StarD5 levels of expression correlate with onset and progression of steatosis and liver fibrosis.

Insufficient expression of steroidogenic acute regulatory-related lipid transfer protein 5 (StarD5) on liver cholesterol/lipid homeostasis is not clearly defined. The ablation of StarD5 was analyzed in mice on a normal or Western diet (WD) to determine its importance in hepatic lipid accumulation and fibrosis compared with wild-type (WT) mice. Rescue experiments in StarD5-/- mice and hepatocytes were performed. In addition to increased hepatic triglyceride (TG)-cholesterol levels, global StarD5-/- mice fed a normal diet displayed reduced plasma triglycerides and liver very low-density lipoprotein (VLDL) secretion as compared with WT counterparts. Insulin levels and homeostatic model assessment for insulin resistance (HOMA-IR) scoring were elevated, demonstrating developing insulin resistance (IR). WD-fed StarD5-/- mice upregulated WW domain containing transcription regulator 1 (TAZ or WWTR1) expression with accelerated liver fibrosis when compared with WD-fed WT mice. Suppression of oxysterol 7α-hydroxylase (CYP7B1) coupled with chronic accumulation of toxic oxysterol levels correlated with presentation of fibrosis. "Hepatocyte-selective" StarD5 overexpression in StarD5-/- mice restored expression, reduced hepatic triglycerides, and improved HOMA-IR. Observations in two additional mouse and one human metabolic dysfunction-associated steatotic liver disease (MASLD) model were supportive. The downregulation of StarD5 with hepatic lipid excess is a previously unappreciated physiological function appearing to promote lipid storage for future needs. Conversely, lingering downregulation of StarD5 with prolonged lipid-cholesterol excess accelerates fatty liver's transition to fibrosis; mediated via dysregulation in the oxysterol signaling pathway.NEW & NOTEWORTHY We have found that deletion of the cholesterol transport protein StarD5 in mice leads to an increase in insulin resistance and lipid accumulation due to the upregulation of lipid synthesis and decrease VLDL secretion from the liver. In addition, deletion of StarD5 increased fibrosis when mice were fed a Western diet. This represents a novel pathway of fibrosis development in the liver.

Insulin dysregulation drives mitochondrial cholesterol metabolite accumulation: initiating hepatic toxicity in nonalcoholic fatty liver disease.

CYP7B1 catalyzes mitochondria-derived cholesterol metabolites such as (25R)26-hydroxycholesterol (26HC) and 3ß-hydroxy-5-cholesten-(25R)26-oic acid (3ßHCA) and facilitates their conversion to bile acids. Disruption of 26HC/3ßHCA metabolism in the absence of CYP7B1 leads to neonatal liver failure. Disrupted 26HC/3ßHCA metabolism with reduced hepatic CYP7B1 expression is also found in nonalcoholic steatohepatitis (NASH). The current study aimed to understand the regulatory mechanism of mitochondrial cholesterol metabolites and their contribution to onset of NASH. We used Cyp7b1-/- mice fed a normal diet (ND), Western diet (WD), or high-cholesterol diet (HCD). Serum and liver cholesterol metabolites as well as hepatic gene expressions were comprehensively analyzed. Interestingly, 26HC/3ßHCA levels were maintained at basal levels in ND-fed Cyp7b1-/- mice livers by the reduced cholesterol transport to mitochondria, and the upregulated glucuronidation and sulfation. However, WD-fed Cyp7b1-/- mice developed insulin resistance (IR) with subsequent 26HC/3ßHCA accumulation due to overwhelmed glucuronidation/sulfation with facilitated mitochondrial cholesterol transport. Meanwhile, Cyp7b1-/- mice fed an HCD did not develop IR or subsequent evidence of liver toxicity. HCD-fed mice livers revealed marked cholesterol accumulation but no 26HC/3ßHCA accumulation. The results suggest 26HC/3ßHCA-induced cytotoxicity occurs when increased cholesterol transport into mitochondria is coupled to decreased 26HC/3ßHCA metabolism driven with IR. Supportive evidence for cholesterol metabolite-driven hepatotoxicity is provided in a diet-induced nonalcoholic fatty liver mouse model and by human specimen analyses. This study uncovers an insulin-mediated regulatory pathway that drives the formation and accumulation of toxic cholesterol metabolites within the hepatocyte mitochondria, mechanistically connecting IR to cholesterol metabolite-induced hepatocyte toxicity which drives nonalcoholic fatty liver disease.

