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.

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.

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.

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.

Novel ABCA1 peptide agonists with antidiabetic action.

Previously, apoE-derived ABCA1 agonist peptides have been shown to possess anti-atherosclerotic and possibly antidiabetic properties. Here we assessed the in vitro and in vivo actions of a second generation of ABCA1 peptide agonists, CS6253 and T6991-2, on glucose homeostasis. The results show that these two peptides improve glucose tolerance in a prediabetic diet-induced obesity mouse model by enhancing insulin secretion. It was further demonstrated that T6991-2 also improved glucose tolerance in leptin-deficient (ob/ob) mice. CS6253 increased insulin secretion both under basal conditions and in response to high glucose stimulation in pancreatic INS-1 ß-cells rendered leptin receptor deficient with specific siRNA. Additional in vitro cell studies suggest that the CS6253 agonist attenuates hepatic gluconeogenesis and glucose transport. It also potentiates insulin-stimulated glucose uptake and utilization. These observed anti-diabetic actions suggest additional benefits of the CS6253 and T6991-2 ABCA1 peptide agonists for cardiovascular disease beyond their direct anti-atherosclerosis properties previously described.

Anti-hyperlipidaemic effects of synthetic analogues of nordihydroguaiaretic acid in dyslipidaemic rats.

BACKGROUND AND PURPOSE: Previous studies have shown that Creosote bush-derived nordihydroguaiaretic acid (NDGA) exerts beneficial actions on the key components of metabolic syndrome including dyslipidaemia, insulin resistance and hypertension in several relevant rodent models. Here, we synthesized and screened a total of 6 anti-hyperlipidaemic analogues of NDGA and tested their efficacy against hepatic lipid metabolism in a high-fructose diet (HFrD) fed dyslipidaemic rat model. EXPERIMENTAL APPROACH: HFrD fed Sprague-Dawley rats treated with NDGA or one of the six analogues were used. Serum samples were analysed for blood metabolites, whereas liver samples were quantified for changes in various mRNA levels by real-time RT-PCR. KEY RESULTS: Oral gavage of HFrD-fed rats for 4 days with NDGA analogues 1 and 2 (100 mg·kg-1 ·day-1 ) suppressed the hepatic triglyceride content, whereas the NDGA analogues 2, 3 and 4, like NDGA, decreased the plasma triglyceride levels by 70-75%. qRT-PCR measurements demonstrated that among NDGA analogues 1, 2, 4 and 5, analogue 4 was the most effective at inhibiting the mRNA levels of some key enzymes and transcription factors involved in lipogenesis. All four analogues almost equally inhibited the key genes involved in triglyceride synthesis and fatty acid elongation. Unlike NDGA, none of the analogues affected the genes of hepatic fatty acid oxidation or transport. CONCLUSIONS AND IMPLICATIONS: Our data suggest that NDGA analogues 1, 2, 4 and 5, particularly analogue 4, exert their anti-hyperlipidaemic actions by negatively targeting genes of key enzymes and transcription factors involved in lipogenesis, triglyceride synthesis and fatty acid elongation. These analogues have therapeutic potential.

Microarray analysis of gene expression in liver, adipose tissue and skeletal muscle in response to chronic dietary administration of NDGA to high-fructose fed dyslipidemic rats.

Nordihydroguaiaretic acid (NDGA), the main metabolite of Creosote Bush, has been shown to have profound effects on the core components of metabolic syndrome, including lowering of blood glucose, free fatty acids and triglyceride levels, attenuating elevated blood pressure in several rodent models of dyslipidemia, and improving body weight, insulin resistance, diabetes and hypertension. In the present study, a high-fructose diet fed rat model of hypertriglyceridemia, dyslipidemia, insulin resistance and hepatic steatosis was employed to investigate the global transcriptional changes in the lipid metabolizing pathways in three insulin sensitive tissues: liver, skeletal muscle and adipose tissue in response to chronic dietary administration of NDGA. Sprague-Dawley male rats (SD) were fed a chow (control) diet, high-fructose diet (HFrD) or HFrD supplemented with NDGA (2.5 g/kg diet) for eight weeks. Dietary administration of NDGA decreased plasma levels of TG, glucose, and insulin, and attenuated hepatic TG accumulation. DNA microarray expression profiling indicated that dietary administration of NDGA upregulated the expression of certain genes involved in fatty acid oxidation and their transcription regulator, PPARα, decreased the expression of a number of lipogenic genes and relevant transcription factors, and differentially impacted the genes of fatty acid transporters, acetyl CoA synthetases, elongases, fatty acid desaturases and lipid clearance proteins in liver, skeletal muscle and adipose tissues. These findings suggest that NDGA ameliorates hypertriglyceridemia and steatosis primarily by inhibiting lipogenesis and enhancing fatty acid catabolism in three major insulin responsive tissues by altering the expression of key enzyme genes and transcription factors involved in de novo lipogenesis and fatty acid oxidation.

