Homocysteine Metabolism in ZDF (Type 2) Diabetic Rats

All procedures were approved by Memorial University’s Institutional Animal Care Committee. Male ZDF rats (ZDF/Gmi fa/fa) aged 5 and 11 weeks and male lean rats (ZDF/Gmi fa/?) of the same ages were obtained from Charles River Laboratories, Indianapolis, Indiana. They were fed Purina 5008 chow ad libitum and had free access to water. On the day of the experiment, animals were anesthetized with an intraperitoneal injection of sodium pentobarbital (65 mg/kg), and blood was collected from the abdominal aorta into heparinized syringes. Before removal of the syringe, a piece of the liver was rapidly removed and freeze-clamped with aluminum tongs precooled in liquid nitrogen. Another portion was removed for immediate enzyme assays. The freeze-clamped liver was stored at −70°C until further use. The blood was centrifuged at 3,700g for 15 min, and the plasma was kept at −20°C until further use.

Plasma tHcy was measured as described (11). Plasma glucose was determined enzymatically (12). Plasma creatinine was measured using a kit (catalog no. 555A) from Sigma Diagnostics (St. Louis, MO). Plasma insulin was measured with a rat insulin enzyme-linked immunosorbent assay kit (catalog no. INSKR020) from Crystal Chem (Chicago, IL) using anti-rat/mouse insulin antibody and a rat insulin standard.

Fresh liver samples were diluted 1:5 with 50 mmol/l K phosphate buffer (pH 7.0) and homogenized with a Polytron (Brinkman Instruments, Toronto, Canada) for 20 s at 50% output. The homogenates were centrifuged at 20,000g for 30 min at 4°C, and the supernatant was removed and used immediately to measure the activities of five of the enzymes of methionine metabolism. Methionine adenosyltransferase (MAT) (13,14), CBS (13,15), cystathionine γ-lyase (CGL) (16), methionine synthase (17), and BHMT (18) were assayed using previously described methods. Methylenetetrahydrofolate reductase (MTHFR) (19) and glycine N-methyltransferase (GNMT) (20) were measured using previously frozen liver tissue. Choline dehydrogenase was measured using liver homogenates (21,22). Protein concentrations were determined by the Biuret method using BSA as a standard. All enzyme assays were determined to be linear with time and with protein.

Choline and its metabolites were extracted from tissue and plasma by the method of Bligh and Dyer (23). Aqueous and organic compounds were separated, analyzed, and quantified directly by high-performance liquid chromatography–mass spectrometry (liquid chromatography–electrospray ionization–isotope dilution mass spectrometry) after the addition of internal standards labeled with stable isotopes that were used to correct for recovery (24). The levels of SAM and S-adenosylhomocysteine (SAH) were measured in freeze-clamped liver tissue (25).

Total RNA was isolated from the fresh liver samples as previously described (26). Two micrograms of RNA were reverse transcribed using a one-step reverse transcription kit (Qiagen, Mississauga, Canada) and was amplified by 27 cycles. An upstream primer (5′-GCCTTCAGGACATCCAGTGT-3′) and a downstream primer (5′-TCTTTCCGGGTCTGCTCACGGGC-3′) corresponding to the type 3 rat CBS cDNA (27) were used to amplify a 1,644-bp PCR fragment. For BHMT, an upstream primer (5′-ACCGATTGCCGGCAAGAA-3′) and a downstream primer (5′-CTGTGCGGATTTGAATTTTTG-3′) corresponding to the rat BHMT cDNA (NM-030850) were used to amplify a 1,216-bp PCR fragment. A 768-bp fragment of the rat β-actin was coamplified using amplimer set primers (CLONTECH, Palo Alto, CA). Once amplified, the PCR products were separated on 0.8% agarose. The bands were stained with ethidium bromide and were visualized under UV illumination.

Hepatocytes were isolated from ZDF fa/fa and ZDF fa/? rats (28). Approximately 5 mg of hepatocytes (dry weight) were preincubated for 20 min at 37°C with Krebs-Henseleit saline with or without 0.3 mmol/l betaine. At the end of the preincubation l-[1-¹⁴C] methionine was added to a final concentration of 0.1 mmol/l and the incubation continued for a further 30 min. The total transsulfuration flux was determined as the sum of the ¹⁴C present in α-ketobutyrate and in CO₂ as previously described (29). In a separate series of experiments, hepatocytes were incubated with 0.1 mmol/l methionine ± 0.3 mmol/l betaine to measure Hcy export (29).