Coffee modulates insulin-hepatocyte nuclear factor-4α-Cyp7b1 pathway and reduces oxysterol-driven liver toxicity in a nonalcoholic fatty liver disease mouse model.

Oxysterol 7α-hydroxylase (CYP7B1) controls the levels of intracellular regulatory oxysterols generated by the "acidic pathway" of cholesterol metabolism. Previously, we demonstrated that an inability to upregulate CYP7B1 in the setting of insulin resistance leads to the accumulation of cholesterol metabolites such as (25R)26-hydroxycholesterol (26HC) that initiate and promote hepatocyte injury; followed by an inflammatory response. The current study demonstrates that dietary coffee improves insulin resistance and restores Cyp7b1 levels in a well-characterized Western diet (WD)-induced nonalcoholic fatty liver disease (NAFLD) mouse model. Ingestion of a WD containing caffeinated (regular) coffee or decaffeinated coffee markedly reduced the serum ALT level and improved insulin resistance. Cyp7b1 mRNA and protein levels were preserved at normal levels in mice fed the coffee containing WD. Additionally, coffee led to upregulated steroid sulfotransferase 2b1 (Sult2b1) mRNA expression. In accordance with the response in these oxysterol metabolic genes, hepatocellular 26HC levels were maintained at physiologically low levels. Moreover, the current study provided evidence that hepatic Cyp7b1 and Sult2b1 responses to insulin signaling can be mediated through a transcriptional factor, hepatocyte nuclear factor (HNF)-4α. We conclude coffee achieves its beneficial effects through the modulation of insulin resistance. Both decaffeinated and caffeinated coffee had beneficial effects, demonstrating caffeine is not fundamental to this effect. The effects of coffee feeding on the insulin-HNF4α-Cyp7b1 signaling pathway, whose dysregulation initiates and contributes to the onset and progression of NASH as triggered by insulin resistance, offer mechanistic insight into approaches for the treatment of NAFLD.NEW & NOTEWORTHY This study demonstrated dietary coffee prevented the accumulation of hepatic oxysterols by maintaining Cyp7b1/Sult2b1 expression in a diet-induced NAFLD mice model. Lowering liver oxysterols markedly reduced inflammation in the coffee-ingested mice. Caffeine is not fundamental to this effect. In addition, this study showed Cyp7b1/Sult2b1 responses to insulin signaling can be mediated through a transcriptional factor, HNF4α. The insulin-HNF4α-Cyp7b1/Sult2b1 signaling pathway, which directly correlates to the onset of NASH triggered by insulin resistance, offers insight into approaches for NAFLD treatment.

Insulin resistance dysregulates CYP7B1 leading to oxysterol accumulation: a pathway for NAFL to NASH transition.

NAFLD is an important public health issue closely associated with the pervasive epidemics of diabetes and obesity. Yet, despite NAFLD being among the most common of chronic liver diseases, the biological factors responsible for its transition from benign nonalcoholic fatty liver (NAFL) to NASH remain unclear. This lack of knowledge leads to a decreased ability to find relevant animal models, predict disease progression, or develop clinical treatments. In the current study, we used multiple mouse models of NAFLD, human correlation data, and selective gene overexpression of steroidogenic acute regulatory protein (StarD1) in mice to elucidate a plausible mechanistic pathway for promoting the transition from NAFL to NASH. We show that oxysterol 7α-hydroxylase (CYP7B1) controls the levels of intracellular regulatory oxysterols generated by the "acidic/alternative" pathway of cholesterol metabolism. Specifically, we report data showing that an inability to upregulate CYP7B1, in the setting of insulin resistance, results in the accumulation of toxic intracellular cholesterol metabolites that promote inflammation and hepatocyte injury. This metabolic pathway, initiated and exacerbated by insulin resistance, offers insight into approaches for the treatment of NAFLD.

