Metabolic Pathway Coupled with Fermentation Process Optimization for High-Level Production of Retinol in Yarrowia lipolytica.

Retinol is a lipid-soluble form of vitamin A that is crucial for human visual and immune functions. The production of retinol through microbial fermentation has been the focus of recent exploration. However, the obtained titer remains limited and the product is often a mixture of retinal, retinol, and retinoic acid, necessitating purification. To achieve efficient biosynthesis of retinol in Yarrowia lipolytica, we improved the metabolic flux of ß-carotene to provide sufficient precursors for retinol in this study. Coupled with the optimization of the expression level of ß-carotene 15,15'-dioxygenase, de novo production of retinol was achieved. Furthermore, Tween 80 was used as an extractant and butylated hydroxytoluene as an antioxidant to extract intracellular retinol and prevent retinol oxidation, respectively. This strategy significantly increased the level of retinol production. By optimizing the enzymes converting retinal to retinol, the proportion of extracellular retinol in the produced retinoids reached 100%, totaling 1042.3 mg/L. Finally, total retinol production reached 5.4 g/L through fed-batch fermentation in a 5 L bioreactor, comprising 4.2 g/L extracellular retinol and 1.2 g/L intracellular retinol. This achievement represents the highest reported titer so far and advances the industrial production of retinol.

Neutrophil-Based Bionic Delivery System Breaks Through the Capillary Barrier of Liver Sinusoidal Endothelial Cells and Inhibits the Activation of Hepatic Stellate Cells.

The capillarization of hepatic sinusoids resulting from the activation of hepatic stellate cells poses a significant challenge, impeding the effective delivery of therapeutic agents to the Disse space for liver fibrosis treatment. Therefore, overcoming these barriers and achieving efficient drug delivery to activated hepatic stellate cells (aHSCs) are pressing challenge. In this study, we developed a synergistic sequential drug delivery approach utilizing neutrophil membrane hybrid liposome@atorvastatin/amlisentan (NCM@AtAm) and vitamin A-neutrophil membrane hybrid liposome @albumin (VNCM@Bai) nanoparticles (NPs) to breach the capillary barrier for targeted HSC cell delivery. Initially, NCM@AtAm NPs were successfully directed to the site of hepatic fibrosis through neutrophil-mediated inflammatory targeting, resulting in the normalization of liver sinusoidal endothelial cells (LSECs) and restoration of fenestrations under the combined influence of At and Am. Elevated tissue levels of the p-Akt protein and endothelial nitric oxide synthase (eNOS) indicated the normalization of LSECs following treatment with At and Am. Subsequently, VNCM@Bai NPs traversed the restored LSEC fenestrations to access the Disse space, facilitating the delivery of Bai into aHSCs under vitamin A guidance. Lastly, both in vitro and in vivo results demonstrated the efficacy of Bai in inhibiting HSC cell activation by modulating the PPAR γ/TGF-ß1 and STAT1/Smad7 signaling pathways, thereby effectively treating liver fibrosis. Overall, our designed synergistic sequential delivery system effectively overcomes the barrier imposed by LSECs, offering a promising therapeutic strategy for liver fibrosis treatment in clinical settings.

LX-2 Stellate Cells Are a Model System for Investigating the Regulation of Hepatic Vitamin A Metabolism and Respond to Tumor Necrosis Factor α and Interleukin 1ß.

