SUMMARY 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.

This article is available online at http://www.jlr.org identifi ed as essential fatty acids that must be consumed in the diet. Once consumed, however, LA and ALA can both be desaturated and elongated into more highly unsaturated fatty acids (HUFA) such as arachidonic (AA), docosapentaenoic (DPA n-6), and docosahexaenoic (DHA) acids via the pathway shown in Fig. 1A . Delta-6 desaturase (D6D) performs the fi rst and rate-limiting step in this process, as well as the last step of desaturation for DHA and DPA n-6 synthesis. The D6D gene FADS2 was cloned in 1999 ( 2 ), and subsequently, a human case of D6D deficiency was identifi ed ( 3 ). The patient exhibited growth retardation accompanied by skin abnormalities, corneal ulceration, and feeding intolerance. Treatment with dietary AA and DHA restored normal growth and eliminated most other symptoms, underscoring the importance of the endogenous synthesis of these HUFAs.AA is a precursor to a host of signaling molecules known as eicosanoids, which include thromboxanes, leukotrienes, prostacyclins, and prostaglandins produced from the oxygenation of AA by cyclooxygenase and lipoxygenase enzymes. However, the symptoms of classic essential fatty acid defi ciency, growth retardation and dermatitis ( 1 ), are attributed to a loss of LA, not AA or eicosanoids. Because LA is an essential component of skin ceramides, LA defi ciency results in the disruption of the skin's water barrier function ( 4 ) and heat loss from skin ( 5 ). These side effects make investigation of AA defi ciency impossible by dietary manipulation without complications from LA defi ciency.DHA is found in large amounts in the retina, brain, and testes ( 6, 7 ). The role of DHA has been largely thought to be structural, increasing the fl uidity of cellular memAbstract Delta-6 desaturase (D6D) catalyzes the fi rst 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 defi ciency without depleting their precursors. In ؊ / ؊ , no in vivo AA synthesis was detected after administration of [U-

This article is available online at http://www.jlr.org Supplementary key words essential fatty acids • arachidonic acid • highly unsaturated fatty acids • very-long-chain polyunsaturated fatty acids • choelsta-3,5-diene • male reproduction • spermiogenesis Delta-6 desaturase (D6D) is the fi rst and rate-limiting enzyme for highly unsaturated fatty acid (HUFA) synthesis that consists of a series of elongation and desaturation reactions ( 1 ). The dietary essential fatty acids 18:2n-6 (linoleic acid) and 18:3n-3 ( ␣ -linolenic acid) are substrates for D6D and precursors of physiologically important HUFAs, such as 20:4n-6 [arachidonic acid (AA)], 22:5n6 [docosapentaenoic acid (DPAn6)], and 22:6n3 [docosahexaenoic acid (DHA)]. D6D is also required for the fi nal desaturation step for the synthesis of DPAn6 and DHA.These HUFAs are present in high concentration in testes and sperm of mammals. DPAn6, a HUFA derived from AA, dramatically increases in rat testes during the sexual maturation stage ( 2 ). In mice, AA, DPAn6, and DHA are abundant in membrane phospholipids of round spermatids ( 3 ) and mature mouse spermatozoa ( 4 ), suggesting an important role for these fatty acids for proper spermatogenesis. In humans, DHA is the main HUFA in sperm ( 5 ). DHA is specifi cally high in the sperm tail when compared with the sperm head in monkeys ( 6 ), implying a role of DHA in sperm tail function. AA may also have a role in male fertility as a precursor to eicosanoids. Prostaglandin E2, for example, has been shown to increase sperm motility ( 7 ), while inhibition of cycloxygenase-2 in mouse vas deferens results in a decrease of sperm motility and fertilAbstract 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 ( Abbreviations: AA, arachidonic acid; CD, cholesta-3,5-diene; D6D, ⌬ 6 desaturase; DHA, docosahexaenoic acid; DPAn6, docosapentaenoic acid; FAME, fatty acid methyl esters; HUFA, highly unsaturated fatty acid; VLPUFA, very-long-chain polyunsaturated fatty acid.

Sterol 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 micro-RNA locus that is activated directly by SREBP-2. Two of the encoded miRs, miR-182 and miR-96, negatively regulate expression of Fbxw7 and Insig-2 respectively, and both are known to negatively affect nuclear SREBP accumulation. Direct manipulation of this miR pathway alters nuclear SREBP levels and endogenous lipid synthesis. Thus, we have uncovered a new mechanism for regulation of intracellular lipid metabolism mediated by the concerted action of a pair of miRs that are expressed from the same SREBP-2 regulated miR locus and each targets a different protein of the multi-step pathway that regulates SREBP function. These studies reveal a miR “operon” analogous to the classic model for genetic control in bacterial regulatory systems.

SUMMARY 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 on active SREBPs. 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.

An omega-3 fatty acid, docosahexaenoic acid (DHA), is enriched in testicular membrane phospholipids, but its function is not well understood. The Fads2 gene encodes an enzyme required for the endogenous synthesis of DHA. Using Fads2-null mice (Fads2-/-), we found in our preceding studies that DHA deficiency caused the arrest of spermiogenesis and male infertility, both of which were reversed by dietary DHA. In this study, we investigated a cellular mechanism underlying the DHA essentiality in spermiogenesis. Periodic acid-Schiff staining and acrosin immunohistochemistry revealed the absence of acrosomes in Fads2-/- round spermatids. Acrosin, an acrosomal marker, was scattered throughout the cytoplasm of the Fads2-/- spermatids, and electron microscopy showed that proacrosomal granules were formed on the trans-face of the Golgi. However, excessive endoplasmic reticulum and vesicles were present on the cis-face of the Golgi in Fads2-/- spermatids. The presence of proacrosomal vesicles but lack of a developed acrosome in Fads2-/- spermatids suggested failed vesicle fusion. Syntaxin 2, a protein involved in vesicle fusion, colocalized with acrosin in the acrosome of wild-type mice. In contrast, syntaxin 2 remained scattered in reticular structures and showed no extensive colocalization with acrosin in the Fads2-/- spermatids, suggesting failed fusion with acrosin-containing vesicles or failed transport and release of syntaxin 2 vesicles from Golgi. Dietary supplementation of DHA in Fads2-/- mice restored an intact acrosome. In conclusion, acrosome biogenesis under DHA deficiency is halted after release of proacrosomal granules. Misplaced syntaxin 2 suggests an essential role of DHA in proper delivery of membrane proteins required for proacrosomal vesicle fusion.

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