Our goal in this report was to assess the impact of ACC inhibition on fatty acid elongation and desaturation in HepG2 and LnCap cells. The rationale for this analysis is based on the fact that malonyl CoA is a substrate for DNL and fatty acid elongation [18
] and a potent allosteric inhibitor of CPT1 activity and mitochondrial β-oxidation (FAO) [15
]. To date, however, no study has reported on the impact of ACC inhibition or ablation on fatty acid elongation. This issue becomes particularly relevant in light of the role fatty acid elongases play in the generation of the diverse array of saturated, mono and polyunsaturated fatty acids in cells. Changes in fatty acid content of cells have broad effects on metabolism, cell signaling, gene expression and impact the onset and progression of chronic diseases.
The increasing obesity epidemic has prompted approaches to manage the accumulation of neutral lipids in tissues because elevated neutral lipid storage promotes inflammation and insulin resistance [44
]. ACC is an attractive drug target because of its effects on DNL and FAO [12
]. In LnCap cells, soraphen A inhibits cell growth and promotes apoptosis [8
]. ACC inhibitors lower cellular malonyl CoA content and impact cholesterol and fatty acid metabolism, insulin sensitivity, cell growth and apoptosis [7
]. Unlike RNAi approaches [50
], however, chemical inhibition of ACC rapidly inhibits fatty acid synthesis () [13
]. Soraphen A was chosen for these studies because the mechanism of soraphen A inhibition of ACC is well defined [37
]. The rapidity of soraphen A inhibition on lipid metabolism minimizes non-specific or adaptive effects brought on by changes in cell fatty acid composition and cell growth. Recombinant adenoviral approaches were used to elevate elongase activity in an effort to assess the impact of this manipulation on DNL and FAO. In agreement with others [8
], soraphen A inhibited DNL and stimulates cholesterol synthesis in HepG2 and LnCap cells ( & ). The new information reported here is: 1) soraphen A inhibits elongation of products derived from DNL ( & ), and exogenous saturated (, & ) and unsaturated fatty acids (– & ). DNL, fatty acid elongation & PUFA synthesis are equally sensitive to soraphen A inhibition, IC50
~ 5 nM (). Soraphen A, however, has no direct effect on fatty acid elongation in isolated microsomes (). 2) Soraphen A augments the accumulation of unsaturated fatty acids derived from saturated and polyunsaturated fatty acid precursors (– & ). 3) Elevated expression of fatty acid elongases, Elovl5 and Elovl6, or fatty acid desaturases (FADS1 and FADS2) failed to override the inhibitory effect of soraphen A on fatty acid elongation, or synthesis of C18–22 MUFA or PUFA ( & ). These results establish that ACC inhibition not only impacts fatty acid elongation, but disrupts pathways where both elongases and desaturases are utilized to generate major MUFA and PUFA found in cells. These studies establish a strong link between ACC and SFA, MUFA and PUFA synthesis through the control of fatty acid elongase activity.
Based on mRNA analysis, ACC1 is the major ACC subtype expressed in HepG2 and LnCap cells (Fig. 1S, supplementary data
). Although soraphen A binds the BC domain of both ACC1 and ACC2 and inhibits ACC activity [37
], soraphen A likely targets ACC1 in both cell types. As such, ACC1 is likely the major source of malonyl CoA for microsomal fatty acid elongation in HepG2 and LnCap cells. Once ACC1 and ACC2-specific inhibitors become available, it will be interesting to determine if ACC2 contributes malonyl CoA to fatty acid elongation.
Addition of soraphen A to high fat diets was found to improve body weight, total body fat, peripheral insulin sensitivity & plasma glucose, insulin and β-hydroxybutyrate levels in C57BL/6 mice [7
]. These authors did not describe effects on tissue or plasma PUFA content. Based on our findings, dietary soraphen A or other ACC inhibitors are predicted to alter tissue and plasma MUFA and PUFA content. Inhibition of ACC affects PPAR regulated gene expression [49
]. PPARα is a well established target of fatty acid regulation[53
]. Changes in cellular fatty acid elongases activity affect cellular fatty acid content which in turn impacts PPARα activity [29
The effect of soraphen A on 16- and 18-carbon saturated and monounsaturated fatty acid synthesis may be beneficial. Soraphen A inhibits the elongation of fatty acids derived from DNL and exogenously supplied 16:0 (–). Inhibition of ACC, however, does not attenuate the formation of 16:1,n-7, a stearoyl CoA desaturation (SCD) product of 16:0. Soraphen A and ablation of SCD1 have a common effect on cell fatty acid composition; both treatments lower tissue content of 18:1 (n-7 and n-9). SCD1 ablation inhibits hepatic triglyceride accumulation and protects against diet-induced obesity [26
]. The soraphen A-induced accumulation of 16:1,n-7 in cells is noteworthy because 16:1,n-7 was recently reported as a lipokine capable of enhancing muscle insulin sensitivity [54
]. Since soraphen A inhibits elongation, and not desaturation, chronic use of ACC inhibitors might inhibit triglyceride synthesis and improve insulin sensitivity.