StarD5: an ER stress protein regulates plasma membrane and intracellular cholesterol homeostasis.

How plasma membrane (PM) cholesterol is controlled is poorly understood. Ablation of the gene encoding the ER stress steroidogenic acute regulatory-related lipid transfer domain (StarD)5 leads to a decrease in PM cholesterol content, a decrease in cholesterol efflux, and an increase in intracellular neutral lipid accumulation in macrophages, the major cell type that expresses StarD5. ER stress increases StarD5 expression in mouse hepatocytes, which results in an increase in accessible PM cholesterol in WT but not in StarD5-/- hepatocytes. StarD5-/- mice store higher levels of cholesterol and triglycerides, which leads to altered expression of cholesterol-regulated genes. In vitro, a recombinant GST-StarD5 protein transfers cholesterol between synthetic liposomes. StarD5 overexpression leads to a marked increase in PM cholesterol. Phasor analysis of 6-dodecanoyl-2-dimethylaminonaphthalene fluorescence lifetime imaging microscopy data revealed an increase in PM fluidity in StarD5-/- macrophages. Taken together, these studies show that StarD5 is a stress-responsive protein that regulates PM cholesterol and intracellular cholesterol homeostasis.

Mitochondrial Cholesterol Metabolites in a Bile Acid Synthetic Pathway Drive Nonalcoholic Fatty Liver Disease: A Revised "Two-Hit" Hypothesis.

The rising prevalence of nonalcoholic fatty liver disease (NAFLD)-related cirrhosis highlights the need for a better understanding of the molecular mechanisms responsible for driving the transition of hepatic steatosis (fatty liver; NAFL) to steatohepatitis (NASH) and fibrosis/cirrhosis. Obesity-related insulin resistance (IR) is a well-known hallmark of early NAFLD progression, yet the mechanism linking aberrant insulin signaling to hepatocyte inflammation has remained unclear. Recently, as a function of more distinctly defining the regulation of mechanistic pathways, hepatocyte toxicity as mediated by hepatic free cholesterol and its metabolites has emerged as fundamental to the subsequent necroinflammation/fibrosis characteristics of NASH. More specifically, aberrant hepatocyte insulin signaling, as found with IR, leads to dysregulation in bile acid biosynthetic pathways with the subsequent intracellular accumulation of mitochondrial CYP27A1-derived cholesterol metabolites, (25R)26-hydroxycholesterol and 3ß-Hydroxy-5-cholesten-(25R)26-oic acid, which appear to be responsible for driving hepatocyte toxicity. These findings bring forth a "two-hit" interpretation as to how NAFL progresses to NAFLD: abnormal hepatocyte insulin signaling, as occurs with IR, develops as a "first hit" that sequentially drives the accumulation of toxic CYP27A1-driven cholesterol metabolites as the "second hit". In the following review, we examine the mechanistic pathway by which mitochondria-derived cholesterol metabolites drive the development of NASH. Insights into mechanistic approaches for effective NASH intervention are provided.

ER stress increases StarD5 expression by stabilizing its mRNA and leads to relocalization of its protein from the nucleus to the membranes.

