Differential proliferative response of cultured fetal and regenerating hepatocytes to growth factors and hormones

Upon epidermal growth factor (EGF) stimulation, fetal (20 days of gestation) and regenerating (44-48 h after partial hepatectomy) rat hepatocytes, isolated and cultured under identical conditions, increased DNA synthesis and entered into S-phase and mitosis, measured as [3H]thymidine incorporation and DNA content per nucleus in a flow cytometer, respectively. Fetal hepatocytes consisted of a homogeneous population of diploid (2C) cells. Two different populations of cells were present in regenerating liver, diploid (2C) and tetraploid (4C) cells, that responded to EGF. Glucagon or norepinephrine did not affect EGF stimulation of DNA synthesis in fetal liver cells, but they potentiated EGF response in regenerating hepatocyte cultures. Glucocorticoid hormones (dexamethasone) inhibited DNA synthesis in fetal hepatocyte cultures, an effect potentiated by the presence of glucagon or norepinephrine. In contrast, in regenerating hepatocytes, dexamethasone increased EGF-induced proliferation. EGF-dependent DNA synthesis was inhibited by TGF-beta in both fetal and regenerating cultured hepatocytes. TGF-beta action was partially suppressed by norepinephrine in regenerating hepatocytes, but was without effect in fetal hepatocyte cultures, whereas a synergistic action between TGF-beta and dexamethasone inhibiting growth in fetal but not in regenerating hepatocytes was found. Taken together, these results may suggest that there are significant differences between fetal and regenerating hepatocyte growth in their response to various hormones.     

Increase in liver glucose transporter mRNA levels during rat liver regeneration

Gene expression of liver facilitated glucose transporter was rapidly induced during the liver regenerating process in rats. It reached maximum of 2.7 times at 8 hr of the regenerating course and returned to normal by 48 hr. The protein synthesis inhibitor, cycloheximide, did not interfere with the increased gene expression of liver facilitated glucose transporter. By contrast, erythrocyte/brain-type glucose transporter mRNA could not be detected in the livers of partially hepatectomized rats and sham-operated rats. The plasma glucose levels were transiently increased within 2 hr of the regenerative course and then decreased to a nadir at 4 hr. These results suggest that the increased gene expression of liver facilitated glucose transporter contributes to the decrease in plasma glucose levels.     

Glucose and cationic amino acid transporter expression in growing chickens (Gallus gallus domesticus)

Tissue glucose transporter (GLUT1-3) and cationic amino acid transporter (CAT1-3) mRNA expression was determined in growing broiler chicks posthatch. In two experiments, tissues were either collected on days 1, 3 and 7 or days 1 and 14 posthatch. Heart and liver were the only tissues expressing a GLUT isoform on day 1. All tissues expressed a GLUT isoform on day 7 except for the thymus. Most tissues expressing a CAT isoform on day 1 decreased mRNA levels through day 7 (P<0.05), except for bursa CAT-1 which tended to increase (P=0.05). The thymus and spleen did not express any CAT isoform mRNA until day 7. The liver was the only tissue expressing GLUT-2 mRNA through day 14. On day 14, GLUT-1, CAT-1 and CAT-2 mRNA were differentially expressed across tissues (P<0.05). High-affinity GLUT and CAT mRNA expression was highest in the heart and bursa, respectively (P<0.05). Total CAT mRNA expression was greatest in the bursa (P<0.05). The thymus had the lowest high affinity GLUT and total CAT mRNA expression on day 14 posthatch. Therefore, T lymphocytes within the thymus may be most susceptible to glucose and cationic amino acid supply.     

Glucose transporter isotypes switch in T-antigen-transformed pancreatic beta cells growing in culture and in mice.