SNARE-Mediated Cholesterol Movement to Mitochondria Supports Steroidogenesis in Rodent Cells.

Vesicular transport involving soluble N-ethylmaleimide sensitive factor attachment protein receptor (SNARE) proteins is known to be responsible for many major cellular activities. In steroidogenic tissues, chronic hormone stimulation results in increased expression of proteins involved in the steroidogenic pathway, whereas acute hormone stimulation prompts the rapid transfer of cholesterol to the inner mitochondrial membrane to be utilized as substrate for steroid hormone production. Several different pathways are involved in supplying cholesterol to mitochondria, but mobilization of stored cholesteryl esters appears to initially constitute the preferred source; however, the mechanisms mediating this cholesterol transfer are not fully understood. To study the potential contribution of SNARE proteins in steroidogenesis, we examined the expression levels of various SNARE proteins in response to hormone stimulation in steroidogenic tissues and cells and established an in vitro mitochondria reconstitution assay system to assess the contribution of various SNARE proteins on cholesterol delivery for steroidogenesis. Our results from reconstitution experiments along with knockdown studies in rat primary granulosa cells and in a Leydig cell line show that soluble N-ethylmaleimide sensitive factor attachment protein-α, synaptosomal-associated protein of 25 kDa, syntaxin-5, and syntaxin-17 facilitate the transport of cholesterol to mitochondria. Thus, although StAR is required for efficient cholesterol movement into mitochondria for steroidogenesis, specific SNAREs participate and are necessary to mediate cholesterol movement to mitochondria.

Effect of Creosote Bush-Derived NDGA on Expression of Genes Involved in Lipid Metabolism in Liver of High-Fructose Fed Rats: Relevance to NDGA Amelioration of Hypertriglyceridemia and Hepatic Steatosis.

Nordihydroguaiaretic acid (NDGA), the main metabolite of Creosote bush, has been shown to have profound effects on the core components of the metabolic syndrome (MetS), lowering blood glucose, free fatty acids (FFA) and triglyceride (TG) levels in several models of dyslipidemia, as well as improving body weight (obesity), insulin resistance, diabetes and hypertension, and ameliorating hepatic steatosis. In the present study, a high-fructose diet (HFrD) fed rat model of hypertriglyceridemia was employed to further delineate the underlying mechanism by which NDGA exerts its anti-hypertriglyceridemic action. In the HFrD treatment group, NDGA administration by oral gavage decreased plasma levels of TG, glucose, FFA, and insulin, increased hepatic mitochondrial fatty acid oxidation and attenuated hepatic TG accumulation. qRT-PCR measurements indicated that NDGA treatment increased the mRNA expression of key fatty acid transport (L-FABP, CD36), and fatty acid oxidation (ACOX1, CPT-2, and PPARα transcription factor) genes and decreased the gene expression of enzymes involved in lipogenesis (FASN, ACC1, SCD1, L-PK and ChREBP and SREBP-1c transcription factors). Western blot analysis indicated that NDGA administration upregulated hepatic insulin signaling (P-Akt), AMPK activity (P-AMPK), MLYCD, and PPARα protein levels, but decreased SCD1, ACC1 and ACC2 protein content and also inactivated ACC1 activity (increased P-ACC1). These findings suggest that NDGA ameliorates hypertriglyceridemia and hepatic steatosis primarily by interfering with lipogenesis and promoting increased channeling of fatty acids towards their oxidation.

The proteome of cholesteryl-ester-enriched versus triacylglycerol-enriched lipid droplets.

