Adiponectin suppresses gluconeogenic gene expression in mouse hepatocytes independent of LKB1-AMPK signaling

Abstract

The adipocyte-derived hormone adiponectin signals from the fat storage depot to regulate metabolism in peripheral tissues. Inversely correlated with body fat levels, adiponectin reduction in obese individuals may play a causal role in the symptoms of metabolic syndrome. Adiponectin lowers serum glucose through suppression of hepatic glucose production, an effect attributed to activation of AMPK. Here, we investigated the signaling pathways that mediate the effects of adiponectin by studying mice with inducible hepatic deletion of LKB1, an upstream regulator of AMPK. We found that loss of LKB1 in the liver partially impaired the ability of adiponectin to lower serum glucose, though other actions of the hormone were preserved, including reduction of gluconeogenic gene expression and hepatic glucose production as assessed by euglycemic hyperinsulinemic clamp. Furthermore, in primary mouse hepatocytes, the absence of LKB1, AMPK, or the transcriptional coactivator CRTC2 did not prevent adiponectin from inhibiting glucose output or reducing gluconeogenic gene expression. These results reveal that whereas some of the hormone's actions in vivo may be LKB1 dependent, substantial LKB1-, AMPK-, and CRTC2-independent signaling pathways also mediate effects of adiponectin.

Figure 1. Loss of hepatic LKB1 leads to elevated serum glucose in fasted and fed–ad libitum mice.

LKB1^lox/lox mice were infected with adeno-associated virus expressing either GFP or Cre recombinase 16 days prior to study. Mice were either fasted for 18 hours prior to sacrifice or fed ad libitum and sacrificed at 10 am. We quantified (A) blood glucose values and (B) serum insulin values from mice under the indicated conditions. (C) Liver tissue was extracted and Western blotted for phosphorylated Akt (T473), total Akt, phosphorylated Foxo1 (T26), total Foxo1, and total LKB1 protein. A.L., ad libitum. (D and E) Western blots were quantified and the phosphorylated/total Akt and Foxo ratio is shown. Total hepatic (F) triglycerides and (G) protein were quantified and (H) liver weight/body weight ratio calculated. *P < 0.05, GFP versus Cre; ^‡P < 0.05, fasting versus fed. All results are expressed as the mean, and error bars represent SEM.

Figure 2. Hepatic loss of LKB1 leads to reduced efficacy of adiponectin.

LKB1^lox/lox mice were infected with adenovirus expressing either GFP or Cre recombinase 16 days prior to study. (A) Western blots of liver tissue 8 hours after injection with either PBS or 34 μg/g body weight adiponectin (adipo) showing excision of LKB1 protein and concomitant reduction in AMPK T172 phosphorylation. (B) Blood glucose levels were obtained from infected mice injected i.p. with either PBS or 34 μg/g body weight adiponectin. (C) Fold change in blood glucose levels of adiponectin versus PBS injected at the indicated time point. *P < 0.05 versus PBS treatment of same genotype at equivalent time points; ^‡P < 0.05 GFP versus Cre. All results are expressed as the mean, and error bars represent SEM.

Figure 3. Hepatic loss of LKB1 alters adiponectin’s effects on hepatic glucose production.

Hyperinsulinemic, euglycemic clamp studies of adenovirus-infected LKB1^lox/lox mice infused i.v. with PBS or 50 ng/min/g body weight of adiponectin were performed as indicated in Methods. Each group consisted of 4 mice. (A) Western blots from liver collected from mice following clamp experiments exhibiting loss of LKB1 protein. (B) Glucose infusion rate (GIR) and (C) hepatic glucose production (HGP) rate determined from clamped animals. (D) The fold reduction in hepatic glucose production due to adiponectin infusion was calculated for both groups of mice. (E) The rate of glucose disposal (Rd) and (F) rate of glucose uptake into white adipose tissue (WAT) and skeletal muscle (SkMus) for each group. *P < 0.05, PBS versus adiponectin; ^‡P < 0.05, GFP versus Cre. All results are expressed as the mean, and error bars represent SEM.

