MicroRNA regulation of adrenal glucocorticoid and androgen biosynthesis.

Steroid hormones are derived from a common precursor molecule, cholesterol, and regulate a wide range of physiologic function including reproduction, salt balance, maintenance of secondary sexual characteristics, response to stress, neuronal function, and various metabolic processes. Among the steroids synthesized by the adrenal and gonadal tissues, adrenal mineralocorticoids, and glucocorticoids are essential for life. The process of steroidogenesis is regulated at multiple levels largely by transcriptional, posttranscriptional, translational, and posttranslational regulation of the steroidogenic enzymes (i.e., cytochrome P450s and hydroxysteroid dehydrogenases), cellular compartmentalization of the steroidogenic enzymes, and cholesterol processing and transport proteins. In recent years, small noncoding RNAs, termed microRNAs (miRNAs) have been recognized as major post-transcriptional regulators of gene expression with essential roles in numerous biological processes and disease pathologies. Although their role in the regulation of steroidogenesis is still emerging, several recent studies have contributed significantly to our understanding of the role miRNAs play in the regulation of the steroidogenic process. This chapter focuses on the recent developments in miRNA regulation of adrenal glucocorticoid and androgen production in humans and rodents.

Early enforcement of cell identity by a functional component of the terminally differentiated state.

How progenitor cells can attain a distinct differentiated cell identity is a challenging problem given the fluctuating signaling environment in which cells exist and that critical transcription factors are often not unique to a differentiation process. Here, we test the hypothesis that a unique differentiated cell identity can result from a core component of the differentiated state doubling up as a signaling protein that also drives differentiation. Using live single-cell imaging in the adipocyte differentiation system, we show that progenitor fat cells (preadipocytes) can only commit to terminally differentiate after up-regulating FABP4, a lipid buffer that is highly enriched in mature adipocytes. Upon induction of adipogenesis in mouse preadipocyte cells, we show that after a long delay, cells first abruptly start to engage a positive feedback between CEBPA and PPARG before then engaging, after a second delay, a positive feedback between FABP4 and PPARG. These sequential positive feedbacks both need to engage in order to drive PPARG levels past the threshold for irreversible differentiation. In the last step before commitment, PPARG transcriptionally increases FABP4 expression while fatty acid-loaded FABP4 increases PPARG activity. Together, our study suggests a control principle for robust cell identity whereby a core component of the differentiated state also promotes differentiation from its own progenitor state.

Mice deficient in ER protein seipin have reduced adrenal cholesteryl ester lipid droplet formation and utilization.

Cholesteryl ester (CE)-rich lipid droplets (LDs) accumulate in steroidogenic tissues under physiological conditions and constitute an important source of cholesterol as the precursor for the synthesis of all steroid hormones. The mechanisms specifically involved in CE-rich LD formation have not been directly studied and are assumed by most to occur in a fashion analogous to triacylglycerol-rich LDs. Seipin is an endoplasmic reticulum protein that forms oligomeric complexes at endoplasmic reticulum-LD contact sites, and seipin deficiency results in severe alterations in LD maturation and morphology as seen in Berardinelli-Seip congenital lipodystrophy type 2. While seipin is critical for triacylglycerol-rich LD formation, no studies have directly addressed whether seipin is important for CE-rich LD biogenesis. To address this issue, mice with deficient expression of seipin specifically in adrenal, testis, and ovary, steroidogenic tissues that accumulate CE-rich LDs under normal physiological conditions, were generated. We found that the steroidogenic-specific seipin-deficient mice displayed a marked reduction in LD and CE accumulation in the adrenals, demonstrating the pivotal role of seipin in CE-rich LD accumulation/formation. Moreover, the reduction in CE-rich LDs was associated with significant defects in adrenal and gonadal steroid hormone production that could not be completely reversed by addition of exogenous lipoprotein cholesterol. We conclude that seipin has a heretofore unappreciated role in intracellular cholesterol trafficking.

Flattening of circadian glucocorticoid oscillations drives acute hyperinsulinemia and adipocyte hypertrophy.