Hepatic stellate cells (HSCs) are the major site of vitamin A (retinol) esterification and subsequent storage as retinyl esters within lipid droplets. However, retinyl esters become depleted in many pathophysiological states, including acute and chronic liver injuries. Recently, using a liver slice culture system as a model of acute liver injury and fibrogenesis, a time-dependent increase and decrease in the apparent formation of the bioactive retinoid all-trans-retinoic acid (atRA) and retinyl palmitate was measured, respectively. This coincided with temporal changes in the gene expression of retinoid-metabolizing enzymes and binding proteins, that preceded HSC activation. However, the underlying mechanisms that promote early changes in retinoid metabolism remain unresolved. We hypothesized that LX-2 cells could be applied to investigate differences in quiescent and activated HSC retinoid metabolism. We demonstrate that the hypermetabolic state of activated stellate cells relative to quiescent stellate cells may be attributed to induction of STRA6, RBP4, and CYP26A1, thereby reducing intracellular concentrations of atRA. We further hypothesized that paracrine and autocrine cytokine signaling regulates HSC vitamin A metabolism in both quiescent and activated cells. In quiescent cells, tumor necrosis factor α dose-dependently downregulated LRAT and CRBP1 mRNA, with EC50 values of 30-50 pg/mL. Likewise, interleukin-1ß decreased LRAT and CRBP1 gene expression but with less potency. In activated stellate cells, multiple enzymes were downregulated, suggesting that the full effects of altered hepatic vitamin A metabolism in chronic conditions require both paracrine and autocrine signaling events. Further, this study suggests the potential for cell type-specific autocrine effects in hepatic retinoid signaling. SIGNIFICANCE STATEMENT: HSCs are the major site of vitamin A storage and important determinants of retinol metabolism during liver fibrogenesis. Here, two LX-2 culture methods were applied as models of hepatic retinoid metabolism to demonstrate the effects of activation status and dose-dependent cytokine exposure on the expression of genes involved in retinoid metabolism. This study suggests that compared to quiescent cells, activated HSCs are hypermetabolic and have reduced apparent formation of retinoic acid, which may alter downstream retinoic acid signaling.

Fatty acid/monoglyceride type and amount modulate fat-soluble vitamin absorption from mixed assemblies in mice.

We investigated the effects of fatty acid/ monoglyceride type and amount on the absorption of fat-soluble vitamins. Micelles or vesicles made with either caprylic acid (CA) + monocaprylin (MC) or oleic acid (OA) + monoolein (MO) at low or high concentrations were infused in bile duct-ligated mice. Retinol + retinyl ester and γ-tocopherol intestinal mucosa contents were higher in mice infused with CA + MC than with OA + MO (up to + 350 % for vitamin A and up to + 62 %, for vitamin E; p < 0.05). Cholecalciferol intestinal mucosa content was the highest in mice infused with micelles with CA + MC at 5 mg/mL (up to + 105 %, p < 0.05). Retinyl ester plasma response was higher with mixed assemblies formed at low concentration of FA + MG compared to high concentration (up to + 1212 %, p < 0.05), while no difference in cholecalciferol and γ-tocopherol plasma responses were measured. No correlation between size or zeta potential and vitamin absorption was found. The impact of FA and MG on fat-soluble vitamin absorption thus differs from one vitamin to another and should be considered to formulate adequate vitamin oral or enteral supplements.

ß-Carotene accelerates the resolution of atherosclerosis in mice.

ß-Carotene oxygenase 1 (BCO1) catalyzes the cleavage of ß-carotene to form vitamin A. Besides its role in vision, vitamin A regulates the expression of genes involved in lipid metabolism and immune cell differentiation. BCO1 activity is associated with the reduction of plasma cholesterol in humans and mice, while dietary ß-carotene reduces hepatic lipid secretion and delays atherosclerosis progression in various experimental models. Here we show that ß-carotene also accelerates atherosclerosis resolution in two independent murine models, independently of changes in body weight gain or plasma lipid profile. Experiments in Bco1-/- mice implicate vitamin A production in the effects of ß-carotene on atherosclerosis resolution. To explore the direct implication of dietary ß-carotene on regulatory T cells (Tregs) differentiation, we utilized anti-CD25 monoclonal antibody infusions. Our data show that ß-carotene favors Treg expansion in the plaque, and that the partial inhibition of Tregs mitigates the effect of ß-carotene on atherosclerosis resolution. Our data highlight the potential of ß-carotene and BCO1 activity in the resolution of atherosclerotic cardiovascular disease.

Retinoic acid signaling pathway in pancreatic stellate cells: Insight into the anti-fibrotic effect and mechanism.

