Prevention of hepatic steatosis and hepatic insulin resistance by knockdown of cAMP response element-binding protein

Abstract

In patients with poorly controlled type 2 diabetes mellitus (T2DM), hepatic insulin resistance and increased gluconeogenesis contribute to fasting and postprandial hyperglycemia. Since cAMP response element-binding protein (CREB) is a key regulator of gluconeogenic gene expression, we hypothesized that decreasing hepatic CREB expression would reduce fasting hyperglycemia in rodent models of T2DM. In order to test this hypothesis, we used a CREB-specific antisense oligonucleotide (ASO) to knock down CREB expression in liver. CREB ASO treatment dramatically reduced fasting plasma glucose concentrations in ZDF rats, ob/ob mice, and an STZ-treated, high-fat-fed rat model of T2DM. Surprisingly, CREB ASO treatment also decreased plasma cholesterol and triglyceride concentrations, as well as hepatic triglyceride content, due to decreases in hepatic lipogenesis. These results suggest that CREB is an attractive therapeutic target for correcting both hepatic insulin resistance and dyslipidemia associated with nonalcoholic fatty liver disease (NAFLD) and T2DM.

Figure 1. CREB ASO improves metabolic parameters in three other diabetic models

Fasting plasma glucose (A), and insulin (B) concentrations (n = 8–21) in the T2DM rat model. Fasting plasma glucose concentrations (C) were decreased with CREB ASO in the ZDF rat model (n = 4–5). Due to the pancreas saving effect of lower glucose values plasma insulin values tended to be increased (D) (n = 4–5). In the lard diet based model, CREB ASO reduced fasting plasma glucose (E), and insulin (F) concentrations (n = 7–9). Lastly, CREB ASO also reduced fasting plasma glucose in an ob/ob mouse model (G) due to reductions in gluconeogenic genes (H) (n = 4–5). * P < 0.05, ** P < 0.005.

Figure 2. CREB ASO treatment reduces hepatic lipid content

Long chain acyl CoAs (A), and liver DAGs (B) were decreased with CREB ASO treatment (n = 8–10). Liver triglycerides were reduced in the fasted and fed states with CREB ASO (C). In-vivo lipogenesis was reduced with CREB ASO as assessed by the incorporation of [1-¹⁴C]-palmitate in to triglycerides (n = 4 per group) (D). C/EBPα (E), C/EBPβ (F), and key lipogenic genes (G) assessed by RT-PCR (n = 4–7). * P < 0.05, ** P < 0.005 for Control ASO vs. CREB ASO, # P < 0.05 for control ASO fasted vs. control ASO fed.

Figure 3. CREB ASO treatment improves hepatic insulin sensitivity in T2DM Rats

Suppression of hepatic glucose production (A) was assessed using the hyperinsulinemic-euglycemic clamp (n = 4–14 per treatment group). In the T2DM rat model CREB ASO reduced membrane PKCε (B) (n = 4 per treatment group), increased IRS-2 tyrosine phosphorylation (C) (n = 9 per treatment group), and improved Akt2 activity (D) (n = 5 per treatment group). * P < 0.05 for CREB ASO vs. control ASO, # P < 0.05 for T2DM control ASO vs. normal chow control ASO.

Figure 4. CREB ASO therapy decreases plasma triglycerides and cholesterol

Plasma triglycerides were decreased with CREB ASO (n = 10–12) (A). The lipoprotein profile showed significant reductions in VLDL, LDL, and HDL with CREB ASO (B). Free hepatic cholesterol was unchanged with CREB ASO (n = 4 per treatment group) (C) but CREB ASO decreased the concentration of hepatic cholesterol esters (D) as assessed by NMR. Synthesis of cholesterol in rat hepatocytes as assessed by relative [¹⁴C] acetic acid incorporation in to sterols (E) (n = 3–7 per treatment group). Amount of bile acids extracted from feces during a 12 hour fast (G) (n = 7 per treatment group). * P < 0.05 for control ASO vs. CREB ASO.