Disruption of circadian glucocorticoid oscillations in Cushing's disease and chronic stress results in obesity and adipocyte hypertrophy, which is believed to be a main source of the harmful effects of obesity. Here, we recapitulate stress due to jet lag or work-life imbalances by flattening glucocorticoid oscillations in mice. Within 3 days, mice achieve a metabolic state with persistently high insulin, but surprisingly low glucose and fatty acids in the bloodstream, that precedes a more than 2-fold increase in brown and white adipose tissue mass within 3 weeks. Transcriptomic and Cd36-knockout mouse analyses show that hyperinsulinemia-mediated de novo fatty acid synthesis and Cd36-mediated fatty acid uptake drive fat mass increases. Intriguingly, this mechanism by which glucocorticoid flattening causes acute hyperinsulinemia and adipocyte hypertrophy is unexpectedly beneficial in preventing high levels of circulating fatty acids and glucose for weeks, thus serving as a protective response to preserve metabolic health during chronic stress.

Hormone sensitive lipase ablation promotes bone regeneration.

There is an inverse relationship between the differentiation of mesenchymal stem cells (MSCs) along either an adipocyte or osteoblast lineage, with lineage differentiation known to be mediated by transcription factors PPARγ and Runx2, respectively. Endogenous ligands for PPARγ are generated during the hydrolysis of triacylglycerols to fatty acids through the actions of lipases such as hormone sensitive lipase (HSL). To examine whether reduced production of endogenous PPARγ ligands would influence bone regeneration, we examined the effects of HSL knockout on fracture repair in mice using a tibial mono-cortical defect as a model. We found an improved rate of fracture repair in HSL-ko mice documented by serial µCT and bone histomorphometry compared to wild-type (WT) mice. Similarly, accelerated rates of bone regeneration were observed with a calvarial model where implantation of bone grafts from HSL-ko mice accelerated bone regeneration at the injury site. Further analysis revealed improved MSC differentiation down osteoblast and chondrocyte lineage with inhibition of HSL. MSC recruitment to the injury site was greater in HSL-ko mice than WT. Finally, we used single cell RNAseq to understand the osteoimmunological differences between WT and HSL-ko mice and found changes in the pre-osteoclast population. Our study shows HSL-ko mice as an interesting model to study improvements to bone injury repair. Furthermore, our study highlights the potential importance of pre-osteoclasts and osteoclasts in bone repair.

Hormone-sensitive lipase protects adipose triglyceride lipase-deficient mice from lethal lipotoxic cardiomyopathy.

Lipid droplets (LDs) are multifunctional organelles that regulate energy storage and cellular homeostasis. The first step of triacylglycerol hydrolysis in LDs is catalyzed by adipose triglyceride lipase (ATGL), deficiency of which results in lethal cardiac steatosis. Although hormone-sensitive lipase (HSL) functions as a diacylglycerol lipase in the heart, we hypothesized that activation of HSL might compensate for ATGL deficiency. To test this hypothesis, we crossed ATGL-KO (AKO) mice and cardiac-specific HSL-overexpressing mice (cHSL) to establish homozygous AKO mice and AKO mice with cardiac-specific HSL overexpression (AKO+cHSL). We found that cardiac triacylglycerol content was 160-fold higher in AKO relative to Wt mice, whereas that of AKO+cHSL mice was comparable to the latter. In addition, AKO cardiac tissues exhibited reduced mRNA expression of PPARα-regulated genes and upregulation of genes involved in inflammation, fibrosis, and cardiac stress. In contrast, AKO+cHSL cardiac tissues exhibited expression levels similar to those observed in Wt mice. AKO cardiac tissues also exhibited macrophage infiltration, apoptosis, interstitial fibrosis, impaired systolic function, and marked increases in ceramide and diacylglycerol contents, whereas no such pathological alterations were observed in AKO+cHSL tissues. Furthermore, electron microscopy revealed considerable LDs, damaged mitochondria, and disrupted intercalated discs in AKO cardiomyocytes, none of which were noted in AKO+cHSL cardiomyocytes. Importantly, the life span of AKO+cHSL mice was comparable to that of Wt mice. HSL overexpression normalizes lipotoxic cardiomyopathy in AKO mice and the findings highlight the applicability of cardiac HSL activation as a therapeutic strategy for ATGL deficiency-associated lipotoxic cardiomyopathies.

