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Comparative Study
, 300 (6), G956-67

Hepatic Steatosis, Inflammation, and ER Stress in Mice Maintained Long Term on a Very Low-Carbohydrate Ketogenic Diet

Affiliations
Comparative Study

Hepatic Steatosis, Inflammation, and ER Stress in Mice Maintained Long Term on a Very Low-Carbohydrate Ketogenic Diet

Joel R Garbow et al. Am J Physiol Gastrointest Liver Physiol.

Abstract

Low-carbohydrate diets are used to manage obesity, seizure disorders, and malignancies of the central nervous system. These diets create a distinctive, but incompletely defined, cellular, molecular, and integrated metabolic state. Here, we determine the systemic and hepatic effects of long-term administration of a very low-carbohydrate, low-protein, and high-fat ketogenic diet, serially comparing these effects to a high-simple-carbohydrate, high-fat Western diet and a low-fat, polysaccharide-rich control chow diet in C57BL/6J mice. Longitudinal measurement of body composition, serum metabolites, and intrahepatic fat content, using in vivo magnetic resonance spectroscopy, reveals that mice fed the ketogenic diet over 12 wk remain lean, euglycemic, and hypoinsulinemic but accumulate hepatic lipid in a temporal pattern very distinct from animals fed the Western diet. Ketogenic diet-fed mice ultimately develop systemic glucose intolerance, hepatic endoplasmic reticulum stress, steatosis, cellular injury, and macrophage accumulation, but surprisingly insulin-induced hepatic Akt phosphorylation and whole-body insulin responsiveness are not impaired. Moreover, whereas hepatic Pparg mRNA abundance is augmented by both high-fat diets, each diet confers splice variant specificity. The distinctive nutrient milieu created by long-term administration of this low-carbohydrate, low-protein ketogenic diet in mice evokes unique signatures of nonalcoholic fatty liver disease and whole-body glucose homeostasis.

