Sterol Synthesis Research Articles

Hyperproduction of 7-dehydrocholesterol by rewiring the post-squalene module in lipid droplets of Saccharomyces cerevisiae

Lipid droplets (LDs) are specialized organelles that store neutral lipids to reduce the negative effects of lipotoxicity on cells. However, many neutral lipids are precursors for the synthesis of sterols and complex terpenoids, and this sequestration often greatly limits the efficient biosynthesis of sterols and complex terpenoids. In this study, taking 7-dehydrocholesterol (7-DHC) synthesis in Saccharomyces cerevisiae as an example, we revealed the blocking mechanism of LD sequestration on the efficient synthesis of metabolic products and found that LDs can sequester a significant amount of squalene, the precursor of 7-DHC, effectively preventing it from being directed toward the post-squalene pathway. Based on this, a post-squalene pathway was reconstructed on LDs, which resulted in a 28.7% increase in the 7-DHC titer, reaching 684.1 mg/L, whereas the squalene titer was reduced by approximately 97%. Subsequently, the triacylglycerol degradation pathway was weakened to release the storage space in LDs, and the esterification pathway was concurrently strengthened to guide 7-DHC storage within LDs, which further increased 7-DHC production, reaching 792.9 mg/L. Finally, by reducing the NADH/NAD+ ratio to alleviate the redox imbalance, the 7-DHC titer reached 867.6 mg/L in shake flask and 5.1 g/L in a 3-L bioreactor, which is the highest reported titer to date. In summary, this study provides new insights into the important role of LDs in sterol synthesis and offers a novel strategy for constructing cell factories for the efficient synthesis of sterol compounds.

Elucidating Bile Acid Tolerance in Saccharomyces cerevisiae: Effects on Sterol Biosynthesis and Transport Protein Expression

The tolerance of Saccharomyces cerevisiae to high concentrations of bile acids is intricately linked to its potential as a probiotic. While the survival of yeast under high concentrations of bile acids has been demonstrated, the specific mechanisms of tolerance remain inadequately elucidated. This study aims to elucidate the tolerance mechanisms of S. cerevisiae CEN.PK2-1C under conditions of elevated bile acid concentrations. Through growth curve analyses and scanning electron microscopy (SEM), we examined the impact of high bile acid concentrations on yeast growth and cellular morphology. Additionally, transcriptomic sequencing and molecular docking analyses were employed to explore differentially expressed genes under high bile acid conditions, with particular emphasis on ATP-binding cassette (ABC) transporters and steroid hormone biosynthesis. Our findings indicate that high concentrations of bile acids induce significant alterations in the sterol synthesis pathway and transporter protein expression in S. cerevisiae. These alterations primarily function to regulate sterol synthesis pathways to maintain cellular structure and sustain growth, while enhanced expression of transport proteins improves tolerance to elevated bile acid levels. This study elucidates the tolerance mechanisms of S. cerevisiae under high bile acid conditions and provides a theoretical foundation for optimizing fermentation processes and process control.

Investigation of the Cholesterol Biosynthesis by Heart-Cut Liquid Chromatography and Mass Spectrometric Detection

The biosynthesis and homeostasis of cholesterol are essential for cellular function. Cholesterol is a major lipid with multiple roles in membrane stability, signaling, or as a precursor for other molecules. Because of the structural similarity of the sterols involved in the biosynthesis, their accurate identification and quantification is still challenging. Moreover, the huge difference in the concentration of cholesterol and its precursors can cause interferences during the detection. To overcome these problems, a heart-cut liquid chromatographic method was developed by evaluating 38 different columns to achieve optimal separation. The method efficiently separates all sterol biosynthesis intermediates, with detection limits in the low nmol/L-range and an upper limit of quantification of 9 mmol/L for cholesterol by using triple quadrupole mass spectrometric detection. Investigation of lung carcinoma cells treated with statins demonstrated the capability to detect a biological response, showing inhibition of sterol synthesis. This technique offers a robust tool for studying cholesterol biosynthesis and its role in disease.