SNAP25 mutation disrupts metabolic homeostasis, steroid hormone production and central neurobehavior.

OBJECTIVE: SNAP-25 is one of the key proteins involved in formation of soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complexes that are at the core of hormonal secretion and synaptic transmission. Altered expression or function of SNAP-25 can contribute to the development of neuropsychiatric and metabolic disease. A dominant negative (DN) I67T missense mutation in the b-isoform of SNAP-25 (DN-SNAP25mut) mice leads to abnormal interactions within the SNARE complex and impaired exocytotic vesicle recycling, yet the significance of this mutation to any association between the central nervous system and metabolic homeostasis is unknown. METHODS: Here we explored aspects of metabolism, steroid hormone production and neurobehavior of DN-SNAP25mut mice. RESULTS: DN-SNAP25mut mice displayed enhanced insulin function through increased Akt phosphorylation, alongside increased adrenal and gonadal hormone production. In addition, increased anxiety behavior and beigeing of white adipose tissue with increased energy expenditure were observed in mutants. CONCLUSIONS: Our results show that SNAP25 plays an important role in bridging central neurological systems with peripheral metabolic homeostasis, and provide potential insights between metabolic disease and neuropsychiatric disorders in humans.

Hormone-sensitive lipase deficiency affects the expression of SR-BI, LDLr, and ABCA1 receptors/transporters involved in cellular cholesterol uptake and efflux and disturbs fertility in mouse testis.

Hormone-sensitive lipase (HSL) hydrolyse acylglycerols, cholesteryl and retinyl esters. HSL is a key lipase in mice testis, as HSL deficiency results in male sterility. The present work study the effects of the deficiency and lack of HSL on the localization and expression of SR-BI, LDLr, and ABCA1 receptors/transporters involved in uptake and efflux of cholesterol in mice testis, to determine the impact of HSL gene dosage on testis morphology, lipid homeostasis and fertility. The results of this work show that the lack of HSL in mice alters testis morphology and spermatogenesis, decreasing sperm counts, sperm motility and increasing the amount of Leydig cells and lipid droplets. They also show that there are differences in the localization of HSL, SR-BI, LDLr and ABCA1 in HSL+/+, HSL+/- and HSL-/- mice. The deficiency or lack of HSL has effects on protein and mRNA expression of genes involved in lipid metabolisms in mouse testis. HSL-/- testis have augmented expression of SR-BI, LDLr, ABCA1 and LXRß, a critical sterol sensor that regulate multiple genes involved in lipid metabolism; whereas LDLr expression decreased in HSL+/- mice. Plin2, Abca1 and Ldlr mRNA levels increased; and LXRα (Nr1h3) and LXRß (Nr1h2) decreased in testis from HSL-/- compared with HSL+/+; with no differences in Scarb1. Together these data suggest that HSL deficiency or lack in mice testis induces lipid homeostasis alterations that affect the cellular localization and expression of key receptors/transporter involved in cellular cholesterol uptake and efflux (SR-BI, LDRr, ABCA1); alters normal cellular function and impact fertility.

Chemerin regulates formation and function of brown adipose tissue: Ablation results in increased insulin resistance with high fat challenge and aging.

