Polyunsaturated Fatty Acid Desaturation Is a Mechanism for Glycolytic NAD+ Recycling

Abstract

The reactions catalyzed by the delta-5 and delta-6 desaturases (D5D/D6D), key enzymes responsible for highly unsaturated fatty acid (HUFA) synthesis, regenerate NAD⁺ from NADH. Here, we show that D5D/D6D provide a mechanism for glycolytic NAD⁺ recycling that permits ongoing glycolysis and cell viability when the cytosolic NAD⁺/NADH ratio is reduced, analogous to lactate fermentation. Although lesser in magnitude than lactate production, this desaturase-mediated NAD⁺ recycling is acutely adaptive when aerobic respiration is impaired in vivo. Notably, inhibition of either HUFA synthesis or lactate fermentation increases the other, underscoring their interdependence. Consistent with this, a type 2 diabetes risk haplotype in SLC16A11 that reduces pyruvate transport (thus limiting lactate production) increases D5D/D6D activity in vitro and in humans, demonstrating a chronic effect of desaturase-mediated NAD⁺ recycling. These findings highlight key biologic roles for D5D/D6D activity independent of their HUFA end products and expand the current paradigm of glycolytic NAD⁺ regeneration.

Keywords: FADS1-3; NAD(+) recycling; SLC16A11; delta-5-desaturase; delta-6-desaturase; highly unsaturated fatty acids; polyunsaturated fatty acids.

Figure 1.. Inhibition of aerobic respiration increases lipid HUFA content in vitro

(A-G) Effect of 24h rotenone (Rot, 200 nM) treatment of IMCD3 cells on (A) media glucose consumption and lactate secretion; (B) media free fatty acid levels, including linoleic acid (LA), alpha-linolenic acid (α-LA), gamma-linolenic acid (γ-LA), arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA); intracellular (C) cholesterol esters, (D) phosphatidylcholines, (E) diacylglycerols, and (F) triacylglycerols (TAGs) measured by LC-MS, differentiated along the x-axis on the basis of total acyl chain carbon and double bond content; and (G) total TAGs measured by colorimetric assay. Values are means ± SEM; *P < 0.05, **P < 0.01; ***P < 0.001; n = 3. See also Figure S1.

Figure 2.. Inhibition of aerobic respiration increases D5D and D6D activity

(A) Time course of rotenone effect on cellular TAGs. (B) Top, western blot of D5D in IMCD3 cells following 2, 6, 12, and 24h of rotenone (200 nM) or vehicle. Representative gel from one of two independent experiments. Bottom, qPCR of Fads1 and Fads2 in IMCD3 cells following 6h rotenone (200 nM) or vehicle. (C) Confocal micrograph of D5D (green), the ER marker reticulon 4 (RTN4, red), or merged image (yellow) in IMCD3 cells following 6h of 200 nM rotenone or vehicle. (D-E) Isotope-labeled (D) free fatty acid products and (E) substrate of D5D and D6D activity in cultured media of IMCD3 cells following 6h and 24h of rotenone (200 nM) or vehicle. (F) Intracellular TAGs in IMCD3 cells following 24h of rotenone (200 nM) with or without the desaturase inhibitors SC-26196 (10 μM) or CP-24879 (30 μM). Values denote means ± SEM; ns, P > 0.05.; **P < 0.01; ***P < 0.001; n = 3. See also Figure S2 and Table S1.

Figure 3.. Increasing the cytosolic NAD⁺/NADH ratio with an NADH oxidase reduces D5D and D6D activity

(A) Schema for D5D and D6D mediated desaturation and NAD⁺ recycling. (B) Effect of rotenone on total cellular NAD⁺/NADH and NADP⁺/NAPDH ratios. NAD⁺/NADH ratio (left) and NADP⁺/NADPH ratio (right) measured using an enzymatic assay of cell lysates from IMCD3 cells treated with rotenone (200 nM) or vehicle for 6 hours. (C) Effect of rotenone (100 nM) treatment of HeLa cells, with or without LbNOX expression, on cytosolic NAD⁺/NADH measured using the ratio of fluorescence intensities excited at 485 nm and 430 nm in SoNar expressing cells. (D) qPCR (left) of FADS1 and FADS2 in HeLa cells with or without LbNOX expression (+/− Dox); western blot (right) of D5D in HeLa cells with or without LbNOX expression (+/− Dox), 6h following rotenone (200 nM) or vehicle. (E and F) Effect of LbNOX expression on HeLa cell TAGs measured by LC-MS (E) relative to vehicle, or (F) with or without rotenone (100 nM at 6h). Values are means ± SEM; ns, P > 0.05; ***P < 0.001; n = 3 for all experiments except n = 10 for (C).

