Hormonal Regulation of Cystathionine β-Synthase Expression in Liver

Homocysteine metabolism is altered in diabetic patients. Cystathionine β-synthase (CBS), a key enzyme involved in the transsulfuration pathway, which irreversibly converts homocysteine to cysteine, catalyzes the condensation of serine and homocysteine to cystathionine. Studies in streptozotocin-induced diabetic rats have shown that CBS enzyme activity is elevated in the liver but not in the kidney, and this effect is reversed by insulin treatment. To determine whether these effects resulted from alterations at the level of gene transcription, CBS mRNA was measured in diabetic and insulin-treated diabetic rats. CBS mRNA levels were found to be markedly higher in streptozotocin-induced diabetic rat livers; these were reduced by insulin administration. In H4IIE cells, a rat hepatoma cell culture model, glucocorticoids increased the cellular levels of CBS enzyme protein and CBS mRNA; insulin inhibited this stimulatory effect. Treatment with insulin also decreased CBS levels in HepG2 cells, a human hepatoma cell line. Nuclear run-on experiments in the rat cells confirmed that stimulation of CBS gene expression by glucocorticoids and the inhibition by insulin occurred at the transcriptional level. Transient transfections of HepG2 cells with a CBS-1b promoter luciferase reporter construct showed that the promoter activity was decreased by 70% after insulin treatment. These results show that insulin has a direct role in regulating homocysteine metabolism. Altered insulin levels in diseases such as diabetes may influence homocysteine metabolism by regulating the hepatic transsulfuration pathway. Homocysteine metabolism is altered in diabetic patients. Cystathionine β-synthase (CBS), a key enzyme involved in the transsulfuration pathway, which irreversibly converts homocysteine to cysteine, catalyzes the condensation of serine and homocysteine to cystathionine. Studies in streptozotocin-induced diabetic rats have shown that CBS enzyme activity is elevated in the liver but not in the kidney, and this effect is reversed by insulin treatment. To determine whether these effects resulted from alterations at the level of gene transcription, CBS mRNA was measured in diabetic and insulin-treated diabetic rats. CBS mRNA levels were found to be markedly higher in streptozotocin-induced diabetic rat livers; these were reduced by insulin administration. In H4IIE cells, a rat hepatoma cell culture model, glucocorticoids increased the cellular levels of CBS enzyme protein and CBS mRNA; insulin inhibited this stimulatory effect. Treatment with insulin also decreased CBS levels in HepG2 cells, a human hepatoma cell line. Nuclear run-on experiments in the rat cells confirmed that stimulation of CBS gene expression by glucocorticoids and the inhibition by insulin occurred at the transcriptional level. Transient transfections of HepG2 cells with a CBS-1b promoter luciferase reporter construct showed that the promoter activity was decreased by 70% after insulin treatment. These results show that insulin has a direct role in regulating homocysteine metabolism. Altered insulin levels in diseases such as diabetes may influence homocysteine metabolism by regulating the hepatic transsulfuration pathway. cystathionine β-synthase high pressure liquid chromatography phosphoenolpyruvate carboxykinase Cystathionine β-synthase (CBS)1 (EC 4.2.1.22) catalyzes the first committed step in cysteine biosynthesis, the irreversible condensation of homocysteine with serine to form cystathionine (1Finkelstein J.D. Martin J.J. J. Biol. Chem. 1984; 259: 9508-9513Abstract Full Text PDF PubMed Google Scholar). Homocysteine, a sulfur-containing nonprotein amino acid, is an intermediate in the metabolism of methionine. It is at a metabolic crossroads between its synthesis from methionine and its removal through the transsulfuration or remethylation pathways (2Finkelstein J.D Eur. J. Pediatr. 1998; 157 Suppl. 