Pancreatic stellate cells (PSCs) are activated following loss of cytoplasmic vitamin A (retinol)-containing lipid droplets, which is a key event in the process of fibrogenesis of chronic pancreatitis (CP) and pancreatic ductal adenocarcinoma (PDCA). PSCs are the major source of cancer-associated fibroblasts (CAFs) that produce stroma to induce PDAC cancer cell growth, invasion, and metastasis. As an active metabolite of retinol, retinoic acid (RA) can regulate target gene expression in PSCs through its nuclear receptor complex (RAR/RXR or RXR/RXR) or transcriptional intermediary factor. Additionally, RA also has extranuclear and non-transcriptional effects. In vitro studies have shown that RA induces PSC deactivation which reduces extracellular matrix production through multiple modes of action, such as inhibiting TßRⅡ, PDGFRß, ß-catenin and Wnt production, downregulating ERK1/2 and JNK phosphorylation and suppressing active TGF-ß1 release. RA alone or in combination with other reagents have been demonstrated to have an effective anti-fibrotic effect on cerulein-induced mouse CP models in vivo studies. Clinical trial data have shown that repurposing all-trans retinoic acid (ATRA) as a stromal-targeting agent for human pancreatic cancer is safe and tolerable, suggesting the possibility of using RA for the treatment of CP and PDCA in humans. This review focuses on RA signaling pathways in PSCs and the effects and mechanisms of RA in PSC-mediated fibrogenesis as well as the anti-fibrotic and anti-tumor effects of RA targeting PSCs or CAFs in vitro and in vivo, highlighting the potential therapies of RA against CP and PDAC.

Bioactive Vitamins and Epigenetic Modifications in Diabetes: A Perspective.

Diabetes is a complex metabolic disease that has been associated with epigenetic changes. External factors such as dietary patterns can induce an imbalance in the pools of micronutrients and macronutrients in the body. Consequently, bioactive vitamins may influence epigenetic mechanisms via several pathways: involvement in the control of gene expression, and in protein synthesis, by acting as coenzymes and co-factors in the metabolism of methyl groups or methylation of DNA and histones. Herein, we present a perspective on the relevance of bioactive vitamins in the epigenetic modifications that occur in diabetes.

Verbascum species as a new source of saffron apocarotenoids and molecular tools for the biotechnological production of crocins and picrocrocin.

Crocins are glucosylated apocarotenoids present in flowers and fruits of a few plant species, including saffron, gardenia, and Buddleja. The biosynthesis of crocins in these plants has been unraveled, and the enzymes engineered for the production of crocins in heterologous systems. Mullein (Verbascum sp.) has been identified as a new source of crocins and picrocrocin. In this work, we have identified eight enzymes involved in the cleavage of carotenoids in two Verbascum species, V. giganteum and V. sinuatum. Four of them were homologous to the previously identified BdCCD4.1 and BdCCD4.3 from Buddleja, involved in the biosynthesis of crocins. These enzymes were analyzed for apocarotenogenic activity in bacteria and Nicotiana benthamiana plants using a virus-driven system. Metabolic analyses of bacterial extracts and N. benthamiana leaves showed the efficient activity of these enzymes to produce crocins using ß-carotene and zeaxanthin as substrates. Accumulations of 0.17% of crocins in N. benthamiana dry leaves were reached in only 2 weeks using a recombinant virus expressing VgCCD4.1, similar to the amounts previously produced using the canonical saffron CsCCD2L. The identification of these enzymes, which display a particularly broad substrate spectrum, opens new avenues for apocarotenoid biotechnological production.

Adipose retinol saturase is regulated by ß-adrenergic signaling and its deletion impairs lipolysis in adipocytes and acute cold tolerance in mice.

OBJECTIVE: Retinol saturase (RetSat) is an endoplasmic reticulum-localized oxidoreductase highly expressed in organs involved in lipid metabolism such as white (WAT) and brown adipose tissue (BAT). Cold exposure was shown to increase RETSAT protein in BAT but its relevance for non-shivering thermogenesis, a process with beneficial effects on metabolic health, is unknown. METHODS: We analyzed the regulation of RetSat expression in white and brown adipocytes and different murine adipose tissue depots upon ß-adrenergic stimulation and cold exposure. RetSat function during the differentiation and ß-adrenergic stimulation of brown adipocytes was dissected by loss-of-function experiments. Mice with BAT-specific deletion of RetSat were generated and exposed to cold. Gene expression in human WAT was analyzed and the effect of RetSat depletion on adipocyte lipolysis investigated. RESULTS: We show that cold exposure induces RetSat expression in both WAT and BAT of mice via ß-adrenergic signaling. In brown adipocytes, RetSat has minor effects on differentiation but is required for maximal thermogenic gene and protein expression upon ß-adrenergic stimulation and mitochondrial respiration. In mice, BAT-specific deletion of RetSat impaired acute but not long-term adaptation to cold exposure. RetSat expression in subcutaneous WAT of humans correlates with the expression of genes related to mitochondrial function. Mechanistically, we found that RetSat depletion impaired ß-agonist-induced lipolysis, a major regulator of thermogenic gene expression in adipocytes. CONCLUSIONS: Thus, RetSat expression is under ß-adrenergic control and determines thermogenic capacity of brown adipocytes and acute cold tolerance in mice. Modulating RetSat activity may allow for therapeutic interventions towards pathologies with inadequate metabolic activity.