Within cells, lipids are stored in the form of lipid droplets (LDs), consisting of a neutral lipid core, surrounded by a phospholipid monolayer and an outer layer of protein. LDs typically accumulate either triacylglycerol (TAG) and diacylglycerol or cholesteryl ester (CE), depending on the type of tissue. Recently, there has been an increased interest in the proteins that surround LDs. LD proteins have been found to be quite diverse, from structural proteins to metabolic enzymes, proteins involved in vesicular transport, and proteins that may play a role in LD formation. Previous proteomics analyses have focused on TAG-enriched LDs, whereas CE-enriched LDs have been largely ignored. Our study has compared the LD proteins from CE-enriched LDs to TAG-enriched LDs in steroidogenic cells. In primary rat granulosa cells loaded with either HDL to produce CE-enriched LDs or fatty acids to produce TAG-enriched LDs, 61 proteins were found to be elevated in CE-enriched LDs and 40 proteins elevated in TAG-enriched LDs with 278 proteins in similar amounts. Protein expression was further validated by selected reaction monitoring (SRM) mass spectrometry (MS). SRM verified expression of 25 of 27 peptides that were previously detected by tandem mass tagging MS. Several proteins were confirmed to be elevated in CE-enriched LDs by SRM including the intermediate filament vimentin. This study is the first to compare the proteins found on CE-enriched LDs with TAG-enriched LDs and constitutes the first step in creating a better understanding of the proteins found on CE-enriched LDs in steroidogenic cells.

Hormonal regulation of microRNA expression in steroid producing cells of the ovary, testis and adrenal gland.

BACKGROUND: Given the emerging roles of miRNAs as potential posttranscriptional/posttranslational regulators of the steroidogenic process in adrenocortical and gonadal cells, we sought to determine miRNA profiles in rat adrenals from animals treated with vehicle, ACTH, 17α-E2 or dexamethasone. Key observations were also confirmed using hormone (Bt2cAMP)-treated mouse Leydig tumor cells, MLTC-1, and primary rat ovarian granulosa cells. METHODOLOGY: RNA was extracted from rat adrenal glands and miRNA profiles were established using microarray and confirmed with qRT-PCR. The expression of some of the hormone-sensitive miRNAs was quantified in MLTC-1 and granulosa cells after stimulation with Bt2cAMP. Targets of hormonally altered miRNAs were explored by qRT-PCR and Western blotting in adrenals and granulosa cells. RESULTS: Adrenals from ACTH, 17α-E2 and dexamethasone treated rats exhibited miRNA profiles distinct from control animals. ACTH up-regulated the expression of miRNA-212, miRNA-182, miRNA-183, miRNA-132, and miRNA-96 and down-regulated the levels of miRNA-466b, miRNA-214, miRNA-503, and miRNA-27a. The levels of miR-212, miRNA-183, miRNA-182, miRNA-132, miRNA-370, miRNA-377, and miRNA-96 were up-regulated, whereas miR-125b, miRNA-200b, miR-122, miRNA-466b, miR-138, miRNA-214, miRNA-503 and miRNA27a were down-regulated in response to 17α-E2 treatment. Dexamethasone treatment decreased miRNA-200b, miR-122, miR-19a, miRNA-466b and miRNA27a levels, but increased miRNA-183 levels. Several adrenal miRNAs are subject to regulation by more than one hormone. Significant cAMP-induced changes in certain miRNAs were also noted in MLTC-1 and granulosa cells. Some of the hormone-induced miRNAs in steroidogenic cells were predicted to target proteins involved in lipid metabolism/steroidogenesis. We also obtained evidence that miR-132 and miRNA-214 inhibit the expression of SREBP-1c and LDLR, respectively. CONCLUSION: Our results demonstrate that expression of a number of miRNAs in steroidogenic cells of the testis, ovary and adrenal glands is subject to hormonal regulation and that miRNAs and their regulation by specific hormones are likely to play a key role in posttranscriptional/posttranslational regulation of steroidogenesis.

Nordihydroguaiaretic acid improves metabolic dysregulation and aberrant hepatic lipid metabolism in mice by both PPARα-dependent and -independent pathways.

