Glycerol-3-phosphate acyltransferase 1 deficiency in ob/ob mice diminishes hepatic steatosis but does not protect against insulin resistance or obesity

Abstract

Objective: Hepatic steatosis is strongly associated with insulin resistance, but a causal role has not been established. In ob/ob mice, sterol regulatory element binding protein 1 (SREBP1) mediates the induction of steatosis by upregulating target genes, including glycerol-3-phosphate acyltransferase-1 (Gpat1), which catalyzes the first and committed step in the pathway of glycerolipid synthesis. We asked whether ob/ob mice lacking Gpat1 would have reduced hepatic steatosis and improved insulin sensitivity.

Research design and methods: Hepatic lipids, insulin sensitivity, and hepatic insulin signaling were compared in lean (Lep(+/?)), lean-Gpat1(-/-), ob/ob (Lep(ob/ob)), and ob/ob-Gpat1(-/-) mice. RESULTS Compared with ob/ob mice, the lack of Gpat1 in ob/ob mice reduced hepatic triacylglycerol (TAG) and diacylglycerol (DAG) content 59 and 74%, respectively, but increased acyl-CoA levels. Despite the reduction in hepatic lipids, fasting glucose and insulin concentrations did not improve, and insulin tolerance remained impaired. In both ob/ob and ob/ob-Gpat1(-/-) mice, insulin resistance was accompanied by elevated hepatic protein kinase C-epsilon activation and blunted insulin-stimulated Akt activation.

Conclusions: These results suggest that decreasing hepatic steatosis alone does not improve insulin resistance, and that factors other than increased hepatic DAG and TAG contribute to hepatic insulin resistance in this genetically obese model. They also show that the SREBP1-mediated induction of hepatic steatosis in ob/ob mice requires Gpat1.

FIG. 1.

A: Total, NEM-resistant (NEM-R), and NEM-sensitive (NEM-S) GPAT specific activities. Liver total particulate fractions (n = 3–4) were assayed for GPAT activity in the presence or absence of 2 mmol/l NEM as described in the “research design and methods” section. B: Hepatic gene expression of Gpat1 was determined by quantitative RT-PCR and expressed as 2^−ΔΔct relative to the endogenous control 18S rRNA and the lean-Gpat1^+/+ group (n = 4). Lean (Lep^+/?) and ob/ob (Lep^ob/ob). Data are LSM ± SE. Significant differences (P < 0.05) are denoted by different letters. SA, specific activity.

FIG. 2.

Obesity-induced hepatic steatosis, but not insulin resistance, was diminished in Gpat1^−/− mice. A: Representative liver sections stained with hematoxylin-eosin from 16-week-old male mice. Images are at ×100 magnification with 100 μm indicated by the white bar in upper left panel. B: Hepatic triacylglycerol content (n = 9–10). Fasting (C) glucose and (D) insulin. Insulin tolerance tests were conducted in 15-week-old male mice. Lean mice (E; n = 6–8) were given 0.5 units insulin/kg body wt by intraperitoneal injection, and ob/ob mice (F; n = 7–9) were administered 1.5 units insulin/kg body wt. Glucose was measured from tail vein blood at times indicated and net areas of the curves (insets) were calculated as described in the “research design and methods” section. Lean (Lep^+/?) and ob/ob (Lep^ob/ob). Data are expressed as LSM ± SE. Significant differences (P < 0.05) are denoted by different letters. (A high-quality digital representation of this figure is available in the online issue.)

FIG. 3.

Changes in hepatic lipids due to lack of Gpat1. A and B: Total hepatic diacylglycerol and species. C and D: Total acyl-CoA and species. E and F: Total ceramide and species (n = 6). Lipids were extracted from livers and measured as described in the “research design and methods” section. Lean (Lep^+/?) and ob/ob (Lep^ob/ob). Data are LSM ± SE. Significant differences (P < 0.05) are denoted by different letters.

FIG. 4.

Hepatic insulin signaling was not improved in Gpat1^−/− mice. A: PKCε protein expression in cytosolic and membrane fractions of livers from 16-week-old mice was determined by Western blot analysis. Bars represent densities of the membrane to cytosolic ratio of PKCε expression relative to the lean-Gpat1^+/+ mice (n = 8). B: Basal and insulin-stimulated phosphorylated Akt expression. Mice were administered either 2.0 units insulin/kg body wt or PBS by intraperitoneal injection for 10 min. Bars represent densities of P-Akt/total Akt relative to the lean-Gpat1^+/+ mice (n = 4–5). Representative blots are shown. Lean (Lep^+/?) and ob/ob (Lep^ob/ob). Data are expressed as LSM ± SE. Significant differences (P < 0.05) among groups are denoted by different letters; * indicates a significant difference (P < 0.05) between basal and insulin-stimulated P-Akt/total Akt within group.

FIG. 5.

Gpat1^−/− mice were not resistant to genetic-induced obesity. A: Weight gain of lean and ob/ob male mice from 4 to 16 weeks of age (n = 12–17). B: Adipose depot weights of lean and ob/ob male mice at 16 weeks of age (n = 10–13). Gonadal white adipose tissue (gWAT), retroperitoneal (rWAT), inguinal (iWAT), and subscapular brown adipose tissue (BAT) depots were measured. Lean (Lep^+/?) and ob/ob (Lep^ob/ob). Data are LSM ± SE. Significant differences (P < 0.05) are denoted by different letters.