Viral Infections and Their Ability to Modulate Endoplasmic Reticulum Stress Response Pathways.

In eukaryotic cells, the endoplasmic reticulum is particularly important in post-translational modification of proteins before they are released extracellularly or sent to another endomembrane system. The correct three-dimensional folding of most proteins occurs in the ER lumen, which has an oxidative environment that is essential for the formation of disulfide bridges, which are important in maintaining protein structure. The ER is a versatile organelle that ensures the correct structure of proteins and is essential in the synthesis of lipids and sterols, in addition to offering support in the maintenance of intracellular calcium. Consequently, the cells needed to respond to demands caused by physiological conditions and pathological disturbances in the organelle homeostasis, leading to proper functioning of the cell or even programmed cell death. Disturbances to the ER function trigger a response to the accumulation of unfolded or misfolded proteins, known as the unfolded protein response. Such disturbances include abiotic stress, pharmacological agents, and intracellular pathogens, such as viruses. When misfolded proteins accumulate in the ER, they can undergo ubiquitination and proteasomal degradation through components of the ER-associated degradation system. Once a prolonged activity of the UPR pathway occurs, indicating that homeostasis cannot be reestablished, components of this pathway induce cell death by apoptosis. Here, we discuss how viruses have evolved ways to counteract UPR responses to maximize replication. This evolutionary viral ability is important to understand cell pathology and should be taken into account when designing therapeutic interventions and vaccines.

Investigation of cold-resistance mechanisms in cryophylactic yeast Metschnikowia pulcherrima based on comparative transcriptome analysis.

Low temperature inhibits the growth of most microorganisms. However, some microbes can grow well in a low temperature, even a freezing temperature. In this study, the mechanisms conferring cold resistance in the cryophylactic yeast Metschnikowia (M.) pulcherrima MS612, an isolate of the epidermis of ice grapes, were investigated based on comparative transcriptome analysis. A total of 6018 genes and 374 differentially expressed genes (> 2-fold, p < 0.05) were identified using RNA-Seq. The differentially expressed genes were mainly involved in carbohydrate and energy metabolism, transport mechanisms, antifreeze protection, lipid synthesis, and signal transduction. M. pulcherrima MS612 maintained normal growth at low temperature (5°C) by enhancing energy metabolism, sterol synthesis, metal ion homeostasis, amino acid and MDR transport, while increased synthesis of glycerol and proline transport to improve its resistance to the freezing temperature (-5°C). Furthermore, cAMP-PKA and ERAD signaling pathways contribute to resist the low temperature and the freezing temperature, respectively. This study provides new insights into cold resistance in cryophylactic microorganisms for maneuvering various metabolism to resist different cold environment.

Integrating QTL mapping and transcriptomics to decipher the genetic architecture of sterol metabolism in Brassica napus L.

Sterols are secondary metabolites commonly found in rapeseed that play crucial physiological roles in plants and also benefit human health. Consequently, unraveling the genetic basis of sterol synthesis in rapeseed is highly important. In this study, 21 individual sterols as well as total sterol (TS) content were detected in a double haploid (DH) population of Brassica napus, and a total of 24 quantitative trait loci (QTL) and 157 mQTL were identified that were associated with TS and different individual sterols. Time-series transcriptomic analysis showed that the differentially expressed genes (DEGs) involved in sterol and lipid biosynthesis pathways were enriched. Additionally, a regulatory network between sterol-related DEGs and transcription factors (TFs) was established using coexpression analysis. Some candidate genes were identified with the integration of transcriptomic analysis and QTL mapping, and the key candidate gene BnSQS1.C03 was selected for further functional analysis. BnSQS1.C03 demonstrated squalene synthase activity in vitro and increased the TS by 3.8% when overexpressed in Arabidopsis. The present results provide new insights into sterol regulatory pathways and a valuable genetic basis for breeding rapeseed varieties with high sterol content in the future.

