The role of dietary arachidonic acid and docosahexaenoic acid in preventing the phenotype observed with highly unsaturated fatty acid deficiency

The physiological roles of highly unsaturated fatty acids (HUFA), mainly arachidonic acid (AA, 20:4ω6) and docosahexaenoic acid (DHA, 22:6ω3), are not completely understood. In order to study specific functions for AA and DHA, a delta-6 desaturase knockout (D6D-/-) mouse was created. D6D is a key enzyme in synthesizing HUFA from the precursor dietary essential fatty acids, linoleic acid (LA, 18:2ω6) or α-linolenic acid (ALA, 18:3ω3). By disrupting D6D expression, LA and ALA provided in the diet will not be metabolized to HUFA. Phenotype of the D6D-/- mouse is therefore specific to lack of AA and/or DHA and consists of ulcerative dermatitis, male infertility, gastrointestinal ulcers, and hepatic lipidosis. New insight on specific AA and DHA roles was established through dietary prevention of HUFA deficiency phenotype. The absence of a D6D isozyme had to be assessed before further characterizing HUFA roles with the D6D-/- mouse model. The presence of a D6D isozyme would interfere with the creation of HUFA deficiency. The primary D6D isozyme candidate was Fads3 gene due to its increased gene expression in D6D-/- liver and homology to the Fads2 gene that encodes for D6D. Cloning and transfection of Fads3 into cultured HEK293 cells confirmed lack of D6D activity (Chapter 3). The order of appearance of D6D-/- phenotype due to HUFA deficiency had yet to be determined. A D6D-/- time course study (Chapter 4) characterized the mouse at different ages in order to follow sequence of HUFA deficiency pathology. The amount of HUFA in D6D-/- at weaning was comparable to control mouse indicating the presence of HUFA stores that most likely result from HUFA passed on from the mother. Subsequent HUFA depletion with age correlated with severity of D6D-/- phenotype. Male infertility, gastrointestinal erosions, and hepatic lipidosis are the first observed HUFA deficiency phenotype to appear at 6 weeks of age, followed by impaired antibody response at 9 weeks, and ulcerative dermatitis by 21 weeks of age. HUFA supplementation studies helped determine specific roles for AA and DHA in preventing HUFA deficiency phenotype. Hepatic lipidosis was prevented by either AA or DHA (Chapter 5). AA essentiality was specific to skin and gastrointestinal function since DHA supplementation was unsuccessful in preventing ulcerative dermatitis or gastrointestinal ulcers (Chapter 6). DHA essentiality was specific to male reproduction as indicated by full restoration of spermatogenesis, sperm counts, and sperm motility (Chapter 7). The role of DHA in spermatogenesis is related to acrosome biogenesis, a process which relies on vesicle fusion (Chapter 8). The immune system (Chapter 9) was further characterized following up on splenomegaly and thymic atrophy observations of the first characterization of the D6D-/-. HUFA deficiency results in decreased antibody response indicating essentiality for HUFA in immune function. In summary, these studies showed for the first time a specific requirement for AA in skin, and of DHA in male reproduction. The mechanism behind DHA requirement in male fertility has been linked to acrosome biogenesis. Future research done with the D6D-/- mouse model will help develop hypothesis on other potential mechanisms behind the essentiality of AA and DHA. Understanding how HUFA maintain tissue homeostasis will help in the development of treatments for diseases that result from an altered essential fatty acid metabolism

SETDB2 Links Glucocorticoid to Lipid Metabolism through Insig2a Regulation

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An SREBP-Responsive microRNA Operon Contributes to a Regulatory Loop for Intracellular Lipid Homeostasis

SummarySterol regulatory element-binding proteins (SREBPs) have evolved as a focal point for linking lipid synthesis with other pathways that regulate cell growth and survival. Here, we have uncovered a polycistrionic microRNA (miRNA) locus that is activated directly by SREBP-2. Two of the encoded miRNAs, miR-182 and miR-96, negatively regulate the expression of Fbxw7 and Insig-2, respectively, and both are known to negatively affect nuclear SREBP accumulation. Direct manipulation of this miRNA pathway alters nuclear SREBP levels and endogenous lipid synthesis. Thus, we have uncovered a mechanism for the regulation of intracellular lipid metabolism mediated by the concerted action of a pair of miRNAs that are expressed from the same SREBP-2-regulated miRNA locus, and each targets a different protein of the multistep pathway that regulates SREBP function. These studies reveal an miRNA “operon” analogous to the classic model for genetic control in bacterial regulatory systems

Setdb2 Links Glucocorticoid To Lipid Metabolism Through Insig2A Regulation

Transcriptional and chromatin regulations mediate the liver response to nutrient availability. The role of chromatin factors involved in hormonal regulation in response to fasting is not fully understood. We have identified SETDB2, a glucocorticoid-induced putative epigenetic modifier, as a positive regulator of GR-mediated gene activation in liver. Insig2a increases during fasting to limit lipid synthesis, but the mechanism of induction is unknown. We show Insig2a induction is GR-SETDB2 dependent. SETDB2 facilitates GR chromatin enrichment and is key to glucocorticoid-dependent enhancer-promoter interactions. INSIG2 is a negative regulator of SREBP, and acute glucocorticoid treatment decreased active SREBP during refeeding or in livers of Ob/Ob mice, both systems of elevated SREBP-1c-driven lipogenesis. Knockdown of SETDB2 or INSIG2 reversed the inhibition of SREBP processing. Overall, these studies identify a GR-SETDB2 regulatory axis of hepatic transcriptional reprogramming and identify SETDB2 as a potential target for metabolic disorders with aberrant glucocorticoid actions