StarD5 belongs to the StarD4 subfamily of steroidogenic acute regulatory lipid transfer (START) domain proteins. In macrophages, StarD5 is found in the cytosol and maintains a loose association with the Golgi. Like StarD1 and StarD4, StarD5 is known to bind cholesterol. However, its function and regulation remain poorly defined. Recently, it has been shown that its mRNA expression is induced in response to different inducers of endoplasmic reticulum (ER) stress. However, the molecular mechanism(s) involved in the induction of StarD5 expression during ER stress is not known. Here we show that in 3T3-L1 cells, the ER stressor thapsigargin increases intracellular free cholesterol due to an increase in HMG-CoA reductase expression. Activation of StarD5 expression is mediated by the transcriptional ER stress factor XBP-1. Additionally, the induction of ER stress stabilizes the StarD5 mRNA. Furthermore, StarD5 protein is mainly localized in the nucleus, and upon ER stress, it redistributes away from the nucleus, localizing prominently to the cytosol and membranes. These results reveal the increase in StarD5 expression and protein redistribution during the cell protective phase of the ER stress, suggesting a role for StarD5 in cholesterol metabolism during the ER stress response.

Subcellular localization and regulation of StarD4 protein in macrophages and fibroblasts.

StarD4 is a member of the StarD4 subfamily of START domain proteins with a characteristic lipid binding pocket specific for cholesterol. The objective of this study was to define StarD4 subcellular localization, regulation, and function. Immunobloting showed that StarD4 is highly expressed in the mouse fibroblast cell line 3T3-L1, in human THP-1 macrophages, Kupffer cells (liver macrophages), and hepatocytes. In 3T3-L1 cells and THP-1 macrophages, StarD4 protein appeared localized to the cytoplasm and the endoplasmic reticulum (ER). More specifically, in THP-1 macrophages StarD4 co-localized to areas of the ER enriched in Acyl-CoA:cholesterol acyltransferase-1 (ACAT-1), and was closely associated with budding lipid droplets. The addition of purified StarD4 recombinant protein to an in vitro assay increased ACAT activity 2-fold, indicating that StarD4 serves as a rate-limiting step in cholesteryl ester formation by delivering cholesterol to ACAT-1-enriched ER. In addition, StarD4 protein was found to be highly regulated and to redistribute in response to sterol levels. In summary, these observations, together with our previous findings demonstrating the ability of increased StarD4 expression to increase bile acid synthesis and cholesteryl ester formation, provide strong evidence for StarD4 as a highly regulated, non-vesicular, directional, intracellular transporter of cholesterol which plays a key role in the maintenance of intracellular cholesterol homeostasis.

25-Hydroxycholesterol-3-sulfate attenuates inflammatory response via PPARγ signaling in human THP-1 macrophages.

The nuclear receptor peroxisome proliferator-activated receptors (PPARs) are important in regulating lipid metabolism and inflammatory responses in macrophages. Activation of PPARγ represses key inflammatory response gene expressions. Recently, we identified a new cholesterol metabolite, 25-hydroxycholesterol-3-sulfate (25HC3S), as a potent regulatory molecule of lipid metabolism. In this paper, we report the effect of 25HC3S and its precursor 25-hydroxycholesterol (25HC) on PPARγ activity and on inflammatory responses. Addition of 25HC3S to human macrophages markedly increased nuclear PPARγ and cytosol IκB and decreased nuclear NF-κB protein levels. PPARγ response element reporter gene assays showed that 25HC3S significantly increased luciferase activities. PPARγ competitor assay showed that the K(i) for 25HC3S was ∼1 µM, similar to those of other known natural ligands. NF-κB-dependent promoter reporter gene assays showed that 25HC3S suppressed TNFα-induced luciferase activities only when cotransfected with pcDNAI-PPARγ plasmid. In addition, 25HC3S decreased LPS-induced expression and release of IL-1ß. In the PPARγ-specific siRNA transfected macrophages or in the presence of PPARγ-specific antagonist, 25HC3S failed to increase IκB and to suppress TNFα and IL-1ß expression. In contrast to 25HC3S, its precursor 25HC, a known liver X receptor ligand, decreased nuclear PPARγ and cytosol IκB and increased nuclear NF-κB protein levels. We conclude that 25HC3S acts in macrophages as a PPARγ ligand and suppresses inflammatory responses via the PPARγ/IκB/NF-κB signaling pathway.