2: 40-44Crossref Google Scholar). Two pyridoxal 5′-phosphate-dependent enzymes comprise the transsulfuration pathway: CBS, which catalyzes the condensation of serine and homocysteine to cystathionine, and cystathionine γ-lyase, which catalyzes the formation of cysteine, α-ketobutyrate, and ammonia (3Krebs H.A. Hems R. Tyler B. Biochem. J. 1986; 158: 341-353Crossref Scopus (162) Google Scholar, 4Kutzbach C. Stokstad E.L. Biochim. Biophys. Acta. 1971; 250: 459-477Crossref PubMed Scopus (275) Google Scholar). The regulation of CBS gene expression is important in a number of physiological situations. Feeding rats a high protein diet or a high methionine diet increases CBS activity (5Yamamoto N. Tanaka T. Noguchi T. J. Nutr. Sci. Vitaminol. (Tokyo). 1995; 41: 197-205Crossref PubMed Scopus (19) Google Scholar). It is also known that flux through the transsulfuration pathway provides cysteine for glutathione synthesis, so that altered CBS levels may be of importance in oxidative stress (6Taoka S. Ohja S. Shan X. Kruger W.D. Banerjee R. J. Biol. Chem. 1998; 273: 25179-25184Abstract Full Text Full Text PDF PubMed Scopus (225) Google Scholar). The well known sparing effect of cysteine on methionine requirements is mediated, at least in part, via alterations in CBS activity. Our own recent work shows that glucagon administration to rats increases hepatic CBS enzyme activity and mRNA levels (7Jacobs R.L. Stead L.M. Brosnan M.E. Brosnan J.T. J. Biol. Chem. 2001; 276: 43740-43747Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). CBS enzyme deficiency, an autosomal recessively inherited disorder, is the leading cause of homocystinuria. Partial deficiency may lead to hyperhomocysteinemia, causing premature peripheral and cerebral occlusive arterial disease (8Brattstrom L. Israelsson B. Lindgarde F. Hultberg B. Metabolism. 1988; 37: 175-178Abstract Full Text PDF PubMed Scopus (212) Google Scholar, 9Boers G.H. Smals A.G. Trijbels F.J. Fowler B. Bakkeren J.A. Schoonderwaldt H.C. Kleijer W.J. Kloppenborg P.W. N. Engl. J. Med. 1985; 313: 709-715Crossref PubMed Scopus (668) Google Scholar). Elevation of plasma homocysteine levels is recognized as an independent risk factor for the development of cardiovascular disease. The plasma concentration of homocysteine is known to be perturbed in diabetes, being increased when renal insufficiency is evident (10Hultberg B. Agardh E. Andersson A. Brattstrom L. Isaksson A. Israelsson B. Agardh C.D. Scand. J. Clin. Lab. Invest. 1991; 5: 277-282Crossref Scopus (145) Google Scholar, 11Hoogeveen E.K. Kostense P.J. Beks P.J. Mackaay A.J. Jakobs C. Bouter L.M. Heine R.J. Stehouwer C.D. Arterioscler. Thromb. Vasc. Biol. 1998; 18: 133-138Crossref PubMed Scopus (347) Google Scholar, 12Hofmann M.A. Kohl B. Zumbach M.S. Borcea V. Bierhaus A. Henkels M. Amiral J. Fiehn W. Ziegler R. Wahl P. Nawroth P.P. Diabetes Care. 1997; 20: 1880-1886Crossref PubMed Scopus (89) Google Scholar, 13Robillon J.F. Canivet B. Candito M. Sadoul J.L. Jullien D. Morand P. Chambon P. Freychet P. Diabetes Metab. 1994; 20: 494-496PubMed Google Scholar). However, patients with insulin-dependent diabetes mellitus with no clinical signs of renal insufficiency, have lower than normal levels of homocysteine (14Bostom A. Brosnan J.T. Hall B. Nadeau M.R. Selhub J. Atherosclerosis. 1995; 116: 59-62Abstract Full Text PDF PubMed Scopus (206) Google Scholar). Our previous work has shown that the plasma homocysteine level was also decreased in the streptozotocin-induced rat diabetic model with a concomitant increase in hepatic CBS enzyme activity; insulin administration reversed these effects (15Jacobs R.L. House J.D. Brosnan M.E. Brosnan J.T. Diabetes. 1998; 47: 1967-1970Crossref PubMed Scopus (171) Google Scholar). Insulin-dependent diabetes mellitus is characterized not only by insufficient circulating levels of insulin but also by elevated levels of counterregulatory hormones such as glucagon and glucocorticoids (15Jacobs R.L. House J.D. Brosnan M.E. Brosnan J.T. Diabetes. 1998; 47: 1967-1970Crossref PubMed Scopus (171) Google Scholar, 16Wilson D.E. Ann. Intern. Med. 1983; 98: 219-227Crossref PubMed Scopus (21) Google Scholar, 17Unger R.H. Diabetes. 