Comprehensive elucidation of the differential physiological kale response to cytokinins under in vitro conditions

BackgroundKale, a versatile cruciferous crop, valued for its pro-health benefits, stress resistance, and potential applications in forage and cosmetics, holds promise for further enhancement of its bioactive compounds through in vitro cultivation methods. Micropropagation techniques use cytokinins (CKs) which are characterized by various proliferative efficiency. Despite the extensive knowledge regarding CKs, there remains a gap in understanding their role in the physiological mechanisms. That is why, here we investigated the effects of three CKs – kinetin (Kin), 6-benzylaminopurine (BAP), and 2-isopentenyladenine (2iP) – on kale physiology, antioxidant status, steroidal metabolism, and membrane integrity under in vitro cultivation.ResultsOur study revealed that while BAP and 2iP stimulated shoot proliferation, they concurrently diminished pigment levels and photosynthetic efficiency. Heightened metabolic activity in response to all CKs was reflected by increased respiratory rate. Despite the differential burst of ROS, the antioxidant properties of kale were associated with the upregulation of guaiacol peroxidase and the scavenging properties of ascorbate rather than glutathione. Notably, CKs fostered the synthesis of sterols, particularly sitosterol, pivotal for cell proliferation and structure of membranes which are strongly disrupted under the action of BAP and 2iP possibly via pathway related to phospholipase D and lipoxygenase which were upregulated. Intriguingly, both CKs treatment spurred the accumulation of sitostenone, known for its ROS scavenging and therapeutic potential. The differential effects of CKs on brassicasterol levels and brassinosteroid (BRs) receptor suggest potential interactions between CKs and BRs.ConclusionBased on the presented results we conclude that the effect evoked by BAP and 2iP in vitro can improve the industrial significance of kale because this treatment makes possible to control proliferation and/or biosynthesis routes of valuable beneficial compounds. Our work offers significant insights into the nuanced effects of CKs on kale physiology and metabolism, illuminating potential avenues for their application in plant biotechnology and medicinal research.

The transcription factor ERF110 promotes cold tolerance by directly regulating sugar and sterol biosynthesis in citrus.

ERFs (ethylene-responsive factors) are known to play a key role in orchestrating cold stress signal transduction. However, the regulatory mechanisms and target genes of most ERFs are far from being well deciphered. In this study, we identified a cold-induced ERF, designated as PtrERF110, from trifoliate orange (Poncirus trifoliata L. Raf., also known as Citrus trifoliata L.), an elite cold-hardy plant. PtrERF110 is a nuclear protein with transcriptional activation activity. Overexpression of PtrERF110 remarkably enhanced cold tolerance in lemon (Citrus limon) and tobacco (Nicotiana tabacum), whereas VIGS (virus-induced gene silencing)-mediated knockdown of PtrERF110 drastically impaired the cold tolerance. RNA sequence analysis revealed that PtrERF110 overexpression resulted in global transcriptional reprogramming of a range of stress-responsive genes. Three of the genes, including PtrERD6L16 (early responsive dehydration 6-like transporters), PtrSPS4 (sucrose phosphate synthase 4), and PtrUGT80B1 (UDP-glucose: sterol glycosyltransferases 80B1), were confirmed as direct targets of PtrERF110. Consistently, PtrERF110-overexpressing plants exhibited higher levels of sugars and sterols compared to their wild type counterparts, whereas the VIGS plants had an opposite trend. Exogenous supply of sucrose restored the cold tolerance of PtrERF110-silencing plants. In addition, knockdown of PtrSPS4, PtrERD6L16, and PtrUGT80B1 substantially impaired the cold tolerance of P. trifoliata. Taken together, our findings indicate that PtrERF110 positively modulates cold tolerance by directly regulating sugar and sterol synthesis through transcriptionally activating PtrERD6L16, PtrSPS4, and PtrUGT80B1. The regulatory modules (ERF110-ERD6L16/SPS4/UGT80B1) unraveled in this study advance our understanding of the molecular mechanisms underlying sugar and sterol accumulation in plants subjected to cold stress.

