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. 2013 Aug 29;8(8):e74806.
doi: 10.1371/journal.pone.0074806. eCollection 2013.

Role of choline deficiency in the Fatty liver phenotype of mice fed a low protein, very low carbohydrate ketogenic diet

Rebecca C Schugar  1 , Xiaojing Huang, Ashley R Moll, Elizabeth M Brunt, Peter A Crawford
Affiliations

Role of choline deficiency in the Fatty liver phenotype of mice fed a low protein, very low carbohydrate ketogenic diet

Rebecca C Schugar et al. PLoS One. .

Abstract

Though widely employed for clinical intervention in obesity, metabolic syndrome, seizure disorders and other neurodegenerative diseases, the mechanisms through which low carbohydrate ketogenic diets exert their ameliorative effects still remain to be elucidated. Rodent models have been used to identify the metabolic and physiologic alterations provoked by ketogenic diets. A commonly used rodent ketogenic diet (Bio-Serv F3666) that is very high in fat (~94% kcal), very low in carbohydrate (~1% kcal), low in protein (~5% kcal), and choline restricted (~300 mg/kg) provokes robust ketosis and weight loss in mice, but through unknown mechanisms, also causes significant hepatic steatosis, inflammation, and cellular injury. To understand the independent and synergistic roles of protein restriction and choline deficiency on the pleiotropic effects of rodent ketogenic diets, we studied four custom diets that differ only in protein (5% kcal vs. 10% kcal) and choline contents (300 mg/kg vs. 5 g/kg). C57BL/6J mice maintained on the two 5% kcal protein diets induced the most significant ketoses, which was only partially diminished by choline replacement. Choline restriction in the setting of 10% kcal protein also caused moderate ketosis and hepatic fat accumulation, which were again attenuated when choline was replete. Key effects of the 5% kcal protein diet - weight loss, hepatic fat accumulation, and mitochondrial ultrastructural disarray and bioenergetic dysfunction - were mitigated by choline repletion. These studies indicate that synergistic effects of protein restriction and choline deficiency influence integrated metabolism and hepatic pathology in mice when nutritional fat content is very high, and support the consideration of dietary choline content in ketogenic diet studies in rodents to limit hepatic mitochondrial dysfunction and fat accumulation.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interests exist.