1976; 25: 136-151Crossref PubMed Google Scholar). Moreover, cyclic AMP- elevating agents and glucocorticoids have been shown to increase the level of CBS enzyme activity in rat hepatoma cells (18Goss S.J. J. Cell Sci. 1996; 82: 309-320Google Scholar). It is therefore possible that the increase in the hepatic CBS activity in diabetic rat liver may be brought about by decreased insulin and/or by increased levels of counterregulatory hormones. The aim of this study was to examine the role of insulin and counterregulatory hormones on the expression of the CBS gene, using a diabetic rat liver model as well as rat and human hepatoma cell lines. All procedures were approved by Memorial University's Institutional Animal Care Committee and were in accordance with the guidelines of the Canadian Council on Animal Care. Male Sprague-Dawley rats weighing 250–350 g were fed laboratory chowad libitum and had free access to water. Diabetes was induced by a single injection of streptozotocin (100 mg/kg body weight; freshly dissolved in 10 mm citrate buffer (pH 4.5)) through the tail vein, under light ether anesthesia; an equal volume of citrate buffer was administered to control rats. The streptozotocin-diabetic rats were treated subcutaneously with insulin (Novolin Ultralente human insulin; Lilly) for 5 days to allow the animals to recover from any nonspecific effects of streptozotocin. Thereafter, insulin was withdrawn, and the rats received saline injections for up to 5 days. At this point, insulin was readministered to untreated-diabetic rats for up to 5 days. Control rats received saline throughout the experiment. The insulin dose (∼40 units/day) was adjusted to maintain blood glucose close to normal values as measured with an Ames Glucometer II, using a drop of blood obtained by tail prick. On the day of the study, animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (65 mg/kg). Blood samples were taken from the abdominal aorta into heparinized tubes and placed on ice for a few minutes until they were centrifuged at 2700 × g for 15 min for plasma separation. The liver was rapidly removed, freeze-clamped in liquid nitrogen, and stored at −70 °C until used. Rat hepatoma H4IIE cells and human hepatoma HepG2 cells were obtained from the American Tissue Culture Collection (Manassas, VA). The CBS-negative a23 Chinese hamster Don fibroblast cell line has been described previously (19Skovby F. Krassikoff N. Francke U. Hum. Genet. 1984; 65: 291-294Crossref PubMed Scopus (57) Google Scholar). H4IIE cells were grown in 75-cm2 culture flasks in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, penicillin (100 units/ml), and streptomycin (100 HepG2 cells and a23 Chinese hamster Don fibroblast cells were in modified Dulbecco's medium with fetal bovine penicillin (100 units/ml), and streptomycin (100 were grown to under in a at H4IIE cells were by culture in medium containing fetal bovine the of cells were with medium containing fetal bovine for The medium was with and/or insulin At the of the were and cells were with Cell was by Culture of Animal Scholar). CBS enzyme activity in rat liver and in H4IIE cells was using the described by Nadeau M.R. J. D. Selhub J. Biochem. J. 1994; PubMed Scopus Google in the of mm a of the The of cystathionine was measured by J. 1983; PubMed Scopus Google Scholar). Homocysteine were using by the of and B. Eur. J. Clin. Chem. Clin. Biochem. 1991; Google Scholar). plasma homocysteine free and glucose was E. F. of Scholar). concentration in the liver samples was by the and by the of R.J. J. Biol. Chem. Full Text PDF PubMed Google in cells, using bovine as CBS enzyme activity in HepG2 cells was measured to the described by PubMed Scopus Google Scholar). H4IIE cells were in buffer mm 10% mm and the and for to and concentration of the cell was by the P. R. A. F. M. E. N. B. and D. Biochem. using bovine as a of protein was by and to The were with in containing with in and with to in The were and by the to a were by using HepG2 cell samples were on a and using a CBS-negative a23 Chinese Don fibroblast cell was as a The were with in saline containing CBS was using in bovine in and with to The was to in in and The was using the and with was by a as previously described P. N. Chem. Scholar). was in buffer at °C for 15 on and to was by The were and with a for