Figure 4.. Modulating the cytosolic NAD⁺/NADH ratio via LDH activity modulates D5D and D6D activity

Schema for LDH mediated NAD⁺ recycling; alpha-ketobutyrate (AKB), alpha-hydroxybutyrate (AHB), NHI-2 (LDH inhibitor). (B-G) Effect of rotenone (200 nM) treatment of IMCD3 cells, with or without AKB (ImM), on (B) intracellular AHB at 24h; (C) cytosolic NAD⁺/NADH measured with SoNar; (D) intracellular TAGs at 24h; (E) intracellular citrate, isocitrate, and succinate at 24h; and (F and G) isotope-labeled free fatty acids in media at 6h and 24h. (H-I) Effect of NHI-2 (30 μM) and rotenone (100 nM) treatment of IMCD3 cells on (H) cytosolic NAD⁺/NADH measured with SoNar, where box plot shows upper and lower quartiles and whiskers show maximum and minimum values; and (I) intracellular TAGs at 24h. Values denote means ± SEM; ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; n = 3 for all experiments except n = 10 for (C, H). See also Figure S3.

Figure 5.. D5D and D6D inhibition reduces cytosolic NAD⁺/NADH, increases LDH activity, and impairs cell proliferation

(A-B) Cytosolic NAD⁺/NADH measured with SoNar in (A) HeLa cells transfected with FADS1 or FADS2 cDNA or shRNA targeting FADS1 or FADS2; and (B) IMCD3 cells treated with rotenone (200 nM) or the desaturase inhibitors SC-26196 (10 μM) and CP-24879 (30 μM). (C-D) Media lactate, pyruvate, and/or pyruvate/lactate ratio from IMCD3 cells treated with (C) rotenone (200 nM) or increasing concentrations of CP-24879 (CP); and (D) CP-24789 (30 μM) with or without AKB (1 mM). (E-H) Cell proliferation at 3 days for (E) IMCD3 cells treated with increasing concentrations of CP-24789; (F) IMCD3 cells treated with vehicle, CP-24789 (30 μM), CP-24789 (30 μM) and pyruvate (1 mM), CP-24789 (30 μM) and AKB (1 mM), rotenone (200 nM), and rotenone (200 nM) and AKB (1 mM); (G) HeLa cells transfected with shRNA targeting FADS1 and FADS2 or control; and (H) IMCD3 cells treated with CP-24789 (30 μM) with AKB (1 mM) and/or a mixture containing 10 μM each of arachidonic acid (AA), eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA). (I) Schema for D5D and D6D activity and NAD⁺ recycling in relation to glycolysis and lactate fermentation. Values are means ± SEM; ns, P > 0.05; *P < 0.05; **P < 0.01; ***P < 0.001; n = 20–30 for (A), n = 10 for (B), n = 3 for (C, D), and n = 4 for (E-H). See also Figure S4.

Figure 6.. Acute increase in HUFA synthesis is adaptive when aerobic respiration is impaired in vivo

(A-C) Effect of 10 h rotenone (2.8 mg/kg, intraperitoneal) treatment in 11-week old C57BL/6J mice on (A) heart, kidney, liver, and skeletal muscle (S. Mus) tissue TAGs measured by LC-MS, (B)total tissue TAGs measured by colorimetric assay, (C) tissue lactate measured by LC-MS. (D and E) Western blot of D5D for (D) different tissue samples from one vehicle (V, Veh) and one rotenone (R, Rot)-treated mouse and (E) kidney homogenates from all vehicle and rotenone-treated mice. Representative gels from one of two independent experiments. (F) Kidney tissue Fadsl and Fads2 mRNA from all vehicle and rotenone-treated mice. Values are means ± SEM; ns, P> 0.05; *P <0.05; **P < 0.01; n = 3 mice per group. (G-J) Effect of co-treatment of CP-24879 (5 mg/kg, intraperitoneal) and rotenone (1.7 mg/kg, intraperitoneal) in 11-week old C57BL/6J mice on (G) NAD⁺/NADH ratio in kidney tissue; (H) renal tubular damage assessed by H&E staining (asterisk: protein casts, arrow head: apoptotic epithelial cells, arrow: tubular necrosis); (I) plasma creatinine and blood urea nitrogen (BUN); and (J) plasma aminotransferases (ALT and AST). Values are means ± SEM; *P < 0.05; **P < 0.01; ***P < 0.001; n = 3 mice per group. See also Figure S5 and S6.

Figure 7.. The SLC16A11 diabetes risk haplotype reduces cytosolic NAD⁺/NADH ratio and increases HUFA synthesis in humans

(A) Western blot of SLC16A11^T2D-V5 and SLC16A11^REF-V5 in HEK293 cells, representative gel from one of two independent experiments. (B) qPCR of SLC16A11 in HEK293 cells expressing empty vector, SLC16A11^T2D-V5, or SLC16A11^ref-V5. (C) Cytosolic NAD+/NADH measured with SoNar in HEK293 cells expressing either SLC16A11^T2D-V5 or SLC16A11^REF-V5. (D) Intracellular TAGs in HEK293 cells expressing SLC16A11^T2D-V5 or SLC16A11^REF-V5 (red), or SLC16A11^T2D-V5 with or without 1mM pyruvate supplementation (blue). Values are means ± SEM; ***P < 0.001; n = 3 for (B, D), and n = 40 for (C). (E) Plasma TAGs among carriers of the SLC16A11 risk haplotype (n=364) relative to controls (n=313). Values are means; *P<0.05; **P < 0.01. (F) Mexico City Diabetes Study clinical characteristics at time of lipid profiling (G) Schema for interaction between SLC16A11, cytosolic NAD⁺/NADH, and HUFA synthesis. See also Figure S7.