Angiotensin II signaling and HB-EGF shedding via metalloproteinase in glomerular mesangial cells.

BACKGROUND: Angiotensin II (Ang II) has been implicated in the development of glomerulosclerosis by stimulating fibronectin (FN) synthesis. The processing and release of heparin binding-endothelin growth factor (HB-EGF) are activated by protein kinase C (PKC) and Ca2+ signaling. We studied the roles of HB-EGF and endothelial growth factor (EGF) receptor (EGFR) in Ang II-induced FN expression using mesangial cells. METHODS: Mesangial cells were prepared from mouse kidneys by the explant method and cells were used at passages 4 and 5. RESULTS: Ang II stimulated FN mRNA levels dose-dependently with a maximal increase (3.4-fold) after 12 hours of incubation. This action was completely inhibited by PKC inhibitors and slightly blocked by Ca2+ chelating agents. FN mRNA accumulation by Ang II was abolished by tyrosine kinase inhibitors, a specific inhibitor for EGFR (AG1478) and extracellular signal-regulated kinase (ERK) inactivation. Addition of neutralizing anti-HB-EGF antibody, as well as pretreatment with heparin or the metalloproteinase inhibitor batimastat abolished induction of FN expression by Ang II. In mesangial cells stably transfected with a chimeric construct containing HB-EGF and alkaline phosphatase (ALP) genes, ALP activity in incubation medium was rapidly increased by Ang II (1.7-fold at 0.5 min) and reached a 4.1-fold increase at two minutes. Ang II phosphorylated EGFR (maximal at 2 min) and ERK (maximal at 8 min) in a PKC- and metalloproteinase-dependent manner. Ang II stimulated the expression and release of transforming growth factor-beta (TGF-beta) via EGFR-mediated signaling, and the released TGF-beta also contributed to Ang II-mediated FN expression via EGFR transactivation. CONCLUSIONS: Ang II-mediated FN expression was regulated by autocrine effects of HB-EGF and TGF-beta, suggesting a novel paradigm for cross-talk between Ang II and growth factor receptor signaling pathways.

Angiotensin II type 2 receptor inhibits epidermal growth factor receptor transactivation by increasing association of SHP-1 tyrosine phosphatase.

Angiotensin (Ang) II has 2 major receptor isoforms, Ang type 1 (AT(1)) and Ang type (AT(2)). AT(1) transphosphorylates epidermal growth factor receptor (EGFR) to activate extracellular signal-regulated kinase (ERK). Although AT(2) was shown to inactivate ERK, the action of AT(2) on EGFR activation remains undefined. Using AT(2)-overexpressing vascular smooth muscle cells from AT(2) transgenic mice, we studied these undefined actions of AT(2). Maximal ERK activity induced by Ang II was increased 1.9- and 2.2-fold by AT(2) inhibition, which was abolished by orthovanadate but not okadaic acid or pertussis toxin. AT(2) inhibited AT(1)-mediated EGFR tyrosine phosphorylation by 63%. The activity of SHP-1 tyrosine phosphatase was significantly upregulated 1 minute after AT(2) stimulation and association of SHP-1 with EGFR was increased, whereas AT(2) failed to tyrosine phosphorylate SHP-1. Stable overexpression of SHP-1-dominant negative mutant completely abolished AT(2)-mediated inhibition of EGFR and ERK activation. AT(1)-mediated c-fos mRNA accumulation was attenuated by 48% by AT(2) stimulation. Induction of fibronectin gene containing an AP-1 responsive element in its 5'-flanking region was decreased by 37% after AT(2) stimulation, corresponding to the results of gel mobility assay with the AP-1 sequence of fibronectin as a probe. These findings suggested that AT(2) inhibits ERK activity by inducing SHP-1 activity, leading to decreases in AP-1 activity and AP-1-regulated gene expression, in which EGFR dephosphorylation plays an important role via association of SHP-1.

Safety and availability of doxazosin in treating hypertensive patients with chronic renal failure.

