Isocaloric manipulation of macronutrients within a high-carbohydrate/moderate-fat diet induces unique effects on hepatic lipogenesis, steatosis and liver injury

Abstract

Diets containing excess carbohydrate and fat promote hepatic steatosis and steatohepatitis in mice. Little is known, however, about the impact of specific carbohydrate/fat combinations on liver outcome. This study was designed to determine whether high-energy diets with identical caloric density but different carbohydrate and fat composition have unique effects on the liver. Four experimental diets were formulated with 60%kcal carbohydrate and 20%kcal fat, each in nearly pure form from a single source: starch-oleate, starch-palmitate, sucrose-oleate and sucrose-palmitate. The diets were fed to mice for 3 or 12 weeks for analysis of lipid metabolism and liver injury. All mice developed hepatic steatosis over 12 weeks, but mice fed the sucrose-palmitate diet accumulated more hepatic lipid than those in the other three experimental groups. The exaggerated lipid accumulation in sucrose-palmitate-fed mice was attributable to a disproportionate rise in hepatic de novo lipogenesis. These mice accrued more hepatic palmitate and exhibited more evidence of liver injury than any of the other experimental groups. Interestingly, lipogenic gene expression in mice fed the custom diets did not correlate with actual de novo lipogenesis. In addition, de novo lipogenesis rose in all mice between 3 and 12 weeks, without feedback inhibition from hepatic steatosis. The pairing of simple sugar (sucrose) and saturated fat (palmitate) in a high-carbohydrate/moderate-fat diet induces more de novo lipogenesis and liver injury than other carbohydrate/fat combinations. Diet-induced liver injury correlates positively with hepatic de novo lipogenesis and is not predictable by isolated analysis of lipogenic gene expression.

Keywords: Fatty liver; Palmitate; Saturated fat; Steatohepatitis; Sucrose; Sugar; Triglyceride.

Figure 1. Metabolic features of mice fed custom high-carbohydrate diets

(A) Graph depicts the weight gain (relative to initial body weight) of mice fed chow or one of four custom isocaloric diets. (B) Graph illustrates average daily food intake (in grams per gram body weight), measured twice weekly over the duration of the 12-wk study. (C) Graph demonstrates white adipose tissue weight as a fraction of body weight at 3 wk and 12 wk. (D) Graph illustrates serum glucose levels in mice at 12 wk. Values represent mean ± SE for n = 10. Numerical symbols indicate significant differences compared to chow (1) or other diet groups as numbered in the legend. St Ol = starch-oleate; St Palm = starch-palmitate; Suc Ol = sucrose-oleate, Suc Palm = sucrose-palmitate.

Figure 2. Hepatic steatosis in mice fed custom high-carbohydrate diets

(A) Photomicrographs illustrate liver histology in mice fed chow or a custom high-carbohydrate diet for 3 or 12 wk. Hematoxylin and eosin stain; bar = 100 μm. (B) Hepatic triglyceride measurements at 3 and 12 wk. (C) Serum triglycerides at 3 and 12 wk. Values represent mean ± SE for n = 10. Numerical symbols indicate significant differences compared to chow (1) or other diet groups as numbered in the legend. St Ol = starch-oleate; St Palm = starch-palmitate; Suc Ol = sucrose-oleate, Suc Palm = sucrose-palmitate.

Figure 3. Hepatic gene expression in mice fed custom high-carbohydrate diets

Graphs depict mRNA encoding genes involved in hepatic lipid synthesis (SREBP1, sterol regulatory element binding protein-1; FAS, fatty acid synthase; SCD1, stearoyl Co-A desaturase-1) and oxidation (PGC1a, peroxisome proliferator activated receptor gamma co-activator-1α; ACOX1, acyl Co-A oxidase-1; CPT1, carnitine palmitoyltransferase-1) after 3 wk and 12 wk on chow or high-carbohydrate diets. Values represent mean ± SE for n = 10. Numerical symbols indicate significant differences compared to chow (1) or other diet groups as numbered in the legend. St Ol = starch-oleate; St Palm = starch-palmitate; Suc Ol = sucrose-oleate, Suc Palm = sucrose-palmitate.

Figure 4. Hepatic DNL in mice fed custom high-carbohydrate diets

(A) Graph depicts hepatic DNL as the amount of newly synthesized palmitate measured by stable isotope incorporation as described in Methods. Values represent mean ± SE for n = 10. Numerical symbols indicate significant differences compared to chow (1) or other diet groups as numbered in the legend. St Ol = starch-oleate; St Palm = starch-palmitate; Suc Ol = sucrose-oleate, Suc Palm = sucrose-palmitate. (B) Scattergrams depict the relationship between hepatic DNL and gene expression of FAS and SCD1.

Figure 5. Fatty acid profile of the hepatic lipids in mice fed custom high-carbohydrate diets

(A) Histograms depict the proportion of SFA, MUFA, and PUFA in the free fatty acid (FFA), diacylglycerol (DAG) and triacylglycerol (TAG) compartments of mouse liver after 3 or 12 wk on the designated diets. (B) Graph illustrates the desaturation index (C16:1/C16:0), an indicator of SCD1 activity, in hepatic triglycerides from mice on the custom diets for 3 or 12 wk. (C) Histograms depict the absolute levels of hepatic palmitate in mouse livers at 3 and 12 wk on the custom diets. Values represent mean ± SE for n = 10. Numerical symbols indicate significant differences compared to chow (1) or other diet groups as numbered in the legend. St Ol = starch-oleate; St Palm = starch-palmitate; Suc Ol = sucrose-oleate, Suc Palm = sucrose-palmitate.

Figure 6. Markers of liver injury in mice fed custom high-carbohydrate diets for 12 wk

(A) Immunohistochemistry for cytokeratin-8 (CK-8) demonstrates brown staining in most hepatocytes but no staining in ballooned hepatocytes in the dietary groups (arrowheads). TUNEL staining shows positive cells only in the sucrose-palmitate group (arrowheads). Bar = 50 μm. (B). Graph depicts histologic ballooning scores (0-3 as described in Methods) at 12 wk. (C). Graph depicts the number of TUNEL-positive cells (counted as described in Methods) in the liver at 12 wk. (D) Graph illustrates serum ALT levels at 12 wk. Values represent mean ± SE for n = 10. Numerical superscripts indicate significant differences compared to individual group(s) as numbered in the legend. St Ol = starch-oleate; St Palm = starch-palmitate; Suc Ol = sucrose-oleate, Suc Palm = sucrose-palmitate.