Figures

Fig. 1.
Fig. 1.
Reduced body weight and percent body fat in mice fed a very low-carbohydrate, high-fat ketogenic diet (KD) for 12 wk. A: body weights at time zero (6 wk of age), after 6 wk of diet (12 wk of age), and after 12 wk of diet (18 wk of age) in chow-, Western diet (WD)-, and KD-fed mice. B: caloric consumption, in kcal/mouse per day, for each diet. Mean consumption was measured on 20 consecutive days. Total body fat percentage (C) and lean mass (D), measured by magnetic resonance spectroscopy (MRS) in living, awake mice. Data are presented as means ± SE; n = 8 mice/group; *P < 0.05; **P < 0.01; ***P < 0.001 by 1-way ANOVA with Tukey's post hoc testing, for (A and B), and by 2-way ANOVA with Bonferroni post hoc testing (C and D). For C and D, open bars = 6-wk duration on diet; closed bars = 12-wk duration on diet.
Fig. 2.
Fig. 2.
Distinct temporal patterns of hepatic lipid accumulation in KD-fed vs. WD-fed mice. A: hepatic lipid content (%), determined by MRS, in anesthetized mice after the indicated number of weeks maintained on the 3 diets. aP < 0.001 vs. chow at same time point; bP < 0.01 (KD vs. WD) at same time point; bbP < 0.001 (KD vs. WD) at same time point, by 2-way ANOVA with Bonferroni post hoc testing. n = 7, 7, and 9 animals/group, in the chow, WD, and KD groups, respectively. B: biochemical measurement of hepatic lipid content after 12 wk of each diet. n = 8 mice/group; *P < 0.05; **P < 0.01 by 1-way ANOVA with Tukey's post hoc testing. Data are presented as means ± SE.
Fig. 3.
Fig. 3.
Nonalcoholic fatty liver disease-like histopathology in KD-fed mice. All micrographs are taken from liver sections of animals fed each diet for 12 wk. A: chow-fed animal, showing normal architecture. Original magnification, ×10. B: WD-fed animal; note periportal steatosis with sparing of zone 3, surrounding central veins. No evidence of inflammation, injury, or regeneration was observed. Original magnification, ×10. CI: sections from KD-fed animals. In C, note sparse subcapsular steatosis. Original magnification, ×10. D: higher-power field, revealing macrovesicular steatosis and inflammation (arrowheads). Original magnification, ×20. E: pyknotic (apoptotic) hepatocytes (arrowhead). Original magnification, ×60. F: mitotic figure (arrowhead). Original magnification, ×60. Inflammatory foci (arrows) are observed in GI, original magnification, ×10, ×20, and ×40, respectively.
Fig. 4.
Fig. 4.
Increased accumulation and size of macrophages in livers of KD-fed mice. Liver sections from chow-fed (A), WD-fed (B), and KD-fed (C) mice. Fluorescent staining for macrophages (F4/80) is bright red. Original magnification of each panel is ×20. D: quantification of number of F4/80+ cells/×20 field in each condition; ***P < 0.001 by ANOVA with Tukey's post hoc testing, for comparison of KD vs. chow and vs. WD, n = 5 ×20 fields from 3 independent animals/condition. E: quantification of cross-sectional area (CSA, in μm2) of F4/80+ cells; ***P < 0.001 by ANOVA with Tukey's post hoc testing, for comparison of KD vs. chow and vs. WD, n = 25 cells/section from 3 replicate animals/condition.
Fig. 5.
Fig. 5.
Induction of endoplasmic reticulum stress in liver of KD-fed mice. A: ethidium bromide-stained agarose gel separates PCR amplicons derived from spliced and unspliced transcripts of Xbp1 in individual liver samples of chow-, WD-, and KD-fed mice, generated using a traditional primer pair that spans the spliced exon (see Supplemental Fig. S2A for a schematic overview and Supplemental Table S1 for primer sequences). Cultured primary neonatal rat cardiomyocytes (NRCM) treated with vehicle (0.1% DMSO) or 1.5 μM thapsigargin (Tg) in DMSO for 180 min. B: immunoblot for CAAT/enhancer binding homologous protein (CHOP) protein (and actin loading control) in NRCM controls (V, DMSO vehicle) and individual liver samples from each diet group. C: immunoblot for p-eukaryotic initiation factor 2α (eIF2α) and total eIF2α in NRCM controls (V, DMSO vehicle) and individual liver samples from each diet group. D: quantification of data presented in A, plotting ratio of spliced-to-unspliced PCR product intensity. E: RT-qPCR measurements of the selectively amplified spliced (Xbp1-S), unspliced (Xbp1-U), and total Xbp1 (Xbp1) cDNAs using selective primer sets (see Supplemental Fig. S2B for a schematic overview, Supplemental Table S1 for primer sequences, and materials and methods for a description). n = 4–5/group. F: quantification of data presented in B, plotting ratio of CHOP/actin. G: quantification of data presented in C, plotting ratio of p-eIF2α/total eIF-2α. *P < 0.05; **P < 0.01 by 1-way ANOVA with Tukey's post hoc testing. Data are presented as means ± SE.
Fig. 6.
Fig. 6.
Development of glucose intolerance, but preserved systemic insulin sensitivity, in KD-fed mice. A and B: intraperitoneal glucose-tolerance tests in mice maintained on each diet for 6 wk (A) and 12 wk (B). AUC, mean area under the curve. C: intraperitoneal insulin-tolerance tests after 12 wk of chow, WD, or the KD. *P < 0.05; ***P < 0.001 by 1-way ANOVA with Tukey's post hoc testing. Data are presented as means ± SE; n = 8 mice/group.
Fig. 7.
Fig. 7.
Preserved hepatic Akt response to insulin in KD-fed mice. Immunoblot comparisons of p-Akt response to insulin in livers of animals fed control chow vs. KD (A) or chow vs. WD (C), for 12 wk. B and D: quantification of p-Akt/total Akt ratios from A and C, respectively. *P < 0.05; **P < 0.01 by 2-way ANOVA with Bonferroni post hoc testing. Data are presented as means ± SE. Protein extracts from livers of chow-fed mice are presented in both A and C, to independently compare chow vs. KD and chow vs. WD.

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