SKN-1/NRF2 upregulation by vitamin A is conserved from nematodes to mammals and is critical for lifespan extension in Caenorhabditis elegans.

Vitamin A (VA) is a micronutrient essential for the physiology of many organisms, but its role in longevity and age-related diseases remains unclear. In this work, we used Caenorhabditis elegans to study the impact of various bioactive compounds on lifespan. We demonstrate that VA extends lifespan and reduces lipofuscin and fat accumulation while increasing resistance to heat and oxidative stress. This resistance can be attributed to high levels of detoxifying enzymes called glutathione S-transferases, induced by the transcription factor skinhead-1 (SKN-1). Notably, VA upregulated the transcript levels of skn-1 or its mammalian ortholog NRF2 in both C. elegans, human cells, and liver tissues of mice. Moreover, the loss-of-function genetic models demonstrated a critical involvement of the SKN-1 pathway in longevity extension by VA. Our study thus provides novel insights into the molecular mechanism of anti-aging and anti-oxidative effects of VA, suggesting that this micronutrient could be used for the prevention and/or treatment of age-related disorders.

Hepatic transcriptome delineates the therapeutic effects of Sanren Tang on high-fat diet-induced non-alcoholic fatty liver disease.

OBJECTIVE: To evaluate the effects of Sanren Tang (SRT, ) on a high-fat diet (HFD)-induced non-alcoholic fatty liver disease (NAFLD) in mice and to investigate the hepatic transcriptome regulated by SRT. METHODS: The primary SRT components were identified using ultra-high-performance liquid chromatography-high-resolution accurate mass spectrometry. The SRT-induced pharmacological effects on HFD-induced NAFLD were evaluated in mice for 16 weeks. Obeticholic acid was used as a control drug. Body weight, food intake, and homeostatic model assessment for insulin resistance (HOMA-IR) index were analysed. Hepatic histological changes were observed in haematoxylin and eosin-stained sections and quantified using the NAFLD activity score (NAS). Serum alanine aminotransferase (ALT) and hepatic triglyceride (TG) levels were measured. Lipids in hepatocytes were visualised by Oil red staining. RNA-sequencing was performed to determine the transcriptome profile of the liver tissue. The differentially expressed genes were validated using real-time polymerase chain reaction and Western blotting. RESULTS: Four principal compounds were identified in the SRT: adenosine, amygdalin, luteoloside, and magnolol. SRT ameliorated hepatic histology and lipid deposition in the NAFLD mice, and decreased HOMA-IR, NAS and ALT, and hepatic TG levels. Hepatic transcriptome analysis revealed 232 HFD-regulated genes that were reversed by SRT simultaneously. Retinol metabolism, cytokine-cytokine receptor interaction, and peroxisome proliferator-activated receptor (PPAR) γ signalling were the top three SRT-regulated pathways in NAFLD. CONCLUSIONS: SRT significantly ameliorated HFD-induced NAFLD, which was correlated with the regulation of genes enriched in the retinol metabolism, cytokine-cytokine receptor interaction, and PPARγ signalling pathways.

Human Skin Aging and the Anti-Aging Properties of Retinol.

The skin is the most-extensive and -abundant tissue in the human body. Like many organs, as we age, human skin experiences gradual atrophy in both the epidermis and dermis. This can be primarily attributed to the diminishing population of epidermal stem cells and the reduction in collagen, which is the primary structural protein in the human body. The alterations occurring in the epidermis and dermis due to the aging process result in disruptions to the structure and functionality of the skin. This creates a microenvironment conducive to age-related skin conditions such as a compromised skin barrier, slowed wound healing, and the onset of skin cancer. This review emphasizes the recent molecular discoveries related to skin aging and evaluates preventive approaches, such as the use of topical retinoids. Topical retinoids have demonstrated promise in enhancing skin texture, diminishing fine lines, and augmenting the thickness of both the epidermal and dermal layers.

Enzymatic vitamin A2 production enables red-shifted optogenetics.