Mechanism and bioinformatics analysis of the effect of berberine-enhanced fluconazole against drug-resistant Candida albicans

Biofilms produced by Candida albicans present a challenge in treatment with antifungal drug. Enhancing the sensitivity to fluconazole (FLC) is a reasonable method for treating FLC-resistant species. Moreover, several lines of evidence have demonstrated that berberine (BBR) can have antimicrobial effects. The aim of this study was to clarify the underlying mechanism of these effects. We conducted a comparative study of the inhibition of FLC-resistant strain growth by FLC treatment alone, BBR treatment alone, and the synergistic effect of combined FLC and BBR treatment. Twenty-four isolated strains showed distinct biofilm formation capabilities. The antifungal effect of combined FLC and BBR treatment in terms of the growth and biofilm formation of Candida albicans species was determined via checkerboard, time-kill, and fluorescence microscopy assays. The synergistic effect of BBR and FLC downregulated the expression of the efflux pump genes CDR1 and MDR, the hyphal gene HWP1, and the adhesion gene ALS3; however, the gene expression of the transcriptional repressor TUP1 was upregulated following treatment with this drug combination. Furthermore, the addition of BBR led to a marked reduction in cell surface hydrophobicity. To identify resistance-related genes and virulence factors through genome-wide sequencing analysis, we investigated the inhibition of related resistance gene expression by the combination of BBR and FLC, as well as the associated signaling pathways and metabolic pathways. The KEGG metabolic map showed that the metabolic genes in this strain are mainly involved in amino acid and carbon metabolism. The metabolic pathway map showed that several ergosterol (ERG) genes were involved in the synthesis of cell membrane sterols, which may be related to drug resistance. In this study, BBR + FLC combination treatment upregulated the expression of the ERG1, ERG3, ERG4, ERG5, ERG24, and ERG25 genes and downregulated the expression of the ERG6 and ERG9 genes compared with fluconazole treatment alone (p < 0.05).

Combination of Bacillus tequilensis with difenoconazole to control pear black spot and the related synergistic mechanism.

Pear black spot (PBS) is caused by Alternaria alternata and causes severe damage worldwide. It is particularly important to screen for synergistic fungicide combinations to address issues associated with the low efficacy of biocontrol agents, high dosage requirements and poor sustained effectiveness of chemical fungicides. In vitro and in vivo studies were performed to determine the efficacy of a treatment for this important disease. Additionally, transcriptomic and metabolomic analyses were performed to determine the main molecular and biochemical mechanisms involved in the interaction. Bacillus tequilensis 2_2a has a significant synergistic effect with difenoconazole, causing hyphal entanglement and spore lysis and inhibiting the formation of PBS lesions in vitro. In the field, the control effect of the combination was greater than 95%. The pathways associated with the synergistic effect on the mycelia of A. alternata were divided into two main types: one included glycolysis, oxidative phosphorylation, and MAPK signal transduction, while the other included glycolysis, the TCA cycle, coenzyme A biosynthesis, sterol synthesis, and fatty acid degradation. Both types of pathways jointly affect the cell cycle. The main functions of the key genes and metabolites that have been verified as being affected are glucose synthesis and oxidative respiration, as well as citric acid synthesis, acetyl-CoA synthesis, and sterol synthesis. Both functions involve intracellular pyridine nucleotide metabolism and adenine nucleotide transformation. This study helps to reveal the synergistic mechanisms underlying the combined efficacy of biological and chemical agents, providing a scientific basis for field applications.