rat CBS of rat CBS M. T. T. J. Biol. Chem. Full Text PDF PubMed Google which was with using the to the The were and with a to control for equal of and at high at was by of the to or were on and levels were with was from H4IIE cells after with hormones as of was using a and by and a to the of rat CBS P. N. Chem. were to a of the rat was using were on were by The was in for in buffer for with in buffer with in and to and the was by in a The were CBS and was using or The and of the CBS-1b promoter luciferase reporter construct have been described previously J. J. E. C. R. L. D. V. W. V. 1998; PubMed Scopus Google Scholar). The control luciferase reporter control containing no and were obtained from was using to the for cells and of the construct and of the well in a culture cells were in direct cell buffer and were for and luciferase using a luciferase and a to the All promoter values were for by the luciferase of the CBS-1b promoter construct to the luciferase values from the control All values as of the to the control reporter the results shown the of at least independent experiments in with the Nuclear run-on were to a previously described M.E. T. R. J.A. in Scholar). H4IIE cells were grown in for were from 5 × Nuclear were with mm of and and with of The was after and by and was by of were at °C for to previously with 5 of The were containing a for rat liver CBS, Tissue Culture and the were to were by were to of the on plasma homocysteine and glucose levels in diabetic rats. glucose levels were markedly higher in to normal levels on insulin treatment. The animals were also blood in diabetic rats was and the of and was was by insulin treatment. shows that plasma homocysteine level in the diabetic rats was only about of that found in control rats. Treatment of diabetic rats with insulin increased plasma homocysteine levels day of but not to the control level after 5 days of treatment. To examine whether the of homocysteine levels in the diabetic rat be to an increase in CBS enzyme activity by an increase in homocysteine through the transsulfuration pathway, measured the activity of this enzyme in from and insulin-treated diabetic rats. CBS activity was elevated in the diabetic this was to control values by insulin To determine whether this in CBS enzyme activity is at the level of transcription, measured CBS mRNA in treated and diabetic rat CBS mRNA levels were higher in diabetic rat with control rat and this was reversed by insulin for the level of CBS mRNA was in diabetic rat decreased the level of CBS mRNA to control values a day of diabetes and insulin on plasma homocysteine and plasma glucose in day days 5 days as with of in a as with of Insulin-dependent diabetes mellitus is not only by a in insulin levels but also by an increase in the circulating levels of the counterregulatory glucocorticoids and glucagon D.E. Ann. Intern. Med. 1983; 98: 219-227Crossref PubMed Scopus (21) Google Scholar, 17Unger R.H. Diabetes. 1976; 25: 136-151Crossref PubMed Google Scholar). To whether insulin and its hormones a direct and effect on liver cells, hepatoma cell lines. The CBS enzyme activity is in H4IIE cells However, CBS activity was elevated in these cells when with the CBS activity in H4IIE cells, with the an increase in CBS enzyme which to normal levels when to insulin To examine whether this increase in enzyme activity in to was to an increased of CBS cell were by increase in the level of CBS protein was evident in H4IIE cells, and this was on the concentration of shows the increase in CBS enzyme protein expression in to of The CBS protein was evident in cells with of increased in a and a at a concentration of H4IIE cells were therefore by a concentration of (100 in the of of insulin to shows that this CBS expression was markedly inhibited by insulin at a concentration of 10 and inhibition of CBS expression was at a concentration of To examine the effects of and insulin on CBS gene was from and control H4IIE was and as described under were by showed the CBS and Control cells only levels of CBS To that the on were CBS and were and with CBS and and showed that was a increase in CBS mRNA levels when H4IIE cells