Optogenetics is a technology using light-sensitive proteins to control signaling pathways and physiological processes in cells and organs and has been applied in neuroscience, cardiovascular sciences, and many other research fields. Most commonly used optogenetic actuators are sensitive to blue and green light, but red-light activation would allow better tissue penetration and less phototoxicity. Cyp27c1 is a recently deorphanized cytochrome P450 enzyme that converts vitamin A1 to vitamin A2, thereby red-shifting the spectral sensitivity of visual pigments and enabling near-infrared vision in some aquatic species.Here, we investigated the ability of Cyp27c1-generated vitamin A2 to induce a shift in spectral sensitivity of the light-gated ion channel Channelrhodopsin-2 (ChR2) and its red-shifted homolog ReaChR. We used patch clamp to measure photocurrents at specific wavelengths in HEK 293 cells expressing ChR2 or ReaChR. Vitamin A2 incubation red-shifted the wavelength for half-maximal currents (λ50%) by 6.8 nm for ChR2 and 12.4 nm for ReaChR. Overexpression of Cyp27c1 in HEK 293 cells showed mitochondrial localization, and HPLC analysis showed conversion of vitamin A1 to vitamin A2. Notably, the λ50% of ChR2 photocurrents was red-shifted by 10.5 nm, and normalized photocurrents at 550 nm were about twofold larger with Cyp27c1 expression. Similarly, Cyp27c1 shifted the λ50% of ReaChR photocurrents by 14.3 nm and increased normalized photocurrents at 650 nm almost threefold.Since vitamin A2 incubation is not a realistic option for in vivo applications and expression of Cyp27c1 leads to a greater red-shift in spectral sensitivity, we propose co-expression of this enzyme as a novel strategy for red-shifted optogenetics.

Heat Stress Impairs Male Reproductive System with Potential Disruption of Retinol Metabolism and Microbial Balance in the Testis of Mice.

BACKGROUND: Heat stress (HS) has a harmful impact on the male reproductive system, primarily by reducing the sperm quality. The testicular microenvironment plays an important role in sperm quality. OBJECTIVES: This study aimed to explore the underlying mechanism by which HS impairs the male reproductive system through the testicular microenvironment. METHODS: Ten-week-old male mice (n = 8 mice/group) were maintained at a normal temperature (25°C, control) or subjected to HS (38°C for 2 h each day, HS) for 2 wk. The epididymides and testes were collected at week 2 to determine sperm quality, histopathology, retinol concentration, the expression of retinol metabolism-related genes, and the testicular microbiome. The testicular microbiome profiles were analyzed using biostatistics and bioinformatics; other data were analyzed using a 2-sided Student's t test. RESULTS: Compared with the control, HS reduced (P < 0.05) sperm count (42.4%) and motility (97.7%) and disrupted the integrity of the blood-testis barrier. Testicular microbial profiling analysis revealed that HS increased the abundance of the genera Asticcacaulis, Enhydrobacter, and Stenotrophomonas (P < 0.05) and decreased the abundance of the genera Enterococcus and Pleomorphomonas (P < 0.05). Notably, the abundance of Asticcacaulis spp. showed a significant negative correlation with sperm count (P < 0.001) and sperm motility (P < 0.001). Moreover, Asticcacaulis spp. correlated significantly with most blood differential metabolites, particularly retinol (P < 0.05). Compared with the control, HS increased serum retinol concentrations (25.3%) but decreased the testis retinol concentration by 23.7%. Meanwhile, HS downregulated (P < 0.05) the expression of 2 genes (STRA6 and RDH10) and a protein (RDH10) involved in retinol metabolism by 27.3%-36.6% in the testis compared with the control. CONCLUSIONS: HS reduced sperm quality, mainly because of an imbalance in the testicular microenvironment potentially caused by an increase in Asticcacaulis spp. and disturbed retinol metabolism. These findings may offer new strategies for improving male reproductive capacity under HS.

Integrated Transcriptome and Metabolomics to Reveal the Mechanism of Adipose Mesenchymal Stem Cells in Treating Liver Fibrosis.