Hypertension accelerates the progression of renal disease in patients with chronic renal failure. Doxazosin, an alpha1-antagonist, is an antihypertensive agent with a long half-life. In this study, 15 patients with chronic renal failure were treated only with doxazosin and diuretics for 6 months and their blood pressure, renal parameters and lipid profile were measured. The initial dose of doxazosin was 2 mg/day and it was titrated until blood pressure was normalized. The average dose was 5.6 mg/day. As expected, systolic and diastolic blood pressure were decreased with treatment (165/91 mmHg to 135/73 mmHg). The drop in blood pressure was associated with an increase in glomerular filtration and a decrease in plasma BUN and creatinine levels. Reduction in mean blood pressure and decrease in proteinuria had a significant positive correlation (r=0.048, p=0.007). Proteinuria was decreased from 1.8 mg/day to 1.3 mg/day with doxazosin treatment and triglycerides also decreased, while HDL-cholesterol was increased. No side effects were observed. These results indicate that doxazosin is an efficient depressor agent with renal protective actions and that higher doses of doxazosin can be safely given to patients with chronic renal failure.

Implantation of bone marrow mononuclear cells into ischemic myocardium enhances collateral perfusion and regional function via side supply of angioblasts, angiogenic ligands, and cytokines.

BACKGROUND: Bone marrow implantation (BMI) was shown to enhance angiogenesis in a rat ischemic heart model. This preclinical study using a swine model was designed to test the safety and therapeutic effectiveness of BMI. METHODS AND RESULTS: BM-derived mononuclear cells (BM-MNCs) were injected into a zone made ischemic by coronary artery ligation. Three weeks after BMI, regional blood flow and capillary densities were significantly higher (4.6- and 2.8-fold, respectively), and cardiac function was improved. Angiography revealed that there was a marked increase (5.7-fold) in number of visible collateral vessels. Implantation of porcine coronary microvascular endothelial cells (CMECs) did not cause any significant increase in capillary densities. Labeled BM-MNCs were incorporated into approximately 31% of neocapillaries and corresponded to approximately 8.7% of macrophages but did not actively survive as myoblasts or fibroblasts. There was no bone formation by osteoblasts or malignant ventricular arrhythmia. Time-dependent changes in plasma levels for cardiac enzymes (troponin I and creatine kinase-MB) did not differ between the BMI, CMEC, and medium-alone implantation groups. BM-MNCs contained 16% of endothelial-lineage cells and expressed basic fibroblast growth factor>>vascular endothelial growth factor>angiopoietin 1 mRNAs, and their cardiac levels were significantly upregulated by BMI. Cardiac interleukin-1beta and tumor necrosis factor-alpha mRNA expression were also induced by BMI but not by CMEC implantation. BM-MNCs were actively differentiated to endothelial cells in vitro and formed network structure with human umbilical vein endothelial cells. CONCLUSIONS: BMI may constitute a novel safety strategy for achieving optimal therapeutic angiogenesis by the natural ability of the BM cells to secrete potent angiogenic ligands and cytokines as well as to be incorporated into foci of neovascularization.

Effect of angiotensin II type 2 receptor on tyrosine kinase Pyk2 and c-Jun NH2-terminal kinase via SHP-1 tyrosine phosphatase activity: evidence from vascular-targeted transgenic mice of AT2 receptor.

Angiotensin II (Ang II) has two major receptor isoforms, AT1 and AT2. AT1 transphosphorylates Ca(2+)-sensitive tyrosine kinase Pyk2 to activate c-Jun NH2-terminal kinase (JNK). Although AT2 inactivates extracellular signal-regulated kinase (ERK) via tyrosine phosphatases (PTP), the action of AT2 on Pyk2 and JNK remains undefined. Using AT2-overexpressing vascular smooth muscle cells (AT2-VSMC) from AT2-transgenic mice, we studied these undefined actions of AT2. AT1-mediated JNK activity was increased 2.2-fold by AT2 inhibition, which was abolished by orthovanadate. AT2 did not affect AT1-mediated Pyk2 phosphorylation, but attenuated c-Jun mRNA accumulation by 32%. The activity of src-homology 2 domain-containing PTP (SHP-1) was significantly upregulated 1 min after AT2 stimulation. Stable overexpression of SHP-1 dominant negative mutant in AT2-VSMC completely abolished AT2-mediated inhibition of JNK activation and c-Jun expression. These findings suggest that AT2 inhibits JNK activity by affecting the downstream signal of Pyk2 in a SHP-1-dependent manner, leading to a decrease in c-Jun expression.

Decreased triglyceride-rich lipoproteins in transgenic skinny mice overexpressing leptin.