Data are presented as means ± SD. Means were compared using Student’s unpaired t test, paired t test, or one-way ANOVA as appropriate. P < 0.05 was taken to indicate a significant difference.

The body weight, hepatosomatic index, plasma insulin, creatinine, and glucose concentrations of these rats have been reported (30). At 5 weeks, the concentrations of plasma glucose (12.1 ± 1.7 vs. 8.4 ± 1.3 mmol/l) and insulin (1.5 ± 0.5 vs. 0.14 ± 0.07 nmol/l) in the ZDF fa/fa and ZDF fa/? rats, respectively, showed that the ZDF fa/fa rats had become insulin resistant. At 11 weeks, the concentrations of plasma glucose (30.3 ± 1.6 vs. 10.9 ± 0.8 mmol/l) and insulin (0.61 ± 0.14 vs. 0.68 ± 0.1 nmol/l) in the ZDF fa/fa and the ZDF fa/? rats, respectively, showed that the ZDF fa/fa rats had become diabetic. Creatinine levels of ZDF fa/fa and control rats at 5 weeks were similar (32.4 ± 3.8 vs. 28.9 ± 3.1 μmol/l), but at 11 weeks were lower in the ZDF fa/fa rats (18.9 ± 2.7 vs. 29.7 ± 6.1 μmol/l)

At 5 weeks, plasma tHcy was about 25% lower in the ZDF fa/fa rats, while at 11 weeks it was reduced by about 60% (Table 1). The plasma tHcy level in the ZDF fa/fa rats at 11 weeks was further reduced from its value at 5 weeks, whereas the ZDF fa/? rats showed an increase in the plasma tHcy level with age. The reduced plasma tHcy at 5 weeks in the ZDF fa/fa rats was accompanied by changes in several enzymes that metabolize methionine (Table 1). Of the enzymes responsible for producing Hcy, MAT and GNMT showed slight but significant increases. CBS, CGL, BHMT, and MTHFR were increased, respectively, by about 79, 35, 80, and 22%. Methionine synthase showed a slight but significant decrease in the ZDF fa/fa rats. At 11 weeks, MAT, GNMT, CBS, CGL, and BHMT all showed significant increases in activity as did choline dehydrogenase.

In addition to the differences in enzyme activity between the control and the experimental animals at each age-group, MAT, CBS, and GNMT all showed significant increases in their activity with age in the ZDF fa/? rats.

At 5 weeks, the increased activity of CBS in the ZDF fa/fa rats was accompanied by a comparable increase in CBS mRNA level (Fig. 2A). The increased BHMT activity observed in the ZDF fa/fa rats at 5 and 11 weeks was also accompanied by comparable increases in mRNA levels (Fig. 2B).

The hepatic levels of SAM and SAH and the SAM-to-SAH ratio are shown in Table 2. At both 5 and 11 weeks, SAM was significantly increased in the ZDF fa/fa rats. The SAM-to-SAH ratio was significantly higher in the ZDF fa/fa rats at 5 weeks. Both SAM and the SAM-to-SAH ratio showed increases with age in ZDF fa/? rats, consistent with the age-related increase in MAT activity.

In a preliminary study, we investigated the effect of betaine on the transsulfuration flux, as measured by the conversion of l-[1-¹⁴C] methionine to ¹⁴CO₂ and ¹⁴C-α-ketobutyrate by isolated hepatocytes from Sprague Dawley rats. All concentrations of betaine used (0.1, 0.3, or 1 mmol/l) significantly reduced the catabolism of methionine, compared with incubations of hepatocytes with methionine alone. We then conducted studies with hepatocytes from ZDF rats using 0.1 mmol/l l-[1-¹⁴C] methionine and 0.3 mmol/l betaine. Incubation with 0.3 mmol/l betaine significantly decreased the transsulfuration flux in hepatocytes of ZDF fa/fa rats at both ages, but the reduction seen in the hepatocytes of ZDF fa/? rats was significant only at 11 weeks of age (Table 3).

Hepatocytes from ZDF fa/fa and ZDF fa/? rats, at both ages, showed a significant reduction in Hcy export when incubated with 0.3 mmol/l betaine (Table 4). However, at both ages the decrease observed in the ZDF fa/fa rats was about twofold greater than that of the ZDF fa/? rats.