StarD4-mediated translocation of 7-hydroperoxycholesterol to isolated mitochondria: deleterious effects and implications for steroidogenesis under oxidative stress conditions.

StAR family proteins, including StarD4, play a key role in steroidogenesis by transporting cholesterol (Ch) into mitochondria for conversion to pregnenolone. Using a model system consisting of peroxidized cholesterol (7 alpha-OOH)-containing liposomes as donors, we showed that human recombinant StarD4 accelerates 7 alpha-OOH transfer to isolated liver mitochondria, and to a greater extent than Ch transfer. StarD4 had no effect on transfer of non-oxidized or peroxidized phosphatidylcholine, consistent with sterol ring specificity. StarD4-accelerated 7 alpha-OOH transfer to mitochondria resulted in greater susceptibility to free radical lipid peroxidation and loss of membrane potential than in a non-StarD4 control. The novel implication of these findings is that in oxidative stress states, inappropriate StAR-mediated trafficking of peroxidized Ch in steroidogenic tissues could result in damage and dysfunction selectively targeted to mitochondria.

Mitochondrial oxysterol biosynthetic pathway gives evidence for CYP7B1 as controller of regulatory oxysterols.

The aim of this paper was to more completely study the mitochondrial CYP27A1 initiated acidic pathway of cholesterol metabolism. The mitochondrial CYP27A1 initiated pathway of cholesterol metabolism (acidic pathway) is known to synthesize two well-described vital regulators of cholesterol/lipid homeostasis, (25R)-26-hydroxycholesterol (26HC) and 25-hydroxycholesterol (25HC). Both 26HC and 25HC have been shown to be subsequently 7α-hydroxylated by Cyp7b1; reducing their regulatory abilities and furthering their metabolism to chenodeoxycholic acid (CDCA). Cholesterol delivery into the inner mitochondria membrane, where CYP27A1 is located, is considered the pathway's only rate-limiting step. To further explore the pathway, we increased cholesterol transport into mitochondrial CYP27A1 by selectively increased expression of the gene encoding the steroidogenic acute transport protein (StarD1). StarD1 overexpression led to an unanticipated marked down-regulation of oxysterol 7α-hydroxylase (Cyp7b1), a marked increase in 26HC, and the formation of a third vital regulatory oxysterol, 24(S)-hydroxycholesterol (24HC), in B6/129 mice livers. To explore the further metabolism of 24HC, as well as, 25HC and 26HC, characterizations of oxysterols and bile acids using three murine models (StarD1 overexpression, Cyp7b1-/-, Cyp27a1-/-) and human Hep G2 cells were conducted. This report describes the discovery of a new mitochondrial-initiated pathway of oxysterol/bile acid biosynthesis. Just as importantly, it provides evidence for CYP7B1 as a key regulator of three vital intracellular regulatory oxysterol levels.

Inhibition of insulin-like growth factor receptor/AKT/mammalian target of rapamycin axis targets colorectal cancer stem cells by attenuating mevalonate-isoprenoid pathway in vitro and in vivo.

We observed a co-upregulation of the insulin-like growth factor receptor (IGF-1R)/AKT/mammalian target of rapamycin (mTOR) [InAT] axis and the mevalonate-isoprenoid biosynthesis (MIB) pathways in colorectal cancer stem cells (CSCs) in an unbiased approach. Hence, we hypothesized that the InAT axis might regulate the MIB pathway to govern colorectal CSCs growth. Stimulation (IGF-1) or inhibition (IGF-1R depletion and pharmacological inhibition of IGF-1R/mTOR) of the InAT axis produced induction or attenuation of CSC growth as well as expression of CSC markers and self-renewal factors respectively. Intriguingly, activation of the InAT axis (IGF-1) caused significant upregulation of the MIB pathway genes (both mRNA and protein); while its inhibition produced the opposite effects in colonospheres. More importantly, supplementation with dimethylallyl- and farnesyl-PP, MIB metabolites downstream of isopentenyl-diphosphate delta isomerase (IDI), but not mevalonate and isopentenyl-pp that are upstream of IDI, resulted in a near-complete reversal of the suppressive effect of the InAT axis inhibitors on CSCs growth. The latter findings suggest a specific regulation of the MIB pathway by the InAT axis distal to the target of statins that inhibit 3-hydroxy-3-methyl-glutaryl-CoA reductase (HMGCR). Effects of IGF-1R inhibition on colonic CSCs proliferation and the MIB pathway were confirmed in an 'in vivo' HCT-116 xenograft model. These observations establish a novel mechanistic link between the InAT axis that is commonly deregulated in colorectal cancer and the MIB pathway in regulation of colonic CSCs growth. Hence, the InAT-MIB corridor is a novel target for developing paradigm shifting optimum anti-CSCs therapies for colorectal cancer.

