Temporal effects of ethanol consumption on energy homeostasis, hepatic steatosis, and insulin sensitivity in mice

Abstract

Background: Alcoholic liver disease (ALD) progresses from steatosis to inflammation, fibrosis, and cirrhosis. Although ALD has been associated with insulin resistance, it is unclear whether insulin resistance coincides with the development of steatosis.

Methods: We studied the temporal relationship of steatosis and glucose homeostasis in mice fed a Lieber-DeCarli liquid control or ethanol (EtOH) diet for 2, 4, or 8 weeks. We studied the effects of alcohol consumption on energy balance, body composition, and hepatic lipids. Glucose tolerance test was performed, and insulin sensitivity was evaluated with hyperinsulinemic-euglycemic clamp.

Results: EtOH-fed mice developed hepatic steatosis over time as compared with control-fed mice despite similar energy intake and expenditure, and gain in body weight and fat. EtOH-fed mice developed glucose intolerance as early as 2 weeks, while insulin resistance developed at 4 weeks. A hyperinsulinemic clamp study at 8 weeks revealed both hepatic and peripheral insulin resistance in EtOH-fed mice. Insulin resistance was associated with hepatic steatosis, increased ceramide levels, and Perilipin 2 expression.

Conclusions: Chronic EtOH consumption leads to the development of hepatic steatosis, impaired glucose tolerance, and insulin resistance. These changes are independent of energy intake or expenditure, weight, whole body fat content, and inflammation. A better understanding of the processes linking EtOH-induced steatosis and abnormal glucose homeostasis may lead to novel therapies targeting the progression of ALD.

Keywords: Alcoholic Steatosis; Ceramides; Insulin Resistance; Metabolic Pheno-typing; Perilipin 2.

Fig. 1. Liver histology of mice fed control or ethanol diets for two, four or eight weeks

Representative hematoxylin and eosin stained liver sections. 40x magnification. Scale bar is 50 μm.

Fig. 2. Distribution of hepatic lipid droplet size in mice fed control or ethanol diets for eight weeks

The histograms show diameters of lipid droplets in control (n=5) (CTRL) and ethanol-fed (ETOH) (n=5) livers. *P<0.0001 vs. control.

Fig. 3. Glucose tolerance testing

Glucose tolerance testing of mice fed control (Ctrl) or ethanol (ETOH) diets for (A) two, (B) four or (C) eight weeks. Data are means ± SEM. N=5/group/time period. *P<0.05 is considered significant. ANOVA at 2 weeks: Treatment P=0.0034, Time P<0.0001; ANOVA at 4 weeks: Treatment P=0.0003, Time P<0.0001; ANOVA at 8 weeks: Treatment P=0.0009, Time P<0.0001.

Fig. 4. Effects of control or ethanol diets on insulin sensitivity as measured by hyperinsulinemic-euglycemic clamp at two weeks, and insulin tolerance test at four weeks

(A) Basal hepatic glucose production (HGP); (B) clamp glucose infusion rate (GIR); (C) clamp HGP; (D) rate of glucose disposal (R_d); (E) insulin tolerance test (ITT) at 4 weeks. Data are means ± SEM; N=5. *P<0.05 is significant. For Fig. 4E ANOVA: Treatment and Time P<0.0001. CTRL=Control-fed; ETOH= ethanol-fed.

Fig. 5. Hyperinsulinemic-euglycemic clamp of mice on control or ethanol diets for eight weeks

A) Basal hepatic glucose production (HGP); (B) clamp glucose infusion rate (GIR); (C) clamp HGP; (D) rate of glucose disposal (R_d); (E) Muscle glucose uptake; (F) Adipose glucose uptake. Data are means ± SEM. N=5. *P<0.05 is considered significant. CTRL=Control-fed; ETOH= ethanol-fed.

Fig. 6. Effects of alcohol on hepatic Akt and p-Akt levels

A) Immunoblots of total Akt, p-Akt, and GPDH of liver lysates of mice fed control or ethanol diets for 8 weeks. B) Quantification of protein levels normalized to GPDH. Data are means ± SEM. *P=0.0017 vs. control. CTRL=Control-fed; ETOH= ethanol-fed; GPDH= glycerol-3-phosphate dehydrogenase.