Creosote bush-derived nordihydroguaiaretic acid (NDGA), a lipoxygenase inhibitor, possesses antioxidant properties and functions as a potent antihyperlipidemic agent in rodent models. Here, we examined the effect of chronic NDGA treatment of ob/ob mice on plasma dyslipidemia, hepatic steatosis, and changes in hepatic gene expression. Feeding ob/ob mice a chow diet supplemented with either low (0.83 g/kg diet) or high-dose (2.5 g/kg diet) NDGA for 16 wk significantly improved plasma triglyceride (TG), inflammatory chemokine levels, hyperinsulinemia, insulin sensitivity, and glucose intolerance. NDGA treatment caused a marked reduction in liver weight and TG content, while enhancing rates of fatty acid oxidation. Microarray analysis of hepatic gene expression demonstrated that NDGA treatment altered genes for lipid metabolism, with genes involved in fatty acid catabolism most significantly increased. NDGA upregulated the mRNA and nuclear protein levels of peroxisome proliferator-activated receptor α (PPARα), and the activated (phosphorylated) form of AMP-activated kinase. NDGA increased PPARα promoter activity in AML12 hepatocytes and also prevented the fatty acid suppression of PPARα expression. In contrast, PPARα siRNA abrogated the stimulatory effect of NDGA on fatty acid catabolism. Likewise, no stimulatory effect of NDGA on hepatic fatty acid oxidation was observed in the livers of PPARα-deficient mice, but the ability of NDGA to reverse fatty liver conditions was unaffected. In conclusion, the beneficial actions of NDGA on dyslipidemia and hepatic steatosis in ob/ob mice are exerted primarily through enhanced fatty acid oxidation via PPARα-dependent pathways. However, PPARα-independent pathways also contribute to NDGA's action to ameliorate hepatic steatosis.

Ablation of vimentin results in defective steroidogenesis.

In steroidogenic tissues, cholesterol must be transported to the inner mitochondrial membrane to be converted to pregnenolone as the first step of steroidogenesis. Whereas steroidogenic acute regulatory protein has been shown to be responsible for the transport of cholesterol from the outer to the inner mitochondrial membrane, the process of how cholesterol moves to mitochondria from the cytoplasm is not clearly defined. The involvement of the cytoskeleton has been suggested; however, no specific mechanism has been confirmed. In this paper, using genetic ablation of an intermediate filament protein in mice, we present data demonstrating a marked defect in adrenal and ovarian steroidogenesis in the absence of vimentin. Cosyntropin-stimulated corticosterone production is decreased 35 and 50% in male and female Vimentin null (Vim(-/-)) mice, respectively, whereas progesterone production is decreased 70% in female Vim(-/-) mice after pregnant mare's serum gonadotropin and human chorionic gonadotropin stimulation, but no abnormalities in human chorionic gonadotropin-stimulated testosterone production is observed in male Vim(-/-) mice. These defects in steroid production are also seen in isolated adrenal and granulosa cells in vitro. Further studies show a defect in the movement of cholesterol from the cytosol to mitochondria in Vim(-/-) cells. Because the mobilization of cholesterol from lipid droplets and its transport to mitochondria is a preferred pathway for the initiation of steroid production in the adrenal and ovary but not the testis and vimentin is a droplet-associated protein, our results suggest that vimentin is involved in the movement of cholesterol from its storage in lipid droplets to mitochondria for steroidogenesis.

A new HDL mimetic peptide that stimulates cellular cholesterol efflux with high efficiency greatly reduces atherosclerosis in mice.

Here, we report the creation of a single-helix peptide (ATI-5261) that stimulates cellular cholesterol efflux with K(m) molar efficiency approximating native apolipoproteins. Anti-atherosclerosis activity of ATI-5261 was evaluated in LDLR(-/-) and apolipoprotein (apo)E(-/-) mice approximately 5-7 months of age, following 13-18 weeks on a high-fat Western diet (HFWD). Treatment of fat-fed LDLR(-/-) mice with daily intraperitoneal injections of ATI-5261 (30 mg/kg) for 6 weeks reduced atherosclerosis by 30%, as judged by lesion area covering the aorta (7.9 +/- 2 vs.11.3 +/- 2.5% control, P = 0.011) and lipid-content of aortic sinus plaque (25 +/- 5.8 vs. 33 +/- 4.9% control, P = 0.014). In apoE(-/-) mice, the peptide administered 30 mg/kg ip on alternate days for 6 weeks reduced atherosclerosis by approximately 45% (lesion area = 15 +/- 7 vs. 25 +/- 8% control, P = 0.00016; plaque lipid-content = 20 +/- 6 vs. 32 +/- 8% control, P < 0.0001). Similar reductions in atherosclerosis were achieved using ATI-5261:POPC complexes. Single intraperitoneal injection of ATI-5261 increased reverse cholesterol transport from macrophage foam-cells to feces over 24-48 h. In summary, relatively short-term treatment of mice with the potent cholesterol efflux peptide ATI-5261 reduced substantial atherosclerosis. This was achieved using an L-amino acid peptide, in the presence of severe hypercholesterolemia/HFWD, and did not require daily injections or formulation with phospholipids when administered via intraperitoneal injection.

