Hypoglycemia and impaired hepatic glucose production in mice with a deletion of the C/EBPbeta gene

Abstract

The transcription factor CCAAT/enhancer-binding protein beta (C/EBPbeta) is enriched in liver and adipose tissue and controls the expression of a wide variety of genes coding for important metabolic pathways, including gluconeogenesis and lipid synthesis. To investigate the role of C/EBPbeta on glucose homeostasis, we studied mice with a targeted deletion of the gene for C/EBPbeta-/- mice. Adult C/EBPbeta-/- mice have hypoglycemia after an 18-hour fast, accompanied by lower hepatic glucose production (40% of that of wild-type mice), with no change in plasma insulin and a lower concentration of plasma free fatty acids (FFA). Glucagon infusion during a pancreatic clamp acutely stimulated hepatic glucose production by 38% in wild-type animals, with no change detected in C/EBPbeta-/- mice. Unexpectedly, both the basal and glucagon-stimulated hepatic cyclic adenosine monophosphate (cAMP) levels were lower in C/EBPbeta-/- mice, indicating an essential role for C/EBPbeta in controlling proximal signal transduction. Fasting hypoglycemia was associated with normal levels of phosphoenolpyruvate carboxykinase (PEPCK) and glucose-6-phosphatase (G6Pase) gene expression, however net liver glycogenolysis was impaired in C/EBPbeta-/- mice. FFA release from isolated adipose tissue in response to epinephrine was 68% lower in C/EBPbeta-/- mice than in control animals; however, N6,O2'-dibutyryladenosine (Bt2) cAMP stimulated a twofold increase in FFA release in C/EBPbeta-/- compared with no further increase in wild-type mice. Because a deletion in the gene for C/EBPbeta reduces blood glucose and circulating FFA, it could be an important therapeutic target for the treatment of non-insulin-dependent diabetes and possibly obesity, based on designing antagonists that decrease C/EBPbeta activity.

Figure 1

Decreased hepatic glucose production and glucagon resistance in adult mice homozygous for a deletion of the C/EBPβ gene (–/–). Previously catheterized mice were fasted 18 h before undergoing a pancreatic clamp as described in Methods. Somatostatin (0.8 μg/kg/min) was infused at a constant rate after the basal glucose turnover determination at 60 min to suppress endogenous insulin and glucagon secretion. After blood sampling at 80 min, glucagon infusion (0.5 μg/kg/min) was initiated for the next 100 min. Hepatic glucose production was calculated under steady-state conditions (as determined previously by repeated blood sampling every 5 min) at min 60, 80, 110, and 170 by dividing the [³H]glucose infusion rate by the mean plasma glucose specific activity. *Significantly greater than C/EBPβ^–/– at each time point; P < 0.05. ^#Significantly less than 60-min or 120-min time point; P < 0.05. Graphs represent the mean ± SEM of eight animals in each group. C/EBPβ, CCAAT/enhancer-binding protein β.

Figure 2

Reduced hepatic cAMP production in mice homozygous for a deletion of the C/EBPβ gene (–/–). C/EBPβ^–/– and WT control mice were lightly anesthetized, and a liver biopsy was freeze-clamped to measure the basal concentration of cAMP. Glucagon was then delivered through the portal vein at 50 μg/kg. One minute after the glucagon injection, a second piece of liver was biopsied for the determination of cAMP levels. The data are mean ± SEM for seven to eight animals per group. *Significantly reduced compared with WT mice; P < 0.05. WT, wild-type.

Figure 3

Liver gene expression in adult WT mice and mice homozygous for a deletion of the C/EBPβ gene (–/–). (a) Ten-week-old female C/EBPβ^–/– and WT mice were fasted overnight before being anesthetized and sacrificed, and the total liver RNA was isolated. The RNA was subjected to Northern blot analysis, and the blot was hybridized sequentially with probes for PEPCK, G6Pase, and glucokinase. Results were normalized to the level of actin mRNA and expressed relative to WT controls. (b) Effect of Bt₂cAMP on the expression of genes for PEPCK, G6Pase, and glucokinase. Mice were fed a high-carbohydrate diet (81% sucrose) for 5 days, followed by intraperitoneal injection with 60 mg Bt₂cAMP/kg of body weight and 25 μg of theophylline in 150 μl of saline, and were sacrificed 2 h later (27). The livers were frozen at –80°C until they were analyzed. RNA was isolated and Northern blot analysis carried out as described in a. The bars represent the mean ± SEM of four to six animals per group. PEPCK, phosphoenolpyruvate carboxykinase; G6Pase, glucose-6-phosphatase; Bt₂, N⁶,O²′-dibutyryladenosine.

Figure 4

Impaired lipolysis in fat-pads from mice homozygous for a deletion of the C/EBPβ gene (–/–). Periovarian fat-pads weighing 50–70 mg were isolated from fed C/EBPβ^–/– and WT control mice and incubated in 1 ml of Krebs-Henseleit buffer with 3% porcine albumin and 5 mM glucose at 37°C in a shaking water bath (30 cycles/min) for 3 h. The medium was gassed with 95% O₂/5% CO₂. Of the two fat-pads from each animal, one was incubated with epinephrine (5 μg/ml) and 0.01% ascorbic acid, while the other was incubated with a maximal dose of Bt₂cAMP (0.43 mg/ml) and theophylline (25 μg/ml). At the end of the 3-h incubation, the medium was frozen at –20°C for FFA and glycerol analysis as described in Methods. The DNA content was quantified in adipose tissue crude homogenates by a spectrofluorometric assay (31). The bars represent the mean ± SEM of 8–10 animals per group. *Significantly reduced compared with WT; P < 0.05. ⁺Significantly increased compared with epinephrine-treated; P < 0.05. FFA, free fatty acids