The constitutively active form of a key cholesterol synthesis enzyme is lipid droplet-localized and upregulated in endometrial cancer tissues

Cholesterol is essential for both normal cell viability and cancer cell proliferation. Aberrant activity of squalene monooxygenase (SM, also known as squalene epoxidase or SQLE), the rate-limiting enzyme of the committed cholesterol synthesis pathway, is accordingly implicated in a growing list of cancers. We previously reported that hypoxia triggers the truncation of SM to a constitutively active form, thus preserving sterol synthesis during oxygen shortfalls. Here, we show SM truncation is upregulated and correlates with the magnitude of hypoxia in endometrial cancer tissues, supporting the in vivo relevance of our earlier work. To further investigate the pathophysiological consequences of SM truncation, we examine using complementary immunofluorescence and cell fractionation approaches its lipid droplet-localized pool and find that it exclusively comprises the truncated enzyme. This partitioning is facilitated by the loss of an endoplasmic reticulum-embedded region at the SM N-terminus, whereas the catalytic domain containing membrane-associated C-terminal helices is spared. Moreover, we determined multiple amphipathic helices contribute to the lipid droplet localization of trunSM. Taken together, our results expand on the striking differences between the two forms of SM and suggest upregulated truncation may contribute to SM-related oncogenesis.

Inhibition of 7-dehydrocholesterol reductase prevents hepatic ferroptosis under an active state of sterol synthesis

Recent evidence indicates ferroptosis is implicated in the pathophysiology of various liver diseases; however, the organ-specific regulation mechanism is poorly understood. Here, we demonstrate 7-dehydrocholesterol reductase (DHCR7), the terminal enzyme of cholesterol biosynthesis, as a regulator of ferroptosis in hepatocytes. Genetic and pharmacological inhibition (with AY9944) of DHCR7 suppress ferroptosis in human hepatocellular carcinoma Huh-7 cells. DHCR7 inhibition increases its substrate, 7-dehydrocholesterol (7-DHC). Furthermore, exogenous 7-DHC supplementation using hydroxypropyl β-cyclodextrin suppresses ferroptosis. A 7-DHC-derived oxysterol metabolite, 3β,5α-dihydroxycholest-7-en-6-one (DHCEO), is increased by the ferroptosis-inducer RSL-3 in DHCR7-deficient cells, suggesting that the ferroptosis-suppressive effect of DHCR7 inhibition is associated with the oxidation of 7-DHC. Electron spin resonance analysis reveals that 7-DHC functions as a radical trapping agent, thus protecting cells from ferroptosis. We further show that AY9944 inhibits hepatic ischemia-reperfusion injury, and genetic ablation of Dhcr7 prevents acetaminophen-induced acute liver failure in mice. These findings provide new insights into the regulatory mechanism of liver ferroptosis and suggest a potential therapeutic option for ferroptosis-related liver diseases.

Squalene production under oxygen limitation by Schizochytrium sp. S31 in different cultivation systems

The triterpene squalene is widely used in the food, cosmetics and pharmaceutical industries due to its antioxidant, antistatic and anti-carcinogenic properties. It is usually obtained from the liver of deep sea sharks, which are facing extinction. Alternative production organisms are marine protists from the family Thraustochytriaceae, which produce and store large quantities of various lipids. Squalene accumulation in thraustochytrids is complex, as it is an intermediate in sterol biosynthesis. Its conversion to squalene 2,3-epoxide is the first step in sterol synthesis and is heavily oxygen dependent. Hence, the oxygen supply during cultivation was investigated in our study. In shake flask cultivations, a reduced oxygen supply led to increased squalene and decreased sterol contents and yields. Oxygen-limited conditions were applied to bioreactor scale, where squalene accumulation and growth of Schizochytrium sp. S31 was determined in batch, fed-batch and continuous cultivation. The highest dry matter (32.03 g/L) was obtained during fed-batch cultivation, whereas batch cultivation yielded the highest biomass productivity (0.2 g/L*h−1). Squalene accumulation benefited from keeping the microorganisms in the growth phase. Therefore, the highest squalene content of 39.67 ± 1.34 mg/g was achieved by continuous cultivation (D = 0.025 h−1) and the highest squalene yield of 1131 mg/L during fed-batch cultivation. Volumetric and specific squalene productivity both reached maxima in the continuous cultivation at D = 0.025 h−1 (6.94 ± 0.27 mg/L*h−1 and 1.00 ± 0.03 mg/g*h−1, respectively). Thus, the choice of a suitable cultivation method under oxygen-limiting conditions depends heavily on the process requirements.Key points• Measurements of respiratory activity and backscatter light of thraustochytrids• Oxygen limitation increased squalene accumulation in Schizochytrium sp. S31• Comparison of different cultivation methods under oxygen-limiting conditions