Partitioning Circadian Transcription by SIRT6 Leads to Segregated Control of Cellular Metabolism

Circadian rhythms are intimately linked to cellular metabolism. Specifically, the NAD(+)-dependent deacetylase SIRT1, the founding member of the sirtuin family, contributes to clock function. Whereas SIRT1 exhibits diversity in deacetylation targets and subcellular localization, SIRT6 is the only constitutively chromatin-associated sirtuin and is prominently present at transcriptionally active genomic loci. Comparison of the hepatic circadian transcriptomes reveals that SIRT6 and SIRT1 separately control transcriptional specificity and therefore define distinctly partitioned classes of circadian genes. SIRT6 interacts with CLOCK:BMAL1 and, differently from SIRT1, governs their chromatin recruitment to circadian gene promoters. Moreover, SIRT6 controls circadian chromatin recruitment of SREBP-1, resulting in the cyclic regulation of genes implicated in fatty acid and cholesterol metabolism. This mechanism parallels a phenotypic disruption in fatty acid metabolism in SIRT6 null mice as revealed by circadian metabolome analyses. Thus, genomic partitioning by two independent sirtuins contributes to differential control of circadian metabolism

SETDB2 Links Glucocorticoid to Lipid Metabolism through Insig2a Regulation

Transcriptional and chromatin regulations mediate the liver response to nutrient availability. The role of chromatin factors involved in hormonal regulation in response to fasting is not fully understood. We have identified SETDB2, a glucocorticoid-induced putative epigenetic modifier, as a positive regulator of GR-mediated gene activation in liver. Insig2a increases during fasting to limit lipid synthesis, but the mechanism of induction is unknown. We show Insig2a induction is GR-SETDB2 dependent. SETDB2 facilitates GR chromatin enrichment and is key to glucocorticoid-dependent enhancer-promoter interactions. INSIG2 is a negative regulator of SREBP, and acute glucocorticoid treatment decreased active SREBP during refeeding or in livers of Ob/Ob mice, both systems of elevated SREBP-1c-driven lipogenesis. Knockdown of SETDB2 or INSIG2 reversed the inhibition of SREBP processing. Overall, these studies identify a GR-SETDB2 regulatory axis of hepatic transcriptional reprogramming and identify SETDB2 as a potential target for metabolic disorders with aberrant glucocorticoid action

Docosahexaenoic acid supplementation fully restores fertility and spermatogenesis in male delta-6 desaturase-null mice

Delta-6 desaturase-null mice (−/−) are unable to synthesize highly unsaturated fatty acids (HUFAs): arachidonic acid (AA), docosahexaenoic acid (DHA), and n6-docosapentaenoic acid (DPAn6). The −/− males exhibit infertility and arrest of spermatogenesis at late spermiogenesis. To determine which HUFA is essential for spermiogenesis, a diet supplemented with either 0.2% (w/w) AA or DHA was fed to wild-type (+/+) and −/− males at weaning until 16 weeks of age (n = 3–5). A breeding success rate of DHA-supplemented −/− was comparable to +/+. DHA-fed −/− showed normal sperm counts and spermiogenesis. Dietary AA was less effective in restoring fertility, sperm count, and spermiogenesis than DHA. Testis fatty acid analysis showed restored DHA in DHA-fed −/−, but DPAn6 remained depleted. In AA-fed −/−, AA was restored at the +/+ level, and 22:4n6, an AA elongated product, accumulated in testis. Cholesta-3,5-diene was present in testis of +/+ and DHA-fed −/−, whereas it diminished in −/− and AA-fed −/−, suggesting impaired sterol metabolism in these groups. Expression of spermiogenesis marker genes was largely normal in all groups. In conclusion, DHA was capable of restoring all observed impairment in male reproduction, whereas 22:4n6 formed from dietary AA may act as an inferior substitute for DHA

Disruption of FADS2 gene in mice impairs male reproduction and causes dermal and intestinal ulceration

Delta-6 desaturase (D6D) catalyzes the first step in the synthesis of highly unsaturated fatty acids (HUFA) such as arachidonic (AA), docosapentaenoic (DPAn-6), and docosahexaenoic (DHA) acids, as well as the last desaturation of DPAn-6 and DHA. We created D6D-null mice (−/−), which enabled us to study HUFA deficiency without depleting their precursors. In −/−, no in vivo AA synthesis was detected after administration of [U-13C]linoleic acid (LA), indicating absence of D6D isozyme. Unexpectedly, all of the −/− developed ulcerative dermatitis when fed a purified diet lacking D6D products but containing ample LA. The −/− also exhibited splenomegaly and ulceration in duodenum and ileocecal junction. Male −/− lacked normal spermatozoa with a severe impairment of spermiogenesis. Tissue HUFAs in −/− declined differentially: liver AA and DHA by 95%, and a smaller decrease in brain and testes. Dietary AA completely prevented dermatitis and intestinal ulcers in −/−. DPAn-6 was absent in −/− brain under AA supplementation, indicating absence of D6D isozyme for DPAn-6 synthesis from AA. This study demonstrated a distinct advantage of the D6D-null mice (−/−) to elucidate (1) AA function without complication of LA deprivation and (2) DHA function in the nervous system without AA depletion or DPAn-6 replacement seen in traditional models.—Stroud, C. K., T. Y. Nara, M. Roqueta-Rivera, E. C. Radlowski, P. Lawrence, Y. Zhang, B. H. Cho, M. Segre, R. A. Hess, J. T. Brenna, W. M. Haschek, and M. T. Nakamura. Disruption of FADS2 gene in mice impairs male reproduction and causes dermal and intestinal ulceration