Apart from its role in inflammation and immunity, chemerin is also involved in white adipocyte biology. To study the role of chemerin in adipocyte metabolism, we examined the function of chemerin in brown adipose tissue. Brown and white adipocyte precursors were differentiated into adipocytes in the presence of Chemerin siRNA. Chemerin-deficient (Chem-/- ) mice were compared to wild-type mice when fed a high-fat diet. Chemerin is expressed during brown adipocyte differentiation and knock down of chemerin mRNA results in decreased brown adipocyte differentiation with reduced fatty acid uptake in brown adipocytes. Chem-/- mice are leaner than wild-type mice but gain more weight when challenged with high-fat diet feeding, resulting in a larger increase in fat deposition. Chem-/- mice develop insulin resistance when on a high-fat diet or due to age. Brown adipose depots in Chem-/- mice weigh more than in wild-type mice, but with decreased mitochondrial content and function. Compared to wild-type mice, male Chem-/- mice have decreased oxygen consumption, CO2 production, energy expenditure, and a lower respiratory exchange ratio. Additionally, body temperature of Chem-/- mice is lower than that of wild-type mice. These results revealed that chemerin is expressed during brown adipocyte differentiation and has a pivotal role in energy metabolism through brown adipose tissue thermogenesis.

SOD2 deficiency-induced oxidative stress attenuates steroidogenesis in mouse ovarian granulosa cells.

This study investigated the effects of SOD2 (MnSOD)-deficiency-induced excessive oxidative stress on ovarian steroidogenesis in vivo and isolated and cultured granulosa cells using WT and Sod2+/- mice. Basal and 48 h eCG-stimulated plasma progesterone levels were decreased ~50% in female Sod2+/- mice, whereas plasma progesterone levels were decreased ~70% in Sod2+/- mice after sequential stimulation with eCG followed by hCG. Sod2+/- deficiency caused about 50% reduction in SOD2 activity in granulosa cells. SOD2-deficiency also caused a marked reduction in progestins and estradiol in isolated granulosa cells. qRT-PCR measurements indicated that the mRNA expression levels of StAR protein and steroidogenic enzymes are decreased in the ovaries of Sod2+/- mice. Further studies showed a defect in the movement of mobilized cytosolic cholesterol to mitochondria. The ovarian membrane from Sod2+/- mice showed higher susceptibility to lipid peroxidation. These data indicates that SOD2-deficiency induced oxidative stress inhibits ovarian granulosa cell steroidogenesis primarily by interfering with cholesterol transport to mitochondria and attenuating the expression of Star protein gene and key steroidogenic enzyme genes.

Identification of p115 as a novel ACSL4 interacting protein and its role in regulating ACSL4 degradation.

Long-chain acyl-CoA synthetase 4 (ACSL4) is an ACSL family member that exhibits unique substrate preference for arachidonic acid. ACSL4 has a functional role in hepatic lipid metabolism, and is dysregulated in non-alcoholic fatty liver disease. Our previous studies demonstrated AA-induced ACSL4 degradation via the ubiquitin-proteasomal pathway (UPP). To characterize this unique mechanism, we applied proteomic approaches coupled with LC-MS/MS and identified the intracellular general vesicular trafficking protein p115 as the prominent ACSL4 interacting protein in HepG2 cells. Importantly, we found that AA greatly enhanced p115-ACSL4 association. Combined AA treatment with p115 knockdown suggested an additive role for p115 in AA-driven ACSL4 degradation. Furthermore, in vivo studies revealed a significant upregulation of p115 protein in the liver of mice fed a high fat diet that has been previously reported to induce downregulation of ACSL4 protein expression. This new finding has revealed a novel inverse correlation between ACSL4 and p115 proteins in the liver. p115 is crucial for ER-Golgi trafficking and Golgi biogenesis. Thus far, p115 has not been reported to interact with UPP proteins nor with FA metabolism enzymes. Overall, our current study provides a novel insight into the connection between ER-Golgi trafficking and UPP machinery with p115 as a critical mediator. SIGNIFICANCE: ACSL4 is uniquely regulated by its own substrate AA, and in this study, we have found that AA leads to an enhanced interaction of ACSL4 with a novel interacting partner, the intracellular vesicle trafficking protein p115. The latter is crucial for Golgi biogenesis and ER-Golgi transport and is not known to be associated with the ubiquitin-proteasome machinery or protein stability regulation until now. This study is the first report of a possible coordination of the protein secretion pathway and the UPP in regulating a key metabolic enzyme. Our study lays the foundation to this unique crosstalk between the two major cellular pathways- secretion and protein degradation and opens up a new avenue to explore this partnership in controlling hepatic lipid metabolism. Overall, the complete elucidation of the AA-mediated ACSL4 regulation will help identify key targets in participating pathways that can be further studied for the development of therapeutics against diseases such as NAFLD, NASH and hepatocarcinoma, which are associated with dysregulated ACSL4 function.