Common origin of sterol biosynthesis points to a feeding strategy shift in Neoproterozoic animals

Steranes preserved in sedimentary rocks serve as molecular fossils, which are thought to record the expansion of eukaryote life through the Neoproterozoic Era ( ~ 1000-541 Ma). Scientists hypothesize that ancient C27 steranes originated from cholesterol, the major sterol produced by living red algae and animals. Similarly, C28 and C29 steranes are thought to be derived from the sterols of prehistoric fungi, green algae, and other microbial eukaryotes. However, recent work on annelid worms–an advanced group of eumetazoan animals–shows that they are also capable of producing C28 and C29 sterols. In this paper, we explore the evolutionary history of the 24-C sterol methyltransferase (smt) gene in animals, which is required to make C28+ sterols. We find evidence that the smt gene was vertically inherited through animals, suggesting early eumetazoans were capable of C28+ sterol synthesis. Our molecular clock of the animal smt gene demonstrates that its diversification coincides with the rise of C28 and C29 steranes in the Neoproterozoic. This study supports the hypothesis that early eumetazoans were capable of making C28+ sterols and that many animal lineages independently abandoned its biosynthesis around the end-Neoproterozoic, coinciding with the rise of abundant eukaryotic prey.

The subset of peroxisomal tail-anchored proteins do not reach peroxisomes via ER, instead mitochondria can be involved.

Peroxisomes are membrane-enclosed organelles with important roles in fatty acid breakdown, bile acid synthesis and biosynthesis of sterols and ether lipids. Defects in peroxisomes result in severe genetic diseases, such as Zellweger syndrome and neonatal adrenoleukodystrophy. However, many aspects of peroxisomal biogenesis are not well understood. Here we investigated delivery of tail-anchored (TA) proteins to peroxisomes in mammalian cells. Using glycosylation assays we showed that peroxisomal TA proteins do not enter the endoplasmic reticulum (ER) in both wild type (WT) and peroxisome-lacking cells. We observed that in cells lacking the essential peroxisome biogenesis factor, PEX19, peroxisomal TA proteins localize mainly to mitochondria. Finally, to investigate peroxisomal TA protein targeting in cells with fully functional peroxisomes we used a proximity biotinylation approach. We showed that while ER-targeted TA construct was exclusively inserted into the ER, peroxisome-targeted TA construct was inserted to both peroxisomes and mitochondria. Thus, in contrast to previous studies, our data suggest that some peroxisomal TA proteins do not insert to the ER prior to their delivery to peroxisomes, instead, mitochondria can be involved.

Biological effects of trans, trans-farnesol in Leishmania amazonensis.

