Hepatic overexpression of glycerol-sn-3-phosphate acyltransferase 1 in rats causes insulin resistance

Abstract

Fatty liver is commonly associated with insulin resistance and type 2 diabetes, but it is unclear whether triacylglycerol accumulation or an excess flux of lipid intermediates in the pathway of triacyglycerol synthesis are sufficient to cause insulin resistance in the absence of genetic or diet-induced obesity. To determine whether increased glycerolipid flux can, by itself, cause hepatic insulin resistance, we used an adenoviral construct to overexpress glycerol-sn-3-phosphate acyltransferase-1 (Ad-GPAT1), the committed step in de novo triacylglycerol synthesis. After 5-7 days, food intake, body weight, and fat pad weight did not differ between Ad-GPAT1 and Ad-enhanced green fluorescent protein control rats, but the chow-fed Ad-GPAT1 rats developed fatty liver, hyperlipidemia, and insulin resistance. Liver was the predominant site of insulin resistance; Ad-GPAT1 rats had 2.5-fold higher hepatic glucose output than controls during a hyperinsulinemic-euglycemic clamp. Hepatic diacylglycerol and lysophosphatidate were elevated in Ad-GPAT1 rats, suggesting a role for these lipid metabolites in the development of hepatic insulin resistance, and hepatic protein kinase Cepsilon was activated, providing a potential mechanism for insulin resistance. Ad-GPAT1-treated rats had 50% lower hepatic NF-kappaB activity and no difference in expression of tumor necrosis factor-alpha and interleukin-beta, consistent with hepatic insulin resistance in the absence of increased hepatic inflammation. Glycogen synthesis and uptake of 2-deoxyglucose were reduced in skeletal muscle, suggesting mild peripheral insulin resistance associated with a higher content of skeletal muscle triacylglycerol. These results indicate that increased flux through the pathway of hepatic de novo triacylglycerol synthesis can cause hepatic and systemic insulin resistance in the absence of obesity or a lipogenic diet.

FIGURE 1. Pathway of de novo glycerolipid synthesis

GPAT isoforms located in mitochondria (GPAT1) and endoplasmic reticulum (ER) (GPAT3) catalyze the initial step in the synthesis of TAG. Acyl-CoA use for β-oxidation or TAG synthesis is regulated reciprocally by carnitine palmitoyltransferase-1 (CPT-1) and GPAT1. LPA, phosphatidic acid (PA), and DAG, TAG, phosphatidylethanolamine (PE), phosphatidylcholine (PC), and fatty acid (FA) were used.

FIGURE 2. GPAT1 activity and expression in rat liver

Rats were treated with 1.0–2.0 × 10¹² particles/ml of Ad-GPAT1 or Ad-EGFP virus for 5–7 days (Ad-EGFP, n = 9; Ad-GPAT1, n = 11). A, GPAT1 enzyme activity was determined in total liver membranes after inhibiting endoplasmic reticulum GPAT with NEM. Results are expressed as mean ± S.E. B, anti-FLAG Western blot of total membranes from primary hepatocytes infected with Ad-GPAT1 (Control), from liver of a rat infected with Ad-EGFP (EGFP), and from liver, gastrocnemius muscle (2 lanes), and heart (2 lanes) of rats infected with Ad-GPAT.

FIGURE 3. Liver, skeletal muscle, and VLDL TAG content was increased in Ad-GPAT1-treated rats

Rats were treated with 1.0–2.0 × 10¹² particles/ml of Ad-GPAT1 or Ad-EGFP virus for 5–7 days and fasted for 4 h before tissue collection. Ad-EGFP, n = 9. and Ad-GPAT1, n = 11, for liver and gastrocnemius. Results are expressed as mean ± S.E. A, liver TAG. B, gastrocnemius muscle TAG. Plasma lipoprotein fractions were separated by fast protein liquid chromatography. Triglyceride and cholesterol are reported as averages (EGFP, n = 4; GPAT, n = 6). C, VLDL TAG (fraction 14; * p < 0.05). D, VLDL cholesterol (fraction 14; p < 0.06).

FIGURE 4. Ad-GPAT1-treated rats were insulin resistant

Rats were treated with 1.0–2.0 × 10¹² particles/ml of Ad-GPAT1 or Ad-EGFP virus for 7 days and food-deprived for 24 h before hyperinsulinemic-euglycemic clamp experiments (Ad-EGFP, n = 8; Ad-GPAT1, n = 8). A, glucose infusion rate. B, basal and insulin-stimulated hepatic glucose output. C, glucose incorporation into liver glycogen (p = 0.36). D, PEPCK and, E, Glc-6-Pase mRNA expression were measured in liver from Ad-GPAT1-treated rats fasted for 4 h or fasted for 24 h followed by hyperinsulinemic-euglycemic clamps for 3 h. PEPCK mRNA expression after clamp (n = 6). Glc-6-Pase mRNA expression after clamp (p = 0.33) (n = 6). F, PEPCK protein expression after clamp (n = 8).

FIGURE 5. Skeletal muscle glucose metabolism was impaired in Ad-GPAT1-treated rats

Rats were food deprived for 24 h before hyperinsulinemic-euglycemic clamp experiments. A, basal and insulin-stimulated hepatic glucose disposal rates. B, 2-deoxyglucose uptake in gastrocnemius muscle. Ad-EGFP, n = 8; Ad-GPAT1, n = 8.

FIGURE 6. Hepatic lipid metabolites were altered in Ad-GPAT1-treated rats

Rats were treated with 1.0–2.0 × 10¹² particles/ml Ad-GPAT1 or Ad-EGFP for 5–7 days and food deprived 4 h before tissues were collected. Liver lipid metabolites were measured by mass spectrometry. A, total acyl-CoA content (inset) and acyl-CoA species (18:0-CoA, *, p < 0.03). B, total LPA content (inset) (**, p < 0.001) and LPA species (*, p < 0.01; **, p < 0.001). C, total DAG (inset) (**, p < 0.001) and DAG species (*, p < 0.05) (**, p < 0.001). Ad-EGFP, n = 9; Ad-GPAT1, n = 11.

FIGURE 7. Hepatic PKCε activity was elevated in Ad-GPAT1-treated rats

Rats were treated with 1.0–2.0 × 10¹² particles/ml Ad-GPAT1 or Ad-EGFP for 5–7 days and fasted for 4 h before tissues were collected. Liver cytosolic and membrane fractions were isolated, and the amount of PKCε in each fraction was determined by Western blot. Ad-EGFP, n = 4; Ad-GPAT1, n = 4.

FIGURE 8. NF-κB activity was lower in Ad-GPAT1-treated rats

Rats were treated with 1.0–2.0 × 10¹² particles/ml Ad-GPAT1 or Ad-EGFP for 5–7 days. Tissues were collected after a 4-h fast and NF-κB activity was measured in rat liver nuclear extracts with an enzyme-linked immunosorbent assay kit. Control (non-viral), n = 4; after a 4-h fast, Ad-EGFP, n = 19; Ad-GPAT1. n = 21. Results are presented as mean ± S.E.