Oxidative stress-induced inhibition of adrenal steroidogenesis requires participation of p38 mitogen-activated protein kinase signaling pathway.

Previous studies from this laboratory identified excessive oxidative stress as an important mediator of age-related decline in steroid hormone production. Here, we investigated whether oxidative stress exerts its antisteroidogenic action through modulation of oxidant-sensitive mitogen-activated protein kinase (MAPK) signaling pathways. To accomplish these studies, we employed a highly responsive mouse adrenocortical cell line, Y1-BS1 cells that secrete large quantities of steroids when stimulated with lipoprotein plus hormone. Treatment of these cells with superoxide, H(2)O(2) or 4-hydroxy-2-nonenal (HNE) significantly inhibited steroid production and increased phosphorylation and activation of p38 MAPK. None of the treatments altered the phosphorylation of either extracellular signal-regulated kinases or c-Jun N-terminal kinases (JNKs). Pretreatment of Y1-BS1 cells with MnTMPyP, a cell-permeable superoxide-dismutase/catalase mimetic reactive oxygen species (ROS scavenger), completely prevented the superoxide- and H(2)O(2)-mediated inhibition of steroid production. Likewise, antioxidant N-acetylcysteine completely blocked the HNE-induced loss of steroidogenic response. Incubation of Y1-BS1 cells with either MnTMPyP or NAC also upregulated Bt(2)cAMP and Bt(2)cAMP+hHDL(3)-stimulated steroid synthesis, indicating that endogenously produced ROS can inhibit steroidogenesis. Inhibition of p38 MAPK with SB203580 or SB202190 upregulated the basal steroid production and also prevented the oxidant-mediated inhibition of steroid production. mRNA measurements by qPCR indicated that Y1-BS1 adrenal cells predominantly express p38 MAPKalpha isoform, along with relatively low-level expression of p38 MAPKgamma. By contrast, little or no expression was detected for p38 MAPKbeta and p38 MAPKdelta isoforms in these cells. Transfection of Y1-BS1 cells with either caMKK3 or caMMK6 construct, the upstream p38 MAPK activators, decreased steroidogenesis, whereas transfection with dnMKK3 or dnMKK6 plasmid DNA increased steroidogenesis. Similarly, transfection of cells with a dnp38 MAPKalpha or dnp38 MAPKbeta construct also increased steroid hormone production; however, the effect was less pronounced after expression of either dnp38 MAPKgamma or dnp38 MAPKdelta construct. These results indicate that activated p38 MAPK mediates oxidant (excessive oxidative stress)-induced inhibition of adrenal steroidogenesis.

Evidence that age-related changes in p38 MAP kinase contribute to the decreased steroid production by the adrenocortical cells from old rats.

The current studies were initiated to investigate whether excessive oxidative stress exerts its antisteroidogenic action through modulation of oxidant-sensitive mitogen-activated protein kinase (MAPK) signaling pathways. Western blot analysis indicated that aging caused increased phosphorylation and activation of rat adrenal p38 MAPK, but not the ERK1/2 or JNK1/2. Lipid peroxidation measurements (an index of cellular oxidative stress) indicated that adrenal membranes from young animals contained only minimal levels of endogenous thiobarbituric acid-reactive substances (TBARS), and exposure of membranes to enzymatic and non-enzymatic pro-oxidants enhanced TBARS formation approximately 12- and 20-fold, respectively. The adrenal membranes from old animals showed much more susceptibility to lipid peroxidation and exhibited roughly 4- to 6-fold higher TBARS formation than young controls both under basal conditions and in response to pro-oxidants. Qualitatively similar results were obtained when lipid peroxide formation was measured using a sensitive FOXRS (ferrous oxidation-xylenol orange-reactive substances) technique. We next tested whether aging-induced excessive oxidative insult alters steroidogenesis through modulation of MAPK signaling pathway. Treatment of adrenocortical cells from old rats with specific p38 MAPK inhibitors restored Bt(2)cAMP-stimulated steroidogenesis approximately 60-70% of the value seen in cells of young animals. Likewise, pretreatment of cells with reactive oxygen species (ROS) scavengers MnTMPyP and N-acetyl cysteine also partially rescued age-induced loss of steroid production. In contrast, simultaneous treatment of cells with ROS scavengers and p38 MAPK inhibitor did not produce any additional effect suggesting that both types of inhibitors exert their stimulatory action through inhibition of p38 MAPK activation. Collectively, these results indicate that p38 MAPK functions as a signaling effector in oxidative stress-induced inhibition of steroidogenesis during aging.