Figures

Figure 1
Figure 1. Metabolic parameters of mice maintained on very high fat, low protein, very low carbohydrate diets.
(A) Body weight responses to 6 weeks of maintenance on the experimental paste diets, compared to chow controls. n=10-15 mice/group. a, p≤0.001; b, p≤0.001; c, p≤0.01. See end of this legend for description of the individual comparisons depicted by each letter. (B) Caloric consumption of diet, normalized per mouse, between weeks two and four of the 6 weeks of maintenance on the diets. n=5-10 mice/group. a, p≤0.001; c, p≤0.05. (C) Caloric consumption of diet, normalized per gram of body weight (BW). n=5-10 mice/group. a, p≤0.001; b, p≤0.01. (D) Percent adiposity after 6 weeks on each of the diets. n=5-10 mice/group. a, p≤0.01; c, p≤0.05. For all panels, data are presented as means±SEM. a, significantly different compared to chow; b, significant difference attributable to decrease in protein content (from 10% kcal to 5% kcal) at a fixed choline content; c, significant difference attributable to restriction in choline content (from 5.3 g/kg to 0.3 g/kg) at a fixed protein content; by 1-way ANOVA with Tukey’s post hoc testing.
Figure 2
Figure 2. Roles of varying protein and choline nutrient contents in very high fat, low protein, very low carbohydrate diet-induced hepatic steatosis.
(A) Liver weight/body weight ratios after 6 weeks on the diets. n=5-10 mice/group. c, p≤0.05 for LP/C- and p≤0.01 for VLP/C-. See end of this legend for description of the individual comparisons depicted by each letter. (B) Biochemical quantification of hepatic TAG content, normalized to liver mass. n=5-10 mice/group. a, p≤0.05 for LP/C- and p≤0.01 for VLP/C-; b, p≤0.05; c, p≤0.05 for LP/C- and p≤0.01 for VLP/C-. (C) Serum alanine aminotransferase (ALT) concentrations. n=4-6 mice/group. c, p≤0.05. Data are presented as means±SEM. a, significant difference compared to chow; b, significant difference attributable to decrease in protein content (from 10% kcal to 5% kcal) at a fixed choline content; c, significant difference attributable to restriction in choline content (from 5.3 g/kg to 0.3 g/kg) at a fixed protein content; by 1-way ANOVA with Tukey’s post hoc testing.
Figure 3
Figure 3. Intrahepatic triglyceride content and hepatic histopathology in mice fed very high fat, low protein, very low carbohydrate diets.
Hepatic histology in mice maintained for 6 weeks on (A–C) standard chow; (D–F) LP/C+ diet, which caused very small amounts of mixed large and small droplet steatosis in hepatocytes restricted to zone 2 (a representative example of zone 2 is displayed in panel F), and no inflammation; (G–I) LP/C- diet, which caused mixed large and small droplet macrovesicular steatosis in a zone 2 distribution (a representative example of zone 2 is displayed in panel I); (J–L) VLP/C+ diet, which exhibited small lipid droplets only at higher power; (M–O) VLP/C- diet, which caused diffuse steatosis that is predominantly small and microvesicular with some macrovesicular droplets. Numerous clusters of inflammatory cells, some of which are likely associated with necrotic hepatocytes, were observed. Livers of mice fed both VLP/C+ (P) and VLP/C- (Q) exhibit inflammatory foci (arrows). (R) Only in livers from VLP/C--fed were mitotic figures observed (arrow). Scale bars: (A, B, D, E, G, H, J, K, M, N, lower power images taken with standard light microscopy, original magnification at 10X or 20X), 100 μm; (C, F, I, L, O, higher power images taken with confocal microscopy, original magnification at 80X), 10 μm; (P, Q, R, medium power images taken with standard light microscopy, original magnification at 40X), 50 μm.
Figure 4
Figure 4. Hepatic macrophage density in mice fed very high fat, low protein, very low carbohydrate diets.
(A) Confocal images of F4/80+ macrophages (scale bars, 50 μm) and (B) quantification of F4/80+ macrophages normalized to the number of DAPI-stained nuclei from liver sections of mice maintained on the indicated diets for 6 weeks. Data are presented as means±SEM. n=3 mice/group with n=3 20X fields quantified per section/mouse.
Figure 5
Figure 5. Hepatic TAG secretion in mice fed very high fat, low protein, very low carbohydrate diets.
Mice from each dietary group (6 weeks each diet) were fasted for 18 h. Blood was collected prior to (0 h) and after intraperitoneal injection of tyloxapol. (A) Serum TAG concentration and (B) areas under the curve (AUC), n=5 mice/group. Data are presented as means±SEM. a, significantly different compared to chow, p≤0.001 by 1-way ANOVA with Tukey’s post hoc testing.
Figure 6
Figure 6. Abnormal mitochondrial ultrastructure in mice fed a choline restricted, very high fat, low protein, very low carbohydrate diet.
(A) Transmission electron micrograph of hepatocytes from mice maintained for 6 weeks on standard chow reveals normal mitochondrial structure. Arrows, mitochondria. (B) Higher power image of mitochondria from mice maintained on standard chow, demonstrating normal cristae. (C) Hepatocyte mitochondria from livers of mice maintained on VLP/C+ exhibited normal cristae folding and evident double membranes. Sparse microvesicular lipid droplets were also evident (white circular structure). Arrows, mitochondria; arrowhead; autophagosome. (D) Higher power image of hepatocyte mitochondria from mice maintained on VLP/C+, showing morphology of the cristae. (E) VLP/C- diet induces massive hepatocyte steatosis (note large circular pale fat droplets), and swollen mitochondria with disorganized and dilated cristae. Hepatocyte nucleus is on the right side of the image. Arrows, mitochondria. (F) Higher power image of hepatocyte mitochondria from mice maintained on VLP/C-, with dilated cristae (arrow) and an autophagosome (arrowhead). (G) High power image of hepatocyte mitochondria from mice maintained on VLP/C-, with dilated cristae (arrows). Scale bars, 500 nm (A–F), 100 nm (G).
Figure 7
Figure 7. Relative hepatic mitochondrial genome content in mice fed very low protein and carbohydrate, very high fat diets.
Quantification of mitochondrial genome copy number (relative abundance) by qPCR using purified liver gDNA from mice maintained on the diets for 6 weeks. Data are presented as means±SEM; n=4-5/group, *p≤0.05 by 1-way ANOVA with Tukey’s post hoc testing.
Figure 8
Figure 8. Respiration studies of hepatic mitochondria isolated from mice fed very low protein and carbohydrate, very high fat diets.
(A) Respiration rates in the basal leak condition (state 2), ADP-stimulated condition (state 3), F1F0-ATPase independent condition (state 4), and uncoupled condition in hepatic mitochondria isolated from chow-fed, VLP/C+-fed, and VLP/C--fed (for 6 weeks) mice using palmitoyl-L-carnitine and malate as substrates. n=4 mice/group. (B) Relative respiratory ratios of basal leak (state 2/state 3), respiratory control (RCR, state 3/state 4), and coupling control (CCR, state 4/uncoupled), derived from panel A. (C) Respiration rates in states 2-4 and while uncoupled in hepatic mitochondria isolated from chow-, VLP/C+, and VLP/C--fed mice that respired using the Complex II-electron donor substrate succinate in the presence of rotenone (Complex I activity inhibitor). n=9 mice/group. (D) Relative respiratory ratios of state 2/state 3, state 3/state 4, and state 4/uncoupled, derived from panel C. (E) Respiration rates in states 2-4 in hepatic mitochondria isolated from chow-, VLP/C+, and VLP/C--fed mice that respired using the Complex III-donor substrate duroquinol plus rotenone. n=9 mice/group. (F) Relative respiratory ratios of state 2/state 3, state 3/state 4, and state 4/uncoupled, derived from panel E. (G) Respiration rates in states 2-4 in hepatic mitochondria isolated from chow-, VLP/C+, and VLP/C--fed mice that respired using the Complex IV-donor substrate combination TMPD/ascorbate, plus rotenone. n=9 mice/group. (H) Relative respiratory ratios of state 2/state 3, state 3/state 4, and state 4/uncoupled, derived from panel G. Data are presented as means±SEM. *p≤0.05; **p≤0.01 by 1-way ANOVA with Tukey’s post hoc testing.
Figure 9
Figure 9. Influence of choline restriction in integrated hepatic mitochondrial fatty acyl-CoA metabolism.
Red text and red arrows highlight hepatic processes known to be impaired by administration of experimental choline deficient diets. Unlike previous observations using lower fat choline deficient versus replete formulations, in this study, choline repletion in ~90% kcal fat diets did not alter triacylglycerol (TAG) secretion as VLDL. However, choline restriction in a ~90% kcal fat diet was associated with mitochondrial structural and functional abnormalities, which were linked to liver fat accumulation and injury. CPT1, carnitine palmitoyltransferase 1; TCAC, tricarboxylic acid cycle; ETC, electron transport chain; ATP, adenosine triphosphate.
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