Abstract
In mammals, a family of five acyl-CoA synthetases (ACSLs), each the product of a separate gene, activates long chain fatty acids to form acyl-CoAs. Because the ACSL isoforms have overlapping preferences for fatty acid chain length and saturation and are expressed in many of the same tissues, the individual function of each isoform has remained uncertain. Thus, we constructed a mouse model with a liver-specific knock-out of ACSL1, a major ACSL isoform in liver. Eliminating ACSL1 in liver resulted in a 50% decrease in total hepatic ACSL activity and a 25-35% decrease in long chain acyl-CoA content. Although the content of triacylglycerol was unchanged in Acsl1(L)(-/-) liver after mice were fed either low or high fat diets, in isolated primary hepatocytes the absence of ACSL1 diminished the incorporation of [(14)C]oleate into triacylglycerol. Further, small but consistent increases were observed in the percentage of 16:0 in phosphatidylcholine and phosphatidylethanolamine and of 18:1 in phosphatidylethanolamine and lysophosphatidylcholine, whereas concomitant decreases were seen in 18:0 in phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine, and lysophosphatidylcholine. In addition, decreases in long chain acylcarnitine content and diminished production of acid-soluble metabolites from [(14)C]oleate suggested that hepatic ACSL1 is important for mitochondrial beta-oxidation of long chain fatty acids. Because the Acsl1(L)(-/-) mice were not protected from developing either high fat diet-induced hepatic steatosis or insulin resistance, our study suggests that lowering the content of hepatic acyl-CoA without a concomitant decrease in triacylglycerol and other lipid intermediates is insufficient to protect against hepatic insulin resistance.
Highlights
It has been suggested that lipid intermediates, including fatty acid (FA), long chain acyl-CoAs, DAG, ceramide, and phosphatidic acid, rather than TAG accumulation per se, might underlie the development of insulin resistance (10 –13)
This model has allowed us to determine whether ACSL1 deficiency protects liver from hepatic steatosis and alters the incorporation of FAs into specific glycerolipids and to learn whether a decrease in long chain acyl-CoA content is sufficient to protect the liver from high fat diet-induced insulin resistance
Acsl1LϪ/Ϫ were crossed with Acsl1Flox/Flox littermates lacking the Alb-Cre transgene (Acsl1Flox/Flox-Alb-Cre0/0) to yield Acsl1LϪ/Ϫ mice and Acsl1Flox/Flox mice
Summary
Generation of Acsl1LϪ/Ϫ Mice—Liver-specific ACSL1 knockout mice (Acsl1LϪ/Ϫ) were created by LoxP-Cre strategy [21]. For high fat diet studies, 8-week-old Acsl1LϪ/Ϫ and control (Acsl1Flox/Flox) mice were fed a high fat Western diet (45% calories from fat (mostly lard), 35% from carbohydrate (sucrose and corn starch) Research Diets. At ϳ25 weeks, mice were killed, and tissues were snap frozen in liquid nitrogen and analyzed for enzyme activity, quantitative real time-PCR, Western blotting, and lipid content. GPAT specific activity was assayed with 20 – 40 g of liver homogenate at room temperature in a 200-l reaction mixture containing 75 mM Tris-HCl (pH 7.5), 4 mM MgCl2, 1 mg/ml bovine serum albumin (essentially FA-free), 1 mM dithiothreitol, 8 mM NaF, 800 M [3H]glycerol 3-phosphate, and 80 M palmitoyl-CoA [26]. A p value Ͻ 0.05 was considered significant unless otherwise indicated
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