High-level expression of the low-Km glucose transporter isoform GLUT-1 is characteristic of many cultured tumor and oncogene-transformed cells. In this study, we tested whether induction of GLUT-1 occurs in tumors in vivo. Normal mouse beta islet cells express the high-Km (approximately 20 mM) glucose transporter isoform GLUT-2 but not the low-Km (1 to 3 mM) GLUT-1. In contrast, a beta cell line derived from an insulinoma arising in a transgenic mouse harboring an insulin-promoted simian virus 40 T-antigen oncogene (beta TC3) expressed very low levels of GLUT-2 but high levels of GLUT-1. GLUT-1 protein was not detectable on the plasma membrane of islets or tumors of the transgenic mice but was induced in high amounts when the tumor-derived beta TC3 cells were grown in tissue culture. GLUT-1 expression in secondary tumors formed after injection of beta TC3 cells into mice was reduced. Thus, high-level expression of GLUT-1 in these tumor cells is characteristic of culture conditions and is not induced by the oncogenic transformation; indeed, overnight culture of normal pancreatic islets causes induction of GLUT-1. We also investigated the relationship between expression of the different glucose transporter isoforms by islet and tumor cells and induction of insulin secretion by glucose. Prehyperplastic transgenic islet cells that expressed normal levels of GLUT-2 and no detectable GLUT-1 exhibited an increased sensitivity to glucose, as evidenced by maximal insulin secretion at lower glucose concentrations, compared with that exhibited by normal islets. Further, hyperplastic islets and primary and secondary tumors expressed low levels of GLUT-2 and no detectable GLUT-1 on the plasma membrane; these cells exhibited high basal insulin secretion and responded poorly to an increase in extracellular glucose. Thus, abnormal glucose-induced secretion of insulin in prehyperplastic islets in mice was independent of changes in GLUT-2 expression and did not require induction of GLUT-1 expression.     

Transforming growth factor-beta (TGF-beta) isoforms in rat liver regeneration: messenger RNA expression and activation of latent TGF-beta.

Expression of transforming growth factor-beta s (TGF-beta s) 1-3 was studied in normal liver and during liver regeneration after partial hepatectomy in the rat to determine whether each of these isoforms might be involved in hepatocyte growth in vivo. Expression of the mRNAs for all three TGF-beta isoforms increases in the regenerating liver. In addition, the levels of expression of the mRNAs for several extracellular matrix proteins, including fibronectin, vitronectin, laminin, and collagen, also increase in the regenerating liver. Immunohistochemical staining analysis shows a similar distribution of all three TGF-beta s in normal and regenerating liver; however, in both tissues, the level of expression of TGF-beta 1 is 8- to 10-fold higher than that of TGF-beta 2 as determined by sandwich enzyme-linked immunosorbent assay. Expression of all three TGF-beta mRNAs is restricted to liver nonparenchymal cells. Although hepatocytes from normal and regenerating livers do not synthesize TGF-beta, they are sensitive to inhibition of growth by all three TGF-beta isoforms. Hepatocytes from regenerating livers are capable of activating latent TGF-beta 1 complexes in vitro, whereas normal hepatocytes are not. The different TGF-beta isoforms may function in an inhibitory paracrine mechanism that is activated during liver regeneration and may also regulate the synthesis of extracellular matrix components in the regenerating liver.     

Resistin modulates glucose uptake and glucose transporter-1 (GLUT-1) expression in trophoblast cells

The adipocytokine resistin impairs glucose tolerance and insulin sensitivity. Here, we examine the effect of resistin on glucose uptake in human trophoblast cells and we demonstrate that transplacental glucose transport is mediated by glucose transporter (GLUT)-1. Furthermore, we evaluate the type of signal transduction induced by resistin in GLUT-1 regulation. BeWo choriocarcinoma cells and primary cytotrophoblast cells were cultured with increasing resistin concentrations for 24 hrs. The main outcome measures include glucose transport assay using [³H]-2-deoxy glucose, GLUT-1 protein expression by Western blot analysis and GLUT-1 mRNA detection by quantitative real-time RT-PCR. Quantitative determination of phospho(p)-ERK1/2 in cell lysates was performed by an Enzyme Immunometric Assay and Western blot analysis. Our data demonstrate a direct effect of resistin on normal cytotrophoblastic and on BeWo cells: resistin modulates glucose uptake, GLUT-1 messenger ribonucleic acid (mRNA) and protein expression in placental cells. We suggest that ERK1/2 phosphorylation is involved in the GLUT-1 regulation induced by resistin. In conclusion, resistin causes activation of both the ERK1 and 2 pathway in trophoblast cells. ERK1 and 2 activation stimulated GLUT-1 synthesis and resulted in increase of placental glucose uptake. High resistin levels (50–100 ng/ml) seem able to affect glucose-uptake, presumably by decreasing the cell surface glucose transporter.     