Farnesol, derived from farnesyl pyrophosphate in the sterols biosynthetic pathway, is a molecule with three unsaturations and four possible isomers. Candida albicans predominantly secretes the trans, trans-farnesol (t, t-FOH) isomer, known for its role in regulating the virulence of various fungi species and modulating morphological transition processes. Notably, the evolutionary divergence in sterol biosynthesis between fungi, including Candida albicans, and trypanosomatids resulted in the synthesis of sterols with the ergostane skeleton, distinct from cholesterol. This study aims to assess the impact of exogenously added trans, trans-farnesol on the proliferative ability of Leishmania amazonensis and to identify its presence in the lipid secretome of the parasite. The study involved the addition of exogenous trans, trans-farnesol to evaluate its interference with the proliferation of L. amazonensis promastigotes. Proliferation, cell cycle, DNA fragmentation, and mitochondrial functionality were assessed as indicators of the effects of trans, trans-farnesol. Additionally, lipid secretome analysis was conducted, focusing on the detection of trans, trans-farnesol and related products derived from the precursor, farnesyl pyrophosphate. In silico analysis was employed to identify the sequence for the farnesene synthase gene responsible for producing these isoprenoids in the Leishmania genome. Exogenously added trans, trans-farnesol was found to interfere with the proliferation of L. amazonensis promastigotes, inhibiting the cell cycle without causing DNA fragmentation or loss of mitochondrial functionality. Despite the absence of trans, trans-farnesol in the culture supernatant, other products derived from farnesyl pyrophosphate, specifically α-farnesene and β-farnesene, were detected starting on the fourth day of culture, continuing to increase until the tenth day. Furthermore, the identification of the farnesene synthase gene in the Leishmania genome through in silico analysis provided insights into the enzymatic basis of isoprenoid production. The findings collectively offer the first insights into the mechanism of action of farnesol on L. amazonensis. While trans, trans-farnesol was not detected in the lipid secretome, the presence of α-farnesene and β-farnesene suggests alternative pathways or modifications in the isoprenoid metabolism of the parasite. The inhibitory effects on proliferation and cell cycle without inducing DNA fragmentation or mitochondrial dysfunction raise questions about the specific targets and pathways affected by exogenous trans, trans-farnesol. The identification of the farnesene synthase gene provides a molecular basis for understanding the synthesis of related isoprenoids in Leishmania. Further exploration of these mechanisms may contribute to the development of novel therapeutic strategies against Leishmania infections.

Effects of Neonatal Hypoxic-Ischemic Injury on Brain Sterol Synthesis and Metabolism.

Neonatal hypoxic-ischemic brain injury (HIBI) results from disruptions to blood supply and oxygen in the perinatal brain. The goal of this study was to measure brain sterol metabolites and plasma oxysterols after injury in a neonatal HIBI mouse model to assess for potential therapeutic targets in the brain biochemistry as well as potential circulating diagnostic biomarkers. Postnatal day 9 CD1-IGS mouse pups were randomized to HIBI induced by carotid artery ligation followed by 30 minutes at 8% oxygen or to sham surgery and normoxia. Brain tissue was collected for sterol analysis by liquid chromatography with tandem mass spectrometry (LC-MS/MS). Plasma was collected for oxysterol analysis by LC-MS/MS. There were minimal changes in brain sterol concentrations in the first 72 hours after HIBI. In severely injured brains, there was a significant increase in desmosterol, 7-DHC, 8-DHC, and cholesterol 24 hours after injury in the ipsilateral tissue. Lanosterol, 24-dehydrolathosterol, and 14-dehydrozymostenol decreased in plasma 24 hours after injury. Severe neonatal HIBI was associated with increased cholesterol and sterol precursors in the cortex at 24 hours after injury. Differences in plasma oxysterols were seen at 24 hours but were not present at 30 minutes after injury, suggesting that these sterol intermediates would be of little value as early diagnostic biomarkers.

High-Carbohydrate Diet Consumption Poses a More Severe Liver Cholesterol Deposition than a High-Fat and High-Calorie Diet in Mice.