The StarD4 subfamily of steroidogenic acute regulatory-related lipid transfer (START) domain proteins: new players in cholesterol metabolism.

Cholesterol levels in the body are maintained through the coordinated regulation of its uptake, synthesis, distribution, storage and efflux. However, the way cholesterol is sorted within cells remains poorly defined. The discovery of the newly described StarD4 subfamily, part of the steroidogenic acute regulatory lipid transfer (START) domain family of proteins, affords an opportunity for the study of intracellular cholesterol movement, metabolism and its disorders. The three members of this intracellular subfamily of proteins (StarD4, StarD5 and StarD6) have a similar lipid binding pocket specific for sterols (cholesterol in particular), but differing regulation and localization. The ability to bind and transport cholesterol through a non-vesicular mean suggests that they play a previously unappreciated role in cholesterol homeostasis.

Identification of novel regulatory cholesterol metabolite, 5-cholesten, 3ß,25-diol, disulfate.

Oxysterol sulfation plays an important role in regulation of lipid metabolism and inflammatory responses. In the present study, we report the discovery of a novel regulatory sulfated oxysterol in nuclei of primary rat hepatocytes after overexpression of the gene encoding mitochondrial cholesterol delivery protein (StarD1). Forty-eight hours after infection of the hepatocytes with recombinant StarD1 adenovirus, a water-soluble oxysterol product was isolated and purified by chemical extraction and reverse-phase HPLC. Tandem mass spectrometry analysis identified the oxysterol as 5-cholesten-3ß, 25-diol, disulfate (25HCDS), and confirmed the structure by comparing with a chemically synthesized compound. Administration of 25HCDS to human THP-1-derived macrophages or HepG2 cells significantly inhibited cholesterol synthesis and markedly decreased lipid levels in vivo in NAFLD mouse models. RT-PCR showed that 25HCDS significantly decreased SREBP-1/2 activities by suppressing expression of their responding genes, including ACC, FAS, and HMG-CoA reductase. Analysis of lipid profiles in the liver tissues showed that administration of 25HCDS significantly decreased cholesterol, free fatty acids, and triglycerides by 30, 25, and 20%, respectively. The results suggest that 25HCDS inhibits lipid biosynthesis via blocking SREBP signaling. We conclude that 25HCDS is a potent regulator of lipid metabolism and propose its biosynthetic pathway.

Analysis of Mammalian Histidine Decarboxylase Dimerization Interface Reveals an Electrostatic Hotspot Important for Catalytic Site Topology and Function.

Selective intervention of mammalian histidine decarboxylase (EC 4.1.1.22) could provide a useful antihistaminic strategy against many different pathologies. It is known that global conformational changes must occur during reaction that involves the monomer-monomer interface of the enzyme. Thus, the dimerization surface is a promising target for histidine decarboxylase inhibition. In this work, a rat apoenzyme structural model is used to analyze the interface of the dimeric active HDC. The dimerization surface mainly involves the fragments 1-213 and 308-371 from both subunits. Part of the overlapping surfaces conforms each catalytic site entrance and the substrate-binding sites. In addition, a cluster of charged residues is located in each overlapping surface, so that both electrostatic hotspots mediate in the interaction between the catalytic sites of the dimeric enzyme. It is experimentally demonstrated that the carboxyl group of aspartate 315 is critical for the proper conformation of the holoenzyme and the progression of the reaction. Comparison to the available information on other evolutionary related enzymes also provides new insights for characterization and intervention of homologous l-amino acid decarboxylases.