Consequences of over-expression of rat Scavenger Receptor, SR-BI, in an adrenal cell model.

BACKGROUND: The plasma membrane scavenger receptor, SR-BI, mediates the 'selective uptake' process by which cholesteryl esters (CE) from exogenously supplied HDL are taken up by target cells. Recent work suggests that dimer and higher order oligomeric forms of the SR-BI protein are important to this process. SR-BI has been shown to be particularly associated with microvilli and microvillar channels found at the cell surface of steroidogenic cells, and a study with the hormone stimulated adrenal gland has shown impressive changes in the size and complexity of the microvillar compartment as the mass of CE uptake (and accompanying steroidogenesis) fluctuates. In the present study, we examine a cell line in which we overexpress the SR-BI protein to determine if morphological, biochemical and functional events associated with SR-BI in a controlled cell system are similar to those observed in the intact mammalian adrenal which is responsive to systemic factors. METHODS: Y1-BS1 mouse adrenocortical cells were transiently transfected using rat SR-BI-pcDNA6-V5-His, rat SR-BI-pcDNA6-cMyc-His or control pcDNA6-V5-His vector construct using a CaPO4 precipitation technique. Twenty four hours after transfection, cells were treated with, or without, Bt2cAMP, and SR-BI expression, CE uptake, and steroidogenesis was measured. SR-BI dimerization and cell surface architectural changes were assessed using immunoelectron microscopic techniques. RESULTS: Overexpression of the scavenger receptor protein, SR-BI, in Y1-BS1 cells results in major alterations in cell surface architecture designed to increase uptake of HDL supplied-CEs. Changes include 1 the formation of crater-like erosions of the surface with multiple double membraned channel structures lining the craters, and 2 dimerized formations of SR-BI lining the newly formed craters and associated double membraned channels. CONCLUSION: These data show that overexpression of the scavenger receptor protein, SR-BI (accompanied by suitable hormone treatment and lipoproteins) in susceptible mammalian cells - is associated with increased cholesterol uptake and SR-BI dimerization within a much enlarged and architecturally complex microvillar compartment. These changes duplicate the structural, biochemical and functional changes related to the uptake of HDL CEs normally signaled by the action of ACTH on intact adrenal tissue.

Dimerization of the scavenger receptor class B type I: formation, function, and localization in diverse cells and tissues.

This study has examined the dimeric/oligomeric forms of scavenger receptor class B type I (SR-BI) and its alternatively spliced form, SR-BII, in a diverse group of cells and tissues: i.e., normal and hormonally altered tissues of mice and rats as well as tissues of transgenic animals and genetically altered steroidogenic and nonsteroidogenic cells overexpressing the SR-B proteins. Using both biochemical and morphological techniques, we have seen that these dimeric and higher order oligomeric forms of SR-BI expression are strongly associated with both functional and morphological expression of the selective HDL cholesteryl ester uptake pathway. Rats and mice show some species differences in expression of SR-BII dimeric forms; this difference does not extend to the use of SR-B cDNA types for transfection purposes. In a separate study, cotransfection of HEK293 cells with cMyc and V5 epitope-tagged SR-BI permitted coprecipitation and quantitative coimmunocytochemical measurements at the electron microscope level, suggesting that much of the newly expressed SR-BI protein in stimulated cells dimerizes and that the SR-BI dimers are localized to the cell surface and specifically to microvillar or double membraned intracellular channels. These combined data suggest that SR-BI self-association represents an integral step in the selective cholesteryl ester uptake process.