were with for stimulation of mRNA expression by glucocorticoids was inhibited by insulin of these cells by resulted in a increase in CBS mRNA about However, no effect was when cells were by a of and by glucagon showed no effect on CBS mRNA Our results show that and insulin effects on CBS enzyme protein concentration and mRNA levels in H4IIE known to by the of of M. Full Text PDF PubMed Scopus Google Scholar). To determine whether glucocorticoids and insulin direct control the of the CBS gene, run-on were on from H4IIE cells that had been with in the and of insulin for show that the were increased in cells that had been with to the was an increase in the CBS after reversed this that the effect of hormones is at the level of and of hormones on CBS in H4IIE from and insulin-treated cells were and for run-on of CBS, and when to the from independent whether the effects of hormones on CBS enzyme activity and expression in cells be in a human cell was a expression of CBS in HepG2 these cells were to insulin CBS enzyme activity was decreased by about Treatment with the counterregulatory had no effect on CBS enzyme activity in these cells These results were to obtained from the rat showed that this in CBS activity is by decreased levels of CBS protein in insulin-treated cells with was no in the level of CBS protein in cells 5 To examine whether insulin its effect by the promoter that CBS gene transcription, HepG2 cells were with the CBS-1b promoter reporter construct to a luciferase reporter gene J. J. E. C. R. L. D. V. W. V. 1998; PubMed Scopus Google Scholar). Treatment of the cells with insulin for resulted in a in luciferase activity 5 that the decreased CBS activity brought about by insulin is through on the CBS gene by glucagon had no effect on CBS promoter activity. It is that homocysteine metabolism is in diabetic patients (15Jacobs R.L. House J.D. Brosnan M.E. Brosnan J.T. Diabetes. 1998; 47: 1967-1970Crossref PubMed Scopus (171) Google Scholar, L. Nutr. Metab. 2001; Google Scholar). with have increased plasma homocysteine levels (10Hultberg B. Agardh E. Andersson A. Brattstrom L. Isaksson A. Israelsson B. Agardh C.D. Scand. J. Clin. Lab. Invest. 1991; 5: 277-282Crossref Scopus (145) Google Scholar, 12Hofmann M.A. Kohl B. Zumbach M.S. Borcea V. Bierhaus A. Henkels M. Amiral J. Fiehn W. Ziegler R. Wahl P. Nawroth P.P. Diabetes Care. 1997; 20: 1880-1886Crossref PubMed Scopus (89) Google Scholar, 13Robillon J.F. Canivet B. Candito M. Sadoul J.L. Jullien D. Morand P. Chambon P. Freychet P. Diabetes Metab. 1994; 20: 494-496PubMed Google Scholar). may be to the that the is an important in the removal of homocysteine L. Nutr. Metab. 2001; Google Scholar, J.D. Brosnan M.E. Brosnan J.T. Biochem. J. 1997; PubMed Scopus Google Scholar). In diabetic patients with normal levels of have lower plasma homocysteine levels J.F. Canivet B. Candito M. Sadoul J.L. Jullien D. Morand P. Chambon P. Freychet P. Diabetes Metab. 1994; 20: 494-496PubMed Google Scholar). The study shows that plasma homocysteine levels decreased in the streptozotocin-induced diabetic rat rats were diabetic as by elevated plasma glucose which were well in the insulin-treated diabetic have previously shown that plasma levels were not elevated in diabetic rats (15Jacobs R.L. House J.D. Brosnan M.E. Brosnan J.T. Diabetes. 1998; 47: 1967-1970Crossref PubMed Scopus (171) Google Scholar). the in plasma homocysteine levels were not to in renal Our show that in diabetic rats is an increased CBS enzyme activity by a concomitant increase in CBS mRNA which be reversed by insulin treatment. the decreased homocysteine levels in the diabetic model with an increase in CBS a key enzyme involved in the of in is by an increase in CBS gene that the hepatic transsulfuration pathway is by insulin at the level of flux through the hepatic transsulfuration pathway is also by work from this which that the hepatic of cysteine is increased in diabetic the level of methionine is J.T. Hall D.E. Brosnan M.E. J. 1983; Google Scholar). enzyme is also which is a of CBS J.D. Martin J.L. Biochem. Biophys. PubMed Scopus Google Scholar). However, have measured the levels of this in freeze-clamped and have found no in streptozotocin-diabetic rats with or insulin diabetic CBS enzyme activity is known to an important role in plasma homocysteine The human CBS gene has been to and is in the of patients M. T. Francke U. J. Hum. Genet. 