• The role of glycerol-3-phosphate acyltransferase 1 in mitochondrial phospholipid biosynthesis of cold-bodied fishes

      Keenan, Kelly Anne; O'Brien, Kristin; Schulte, Marvin; López, Andrés; Harris, Michael (2015-08)
      Mitochondrial biogenesis is induced by low temperature in many fish species. For example, cold acclimation of Gasterosteus aculeatus (threespine stickleback) increases mitochondrial densities in oxidative skeletal muscle. Oxidative muscles of Antarctic icefishes (suborder Notothenioidei) also have high mitochondrial densities characterized by higher densities of phospholipids compared to red-blooded notothenioids. Mitochondrial biogenesis has been well studied in mammals yet it is unknown how mitochondrial phospholipid synthesis is regulated. I hypothesized that both activity and mRNA levels of glycerol-3-phosphate acyltransferase (GPAT), the rate-limiting enzyme in glycerolipid biosynthesis, would increase in oxidative muscle of stickleback, where mitochondrial biogenesis occurs, but not in liver, in response to cold acclimation, and that GPAT1 and /or GPAT2 mRNA levels would be higher in hearts of icefishes compared to red-blooded species. To test these hypotheses, maximal activity of GPAT and mRNA levels of GPAT1 and GPAT2 were measured in liver and oxidative muscle of coldand warm- acclimated stickleback. GPAT1 and GPAT2 mRNA levels were also quantified in hearts and livers of red- and white-blooded Antarctic notothenioids. Additionally, cDNA of GPAT1 was sequenced in Antarctic and sub-Antarctic notothenioids to gain insight to the evolution of a mitochondrial membrane protein and identify candidate amino acid residues responsible for maintaining function at cold temperature. GPAT activity increased in oxidative muscle but not in liver, and transcript levels of GPAT1 increased in liver but not in oxidative muscle, in response to cold acclimation in stickleback. GPAT2 transcripts were undetectable in both tissues. GPAT1 mRNA levels were highest in liver of red-blooded Antarctic notothenioids and did not differ in hearts between red- and white-blooded fishes, and GPAT2 transcripts were undetectable. GPAT protein levels may not change concurrently with GPAT1 and GPAT 2 mRNA levels because GPAT3 or 1-acylglycerol-3-phosphate acyltransferase (AGPAT), the enzyme subsequent to GPAT, may be involved in regulating phospholipid synthesis during mitochondrial biogenesis. The amino acid sequence of GPAT1 is highly conserved (97.94-98.06%) among Antarctic and sub-Antarctic notothenioids, with three potential sites in the cytosolic region that may be important for maintaining function at cold temperature: Ser415Ala, Asp603Glu and Thr648Ala.