Divergent effects of cycloheximide on the induction of class II and class III cytochrome P450 mRNAs in cultures of adult rat hepatocytes

We have previously reported that when hepatocytes isolated from adult male rats are cultured in serum-free medium on matrigel, a reconstituted basement membrane gel, it is possible to elicit a stimulation of gene expression for both Class II cytochrome P450b/e and Class III cytochrome P450p by phenobarbital treatment (E.G. Schuetz et al., 1990 J. Biol. Chem. 265, 1188-1192). In the present study, an investigation of the requirement of protein synthesis for the rise in mRNAs for these cytochromes, pretreatment of the cells with cycloheximide prior to adding phenobarbital or "phenobarbital-like" inducers to the culture medium inhibited induction of P450b/e mRNA (46-90%), whereas the accumulation of P450p mRNA was enhanced (2- to 19-fold). Heme depletion did not appear to explain these observations because the inhibitory effects of cycloheximide on the induction of P450b/e mRNA were not overcome by supplementation of the medium with exogenous heme or with delta-aminolevulinic acid. Because Class IIIA P450s are regulated by gender as well as by phenobarbital, we examined the basal expression of P450p mRNA in cultures of hepatocytes derived from male rats and found that cycloheximide treatment was without effect. However, in cultures of hepatocytes isolated from female rats, where P450p mRNA is barely detectable, cycloheximide treatment greatly enhanced expression of P450p mRNA. As was observed in the cultured cells, the treatment of living female rats with cycloheximide also increased the amounts of P450p mRNA to levels comparable to those found in livers of untreated male rats. Analysis of Northern blots hybridized with oligonucleotides specific for P450PCN1(IIIA1) and P450PCN2(IIIA2), respectively, revealed that untreated male rat liver and cultures of hepatocytes prepared from these animals expressed readily detectable amounts of P450PCN1(IIIA1) mRNA. Such analyses confirmed that cycloheximide treatment selectively increased P450PCN1(IIIA1) mRNA in female rat liver, whereas the amount of mRNA for P450PCN2(IIIA2), a closely related male-specific family member, was unaffected. We conclude that the pathways for the induction of P450b/e and P450p by phenobarbital, and the pathways for the gender-specific basal expression of P450PCN1(IIIA1) and P450PCN2(IIIA2) are not the same and can be distinguished by their differential response to inhibition of ongoing protein synthesis.     

Regulation of glucose transport in Clone 9 cells by thyroid hormone.

Triiodothyronine (T3) is found to stimulate cytochalasin B-inhibitable glucose transport in Clone 9 cells, a 'non-transformed' rat liver cell line. After an initial lag period of more than 3 h, glucose transport rate is significantly increased at 6 h and reaches more than 3-times the control rate at 24 h. The enhancement of glucose transport by T3 is due to an increase in transport Vmax and occurs in the absence of a change in either the Km for glucose transport (approximately 3 mM) or the Ki for inhibition of transport by cytochalasin B ((1-2).10(-7) M). Consistent with the observed Ki for cytochalasin B, Northern blot analysis of RNA from control and T3-treated cells employing cDNA probes encoding GTs of the human erythrocyte/rat brain/HepG2 cell transporter (GLUT-1), rat muscle/fat cell transporter (GLUT-4), and rat liver transporter (GLUT-2) types indicates expression of only the GLUT-1 mRNA isoform in these cells. The abundance of GLUT-1 mRNA increases approx. 1.9-fold after 24 h of T3 treatment and is accompanied by an approx. 1.3-fold increase in the abundance of GLUT-1 in whole-cell extracts as demonstrated by Western blot analysis employing a polyclonal antibody directed against the 13 amino acid C-terminal peptide of GLUT-1. The more than 3-fold stimulation of glucose transport at 24 h substantially exceeds the fractional increment in transporter abundance suggesting that, in addition to increasing total GLUT-1 abundance, exposure to T3 may result in a translocation of transporters to the plasma membrane or an activation of pre-existing membrane transporter sites.     