Regulation of hepatocyte lipid metabolism and inflammatory response by 25-hydroxycholesterol and 25-hydroxycholesterol-3-sulfate.

Dysregulation of lipid metabolism is frequently associated with inflammatory conditions. The mechanism of this association is still not clearly defined. Recently, we identified a nuclear oxysterol, 25-hydroxycholesterol-3-sulfate (25HC3S), as an important regulatory molecule involved in lipid metabolism in hepatocytes. The present study shows that 25HC3S and its precursor, 25-hydroxycholesterol (25HC), diametrically regulate lipid metabolism and inflammatory response via LXR/SREBP-1 and IkappaBalpha/NFkappaB signaling in hepatocytes. Addition of 25HC3S to primary rat hepatocytes decreased nuclear LXR and SREBP-1 protein levels, down-regulated their target genes, acetyl CoA carboxylase 1 (ACC1), fatty acid synthase (FAS), and SREBP-2 target gene HMG reductase, key enzymes involved in fatty acid and cholesterol biosynthesis. 25HC3S reduced TNFalpha-induced inflammatory response by increasing cytoplasmic IkappaBalpha levels, decreasing NFkappaB nuclear translocation, and consequently repressing expression of NFkappaB-dependent genes, IL-1beta, TNFalpha, and TRAF1. NFkappaB-dependent promoter reporter gene assay showed that 25HC3S suppressed luciferase activity in the hepatocytes. In contrast, 25HC elicited opposite effects by increasing nuclear LXR and SREBP-1 protein levels, and by increasing ACC1 and FAS mRNA levels. 25HC also decreased cytoplasmic IkappaBalpha levels and further increased TNFalpha-induced NFkappaB activation. The current findings suggest that 25HC and 25HC3S serve as potent regulators in cross-talk of lipid metabolism and inflammatory response in the hepatocytes.

Intracellular cholesterol transporter StarD4 binds free cholesterol and increases cholesteryl ester formation.

StarD4 protein is a member of the StarD4 subfamily of steroidogenic acute regulatory-related lipid transfer (START) domain proteins that includes StarD5 and StarD6, proteins whose functions remain poorly defined. The objective of this study was to isolate and characterize StarD4's sterol binding and to determine in a hepatocyte culture model its sterol transport capabilities. Utilizing purified full-length StarD4, in vitro binding assays demonstrated a concentration-dependent binding of [(14)C]cholesterol by StarD4 similar to that of the cholesterol binding START domain proteins StarD1 and StarD5. Other tested sterols showed no detectable binding to StarD4, except for 7alpha-hydroxycholesterol, for which StarD4 demonstrated weak binding on lipid protein overlay assays. Subsequently, an isolated mouse hepatocyte model was used to study the ability of StarD4 to bind/mobilize/distribute cellular cholesterol. Increased expression of StarD4 in primary mouse hepatocytes led to a marked increase in the intracellular cholesteryl ester concentration and in the rates of bile acid synthesis. The ability and specificity of StarD4 to bind cholesterol and, as a function of its level of expression, to direct endogenous cellular cholesterol suggest that StarD4 plays an important role as a directional cholesterol transporter in the maintenance of cellular cholesterol homeostasis.

25-Hydroxycholesterol-3-sulfate regulates macrophage lipid metabolism via the LXR/SREBP-1 signaling pathway.

