Protein Kinase A Subunit Balance Regulates Lipid Metabolism in Caenorhabditis elegans and Mammalian Adipocytes

Abstract

Protein kinase A (PKA) is a cyclic AMP (cAMP)-dependent protein kinase composed of catalytic and regulatory subunits and involved in various physiological phenomena, including lipid metabolism. Here we demonstrated that the stoichiometric balance between catalytic and regulatory subunits is crucial for maintaining basal PKA activity and lipid homeostasis. To uncover the potential roles of each PKA subunit, Caenorhabditis elegans was used to investigate the effects of PKA subunit deficiency. In worms, suppression of PKA via RNAi resulted in severe phenotypes, including shortened life span, decreased egg laying, reduced locomotion, and altered lipid distribution. Similarly, in mammalian adipocytes, suppression of PKA regulatory subunits RIα and RIIβ via siRNAs potently stimulated PKA activity, leading to potentiated lipolysis without increasing cAMP levels. Nevertheless, insulin exerted anti-lipolytic effects and restored lipid droplet integrity by antagonizing PKA action. Together, these data implicate the importance of subunit stoichiometry as another regulatory mechanism of PKA activity and lipid metabolism.

Keywords: adipose triglyceride lipase (ATGL); lipid droplet; lipolysis; protein kinase A (PKA); stoichiometry.

FIGURE 1.

PKA subunit knockdown modulates PKA activity in C. elegans. A, in the basal state, catalytic subunits [C] of PKA are efficiently suppressed by regulatory subunits [R]. When the levels of regulatory subunits decrease, catalytic subunits are derepressed and stimulate PKA's kinase activity and downstream signaling. B, simple inhibitor model of PKA subunit stoichiometry and activity. [C]_a, free catalytic subunits; [C]_t, total catalytic subunits; [CR], catalytic subunits bound to regulatory subunits; [R], free regulatory subunits; [R]_t, total regulatory subunits; K_d, dissociation constant. C, graphical expression of the equation in B under the condition that K_d = 0.1 nm. D, transcriptional GFP reporter strains of kin-1 and kin-2 promoters visualized with fluorescence microscopy. E, relative mRNA levels of kin-1 and kin-2. F, PKA activity assays using the Kemptide substrate and total protein extracts obtained from young adult worms after RNAi in the presence or absence of dibutyryl-cAMP (db-cAMP) and protein kinase A inhibitor peptide (PKI). Error bars represent standard deviations; **, p < 0.01.

FIGURE 2.

PKA subunit alteration induces redistribution of lipids in C. elegans. A, differential interference contrast images of young adult worms grown on control, kin-1 RNAi, and kin-2 RNAi plates. B, life span assay (n = 60–90). C, bending rates of young adult worms in liquid media (n = 24). D, pumping rates of young adult worms in control, kin-1 RNAi, and kin-2 RNAi plates. E, number of eggs laid/h by young adult worms on control, kin-1 RNAi, and kin-2 RNAi plates (n = 10). F, triglyceride levels in young adult worms grown on control, kin-1 RNAi, and kin-2 RNAi plates. Triglyceride levels were normalized to total protein. G, images of C. elegans stained with BODIPY-conjugated fatty acid, Nile Red, and Oil Red O. BODIPY-conjugated fatty acid and Nile Red staining were performed on live worms, whereas Oil Red O stain was completed after fixation. H, high magnification observation of Oil Red O-stained images of control and kin-2 RNAi worms. White dashed lines indicate intestinal cells. I, confocal microscopic images of atgl-1(hj67) worms grown under control, kin-1 RNAi, and kin-2 RNAi conditions. J, mRNA levels of fil-1, cpt-3, and atgl-1 measured by qRT-PCR and normalized to act-1/3 mRNA. **, p < 0.01 versus control.

FIGURE 3.