Leptin is an adipocyte-derived circulating satiety factor with a variety of biological effects. Evidence has accumulated suggesting that leptin may modulate glucose and lipid metabolism. In the present study, we examined lipid metabolism in transgenic skinny mice with elevated plasma leptin concentrations. The plasma concentrations of triglycerides and free fatty acids in transgenic skinny mice were 71.5 (P < 0.01) and 89.1% (P < 0.05) of those in their nontransgenic littermates, respectively. Separation of plasma into lipoprotein classes by ultracentrifugation revealed that very low density lipoprotein-triglyceride concentrations were markedly reduced in transgenic skinny mice relative to the controls. The clearance of triglycerides estimated by a fat-loading test was enhanced in transgenic skinny mice; the triglyceride concentration in transgenic skinny mice 3 h after fat loading was 39.7% (P < 0.05) of that of their nontransgenic littermates. Postheparin plasma lipoprotein lipase activity increased 1.4-fold (P < 0.05) in transgenic skinny mice. Our data demonstrated a significant reduction in plasma triglyceride concentrations, accompanied by increased lipoprotein lipase activity in transgenic skinny mice overexpressing leptin, suggesting that leptin plays a role in long-term triglyceride metabolism.

Identification of intron and exon sequences involved in alternative splicing of insulin receptor pre-mRNA.

The insulin receptor exists as two isoforms, A and B, that result from alternative splicing of exon 11 in the primary transcript. We have shown previously that the alternative splicing is developmentally and hormonally regulated. Consequently, these studies were instigated to identify sequences within the primary RNA transcript that regulate the alternative splicing. Minigenes containing exons 10, 11, and 12 and the intervening introns were constructed and transfected into HepG2 cells, which contain both isoforms of the insulin receptor. The cells were able to splice the minigene transcript to give both A (- exon 11) and B-like (+ exon 11) RNAs. A series of internal deletions within intron 10 were tested for their ability to give A and B RNAs. Intron 10 contained two sequences that modulated exon 11 inclusion; a 48-nucleotide purine-rich sequence at the 5' end of intron 10 that functions as a splicing enhancer and causes an increase in exon 11 inclusion, and a 43-nucleotide sequence at the 3' end of intron 10 upstream of the branch point sequence that favors skipping of exon 11. Increasing the length of the polypyrimidine tract at the 3' end of intron 10 caused exon 11 to be spliced constitutively, indicating that a weak splice site is required for alternative splicing. Finally, point mutations, insertions, and deletions within exon 11 itself were able to regulate inclusion of the exon both positively and negatively.

14-3-3beta protein associates with insulin receptor substrate 1 and decreases insulin-stimulated phosphatidylinositol 3'-kinase activity in 3T3L1 adipocytes.

The 14-3-3 protein family has been implicated in growth factor signaling. We investigated whether 14-3-3 protein is involved in insulin signaling in 3T3L1 adipocytes. A significant amount of insulin receptor substrate 1 (IRS-1) was immunodetected in the immunoprecipitate with anti-14-3-3beta antibody at the basal condition. 100 nM insulin increased the amount of IRS-1 in the immunoprecipitate 2.5-fold. The effect of insulin was abolished by 100 nM wortmannin. An in vitro binding study revealed that glutathione S-transferase-14-3-3beta fusion protein directly associates with recombinant IRS-1. Pretreatment of recombinant IRS-1 with alkaline phosphatase clearly decreased this association. Because the recombinant IRS-1 was not phosphorylated on its tyrosine residues, the results suggest that serine/threonine phosphorylation of IRS-1 is responsible for the association. When the cells are treated with insulin, phosphatidylinositol 3'-kinase (PI3K) is supposed to complex either 14-3-3beta-IRS-1 or IRS-1. The 14-3-3beta-IRS-1-PI3K and IRS-1-PI3K complexes were separately prepared by a sequential immunoprecipitation, first with anti-14-3-3beta and then with anti-IRS-1 antibodies. The specific activity of the PI3K in the former was approximately half of that in the latter, suggesting that 14-3-3beta protein bound to IRS-1 inhibits insulin-stimulated lipid kinase activity of PI3K in 3T3L1 adipocytes.

Differential activation of mitogen-activated protein kinase by insulin and epidermal growth factor in 3T3-L1 adipocytes: a possible involvement of PI3-kinase in the activation of the MAP kinase by insulin.