Figure 4. Adiponectin-induced gene expression following adiponectin tolerance test is not LKB1 dependent.

Adenovirus-infected LKB1^lox/lox mice were injected i.p. with PBS or 34 μg/g body weight adiponectin, and 6 hours later, their livers were collected for mRNA analysis. The indicated mRNAs were quantified and expressed relative to Tbp mRNA levels. The data are represented normalized to the GFP-infected, PBS-injected group. *P < 0.05 PBS versus adiponectin; ^‡P < 0.05 GFP versus Cre. All results are expressed as the mean, and error bars represent SEM.

Figure 5. Adiponectin signaling in LKB1-deficient primary hepatocytes.

(A) Primary hepatocytes isolated from adenovirus-infected LKB1^lox/lox mice were treated with 30 μg/ml adiponectin (Adipo) for 5 or 15 minutes, 1 mM AICAR for 1 hour, or 1 mM phenformin (Phen) for 1 hour, and Western blots examining AMPK phosphorylation (pAMPK), total and phosphorylated ACC (tACC and pACC), total and phosphorylated Raptor (tRaptor and pRaptor), and LKB1 proteins were performed. (B) Western blots from hepatocytes isolated from adenovirus-infected LKB1^lox/lox mice were treated with 30 μg/ml adiponectin, 500 μM AICAR, and 100 μM db-cAMP for 6 hours and probed for CRTC2, total AMPK (tAMPK) and phosphorylated AMPK, LKB1, total and phosphorylated ACC, and tubulin. (C–E) Primary hepatocytes isolated from adenovirus-infected LKB1^lox/lox mice were treated with 30 μg/ml adiponectin, 500 μM AICAR, and 100 μM db-cAMP for 6 hours. Total RNA was isolated and (C) Ppargc1a, (D) Pck1, and (E) G6pc mRNA was quantified and expressed relative to cyclophilin A mRNA and normalized to basal mRNA from GFP-infected hepatocytes. *P < 0.05 vs. db-cAMP; ^‡P < 0.05 GFP versus Cre. (F) Primary hepatocytes isolated from adenovirus-infected LKB1^lox/lox mice were treated with 30 μg/ml adiponectin, 500 μM AICAR, and 100 μM db-cAMP for 6 hours in glucose-free medium, and glucose production was measured and normalized to total protein. *P < 0.05 vs. untreated with adiponectin or AICAR. All results are expressed as the mean, and error bars represent SEM.

Figure 6. Adiponectin signaling in AMPK α1/α2–deficient primary hepatocytes.

(A) Western blots from hepatocytes isolated from AAV-TBG-GFP– or AAV-TBG-Cre–infected Ampkα1^lox/lox;Ampkα2^lox/lox mice were treated with 30 μg/ml adiponectin, 500 μM AICAR, and 100 μM db-cAMP for 6 hours and probed for CRTC2, total and phosphorylated AMPK, total and phosphorylated ACC, and total and phosphorylated Raptor. (B–D) Primary hepatocytes isolated from AAV-TBG-GFP– or AAV-TBG-Cre–infected Ampkα1^lox/lox;Ampkα2^lox/lox mice were treated with 30 μg/ml adiponectin, 500 μM AICAR, and 100 μM db-cAMP for 6 hours. Total RNA was isolated and (B) Ppargc1a, (C) Pck1, and (D) G6pc mRNA was quantified and expressed relative to cyclophilin A mRNA and normalized to basal mRNA from GFP-infected hepatocytes. *P < 0.05 versus db-cAMP. (E) Primary hepatocytes isolated from AAV-TBG-GFP– or AAV-TBG-Cre–infected Ampkα1^lox/lox;Ampkα2^lox/lox mice were treated with 30 μg/ml adiponectin, 500 μM AICAR, and 100 μM db-cAMP for 6 hours in glucose-free medium. Glucose production was measured and normalized to total protein. *P < 0.05 versus untreated with adiponectin or AICAR. All results are expressed as the mean, and error bars represent SEM.