Tissue distribution of PKA subunit genes in mammals. A, relative mRNA levels of PKA subunit genes analyzed by qRT-PCR. Cyclophilin mRNA levels were considered as 1. B, mRNA levels of PKA subunit genes measured by qRT-PCR during adipocyte differentiation of 3T3-L1 cells. mRNA levels were normalized to cyclophilin mRNA levels, which were considered as 1.

FIGURE 4.

Knockdown of PKA RIα and RIIβ subunits activates PKA and lipolysis without cAMP increase in adipocytes. A–C, glycerol concentration in media from differentiated 3T3-L1 adipocytes after siRNA transfection. D, Nile Red staining images merged with differential interference contrast of differentiated 3T3-L1 adipocytes 48 h after siRNA transfection. E, Western blotting data showing phosphorylated Plin1 protein levels using anti-phospho-PKA substrate antibody. β-Actin was used as a loading control. F, intracellular cAMP levels were measured in adipocytes transfected with siRNAs. ISO, 1 μm isoproterenol was added to differentiated 3T3-L1 adipocytes for 15 min. G, glycerol concentration of media from fully differentiated 3T3-L1 adipocytes in the presence or absence of 1 μm ISO. H, glycerol concentration in media from hADSCs after siRNA transfection. Glycerol concentrations were normalized to total protein and time. I, BODIPY staining images of hADSCs. Error bars represent standard deviations; scale bars, 10 μm; **, p < 0.01 versus control; N.S., not significant.

FIGURE 5.

Knockdown of Atgl suppresses PKA activation-induced lipolysis. A, glycerol concentration in media from differentiated 3T3-L1 adipocytes 24 h after siRNA transfection. B, representative images of 3T3-L1 adipocytes 48 h after siRNA transfection. BODIPY 493/503 staining after fixation. C, Western blotting of lipolysis-related proteins in differentiated 3T3-L1 adipocytes 48 h after siRNA transfection. β-Actin protein was used as a loading control. Error bars represent standard deviations. D, measurement of glycerol concentration in media from differentiated 3T3-L1 adipocytes in the presence or absence of lipase inhibitors. Glycerol concentrations were normalized to total protein and time. Scale bars, 10 μm; **, p < 0.01.

FIGURE 6.

Insulin suppresses lipolysis in PKA-activated adipocytes. A, glycerol concentration in media from fully differentiated 3T3-L1 adipocytes 24 h after siRNA transfection. B, representative images of 3T3-L1 adipocytes 48 h after siRNA transfection. Cells were treated with insulin (100 nm) for 24 h and then stained with BODIPY 493/503 after fixation. C, lipid droplet diameters measured in confocal images using ImageJ software. D, Western blot of lipolysis-related proteins in differentiated 3T3-L1 adipocytes 48 h after siRNA transfection. β-Actin protein was used as a loading control. E, glycerol concentrations in media from fully differentiated 3T3-L1 adipocytes 24 h after siRNA transfection. Glycerol concentrations were normalized to total protein and time. F, representative images of 3T3-L1 adipocytes 48 h after siRNA transfection. Cells were treated with insulin (100 nm) for 24 h then stained with BODIPY 493/503 after fixation. G, Western blot of phospho-Akt, Akt, and Fsp27 in differentiated 3T3-L1 adipocytes 48 h after siRNA transfection. Cells were treated with insulin (100 nm) for 15 min after 3 h of serum starvation. β-Actin protein was used as a loading control. Error bars represent standard deviations. Scale bars, 10 μm; *, p < 0.05; **, p < 0.01.

FIGURE 7.

Working model. A, in the basal state, PKA catalytic subunits [C] are efficiently suppressed by regulatory subunits [R]. B, upon hormonal stimulation, cAMP levels increased and bind regulatory subunits, which lead to the dissociation of regulatory and catalytic subunits. C, suppression of regulatory subunits promotes lipolysis by increasing the amount of free catalytic subunits, thereby increasing PKA activity without cAMP changes. AC, adenylyl cyclase.