1988; Google Scholar, M. S. P. S.J. J. Hum. Genet. 2001; Full Text Full Text PDF PubMed Scopus (212) Google Scholar). These patients have lower than normal plasma homocysteine B. M. M. M. P. D. 1988; 2: PubMed Scopus Google Scholar). CBS have also been M. J. R. M.R. N. Sci. U. S. A. 1995; PubMed Scopus Google Scholar). have a in CBS expression and the normal homocysteine levels B. M. M. M. P. D. 1988; 2: PubMed Scopus Google Scholar). also in S. N. M.R. J. PubMed Google Scholar, M.A. A. J.A. M.A. M. S. R. M. P.J. R. J. J. Clin. Invest. PubMed Scopus Google Scholar). work from N. S. C. J. Sci. U. S. A. 2001; 98: PubMed Scopus Google Scholar, N. S. C. A. J. Arterioscler. Thromb. Vasc. Biol. PubMed Scopus Google has that the effects of homocysteine at least by oxidative of showed that the in be by the of glutathione or by increased cellular N. S. C. J. Sci. U. S. A. 2001; 98: PubMed Scopus Google Scholar, N. S. C. A. J. Arterioscler. Thromb. Vasc. Biol. PubMed Scopus Google Scholar). CBS expression an important role in plasma homocysteine levels and is confirmed in study, diabetes increases CBS insulin and of these in plasma homocysteine Our that the effects of glucocorticoids and insulin on CBS expression at the transcriptional level. in enzyme activity by in the levels of CBS protein and mRNA in H4IIE cells, run-on showed that stimulation of CBS expression by glucocorticoids and inhibition by insulin occurred at the level of In inhibited the of CBS mRNA in H4IIE cells not of a CBS-1b construct into HepG2 cells showed that insulin the promoter activity. the in CBS enzyme activity and the of CBS a direct regulation of the promoter activity by The expression of CBS is in rat liver cells (15Jacobs R.L. House J.D. Brosnan M.E. Brosnan J.T. Diabetes. 1998; 47: 1967-1970Crossref PubMed Scopus (171) Google but in H4IIE cells was In these cells, CBS expression was markedly by a has shown that the level of CBS enzyme activity in and cells hepatoma was elevated when with a of and cyclic (18Goss S.J. J. Cell Sci. 1996; 82: 309-320Google Scholar). However, the control of CBS gene expression was not Our results that CBS enzyme protein and mRNA be induced by in H4IIE of H4IIE and HepG2 cells by glucagon had no effect on CBS gene in cells was a but stimulation of CBS by a To this is the first that shows that insulin a direct effect on the CBS an important role in the control of hepatic enzymes of enzymes involved in amino metabolism. phosphoenolpyruvate carboxykinase and enzymes of the E. W. M. D. B. Eur. J. Biochem. PubMed Scopus Google Scholar, 1988; 2: PubMed Scopus Google Scholar). of these is by glucocorticoids and and these by insulin Biochem. J. 1991; PubMed Scopus Google Scholar, J. Biol. Chem. 1984; 259: Full Text PDF PubMed Google Scholar, Sci. U. S. A. PubMed Scopus Google Scholar). of expression with that of CBS is of In hepatic is induced in diabetes and by insulin treatment. In rat liver cells, and CBS by the effect on is The well characterized insulin the so on the of and is also on the human CBS gene promoter at to with to the of the CBS J. J. E. C. R. L. D. V. W. V. 1998; PubMed Scopus Google Scholar, Biochem. 2001; PubMed Scopus Google Scholar). also found on the of rat CBS E. J. M. 1996; PubMed Scopus Google Scholar). However, this is from expression is by insulin have that the regulation of CBS is to recent by have shown that the gene is by the and M. E. V. R.H. J. PubMed Scopus Google Scholar, J. Biol. Chem. 2001; 276: Full Text Full Text PDF PubMed Scopus Google Scholar). has been to a role in the regulation of CBS J. of Homocysteine Homocysteine and a number of have been described insulin and glucocorticoids the expression of B. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, Noguchi T. N. Biochem. Biophys. 2001; PubMed Scopus (19) Google Scholar). work is to the by which insulin the expression of

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