In the past few decades, many researchers believed that a high-fat and high-calorie diet is the most critical factor leading to metabolic diseases. However, increasing evidence shows a high-carbohydrate and low-fat diet may also be a significant risk factor. It needs a comprehensive evaluation to prove which viewpoint is more persuasive. We systematically compared the effects of high-fat and high-calorie diets and high-carbohydrate and low-fat ones on glycolipid metabolism in mice to evaluate and compare the effects of different dietary patterns on metabolic changes in mice. Sixty 8-week-old male C57BL/6 mice were divided into four groups after acclimatization and 15% (F-15), 25% (F-25), 35% (F-35), and 45% (F-45) of their dietary energy was derived from fat for 24 weeks. The body weight, body-fat percentage, fasting blood glucose, lipid content in the serum, and triglyceride content in the livers of mice showed a significantly positive correlation with dietary oil supplementation. Interestingly, the total cholesterol content in the livers of mice in the F-15 group was significantly higher than that in other groups (p < 0.05). Compared with the F-45 group, the mRNA expression of sterol synthesis and absorption-related genes (e.g., Asgr1, mTorc1, Ucp20, Srebp2, Hmgcr, and Ldlr), liver fibrosis-related genes (e.g., Col4a1 and Adamts1) and inflammation-related genes (e.g., Il-1β and Il-6) were significantly higher in the F-15 group. Compared with the F-45 group, the relative abundance of unclassified_f_Lachnospiraceae and Akkermansia was decreased in the F-15 group. While unclassified_f_Lachnospiraceae and Akkermansia are potentially beneficial bacteria, they have the ability to produce short-chain fatty acids and modulate cholesterol metabolism. In addition, the relative abundance of unclassified_f_Lachnospiraceae and Akkermansia was significantly positively correlated with fatty acid transporters expression and negatively correlated with that of cholesteryl acyltransferase 1 and cholesterol synthesis-related genes. In conclusion, our study delineated how a high-fat and high-calorie diet (fat supplied higher than or equal to 35%) induced obesity and hepatic lipid deposition in mice. Although the high-carbohydrate and low-fat diet did not cause weight gain in mice, it induced cholesterol deposition in the liver. The mechanism is mainly through the induction of endogenous synthesis of cholesterol in mice liver through the ASGR1-mTORC1-USP20-HMGCR signaling pathway. The appropriate oil and carbon water ratio (dietary energy supply from fat of 25%) showed the best gluco-lipid metabolic homeostasis in mice.

The yeast mRNA-binding protein Cth2 post-transcriptionally modulates ergosterol biosynthesis in response to iron deficiency

Sterol synthesis is an iron-dependent metabolic pathway in eukaryotes. Consequently, fungal ergosterol biosynthesis (ERG) is down-regulated in response to iron deficiency. In this report, we show that, upon iron limitation or overexpression of the iron-regulated mRNA-binding protein Cth2, the yeast Saccharomyces cerevisiae down-regulates the three initial enzymatic steps of ergosterol synthesis (ERG1, ERG7 and ERG11). Mechanistically, we show that Cth2 protein limits the translation and promotes the decrease in the mRNA levels of these specific ERG genes, which contain consensus Cth2-binding sites defined as AU-rich elements (AREs). Thus, expression of CTH2 leads to the accumulation of initial sterol intermediates, such as squalene, and to the drop of ergosterol levels. Changes in CTH2 expression levels disturb the response of yeast cells to stresses related to membrane integrity such as high ethanol and sorbitol concentrations. Therefore, CTH2 should be considered as a critical regulatory factor of ergosterol biosynthesis during iron deficiency.

The Effect of MSTN Mutation on Bile Acid Metabolism and Lipid Metabolism in Cattle.

Myostatin (MSTN) is a negative regulator of skeletal muscle genesis during development. MSTN mutation leads to increased lean meat production and reduced fat deposition in livestock. However, the mechanism by which MSTN promotes myogenesis by regulating metabolism is not clear. In this study, we compared the metabolomics of the livers of wild-type (WT) and MSTN mutation cattle (MT), and found changes in the content and proportion of fatty acids and bile acids in MT cattle. The differential metabolites were enriched in sterol synthesis and primary bile acid synthesis. We further analyzed the expression of genes involved in the regulation of lipid and bile acid metabolism, and found that the loss of MSTN may alter lipid synthesis and bile acid metabolism. This study provides new basic data for MSTN mutations in beef cattle breeding.