Table 5 shows the liver concentrations of choline and related metabolites. The liver betaine concentration was lower in the ZDF fa/fa rats at both 5 and 11 weeks, while there was no difference in the choline concentration. One of the most remarkable findings was the high level of betaine found in the liver of the young animals and the reduction that occurs with age. Hepatic glycerophosphorylcholine was significantly higher in the ZDF fa/fa rats at 5 weeks, though at 11 weeks it was significantly lower. Phosphorylcholine was significantly higher in the ZDF fa/fa rats at both ages. Both glycerophosphorylcholine and phosphorylcholine levels increased with age in the ZDF fa/? rats. Phosphatidylcholine was significantly decreased in ZDF fa/fa rats at 11 weeks, while sphingomyelin was significantly reduced at both 5 and 11 weeks.

Table 6 shows choline and related metabolite concentrations in the plasma. The only significant difference in plasma metabolites between the different rats was an increased phosphatidylcholine level in the ZDF fa/fa rats at 11 weeks. However, the plasma betaine concentration decreased with age in both groups of rats while the concentration of phosphatidylcholine increased in the ZDF fa/fa rats from 5 to 11 weeks.

This study describes the altered metabolism of methionine and Hcy that occurs in insulin resistance and in early type 2 diabetes. It is clear that the hormonal changes that are prevalent in these two disease states act at several sites in the pathway of methionine metabolism. Hyperphagia in the diabetic rats may also contribute to these metabolic changes. Indeed, we observed a twofold increase in food intake per 100 g body weight at the diabetic stage. However, there was no difference at 5 weeks. The increased hepatic CBS and BHMT activity at 5 weeks cannot, therefore, be attributed to increased protein intake. In addition, we have shown a direct effect of insulin in repressing CBS expression in both human and rat cultured hepatocytes (42). Our finding of lowered plasma tHcy in the 5-week ZDF fa/fa rats shows that insulin resistance alone can decrease plasma tHcy levels.

The most striking finding in the present study concerns the role of betaine and BHMT in determining plasma tHcy. Not only was BHMT activity and mRNA levels elevated but the hepatic betaine concentration was remarkably reduced. Furthermore, this increased BHMT was clearly effective in decreasing hepatic Hcy output. This is evident from the data showing that addition of betaine to hepatocytes from ZDF fa/fa rats virtually eliminated Hcy export from cells compared with a lesser effect in hepatocytes from ZDF fa/? rats (Table 4). Increased remethylation of Hcy by BHMT also reduces the Hcy available for flux through the transsulfuration pathway, which decreased in these cells (Table 3). Although the importance of the BHMT-catalyzed remethylation reaction in determining Hcy levels has not received as much attention as the folate-dependent remethylation pathway, it should be recalled that Schwahn et al. (31) found a highly significant negative correlation between plasma betaine and plasma tHcy concentrations in humans. Supplementation with betaine was shown to lower plasma tHcy in humans (32) and in MTHFR +/+, +/−, and −/− mice (31). Because of the elevated BHMT activity, betaine may be particularly efficacious in reducing plasma tHcy in the later stages of type 2 diabetes when renal complications bring about hyperhomocysteinemia.

Our data suggest that there is enhanced utilization of betaine in the ZDF fa/fa rat. The observed decrease in hepatic phosphatidylcholine concentration (Table 5) is consistent with enhanced utilization of choline to form betaine in the livers of these rats. Sphingomyelin, which is derived from phosphatidylcholine, is also reduced in fa/fa liver. The increase in phosphorylcholine concentrations may reflect increased activity of the pathway for phosphatidylcholine biosynthesis that occurs when tissue phosphatidylcholine concentrations fall. The significant increase in plasma phosphatidylcholine concentrations in ZDF fa/fa rats at 11 weeks (Table 6) probably reflects increased secretion of lipoproteins from liver.

Another remarkable feature of betaine metabolism is the extraordinarily high hepatic betaine concentration found in young rats. In the control animals (ZDF fa/?), the hepatic betaine level was 17.6 μmol/g at 5 weeks; this decreased to 5.3 μmol/g at 11 weeks. Assuming that hepatic betaine is evenly distributed in intracellular water and that rat liver contains 0.45 ml of cell water per gram (35), we can calculate a betaine concentration in the 5-week-old control animals of 39 mmol/l. This should be compared with total osmolarity of about 305–310 mOs mmol/l in mammalian tissues (except for the renal medulla).

Davies et al. (36) have reported a very large urinary excretion of betaine in normal neonates and in young rats after weaning. High betaine concentrations are found in mammalian kidneys, particularly in the renal medulla. Renal betaine has been characterized as a compatible osmolyte (together with other trimethylamines and polyols) where it can offset the high extracellular osmolarity that arises as a result of the urinary concentrating mechanisms. In addition, these osmolytes may protect these cells from adverse effects of urea, which is present at high concentrations (37).