In contrast, ACC inhibition of PUFA synthesis raises concern (–). Soraphen A does not inhibit 18:2,n-6 desaturation by FADS2, its blocks the subsequent elongation of 18:3,n-6 to 20:3,n-6 a substrate for FADS1. Soraphen A blocks arachidonic (20:4,n-6) synthesis. Since the same enzymes are used for both n-3 and n-6 PUFA synthesis, we predict that dietary 18:3,n-3 will not be converted to 22:6,n-3. 20:4,n-6 and 22:6,n-3 are major PUFA that affect multiple cell function through diverse mechanisms, including membrane fluidity, cholesterol content & lipid raft integrity, eicosanoids & docosanoids, cytokine & adhesion molecule production, as well as controlling several cell signaling mechanisms & and effects on gene expression [53
]. An absence of essential fatty acids in the diet (i.e., essential fatty acid deficiency) or global ablation of a FADS2 leads to major problems with skin, reproduction, vision and learning [56
]. Disruption of PUFA synthesis through inhibition of ACC is likely to significantly affect multiple physiological systems.
Previous efforts to alter malonyl CoA metabolism involved over expression of malonyl CoA decarboxylase (MCD) (); elevated expression of MCD lowers hepatic malonyl CoA by 60%, reverses hepatic steatosis and improves whole body insulin sensitivity in high fat-fed mice [59
]. Since elevated hepatic Elovl5 activity lowers hepatic and plasma triglyceride content [29
] and lowers plasma glucose and insulin in high fat fed mice [60
], we speculated that elevated elongase activity in HepG2 cells might induce FAO. Elevation of Elovl5 and Elovl6 expression in HepG2 cells did not induce FAO of 16:0 or 18:2,n-6 (). Supra-physiological levels of Elovl5 stimulated 18:2,n-6 elongation to 20:2,n-6 by ~10-fold and FAO by ~2-fold (). Despite the fact that hepatic fatty acid elongases are regulated by age, diet and drugs [24
], the changes seen in Elovl5 & Elovl6 activity in vivo
do not approach the changes described in . Our results do not support a role for Elovl5 or Elovl6 in the suppression of cellular malonyl CoA to levels sufficient to activate FAO. The finding that elevated FADS2 stimulates both PUFA synthesis and FAO () suggests that altered PUFA synthesis might be a factor controlling hepatic lipid and carbohydrate metabolism. Further study will be required to define this mechanism.
Schematic illustrating effects of soraphen A on lipid metabolism
Finally, HepG2 cells have low FADS2 expression (Fig. 1S, supplementary data
) and 20:2,n-6 is the predominant product of 18:2,n-6 metabolism ( & ). Infection of HepG2 cells with Elovl5 increases 18:2,n-6 conversion to 20:2,n-6. This fatty acid was recently described in studies with the FADS2 null mouse [42
]. LnCap cells, in contrast, express high levels of FADS2 and generate robust levels of 20:3,n-6 and 20:4,n-6 from 18:2,n-6 metabolism. The paucity of FADS2 activity is not unique to HepG2 cells. Other hepatoma cell lines (LO2, McA-RH777) as well as rat primary hepatocytes have low FADS2 expression and FADS2-mediated desaturation [61
]. Results with primary cells and established cell lines contrast with the level of expression of FADS2 in rat, mouse & human liver [24
]. The reason for the deficient FADS2 expression in hepatoma cell lines and rat primary hepatocytes is unknown. Since Ad-FADS2 infection of HepG2 cells restores desaturation of 18:2,n-6, other factors required for fatty acid desaturation are not limiting (). Defining the impact of pharmacological regulators of fatty acid synthesis requires using cells with the capacity for DNL, MUFA and PUFA synthesis.
In summary, inhibition of ACC activity increases FAO and fatty acid desaturation, but blocks DNL and fatty acid elongation. Inhibition of ACC leads to a significant shift in fatty acid metabolism where SFA and essential fatty acids are desaturated but not elongated. This impairs the formation of key fatty acids e.g., 18:1,n-9, 20:4,n-6 and 22:6,n-3, and promotes the accumulation of 16:1,n-7 and 18:3,n-6. How such changes in tissue fatty acid profiles impact cell function remain to be determined. ACC plays an important role in SFA, MUFA and PUFA synthesis through mechanism that are independent of its role in DNL and FAO.