Slc43a3 is a regulator of free fatty acid flux.

Adipocytes take up long chain FAs through diffusion and protein-mediated transport, whereas FA efflux is considered to occur by diffusion. To identify potential membrane proteins that are involved in regulating FA flux in adipocytes, the expression levels of 55 membrane transporters without known function were screened in subcutaneous adipose samples from obese patients before and after bariatric surgery using branched DNA methodology. Among the 33 solute carrier (SLC) transporter family members screened, the expression of 14 members showed significant changes before and after bariatric surgery. One of them, Slc43a3, increased about 2.5-fold after bariatric surgery. Further investigation demonstrated that Slc43a3 is highly expressed in murine adipose tissue and induced during adipocyte differentiation in primary preadipocytes and in OP9 cells. Knockdown of Slc43a3 with siRNA in differentiated OP9 adipocytes reduced both basal and forskolin-stimulated FA efflux, while also increasing FA uptake and lipid droplet accumulation. In contrast, overexpression of Slc43a3 decreased FA uptake in differentiated OP9 cells and resulted in decreased lipid droplet accumulation. Therefore, Slc43a3 seems to regulate FA flux in adipocytes, functioning as a positive regulator of FA efflux and as a negative regulator of FA uptake.

FXR activation promotes intestinal cholesterol excretion and attenuates hyperlipidemia in SR-B1-deficient mice fed a high-fat and high-cholesterol diet.

Obeticholic acid (OCA) activates the farnesoid X receptor (FXR) to lower circulating total cholesterol (TC) and high density lipoprotein-cholesterol (HDL-C) concentrations and to stimulate fecal cholesterol excretion in mice by increasing hepatic SR-B1 expression. Here we show that hepatic SR-B1 depletion by an adenovirus expressing Sr-b1 shRNA (Ad-shSR-B1) attenuated these beneficial effects of OCA in mice on a chow diet. The mRNA levels of ABC cholesterol transporter genes (Abca1, Abcg1, Abcg5, and Abcg8) were unchanged in the liver of hepatic SR-B1-depleted mice regardless of OCA treatment; however, a modest increase in Abca1, Abcg5, and Abcg8 mRNA levels was observed in the ileum of vehicle-treated control mice and Abca1 and Abcg8 mRNA levels were increased more by OCA administration. OCA treatment of Sr-b1 knock out (KO) mice (Sr-b1-/-) fed a normal chow diet (NCD) displayed a similar lack of transhepatic cholesterol movement, as well as a modest increase in the levels of ileum cholesterol transporter expression. However, OCA treatment of Sr-b1 KO mice fed a cholesterol-enriched diet reduced circulating cholesterol and increased fecal cholesterol output to comparable degrees to that of wild-type (WT) mice, and these effects were accompanied by substantial elevations of mRNA levels of Abca1, Abcg1, Abcg5, and Abcg8 in the ileum of Sr-b1 KO mice. Our studies suggest that FXR activation stimulates intestinal cholesterol excretion and reduces diet-induced hyperlipidemia through increased expression of ileal cholesterol transporters when hepatic SR-B1-mediated cholesterol movement is absent.

Molecular changes in hepatic metabolism in ZDSD rats-A new polygenic rodent model of obesity, metabolic syndrome, and diabetes.