Transforming growth factor-beta 1 stimulates glucose uptake and the expression of glucose transporter mRNA in quiescent Swiss mouse 3T3 cells

Transforming growth factor-beta 1 (TGF-beta 1) is a multifunctional polypeptide that regulates the proliferation and differentiation of various types of animal cells. TGF-beta 1 stimulated glucose uptake and the expression of a brain-type glucose transporter (GLUT1) mRNA in quiescent mouse 3T3 cells. TGF-beta 1 also synergistically stimulated these activities when given together with calf serum, phorbol ester, fibroblast growth factor, or epidermal growth factor. The increases in glucose uptake and the GLUT1 mRNA level were induced by picomolar concentrations of TGF-beta 1 within 3 h of stimulation, reached a peak between 6 and 9 h, and then decreased gradually to basal levels before an increase in DNA synthesis. The stimulation of GLUT1 mRNA expression was completely abolished by actinomycin D, but was not affected by cycloheximide, suggesting that new protein synthesis was not required for the expression of GLUT1 mRNA. TGF-beta 1 had little mitogenic activity and did not affect serum-induced DNA synthesis in quiescent 3T3 cells. However, it stimulated DNA synthesis synergistically when given with fibroblast growth factor, epidermal growth factor, phorbol ester, or insulin. These results suggest that TGF-beta 1 mediates the stimulation of glucose uptake, GLUT1 mRNA expression, and DNA synthesis via a pathway(s) and cellular components distinct from those for other growth factors. The possible role of the TGF-beta 1-induced stimulation of glucose transport activity in the control of mouse fibroblast proliferation is also discussed.     

Expression of glucose transporter-2, glucokinase and mitochondrial glycerolphosphate dehydrogenase in pancreatic islets during rat ontogenesis.

To gain better insight into the insulin secretory activity of fetal beta cells in response to glucose, the expression of glucose transporter 2 (GLUT-2), glucokinase and mitochondrial glycerol phosphate dehydrogenase (mGDH) were studied. Expression of GLUT-2 mRNA and protein in pancreatic islets and liver was significantly lower in fetal and suckling rats than in adult rats. The glucokinase content of fetal islets was significantly higher than of suckling and adult rats, and in liver the enzyme appeared for the first time on about day 20 of extrauterine life. The highest content of hexokinase I was found in fetal islets, after which it decreased progressively to the adult values. Glucokinase mRNA was abundantly expressed in the islets of all the experimental groups, whereas in liver it was only present in adults and 20-day-old suckling rats. In fetal islets, GLUT-2 and glucokinase protein and their mRNA increased as a function of increasing glucose concentration, whereas reduced mitochondrial citrate synthase, succinate dehydrogenase and cytochrome c oxidase activities and mGDH expression were observed. These findings, together with those reported by others, may help to explain the decreased insulin secretory activity of fetal beta cells in response to glucose.     

Differential regulation of the expression of transforming growth factor-beta mRNAs by growth factors and retinoic acid in chicken embryo chondrocytes, myocytes, and fibroblasts.

