193 - Pde3a Deficiency Plays a Novel Role in Lipolysis
Saturday, April 29, 2023
3:30 PM – 6:00 PM ET
Poster Number: 193 Publication Number: 193.213
Gabrielle McGeorge, Nationwide Children's Hospital, Westerville, OH, United States; Jenna L. McPeek, Nationwide Children's Hospital, Westerville, OH, United States; Leif D. Nelin, Nationwide Children's Hospital, Columbus, OH, United States; Yusen Liu, Abigail Wexner Research Institute at Nationwide Children's Hospital, Columbus, OH, United States; Bernadette Chen, Nationwide Children's Hospital, Columbus, OH, United States
Research Associate Nationwide Children's Hospital Westerville, Ohio, United States
Background: PDE3A and PDE3B hydrolyze cAMP and cGMP, which are critical second messengers important in cellular function. While PDE3B has been shown to have an anti-lipolytic effect, the role of PDE3A on lipolysis is not known. We have previously found that Pde3a-/- mice (3A-KO) have decreased adiposity, histopathologic liver disease, and increased serum-free FA (FFA) levels, while Pde3b-/- mice (3B-KO) have no obvious phenotype. Objective: To elucidate the etiology of high circulating FFA in 3A-KO mice and their effects on the liver. Design/Methods: 3-5 wk-old C57bl/6J (WT), 3A-KO and 3B-KO mice were sacrificed (n=3 per gender). White adipose tissue (AT) was isolated and protein quantified for PDE3A, PDE3B, hormone-sensitive lipase (HSL), adipose triglyceride lipase (ATGL), adiponectin, and b-actin by Western blot. Pre-adipocytes were isolated from WT mice, differentiated, and transfected with scramble, PDE3A (siPDE3A), or PDE3B (siPDE3B) siRNA. Media was collected and FFA measured. Hepatocytes were isolated from WT mice and transfected with scramble siRNA (scramble-WT) or siPDE3A. Scramble-WT hepatocytes were incubated for 48 hours with conditioned media from the siPDE3A-transfected adipocytes (scramble-WT-CM). Protein was harvested and quantified for fatty acid synthase (FAS), sterol regulatory element binding transcription factor 1 (SREB1c), cleaved and total caspase-3, diacylglycerol O-acyltransferase 2 (DGAT-2), pyruvate dehydrogenase kinase (PDK) 1-4, and b-actin by Western blot. Hepatocyte intracellular cAMP levels were measured. Results: PDE3A protein was increased in 3B-KO AT and siPDE3B adipocytes, whereas PDE3B was decreased in the siPDE3A adipocytes (p< 0.001). PDE3A deficient AT and adipocytes had increased HSL and ATGL and decreased adiponectin (p< 0.001) compared to PDE3B-deficient AT/cells. FFA levels in the media were significantly higher in siPDE3A, and lower in siPDE3B adipocytes than in scramble-WT. FAS and SREB1c were decreased, while DGAT-2, PDK 1-4, caspase 3 protein levels and intracellular cAMP levels were increased in both the scramble-WT-CM and siPDE3A compared to scramble-WT (p< 0.01).
Conclusion(s): These data suggest that the liver findings and potentially other organs affected in 3A-KO mice may be due to elevated cAMP levels in AT, thereby activating lipolysis to cause excess circulating FFA. We hypothesize that the FFA overwhelm the cells housed in organs highly utilizing FA oxidation and produce lipotoxic metabolites that cause tissue damage and mitochondrial dysfunction.