The oxysterol receptor LXR is a key transcriptional regulator of lipid metabolism. LXR increases expression of SREBP-1, which in turn regulates at least 32 genes involved in lipid synthesis and transport. We recently identified 25-hydroxycholesterol-3-sulfate (25HC3S) as an important regulatory molecule in the liver. We have now studied the effects of 25HC3S and its precursor, 25-hydroxycholesterol (25HC), on lipid metabolism as mediated by the LXR/SREBP-1 signaling in macrophages. Addition of 25HC3S to human THP-1-derived macrophages markedly decreased nuclear LXR protein levels. 25HC3S administration was followed by dose- and time-dependent decreases in SREBP-1 mature protein and mRNA levels. 25HC3S decreased the expression of SREBP-1-responsive genes, acetyl-CoA carboxylase-1, and fatty acid synthase (FAS) as well as HMGR and LDLR, which are key proteins involved in lipid metabolism. Subsequently, 25HC3S decreased intracellular lipids and increased cell proliferation. In contrast to 25HC3S, 25HC acted as an LXR ligand, increasing ABCA1, ABCG1, SREBP-1, and FAS mRNA levels. In the presence of 25HC3S, 25HC, and LXR agonist T0901317, stimulation of LXR targeting gene expression was repressed. We conclude that 25HC3S acts in macrophages as a cholesterol satiety signal, downregulating cholesterol and fatty acid synthetic pathways via inhibition of LXR/SREBP signaling. A possible role of oxysterol sulfation is proposed.

Sulfated oxysterol, 25HC3S, is a potent regulator of lipid metabolism in human hepatocytes.

Recently, a novel oxysterol, 5-cholesten-3beta, 25-diol 3-sulfate (25HC3S) was identified in primary rat hepatocytes following overexpression of the cholesterol transport protein, StarD1. This oxysterol was also detected in human liver nuclei. In the present study, 25HC3S was chemically synthesized. Addition of 25HC3S (6 microM) to human hepatocytes markedly inhibited cholesterol biosynthesis. Quantitative RT-PCR and Western blot analysis showed that 25HC3S markedly decreased HMG-CoA reductase mRNA and protein levels. Coincidently, 25HC3S inhibited the activation of sterol regulatory element binding proteins (SREBPs), suggesting that inhibition of cholesterol biosynthesis occurred via blocking SREBP-1 activation, and subsequently by inhibiting the expression of HMG CoA reductase. 25HC3S also decreased SREBP-1 mRNA levels and inhibited the expression of target genes encoding acetyl CoA carboxylase-1 (ACC-1) and fatty acid synthase (FAS). In contrast, 25-hydroxycholesterol increased SREBP1 and FAS mRNA levels in primary human hepatocytes. The results imply that 25HC3S is a potent regulator of SREBP mediated lipid metabolism.

Localization of StarD5 cholesterol binding protein.

Human StarD5 belongs to the StarD4 subfamily of START (for steroidogenic acute regulatory lipid transfer) domain proteins. We previously reported that StarD5 is located in the cytosolic fraction of human liver and binds cholesterol and 25-hydroxycholesterol. After overexpression of the gene encoding StarD5 in primary rat hepatocytes, free cholesterol accumulated in intracellular membranes. These findings suggested StarD5 to be a directional cytosolic sterol transporter. The objective of this study was to determine the localization of StarD5 in human liver. Western blot analysis confirmed StarD5's presence in the liver but not in human hepatocytes. Immunohistochemistry studies showed StarD5 localized within sinusoidal lining cells in the human liver and colocalized with CD68, a marker for Kupffer cells. Western blot analyses identified the presence of StarD5 in monocytes and macrophages as well as mast cells, basophils, and promyelocytic cells, but not in human hepatocytes, endothelial cells, fibroblasts, osteocytes, astrocytes, or brain tissue. Cell fractionation and immunocytochemistry studies on THP-1 macrophages localized StarD5 to the cytosol and supported an association with the Golgi. The presence of this cholesterol/25-hydroxycholesterol-binding protein in cells related to inflammatory processes provides new clues to the role of this protein in free sterol transport in the cells and in lipid-mediated atherogenesis.