In recent years, the prevalence of obesity, metabolic syndrome and type 2 diabetes is increasing dramatically. They share pathophysiological mechanisms and often lead to cardiovascular diseases. The ZDSD rat was suggested as a new animal model to study diabetes and the metabolic syndrome. In the current study, we have further characterized metabolic and hepatic gene expression changes in ZDSD rats. Immuno-histochemical staining of insulin and glucagon on pancreas sections of ZDSD and control SD rats revealed that ZDSD rats have severe damage to their islet structures as early as 15 weeks of age. Animals were followed till they were 26 weeks old, where they exhibited obesity, hypertension, hyperglycemia, dyslipidemia, insulin resistance and diabetes. We found that gene expressions involved in glucose metabolism, lipid metabolism and amino acid metabolism were changed significantly in ZDSD rats. Elevated levels of ER stress markers correlated with the dysregulation of hepatic lipid metabolism in ZDSD rats. Key proteins participating in unfolded protein response pathways were also upregulated and likely contribute to the pathogenesis of dyslipidemia and insulin resistance. Based on its intact leptin system, its insulin deficiency, as well as its timeline of disease development without diet manipulation, this insulin resistant, dyslipidemic, hypertensive, and diabetic rat represents an additional, unique polygenic animal model that could be very useful to study human diabetes.

The role of miRNAs in regulating adrenal and gonadal steroidogenesis.

miRNAs are endogenous noncoding single-stranded small RNAs of ~22 nucleotides in length that post-transcriptionally repress the expression of their various target genes. They contribute to the regulation of a variety of physiologic processes including embryonic development, differentiation and proliferation, apoptosis, metabolism, hemostasis and inflammation. In addition, aberrant miRNA expression is implicated in the pathogenesis of numerous diseases including cancer, hepatitis, cardiovascular diseases and metabolic diseases. Steroid hormones regulate virtually every aspect of metabolism, and acute and chronic steroid hormone biosynthesis is primarily regulated by tissue-specific trophic hormones involving transcriptional and translational events. In addition, it is becoming increasingly clear that steroidogenic pathways are also subject to post-transcriptional and post-translational regulations including processes such as phosphorylation/dephosphorylation, protein‒protein interactions and regulation by specific miRNAs, although the latter is in its infancy state. Here, we summarize the recent advances in miRNA-mediated regulation of steroidogenesis with emphasis on adrenal and gonadal steroidogenesis.

Scavenger receptor class B, type 1 facilitates cellular fatty acid uptake.

SR-B1 belongs to the class B scavenger receptor, or CD36 super family. SR-B1 and CD36 share an affinity for a wide array of ligands. Although they exhibit similar ligand binding specificity, SR-B1 and CD36 have some very specific lipid transport functions. Whereas SR-B1 primarily facilitates the selective delivery of cholesteryl esters (CEs) and cholesterol from HDL particles to the liver and non-placental steroidogenic tissues, as well as participating in cholesterol efflux from cells, CD36 primarily mediates the uptake of long-chain fatty acids in high fatty acid-requiring organs such as the heart, skeletal muscle and adipose tissue. However, CD36 also mediates cholesterol efflux and facilitates selective lipoprotein-CE delivery, although less efficiently than SR-B1. Interestingly, the ability or efficiency of SR-B1 to mediate fatty acid uptake has not been reported. In this paper, using overexpression and siRNA-mediated knockdown of SR-B1, we show that SR-B1 possesses the ability to facilitate fatty acid uptake. Moreover, this function is not blocked by BLT-1, a specific chemical inhibitor of HDL-CE uptake activity of SR-B1, nor by sulfo-N-succinimidyl oleate, which inhibits fatty acid uptake by CD36. Attenuated fatty acid uptake was also observed in primary adipocytes isolated from SR-B1 knockout mice. In conclusion, facilitation of fatty acid uptake is an additional function that is mediated by SR-B1.

Cardiac overexpression of perilipin 2 induces atrial steatosis, connexin 43 remodeling, and atrial fibrillation in aged mice.