Mitogen-activated protein (MAP) kinase plays crucial roles in cell growth and differentiation. It has recently been shown that the MAP kinase cascade in growth factor signaling diverges and cross-talks with other signaling pathways. In the present study, we examined the effects of wortmannin, a specific inhibitor of phosphatidylinositol 3-kinase (PI3-kinase), on the activation of Ras, Raf-1 kinase, and MAP kinase by insulin and epidermal growth factor (EGF). The effect of LY294002, a structurally distinct PI3-kinase inhibitor, on the activation of Raf-1 kinase by both ligands was also examined. In 3T3-L1 adipocytes, 25 nmol/l wortmannin inhibited the insulin-induced activation of Raf-1 kinase to the basal level, whereas the same dose of wortmannin had little effect on the EGF-induced activation of Raf-1 kinase. One hundred micromol/l LY294002 blocked insulin-induced activation of Raf-1 kinase without affecting EGF-induced activation of this kinase. Twenty-five nmol/l wortmannin inhibited the insulin-induced activation of MAP kinase to the basal level with no effect on the EGF-induced activation of this kinase. But the same dose of wortmannin did not affect the formation of guanosine 5'-triphosphate (GTP)-bound Ras stimulated by either ligand. In KB cells, results similar to those in 3T3-L1 adipocytes were obtained. In contrast, in Chinese hamster ovary cells overexpressing the human insulin receptor (CHO-HIR cells), neither wortmannin nor LY294002 inhibited the insulin-induced activation of Raf-1 kinase, and wortmannin had little effect on the activation of MAP kinase by insulin. These results indicate that 1) PI3-kinase or wortmannin-sensitive molecules are involved in the interaction between activated Ras and Raf-1 kinase in the insulin signaling in 3T3-L1 adipocytes, 2) the involvement of PI3-kinase or wortmannin-sensitive molecules in the insulin-induced activation of MAP kinase appears to be cell-type specific, and 3) differential mechanisms to activate Raf-1 kinase and MAP kinase by insulin and EGF exist.

Reduced expression of the leptin gene (ob) by catecholamine through a G(S) protein-coupled pathway in 3T3-L1 adipocytes.

Leptin, a recently identified hormone, is believed to reduce appetite and maintain body weight. The mRNA of leptin is expressed only in mature adipose cells. To clarify the regulation of leptin gene expression in adipocytes, 3T3-L1 adipocytes were treated for 16 h with various agents known to modulate lipid metabolism, and then the leptin mRNA was measured by the reverse transcription-polymerase chain reaction method. Interestingly, both norepinephrine and isoproterenol reduced the level of leptin mRNA to about 20% of that found in untreated control cells in a dose-dependent fashion. The maximum reduction occurred at 100 nmol/l of either norepinephrine or isoproterenol, and the half-maximal effect was observed at approximately 3 nmol/l norepinephrine and approximately 1 nmol/l isoproterenol. Propranolol reversed about 50% of the reduction by either norepinephrine or isoproterenol. In contrast, phentolamine did not inhibit the reduction by either norepinephrine or isoproterenol. Moreover, both cholera toxin and dibutyryl cAMP decreased the level of leptin mRNA to about 10% of that in control cells in a dose-dependent fashion. The maximum effect was elicited at 100 ng/ml cholera toxin and 100 micromol/l dibutyryl cAMP. The concentration producing the half-maximal effect was approximately 1 ng/ml cholera toxin and approximately 50 micromol/l dibutyryl cAMP. Dibutyryl cGMP, however, did not affect leptin gene expression. These results suggest that a signaling pathway that results in the activation of protein kinase A regulates leptin gene expression in 3T3-L1 adipocytes.

The B isoform of the insulin receptor signals more efficiently than the A isoform in HepG2 cells.

We have demonstrated previously that dexamethasone treatment of HepG2 cells caused an enhancement of insulin's metabolic effects (Kosaki, A., and Webster, N. J. (1993) J. Biol. Chem. 268, 21990-21996). This correlated with increased expression of the mRNA encoding the B isoform of the insulin receptor (IR). In the present study, we have demonstrated that dexamethasone treatment caused in addition an enhancement in insulin-stimulated immediate-early gene expression (c-fos and egr-1). Dexamethasone treatment caused an increase in in vivo IR autophosphorylation and insulin receptor substrate-1 (IRS-1) phosphorylation both early events in the insulin signaling pathway. Furthermore, the IRS-1 phosphorylation was distinctly left shifted, although the level of IRS-1 protein was unchanged. Total cellular tyrosine phosphatase activity was unaltered when assayed with 32P-labeled IR and IRS-1. Studies in vitro on wheat-germ agglutinin-purified receptors showed that the B isoform of the IR had increased kinase activity both toward itself and the exogenous substrates poly.glu4:tyr1 and recombinant IRS-1 protein. In addition, two-dimensional tryptic phosphopeptide maps indicated that the B isoform has an additional phosphopeptide that is not seen for the A isoform. In conclusion, it appears that the B isoform of the IR signals more efficiently than the A isoform in HepG2 cells.

