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

Schugar RC, Huang X, Moll AR, Brunt EM, Crawford PA - PLoS ONE (2013)

Bottom Line: C57BL/6J mice maintained on the two 5% kcal protein diets induced the most significant ketoses, which was only partially diminished by choline replacement.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Center for Cardiovascular Research, Washington University, St. Louis, Missouri, USA.

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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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.
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pone-0074806-g001: 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.

Mentions: As previously observed in mice maintained on Bio-Serv F3666, but unlike mice maintained on standard chow or the other three experimental diets, mice maintained on VLP/C- diet for 6 weeks failed to gain weight (Figure 1A). While mice maintained on VLP/C+ gained weight over the 6 week period, the extent of weight gain was ~60% less than that of mice maintained on standard chow, LP/C+, or LP/C-. Differences in weight gain between VLP/C+ and VLP/C- fed cohorts may be partially attributable to diminished caloric consumption by mice fed VLP/C- (Figure 1B), but normalizing caloric consumption to body weight revealed no differences between these two diet cohorts (Figure 1C). Independent of whether caloric consumption was normalized per animal or to body mass, caloric intake was increased in all cohorts of mice fed these paste diets, compared to chow-fed mice. Together, these observations are consistent with increased energy expenditure of mice maintained on these very high fat diets (compared to standard chow), which was observed in studies of mice maintained on Bio-Serv F3666 [15,21]. Irrespective of choline content, LP diet-ingesting cohorts both gained significantly more weight than mice in the VLP-ingesting cohorts, indicating that increased protein content of these two diets supports weight gain [34]. Percent adiposity, measured by MRI, was commensurate with the chow-fed cohort for mice maintained on LP/C-, VLP/C+, and VLP/C- (Figure 1D), while mice fed LP/C+ displayed a significant increase in adiposity relative to the other cohorts.


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

Schugar RC, Huang X, Moll AR, Brunt EM, Crawford PA - PLoS ONE (2013)

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.
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Related In: Results  -  Collection

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pone-0074806-g001: 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.
Mentions: As previously observed in mice maintained on Bio-Serv F3666, but unlike mice maintained on standard chow or the other three experimental diets, mice maintained on VLP/C- diet for 6 weeks failed to gain weight (Figure 1A). While mice maintained on VLP/C+ gained weight over the 6 week period, the extent of weight gain was ~60% less than that of mice maintained on standard chow, LP/C+, or LP/C-. Differences in weight gain between VLP/C+ and VLP/C- fed cohorts may be partially attributable to diminished caloric consumption by mice fed VLP/C- (Figure 1B), but normalizing caloric consumption to body weight revealed no differences between these two diet cohorts (Figure 1C). Independent of whether caloric consumption was normalized per animal or to body mass, caloric intake was increased in all cohorts of mice fed these paste diets, compared to chow-fed mice. Together, these observations are consistent with increased energy expenditure of mice maintained on these very high fat diets (compared to standard chow), which was observed in studies of mice maintained on Bio-Serv F3666 [15,21]. Irrespective of choline content, LP diet-ingesting cohorts both gained significantly more weight than mice in the VLP-ingesting cohorts, indicating that increased protein content of these two diets supports weight gain [34]. Percent adiposity, measured by MRI, was commensurate with the chow-fed cohort for mice maintained on LP/C-, VLP/C+, and VLP/C- (Figure 1D), while mice fed LP/C+ displayed a significant increase in adiposity relative to the other cohorts.

Bottom Line: C57BL/6J mice maintained on the two 5% kcal protein diets induced the most significant ketoses, which was only partially diminished by choline replacement.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Center for Cardiovascular Research, Washington University, St. Louis, Missouri, USA.

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.

Show MeSH
Related in: MedlinePlus