Transforming growth factor-beta (TGF-beta) autoregulates its expression in several mammalian cell types. We now report that addition of TGF-beta s 1, 2, and 3 to primary chicken embryo cells differentially affects expression of the messenger RNAs for the different TGF-beta isoforms depending on the cell type. In cultured sternal chondrocytes, addition of TGF-beta s 1, 2, or 3 results in an increase in the steady-state levels of the messenger RNAs for TGF-beta s 2 and 3, but does not change expression of TGF-beta 4 mRNA. In contrast, in cultured cardiac myocytes, addition of TGF-beta s 1, 2, or 3 results in an increase in expression of TGF-beta s 3 and 4 mRNAs, but does not change expression of TGF-beta 2 mRNA. Moreover, expression of TGF-beta s 2, 3, and 4 mRNAs is not affected by addition of any of the TGF-beta s to fibroblasts. Addition of platelet-derived growth factor (PDGF), epidermal growth factor (EGF), or interleukin-1 (IL-1) to these chicken cells also has differential effects on expression of the different TGF-beta mRNAs depending on the cell type. Retinoic acid also has contrasting effects on chondrocytes and myocytes either increasing or decreasing, respectively, expression of TGF-beta s 2 and 3 mRNAs and TGF-beta 2 protein. Our results indicate a complex pattern of regulation of the different TGF-beta genes by themselves as well as by PDGF, EGF, IL-1, dexamethasone, TPA, and retinoic acid in chicken embryo cells.     

Transforming growth factor-beta down-regulates apolipoprotein M in HepG2 cells

Apolipoprotein M (apoM) is a novel apolipoprotein presented mostly in high-density lipoprotein (HDL) in human plasma, and is exclusively expressed in liver and in kidney. The pathophysiological function of apoM has not yet been elucidated. Apolipoprotein B (apoB), the characteristic apolipoprotein of low-density lipoprotein (LDL), is like apoM, a very hydrophobic protein, and thereafter they both must co-circulate with lipoprotein particles in plasma. The cytokine, transforming growth factor-beta (TGF-beta), has been shown to decreased apoB secretion in HepG2 cells, and we hypothesized that TGF-beta may have the same effects on apoM expression in HepG2 cells. In the present study, we used real-time RT-PCR to analyze apoM and apoB mRNA levels during administration of TGF-beta, as well as TGF-alpha, epidermal growth factor (EGF) and hepatic growth factor (HGF). TGF-beta significantly inhibited both apoM and apoB mRNA expression in HepG2 cells. The inhibitory effects of TGF-beta were dose-dependent, i.e. 1 ng/ml of TGF-beta decreased apoM mRNA levels by 30%, and 10 or 100 ng/ml of TGF-beta decreased apoM mRNA levels more than 65%. The effect of TGF-beta on apoB mRNA expression was slightly weaker than that of apoM, with a maximum effect at 10 or 100 ng/ml TGF-beta where apoB mRNA levels decreased about 55%. The inhibitory effects of TGF-beta on apoM and apoB mRNA levels also increased with increasing incubation time, where the maximum effect was obtained at 24 h. Moreover TGF-alpha, EGF and HGF all decreased both apoM and apoB mRNA levels, but to a less extent than TGF-beta. Further, all four cytokines had more pronounced effects on apoM mRNA expression than apoB mRNA expression. The present study suggested that apoM, like apoB, may be involved in the hepatic lipoprotein assembly in vivo.     

Regulation of glucose transporter messenger RNA levels in rat adipose tissue by insulin

Analysis of glucose transporter mRNA levels in adipose tissue from streptozotocin (STZ)-induced diabetic rats demonstrated a specific decrease (10-fold) in adipose tissue GLUT-4 mRNA with no significant effect on GLUT-1 mRNA levels. Treatment of STZ-diabetic rats with twice daily injections of insulin for 1-3 days resulted in a 16-fold increase in the relative amount of GLUT-4 mRNA to levels approximately 2-fold greater than those in control animals. However, after 7 days of insulin therapy the amount of GLUT-4 mRNA decreased approximately 2-fold back to the levels in the control animals. Normalization of the STZ-induced serum hyperglycemia by phlorizin treatment, which inhibits renal tubular reabsorption of glucose, had no effect on GLUT-4 mRNA in the absence of insulin. Similar to STZ-diabetes, fasting for 48 h also reduced adipose GLUT-4 mRNA levels. Parenteral administration of insulin with glucose over 7.5 h, but not glucose alone, increased the levels of the GLUT-4 mRNA 3- to 4-fold. These studies demonstrate that the relative glycemic state does not influence GLUT-4 glucose transporter mRNA expression in vivo and strongly suggests that insulin is a major factor regulating the levels of GLUT-4 mRNA in adipose tissue.