulin-like growth factor-1 (IGF-1) on plasminogen activator inhibitor-1 (PAI-1) release from primary cultures of human preadipocytes and adipocytes has been investigated. Initial experiments measuring basal PAI-1 release (ng/ml) indicated variability between individual cultures. Using a novel technique for adipocyte quantitation, additional experiments were performed to determine PAI-1 release per cell, indicating a significant reduction with differentiation. Insulin and IGF-1 over a range of concentrations had no effect on PAI-1 release, and RT-PCR of PAI-1 mRNA following treatment with insulin and IGF-1 also indicated similar expression between treatments. The cultures did exhibit insulin-stimulated glyceraldehyde-3-phosphate dehydrogenase (GAPDH) expression and leptin synthesis following differentiation to the adipocyte phenotype. This is the first report of PAI-1 secretion by primary cultures of human preadipocytes and adipocytes, indicating PAI-1 release independent of insulin and IGF-1 and implicating other factors in the elevated plasma PAI-1 observed with insulin resistance.