Effect of dexamethasone on the alternative splicing of the insulin receptor mRNA and insulin action in HepG2 hepatoma cells.

We have shown that culturing HepG2 cells in Ham's F-12 medium supplemented with calf serum, dexamethasone, and triiodothyronine causes an increase in the insulin sensitivity and responsiveness for glucose incorporation into glycogen. This correlates with increased expression of the mRNA encoding the B isoform of the insulin receptor. Of all the components in the medium, we found that dexamethasone exerted the greatest effect. Dexamethasone alone could cause both a switch in expression from the A to the B isoform of the insulin receptor and also an increase in insulin sensitivity for both glucose incorporation into glycogen and 2-deoxyglucose transport. In addition, we found that expression of the B isoform of the insulin receptor is developmentally regulated in the 3T3-L1 adipocyte leading to the suggestion that the alternatively spliced B isoform of the insulin receptor may play an important role in signal transduction in insulin target tissues such as liver, fat, and muscle.

Effects of ML-9 on insulin stimulation of glucose transport in 3T3-L1 adipocytes.

Treatment of 3T3-L1 adipocytes with insulin resulted in activation of 2-deoxyglucose transport activity and translocation of glucose transporters (GLUT4 and GLUT1) from the cytoplasmic space to the plasma membrane. ML-9 (a myosin light chain kinase inhibitor) inhibited insulin stimulation of 2-deoxyglucose transport activity by 80% at 100 microM (IC50 = 27 microM) without affecting 2-deoxyglucose transport activity in the basal state. The inhibition was independent of extracellular Ca2+ concentration and almost fully reversible at 40 microM ML-9. ML-9 did not inhibit insulin-stimulated tyrosine phosphorylation of 95-kDa protein in the wheat germ agglutinin-purified preparation and of 95- and 160-kDa proteins in intact cells. However, ML-9 inhibited insulin-induced translocation of both GLUT4 and GLUT1 in a dose-dependent manner. The dose-response curves were similar to those observed for the inhibition of insulin stimulation of 2-deoxyglucose transport activity. Neither insulin nor ML-9 affected the phosphorylation state of both heavy and light chains of myosin. Therefore, it seems likely that ML-9 inhibits the insulin-induced translocation of glucose transporters at a step beyond the insulin receptor kinase activity by a mechanism different from that affecting phosphorylation of the myosin light chain. Phosphorylating activity of microtubule-associated protein 2 and myelin basic protein was stimulated by insulin, and this stimulation was not affected by ML-9. ML-9, however, inhibited the phosphorylating activity in vitro and insulin stimulation of the phosphorylating activity of ribosomal protein S6 in intact cells in a dose-dependent manner similar to that observed for the inhibition of insulin stimulation of glucose transport. These results suggest that mitogen-activated protein kinase may be one of the constituents in intracellular insulin signaling to the glucose transport system.

Preparation of anti-phosphoserine and anti-phosphothreonine antibodies and their application in the study of insulin- and EGF-induced phosphorylation.

We prepared antibodies against phosphoserine (P-Ser) and phosphothreonine (P-Thr) by immunizing rabbits with P-Ser or P-Thr conjugated to bovine serum albumin. The antibodies (anti-P-Ser and anti-P-Thr) were purified using P-Ser or P-Thr affinity columns. Anti-P-Thr was highly specific for P-Thr, while anti-P-Ser showed weak cross-reactivity with P-Thr. We showed that these antibodies can immunodetect serine/threonine phosphorylated insulin and epidermal growth factor (EGF) receptors and several proteins which are phosphorylated on serine/threonine residues in response to insulin or EGF stimulation. The antibodies will certainly provide a good tool for discovering novel kinases and substrates involved in signal transduction.

Insulin resistance in Werner's syndrome.

