Impaired bile acid metabolism with defectives of mitochondrial-tRNA taurine modification and bile acid taurine conjugation in the taurine depleted cats

Abstract

Taurine that conjugates with bile acid (BA) and mitochondrial-tRNA (mt-tRNA) is a conditional essential amino acid in humans, similarly to cats. To better understand the influence of acquired depletion of taurine on BA metabolism, the profiling of BAs and its intermediates, BA metabolism-enzyme expression, and taurine modified mt-tRNAs were evaluated in the taurine deficient diet-supplemented cats. In the taurine depleted cats, taurine-conjugated bile acids in bile and taurine-modified mt-tRNA in liver were significantly decreased, whereas unconjugated BA in serum was markedly increased. Impaired bile acid metabolism in the liver was induced accompanied with the decreases of mitochondrial cholesterol 27-hydroxylase expression and mitochondrial activity. Consequently, total bile acid concentration in bile was significantly decreased by the low activity of mitochondrial bile acid synthesis. These results implied that the insufficient dietary taurine intake causes impaired bile acid metabolism, and in turn, a risk for the various diseases similar to the mitochondrial diseases would be enhanced.

Figure 1

Taurine concentration in serum and liver, and histological observations of hepatic tissue. (A) Progress of serum taurine concentration during the experimental diet feeding for 30 weeks. (B) Taurine and glycine concentrations in the liver after the 30-week feeding. (C) H&E stain of hepatic tissue; (Ca & Cb) Overall hepatic lobule (objective × 4) and the portal region (×10) images in the Control group. (Cc & Cd) Overall hepatic lobule (×4) and the portal region (×10) images in the Depletion group. C and P in Fig. 1Cb and Cd show the central vain and portal vain, respectively. Data are the mean ± SE. *P < 0.05, **P < 0.01, ^†P < 0.001 versus the Control group at each point by unpaired Student t-test.

Figure 2

Bile acid profiles in bile. (A) Proportion of unconjugated and taurine/glycine-conjugated forms of BAs (the sum of all BA types). (B) TBA concentration. (C) Concentrations of the respective types of BAs (the sum of unconjugated and conjugated forms). (D) Proportion of BA types (the sum of unconjugated and conjugated forms). (E) Ratios of the primary BAs to the secondary BAs (1^st/2^nd BA) and the CA-derived BAs to the CDCA-derived BAs (CA⁺/CDCA⁺) are presented. Data are the mean ± SE.*P < 0.05, **P < 0.01, ^‡P < 0.0001 versus the Control group by unpaired Student t-test. Abbreviations: BA; bile acid, CA, cholic acid; CDCA, chenodeoxycholic acid, Control; the taurine-contained diet supplemented group; DCA, deoxycholic acid, Depletion; the taurine-deficient diet supplemented group, Gly-conjugated; the sum of glycine conjugated bile acids, LCA, lithocholic acid; Tau-conjugated; the sum of taurine conjugated bile acids, TBA; total bile acid, UDCA, ursodeoxycholic acid, Unconjugated; the sum of unconjugated bile acids.

Figure 3

Bile acid profiles in serum. (A) Proportion of unconjugated and taurine/glycine-conjugated forms of BAs. (B) TBA concentration. (C) Concentrations of the respective types of BAs. (D) Proportion of BA types. (E) Ratios of 1^st/2^nd BA and CA⁺/CDCA⁺. Data are the mean ± SE. *P < 0.05, **P < 0.01 versus the Control group by unpaired Student t-test. Abbreviations: See the legend of Fig. 2 for abbreviations.

Figure 4

Free-cholesterol and oxysterol concentrations in the liver tissue. Bile acids are synthesized from cholesterol in the liver via two main pathways. In one pathway, cholesterol is initially converted to 7αHC by microsomal CYP7A1, which is the rate-limiting enzyme in BA synthesis. After several steps, the intermediate is then metabolized by mitochondrial CYP27A1 to finally yield CA. In another pathway, cholesterol is initially converted to 27HC by CYP27A1, and ultimately metabolized to CDCA. CDCA is also yielded by CYP27A1 from intermediates (e.g., 7αHC and C4) in the CA synthetic pathway. 7αHC, C4, and 27HC are the key intermediates through CYP7A1 and CYP27A1 in BA synthetic pathway. Lathosterol, 7-DHC, and Desmosterol are the intermediates in cholesterol synthesis. Data are the mean ± SE. *P < 0.05, **P < 0.01, ^†P < 0.001 versus the Control group by unpaired Student t-test. Abbreviations: 3β-HSD; 3β-hydroxysteroid dehydrogenase, 7αHC; 7α-hydroxycholesterol, 7DHC; 7-dehydroxycholesterol, 27HC; 27-hydroxycholesterol, C4; 7α-hydroxy-4-cholesten-3-one, CYP7A1; cholesterol 7 alpha-hydroxylase, CYP7B1; 25-hydroxycholesterol 7-alpha-hydroxylase, CYP8B1; 7α-hydroxy-4-cholesten-3-one 12α-hydroxylase, CYP27A1; cholesterol 27-hydroxylase, Free Chol; free-cholesterol, Triol; 5β-cholestane-3α,7α,12α-triol, and see the legend of Fig. 2 for other abbreviations.

Figure 5

Bile acid synthesis-related protein expressions, taurine modified mt-tRNA levels and mitochondrial activity in the liver tissue. (A,B) The BA synthesis proteins expressed in the cytosolic (A) and mitochondrial (B) fractions analyzed by Western blotting. The protein expressions in the cytosolic and mitochondrial fractions were standardized by β-actin and VDAC-1 proteins, respectively. (C) Immunohistochemical stain of CYP27A1 in the liver tissue. (D) τm⁵U and τm⁵S²U levels in the liver. (E) Cytochrome c oxidase activity in the liver. The mt-tRNA level and mitochondrial activity were expressed as per the protein level. Data are presented as the mean ± SE. *P < 0.05, **P < 0.01, ^†P < 0.001, ^†P < 0.001versus the Control group by unpaired Student t-test. Abbreviations: COX-IV; cytochrome c oxidase subunit 4, mt-tRNA; mitochondrial-tRNA τm⁵U; 5-taurinemethyluridine, τm⁵S²U; 5-taurinemethyl 2-thiouridine, and see the legends of Figs. 2 and 4 for other abbreviations.

Figure 6

Defective BA metabolism in taurine depletion by impairments in mt-tRNA modification and in BA conjugation. Taurine depletion decreases the taurine modified mt-tRNAs, and in turn, CYP27A1 protein located on the mitochondrial inner membrane is decreased. Bile acid syntheses, particularly in CDCA synthesis, are decreased according to the accumulated substrates and decreased metabolites of CYP27A1. Bile acid excretion into bile is decreased, and instead, the excretion into the peripheral circulation is increased.