Atrial fibrillation (AF) is prevalent in patients with obesity and diabetes, and such patients often exhibit cardiac steatosis. Since the role of cardiac steatosis per se in the induction of AF has not been elucidated, the present study was designed to explore the relation between cardiac steatosis and AF. Transgenic (Tg) mice with cardiac-specific overexpression of perilipin 2 (PLIN2) were housed in the laboratory for more than 12 mo before the study. Electron microscopy of the atria of PLIN2-Tg mice showed accumulation of small lipid droplets around mitochondrial chains, and five- to ninefold greater atrial triacylglycerol (TAG) content compared with wild-type (WT) mice. Electrocardiography showed significantly longer RR intervals in PLIN2-Tg mice than in WT mice. Transesophageal electrical burst pacing resulted in significantly higher prevalence of sustained (>5 min) AF (69%) in PLIN2-Tg mice than in WT mice (24%), although it was comparable in younger (4-mo-old) mice. Connexin 43 (Cx43), a gap junction protein, was localized at the intercalated disks in WT atria but was heterogeneously distributed on the lateral side of cardiomyocytes in PLIN2-Tg atria. Langendorff-perfused hearts using the optical mapping technique showed slower and heterogeneous impulse propagation in PLIN2-Tg atria compared with WT atria. Cardiac overexpression of hormone-sensitive lipase in PLIN2-Tg mice resulted in atrial TAG depletion and amelioration of AF susceptibility. The results suggest that PLIN2-induced steatosis is associated with Cx43 remodeling, impaired conduction propagation, and higher incidence of AF in aged mice. Therapies targeting cardiac steatosis could be potentially beneficial against AF in patients with obesity or diabetes.

Tissue-Specific Ablation of ACSL4 Results in Disturbed Steroidogenesis.

ACSL4 is a member of the ACSL family that catalyzes the conversion of long-chain fatty acids to acyl-coenzyme As, which are essential for fatty-acid incorporation and utilization in diverse metabolic pathways, including cholesteryl ester synthesis. Steroidogenic tissues such as the adrenal gland are particularly enriched in cholesteryl esters of long-chain polyunsaturated fatty acids, which constitute an important pool supplying cholesterol for steroid synthesis. The current studies addressed whether ACSL4 is required for normal steroidogenesis. CYP11A1 promoter‒mediated Cre was used to generate steroid tissue‒specific ACSL4 knockout (KO) mice. Results demonstrated that ACSL4 plays an important role in adrenal cholesteryl ester formation, as well as in determining the fatty acyl composition of adrenal cholesteryl esters, with ACSL4 deficiency leading to reductions in cholesteryl ester storage and alterations in cholesteryl ester composition. Statistically significant reductions in corticosterone and testosterone production, but not progesterone production, were observed in vivo, and these deficits were accentuated in ex vivo and in vitro studies of isolated steroid tissues and cells from ACSL4-deficient mice. However, these effects on steroid production appear to be due to reductions in cholesteryl ester stores rather than disturbances in signaling pathways. We conclude that ACSL4 is dispensable for normal steroidogenesis.

Creosote bush-derived NDGA attenuates molecular and pathological changes in a novel mouse model of non-alcoholic steatohepatitis (NASH).

Creosote bush (Larrea tridentata)-derived nordihydroguaiaretic acid (NDGA) was shown to have profound effects on the core components of metabolic syndrome. This study investigated the in vivo potential of NDGA for prevention or attenuation of the pathophysiologic abnormalities of NASH. A novel dietary NASH model with feeding C57BL/6J mice with a high trans-fat, high cholesterol and high fructose (HTF) diet, was used. The HTF diet fed mice exhibited obesity, insulin resistance, hepatic steatosis, fibrosis, inflammation, ER stress, oxidative stress, and liver injury. NDGA attenuated these metabolic abnormalities as well as hepatic steatosis and fibrosis together with attenuated expression of genes encoding fibrosis, progenitor and macrophage markers with no effect on the levels of mRNAs for lipogenic enzymes. NDGA increased expression of fatty acid oxidation genes. In conclusion, NDGA exerts anti-NASH/anti-fibrotic actions and raises the therapeutic potential of NDGA for treatment of NASH patients with fibrosis and other associated complications.