Insulin resistance in Werner's syndrome (WS) was studied using the glucose clamp technique, and compared with physiologically aged and young subjects. Fasting immuno-reactive insulin (IRI) was increased in patients with Werner's syndrome compared with aged and young subjects. Metabolic clearance rate (MCR) of glucose was decreased in the aged and WS. A rightward shift of the dose-response curves of insulin and MCR of glucose was observed in the aged and WS with a more pronounced shift in the latter. MCR of insulin was also decreased in WS. [125I]insulin binding to erythrocytes was similar in the three groups. These results suggest that insulin resistance associated with WS is due to a post-binding defect manifested by a rightward shift of the dose-response curve of insulin-induced glucose disposal and a decrease in insulin clearance rate.

Monoclonal antibodies possibly recognize conformational changes in the human erythrocyte glucose transporter.

Two monoclonal antibodies (MAG17 and MAG20) were raised against the human erythrocyte glucose transporter, which was purified on an immunoaffinity column using a polyclonal antibody to the C-terminal peptide (residues 477-492) of the glucose transporter of HepG2 cells. To obtain antibodies which recognize the native glucose transporter integrated in the membrane, hybridomas were screened both by e.l.i.s.a. with purified glucose transporter and by dot-blotting with erythrocyte membranes. The antibodies immunoprecipitated D-glucose-inhibitable [3H]cytochalasin B-photoaffinity-labelled glucose transporters, but did not recognize the transporter on Western blotting. The presence of the C-terminal peptide did not inhibit the binding of these antibodies to the glucose transporter, suggesting that the antibodies recognized sites different from the transporter C-terminus. D-Glucose (0.1-100 microM) inhibited the binding of MAG17 and MAG20 to the transporter by 50%, indicating that the conformation of the epitopes was altered allosterically by D-glucose. Cytochalasin B inhibited the binding of MAG17 to the transporter, but enhanced the binding of MAG20 at low concentrations (less than 0.02 microM). These data suggest that the glucose transporter has high- and low-affinity binding sites for D-glucose and cytochalasin B, and that binding of D-glucose and cytochalasin B induces conformational changes in the transporter. Monoclonal antibodies which recognize the tertiary structure of the glucose transporter can be used for investigating its function and structure when integrated in the membrane.

Insulin-stimulated glucose uptake and fasting blood glucose.

Changes in insulin-stimulated glucose metabolism were studied in young and aged subjects, subjects with impaired glucose tolerance, and patients with NIDDM by means of the glucose clamp technique. The diabetic group includes obese and non-obese patients treated without insulin and non-obese patients treated with insulin. The glucose disposal rate (GDR) was decreased in aged subjects (5.8 +/- 0.4 mg/kg/min) compared with young controls (7.4 +/- 0.3 mg/kg/min). In patients with IGT, it was further decreased to 3.6 +/- 0.5 mg/kg/min, which was comparable to the rate in NIDDM without insulin treatment (3.3 +/- 0.4 mg/kg/min). There were no differences in the GDR between obese (3.0 +/- 0.3 mg/kg/min) and non-obese (3.4 +/- 0.6 mg/kg/min) diabetic patients. In insulin-treated diabetic patients, GDR ranged widely, but the mean value was partially normalized (5.2 +/- 0.9 mg/kg/min). In the diabetic group, no correlation was observed between fasting blood glucose and GDR. These results suggest that in the course of developing NIDDM, a decrease in insulin-stimulated glucose uptake precedes a rise in fasting blood glucose. Thus, as previously reported for Caucasian NIDDM patients, resistance to insulin-stimulated glucose uptake may be one of the basic defects in Japanese patients with NIDDM. The degree of glycemia, however, is not directly related to the magnitude of the defect in insulin action.

Regulation of glucose transporter synthesis in cultured human skin fibroblasts.

The present study was designed to see the effects of glucose on glucose transporter expression and glucose transport activity using cultured human skin fibroblasts. When the cells were incubated with various concentrations of glucose (11.1-44.4 mM), no differences were found in the HepG2 glucose transporter mRNA, protein levels and basal and insulin-stimulated 2-deoxyglucose uptake. Glucose deprivation, however, resulted in approximately 4-fold increases in the mRNA and 3-fold increases in the protein and the basal 2-deoxyglucose uptake. Chronic exposure to insulin increased the glucose transporter protein levels to similar degrees in the cells incubated with 11.1, 22.2 and 44.4 mM glucose accompanied by increases in the glucose transport activity. Effects of insulin on the glucose transporter mRNA and protein levels, however, were not evident in the glucose-deprived cells. It is concluded that glucose transport activity correlates closely with HepG2 glucose transporter expression in cultured human fibroblasts and that glucose (11.1-44.4 mM) does not affect the glucose transporter expression and glucose transport activity.