A very low carbohydrate ketogenic diet prevents the progression of hepatic steatosis caused by hyperglycemia in a juvenile obese mouse model

Abstract

Objective: To investigate whether the improvement in hyperglycemia by dietary control influences hyperglycemia-induced pathologies in tissues of juvenile obese (ob/ob) mice.

Design: Five-week-old ob/ob mice were fed a very low carbohydrate ketogenic diet (KD) for 7 weeks. The blood glucose levels and body weight were monitored during this period. Biochemical parameters in the serum and tissue pathologies of the mice were analyzed at the end of the 7-week period.

Results: The hyperglycemic phenotype of the ob/ob mice was improved by KD feeding for 7 weeks. Surprisingly, we found that KD feeding also drastically reduced the hepatic steatosis phenotype in ob/ob mice, while their obesity phenotype was unaltered. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis revealed that several proteins found in the liver of ob/ob mice fed a regular chow diet were undetectable after being fed KD. Liquid chromatography with tandem mass spectrometry (LC-MS/MS) MASCOT search and western blot analysis revealed that the proteins absent from the mice fed KD included fatty acid synthase (FAS) and acetyl-CoA carboxylase 1 (ACC1), which are key enzymes for lipogenesis in the liver. Fatty acid analysis supported the results because the ratio of C18:1, which is a major product of lipogenesis, was reduced by KD feeding. However, C18:2, which cannot be synthesized in mammalian cells but is present in the KD, was found to be a major component in the liver of KD-fed ob/ob mice.

Conclusion: Hyperglycemia promotes hepatic steatosis via the lipogenic pathway in the liver of juvenile ob/ob mice. However, the development of steatosis is prevented by feeding KD owing to an improvement in hyperglycemia. We found that the progression of steatosis is reflected by the composition of fatty acids in the total lipids of the liver and serum.

Figure 1

Effect of KD feeding on the blood glucose level and body weight of ob/ob mice. Blood glucose levels (a) and body weights (b) of the chow- or KD-fed ob/ob mice during the experimental period (5–12 weeks of age). The parameters for chow-fed wild-type mice (C57BL/6J) are shown as a reference. Filled squares with solid line, chow-fed ob/ob mice; open squares with dashed line, KD-fed ob/ob mice; gray squares with dotted line, chow-fed wild-type mice. Means±s.e., n=9 from two independent experiments. (c) The average daily quantity (kcal) of diet intake in chow- or KD-fed ob/ob mice group during the experimental period. Means±s.e., n=4.

Figure 2

Histological analysis of the liver from ob/ob mice. (a) Representative morphology of the liver from chow-fed (left) and KD-fed (right) ob/ob mice. (b) Paraffin sections prepared from chow-fed (left panel) or KD-fed (right panel) ob/ob mice were stained with hematoxylin–eosin. (c) Cryosections prepared from chow-fed (left panel) or KD-fed (right panel) ob/ob mice were stained with oil red O. Scale bars indicate 10 mm (a) or 100 μm (b and c).

Figure 3

Protein analysis of liver extracts from ob/ob mice. Individual liver extracts of chow-fed (lanes 1–5) and KD-fed ob/ob mice (lanes 6–10) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and stained with Coomassie Brilliant Blue (a) or analyzed by western blotting (b). (a) The arrow (X) highlights the accumulated protein band in chow-fed ob/ob mice (lanes 1–5). The liver extract of chow-fed wild-type mice (lane 0) was loaded as a reference. Lane M, size marker. (b) Protein expression of FAS (upper panel), ACC1 (middle panel) and actin (lower panel).

Figure 4

The expression levels of FAS, ACC1 and actin were measured by ELISA. Individual protein extracts of the liver in chow-fed (closed bars) or KD-fed (open bars) ob/ob mice were analyzed by ELISA. Protein extracts prepared from the liver of chow-fed wild-type mice were indicated as a reference (gray bars). Means±s.d., n=9 from two independent experiments. *P<0.001 chow-fed vs KD-fed ob/ob mice.