Wettstein et al. (38) have showed that betaine plays a role in cell volume homeostasis in perfused livers of adult rats. Whether this is also the function of the high betaine concentrations in neonatal liver is a focus of our current work. We must also comment on the ratio of hepatocellular to plasma betaine. Plasma betaine was fairly constant, in the 90–170 μmol/l range, in all of the rats (Table 6). The liver-to-plasma concentration ratios for betaine were about 230, 130, 100, and 60, respectively, for ZDF fa/? and ZDF fa/fa rats at 5 and 11 weeks of age. How such gradients are produced and maintained are important issues for future work.

Diabetes is characterized not only by the lack of insulin or the resistance to the action of insulin, but also by increases in the counter-regulatory hormones glucocorticoid and glucagon (39,40). Hepatic MAT activity has been shown to be increased by glucocorticoid administration (41). Finkelstein et al. (33) examined the effects of administration of a number of hormones on BHMT activity. The principal findings were that hydrocortisone increased BHMT activity, whereas thyroxin decreased it. This group also demonstrated increased activity in alloxan diabetic rats. This latter finding has been confirmed by Nieman et al. (34) in streptozotocin-induced diabetic rats. We are currently exploring the possibility that insulin may regulate BHMT expression. Insulin treatment of streptozotocin-induced diabetic rats was shown to restore the increased activity of CBS to normal levels (9) by acting at the level of CBS gene transcription to repress CBS promoter activity (42). Cyclic AMP, the intracellular messenger of glucagon, and glucocorticoids increase CBS expression in rat hepatoma cells (43). The increased activity of CBS, brought about both by allosteric activation by the increased hepatic SAM levels (44) and by increased expression, does not imply an increased transsulfuration flux. This flux is determined by the rate at which methionine enters into hepatic metabolism, which will be largely determined by dietary methionine consumption. It seems that the major effect of increased CBS activity is to decrease the steady-state Hcy concentration at which transsulfuration occurs (45). In this way it contributes to the decreased plasma tHcy concentration. Similarly, the increased activity of BHMT does not necessarily imply an increased remethylation flux in vivo but, rather, it decreases the steady-state Hcy concentration at which remethylation via BHMT occurs.

It is also possible that alterations in renal function may contribute, somewhat, to the decreased plasma tHcy, at least when the rats are diabetic. The kidney is a major site for the removal and subsequent metabolism of Hcy (46). Hyperfiltration is a characteristic sign of renal dysfunction in early diabetes (47). Such hyperfiltration has been reported to occur in ZDF fa/fa rats from about 7 weeks of age and to continue until they are about 3 months old (48). Such an occurrence probably accounts for the decreased plasma creatinine seen at 11 weeks. The delivery of an increased quantity of Hcy to the kidney via filtration could result in increased renal catabolism of Hcy and contribute to its reduced plasma concentration. However, this cannot contribute to the decreased Hcy at the insulin-resistant stage where creatinine levels were unchanged.

It is now clear that plasma tHcy levels decrease in both type 1 and type 2 diabetes when there is no renal damage. The present work showed that this is true even at the prediabetic, insulin-resistant stage. However, literature reports on the phenomenon show a variety of responses of Hcy to insulin resistance (5,49,50). This variability is likely to result from different degrees of insulin resistance as well as other factors such as impaired renal function. Our results, which at 5 weeks were obtained in an animal model in which there was no evidence of impaired renal function as well as a consistent degree of insulin resistance, agree remarkably well with Rosolova’s study of the relationship between Hcy and insulin resistance in healthy human subjects (5).

In summary, this study shows a decreased plasma tHcy level in both insulin-resistant and type 2 diabetic rats. We show increased activities of BHMT and CBS in these states, as well as increased mRNA levels for these enzymes. We also show increased hepatic SAM levels, which will activate CBS. We report experiments in hepatocytes that directly demonstrate the role of betaine metabolism in enhanced Hcy removal. These results emphasize the importance of BHMT in regulating Hcy metabolism.

This work was supported by a grant from the Canadian Diabetes Association in honor of the late Neil M. Miller (M.E.B. and J.T.B.) and by a grant from the National Institutes of Health (DK55865 to S.H.Z.). E.P.W. received a graduate fellowship from the School of Graduate Studies, Memorial University of Newfoundland. J.T.B. is a Senior Investigator of the Canadian Institutes of Health Research.