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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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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).
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pone-0074806-g006: 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).

Mentions: Among the paste diet cohorts, mice maintained on the VLP/C+ and VLP/C- diets exhibited the greatest difference in intrahepatic fat content, both histologically and biochemically, despite similar VLDL secretion capacity and equal caloric contents of protein, carbohydrate, and fat. Because adequate choline content is essential for normal mitochondrial structure and function in other macronutrient contexts [36–40], we determined whether choline restriction in the VLP dietary context was associated with abnormalities of mitochondrial ultrastructure. As expected, transmission electron microscopy of liver sections from chow-fed mice revealed that hepatocyte mitochondria exhibit smooth outer and inner membranes with tightly-organized cristae (Figure 6A-B). Most hepatocyte mitochondria of VLP/C+-fed mice also exhibited normal ultrastructure, although a small subset of hepatocyte mitochondria lacked organized cristae (Figure 6C-D). Moreover, sparse microvesicular lipid droplets were evident in hepatocytes of VLP/C+-fed mice, a feature that was not observed in hematoxylin and eosin-stained sections for light microscopy. Unlike hepatocyte mitochondria in chow-fed and VLP/C+-fed mice, all hepatocyte mitochondria of VLP/C--fed mice exhibited swollen and disorganized cristae, and many lacked definitive cristae entirely (Figure 6E–G). Additionally, the spatial organization of mitochondria within the cytoplasm was markedly disrupted by the presence of extensive lipid droplets in hepatocytes of VLP/C--fed mice.


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)

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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3756977&req=5

pone-0074806-g006: 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).
Mentions: Among the paste diet cohorts, mice maintained on the VLP/C+ and VLP/C- diets exhibited the greatest difference in intrahepatic fat content, both histologically and biochemically, despite similar VLDL secretion capacity and equal caloric contents of protein, carbohydrate, and fat. Because adequate choline content is essential for normal mitochondrial structure and function in other macronutrient contexts [36–40], we determined whether choline restriction in the VLP dietary context was associated with abnormalities of mitochondrial ultrastructure. As expected, transmission electron microscopy of liver sections from chow-fed mice revealed that hepatocyte mitochondria exhibit smooth outer and inner membranes with tightly-organized cristae (Figure 6A-B). Most hepatocyte mitochondria of VLP/C+-fed mice also exhibited normal ultrastructure, although a small subset of hepatocyte mitochondria lacked organized cristae (Figure 6C-D). Moreover, sparse microvesicular lipid droplets were evident in hepatocytes of VLP/C+-fed mice, a feature that was not observed in hematoxylin and eosin-stained sections for light microscopy. Unlike hepatocyte mitochondria in chow-fed and VLP/C+-fed mice, all hepatocyte mitochondria of VLP/C--fed mice exhibited swollen and disorganized cristae, and many lacked definitive cristae entirely (Figure 6E–G). Additionally, the spatial organization of mitochondria within the cytoplasm was markedly disrupted by the presence of extensive lipid droplets in hepatocytes of VLP/C--fed mice.

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