Regulation of basal expression of hepatic PEPCK and G6Pase by AKT2

Abstract

Hepatic glucose metabolism signaling downstream of insulin can diverge to multiple pathways including AKT. Genetic studies suggest that AKT is necessary for insulin to suppress gluconeogenesis. To specifically address the role of AKT2, the dominant liver isoform of AKT in the regulation of gluconeogenesis genes, we generated hepatocytes lacking AKT2 (Akt2-/-). We found that, in the absence of insulin signal, AKT2 is required for maintaining the basal level expression of phosphoenolpyruvate carboxyl kinase (PEPCK) and to a lesser extent G6Pase, two key rate-limiting enzymes for gluconeogenesis that support glucose excursion due to pyruvate loading. We further showed that this function of AKT2 is mediated by the phosphorylation of cyclic AMP response element binding (CREB). Phosphorylation of CREB by AKT2 is needed for CREB to induce the expression of PEPCK and likely represents a priming event for unstimulated cells to poise to receive glucagon and other signals. The inhibition of gluconeogenesis by insulin is also dependent on the reduced FOXO1 transcriptional activity at the promoter of PEPCK. When insulin signal is absent, this activity appears to be inhibited by AKT2 in manner that is independent of its phosphorylation by AKT. Together, this action of AKT2 on FOXO1 and CREB to maintain basal gluconeogenesis activity may provide fine-tuning for insulin and glucocorticoid/glucagon to regulate gluconeogenesis in a timely manner to meet metabolic needs.

Keywords: AKT2; CREB; FOXO1; G6Pase; PEPCK; gluconeogenesis.

Figure 1.. AKT2 regulates basal expression of PEPCK And G6Pase.

(A) Hepatocyte cell lines established from Akt2^−/− mouse liver. Top, representative image of hepatocytes established from control (akt2^+/+) and Akt2^−/− mouse liver. Bottom left, expression of albumin in hepatocyte cell lines established from the mouse livers. Bottom right, expression of AKT2, phospho-AKT, and p-GSK3 in hepatocytes with or without ectopic expression of AKT2. (B) Akt2^+/+ and Akt2^−/− hepatocytes are treated with vehicle (open bar) or 100 nM insulin (solid bar) for 6 h. Expression of PEPCK and G6Pase were determined using quantitative PCR analysis. n = 3. Data presented as mean ± SEM. P ≤ 0.05. a, different from Vehicle control treated Akt2^+/+ cells; b, different from insulin treated Akt2^+/+ cells; c. different from vehicle treated Akt2^−/− cells. Experiments repeated at least three times. (C) Akt2^−/− hepatocytes were transiently transfected with Myr-AKT2. Expression of PEPCK and G6Pase were determined using quantitative PCR analysis. n = 3. Data presented as mean ± SEM. * P ≤ 0.05. Different from vector transfected Akt2^−/− cells. Experiments repeated at least three times.

Figure 2.. AKT2 loss suppresses glucose excursion induced by pyruvate in the absence of insulin/nutrient signal.

(A) Fasting plasma glucose levels determined in Akt2^+/+ (A^+/+) and Akt2^−/− (A^−/−) mice fasted for 16 h. (B) Random fed plasma glucose levels. (C) Insulin tolerance test (ITT) performed in Akt2^+/+ (A^+/+) and Akt2^−/− (A^−/−) mice fasted for 6 h. (D) Expression of PEPCK and G6Pase in Akt2^+/+ and Akt2^−/− mice fasted for 16 h. n = 3. (E) Pyruvate tolerant test (PTT) in Akt2^+/+ (A^+/+) and Akt2^−/− (A^−/−) mice fasted for 16 h. Data presented as mean ± SEM. * P ≤ 0.05.

Figure 3.. FOXO1 does not mediate AKT2-regulated induction of PEPCK.

FOXO-AAA is transiently transfected to Akt2^+/+ and Akt2^−/−. PEPCK expression is determined using quantitative PCR analysis. n = 3. Data presented as mean ± SEM. * P ≤ 0.05. Different from vector transfected cells of the same genotype. Experiments repeated at least three times.

Figure 4.. CREB phosphorylation mediates AKT2-regulated induction of PEPCK.

(A) CREB phosphorylation induced by PTEN loss is regulated by AKT2. Liver lysates from mice with different genotypes were analysis for p-CREB. PM, Pten deleted; DM, Pten and Akt2 deleted; AM, Akt2 deleted. Two biological replicas are included for each genotype group. Protein levels of p-AKT, PTEN and p-GSK3 are analyzed and showed up-regulation of AKT and GSK-3 with PTEN loss and inhibition when AKT2 is lost. Phosphorylation of CREB is only observed when PTEN is lost. AKT2 loss together with PTEN blocked this phosphorylation in the DM livers. (B) Akt2^+/+ and Akt2^−/− cell were treated with forskolin (100 μM) for 24 h to induce cAMP and CREB phosphorylation or vehicle as controls. Phosphorylation of CREB is detected and showed to be lower when AKT2 is lost. When forskolin is added to induce cAMP, protein expression of p-CREB is evaluated in both Akt2^+/+ and Akt2^−/− cells even though its levels remains lower in the Akt2^−/− cells. Vinculin is probed as loading control. (C) Introduction of CREB-133A and wtCREB to Akt2^+/+ and Akt2^−/− hepatocytes. Expression of PEPCK is analyzed using quantitative PCR analysis. Left panel, CREB-S133A is introduced to Akt2^+/+ cells. Middle panel, wtCREB is introduced to Akt2^−/− cells; Right panel, wtCREB is introduced to Akt2^+/+ cells. n = 3. Data presented as mean ± SEM. * P ≤ 0.05. Different from vector transfected cells of the same genotype. Experiments repeated at least three times.

Figure 5.. Schematic for AKT2 regulated PEPCK expression mediated by CREB phosphorylation and FOXO1 activity.

In the absence of insulin signal, AKT2 sustains the expression of PEPCK (and to a lesser extent G6Pase) by inducing the phosphorylation of CREB. This action likely primes the PEPCK promoter for further response to glucagon and glucocorticoid induced phosphorylation of CREB mediated by cAMP-PKA. AKT2 also keeps the FOXO1 transcriptional activity at an inhibitory state. This, however is independent of its phosphorylation on FOXO1 in the absence of insulin.