Methionine-deficient diet induces post-transcriptional downregulation of cystathionine β-synthase

Abstract

Objective: Elevated plasma total homocysteine (tHcy) is a risk factor for a variety of human diseases. Homocysteine is formed from methionine and has two primary metabolic fates: remethylation to form methionine or commitment to the transsulfuration pathway by the action of cystathionine β-synthase (CBS). We have examined the metabolic response in mice of a shift from a methionine-replete to a methionine-free diet.

Methods and results: We found that shifting 3-mo-old C57BL6 mice to a methionine-free diet caused a transient increase in tHcy and an increase in the tHcy/methionine ratio. Because CBS is a key regulator of tHcy, we examined CBS protein levels and found that within 3 d on the methionine-deficient diet, animals had a 50% reduction in the levels of liver CBS protein and enzyme activity. Examination of CBS mRNA and studies of transgenic animals that express CBS from a heterologous promoter indicated that this reduction is occurring post-transcriptionally. Loss of CBS protein was unrelated to intracellular levels of S-adenosylmethionine, a known regulator of CBS activity and stability.

Conclusion: Our results imply that methionine deprivation induces a metabolic state in which methionine is effectively conserved in tissue by shutdown of the transsulfuration pathway by an S-adenosylmethionine-independent mechanism that signals a rapid downregulation of CBS protein.

Figure 1. Effect of M⁻ diet on serum methionine (A), serum tHcy (B), and serum tHcy/methionine ratio (C)

C57BL6 mice were placed on M⁻ diet and serum was collected after 3, 7, 11, and 33 days and methionine and tHcy were measured as described in Methods. Control animals (+Met) were maintained on standard methionine containing diet. Bars show SEM for each group with each group having a minimum of seven animals. Single asterisk indicates distribution is significantly different from control at p<0.05.

Figure 2. Depletion of CBS in response to methionine deprivation

(A) C57BL6 animals were fed M⁻ diet for the indicated number of days. Four mice from each time point were analyzed for liver CBS protein by Western blot. (B) Data from (A) were analyzed by densitometry and normalized to actin. Bars show relative CBS levels and error bars show standard deviation. Asterisk indicates P<0.01 compared to M⁺ (day 0) control mice. (C) Eight C57BL6 mice fed either M⁻ or M⁺ diet for 33 days were analyzed for liver CBS by Western blot. (D) Densitometry of results shown in C. Asterisk indicates P<0.0008.

Figure 3. CBS mRNA quantitation by qRT-PCR

C57BL6 Mouse liver mRNA was isolated from animals fed either a M⁺ diet or an M⁻ diet for the indicated number of days and was then analyzed using TaqMan based qRT-PCR as described in Materials and Methods. Results are shown as relative RNA levels normalized to M⁺ animals. Error bars show the SEM (n =5).

Figure 4. Effect of methionine-free diet on Tg-hCBS Cbs^−/− animals

Eight Tg-hCBS Cbs^−/−animals were placed either on M⁺ or M⁻ diet with zinc water. (A) After seven days serum was analyzed for tHcy and Methionine. Error bars show SEM (n=4). (B) Western blot analysis of livers of same animals after 10 days. The arrow shows the location of human CBS.

Figure 5. Analysis of methionine-related metabolites in the liver of M⁻ diet

C57BL6 animals were placed on M⁻ diet for either 3, 7, or 11 days and free pools of liver amino acids were analyzed. (A) S-adenosylmethionine (SAM) and S-adenosylhomocysteine (SAH) levels were determined as described in Materials and Methods. Error bars show SEM (n=5). (B) Cysteine and methionine in liver lysates. All values are nmoles/mg protein. Error bars show SEM (n=5).

Figure 6. Immunoblot analysis of Tg-S466L expressing mice on M⁺ and M⁻ diet

Mice on either M⁺ or M⁻ diet for two weeks were sacrificed and examined for mouse and human CBS by immunoblot. The transgene status of the mice is indicated above. The S466L human CBS protein runs slightly above the endogenous mouse CBS (mCBS).