Liver fibrosis (LF) is a late-stage process observed in various chronic liver diseases with bile and retinol metabolism closely associated with it. Adipose-derived mesenchymal stem cells (ADMSCs) have shown significant therapeutic potential in treating LF. In this study, the transplantation of ADMSCs was applied to a CCl4-induced LF model to investigate its molecular mechanism through a multi-omics joint analysis. The findings reveal that ADMSCs effectively reduced levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TBIL), gamma-glutamyltransferase (GGT), Interleukin-6 (IL-6), tumor necrosis factor-alpha (TNF-α), and α-Smooth muscle actin (α-SMA), thereby mitigating liver lesions, preventing liver parenchymal necrosis, and improving liver collagen deposition. Furthermore, 4751 differentially expressed genes (DEGs) and 270 differentially expressed metabolites (DMs) were detected via transcriptome and metabolomics analysis. Conjoint analysis showed that ADMSCs up-regulated the expression of Cyp7a1, Baat, Cyp27a1, Adh7, Slco1a4, Aldh1a1, and Adh7 genes to promote primary bile acids (TCDCA: Taurochenodeoxycholic acid; GCDCA: Glycochenodeoxycholic acid; GCA: glycocholic acid, TCA: Taurocholic acid) synthesis, secretion and retinol metabolism. This suggests that ADMSCs play a therapeutic role in maintaining bile acid (BA) homeostasis and correcting disturbances in retinol metabolism.

The Functional Characteristics and Soluble Expression of Saffron CsCCD2.

Crocins are important natural products predominantly obtained from the stigma of saffron, and that can be utilized as a medicinal compound, spice, and colorant with significant promise in the pharmaceutical, food, and cosmetic industries. Carotenoid cleavage dioxygenase 2 (CsCCD2) is a crucial limiting enzyme that has been reported to be responsible for the cleavage of zeaxanthin in the crocin biosynthetic pathway. However, the catalytic activity of CsCCD2 on ß-carotene/lycopene remains elusive, and the soluble expression of CsCCD2 remains a big challenge. In this study, we reported the functional characteristics of CsCCD2, that can catalyze not only zeaxanthin cleavage but also ß-carotene and lycopene cleavage. The molecular basis of the divergent functionality of CsCCD2 was elucidated using bioinformatic analysis and truncation studies. The protein expression optimization results demonstrated that the use of a maltose-binding protein (MBP) tag and the optimization of the induction conditions resulted in the production of more soluble protein. Correspondingly, the catalytic efficiency of soluble CsCCD2 was higher than that of the insoluble one, and the results further validated its functional verification. This study not only broadened the substrate profile of CsCCD2, but also achieved the soluble expression of CsCCD2. It provides a firm platform for CsCCD2 crystal structure resolution and facilitates the synthesis of crocetin and crocins.

Discovery of Nonretinoid Inhibitors of CRBP1: Structural and Dynamic Insights for Ligand-Binding Mechanisms.

The dysregulation of retinoid metabolism has been linked to prevalent ocular diseases including age-related macular degeneration and Stargardt disease. Modulating retinoid metabolism through pharmacological approaches holds promise for the treatment of these eye diseases. Cellular retinol-binding protein 1 (CRBP1) is the primary transporter of all-trans-retinol (atROL) in the eye, and its inhibition has recently been shown to protect mouse retinas from light-induced retinal damage. In this report, we employed high-throughput screening to identify new chemical scaffolds for competitive, nonretinoid inhibitors of CRBP1. To understand the mechanisms of interaction between CRBP1 and these inhibitors, we solved high-resolution X-ray crystal structures of the protein in complex with six selected compounds. By combining protein crystallography with hydrogen/deuterium exchange mass spectrometry, we quantified the conformational changes in CRBP1 caused by different inhibitors and correlated their magnitude with apparent binding affinities. Furthermore, using molecular dynamic simulations, we provided evidence for the functional significance of the "closed" conformation of CRBP1 in retaining ligands within the binding pocket. Collectively, our study outlines the molecular foundations for understanding the mechanism of high-affinity interactions between small molecules and CRBPs, offering a framework for the rational design of improved inhibitors for this class of lipid-binding proteins.

A novel mouse model of heart failure with preserved ejection fraction after chronic kidney disease induced by retinol through JAK/STAT pathway.

Heart failure is the leading cardiovascular comorbidity in chronic kidney disease (CKD) patients. Among the types of heart failure according to ejection fraction, heart failure with preserved ejection fraction (HFpEF) is the most common type of heart failure in CKD patients. However, the specific animal model of HFpEF afer CKD is currently missing. In this study, we determined the heart failure characteristics and dynamic progression in CKD mice. Based on these features, we established the practical HFpEF after CKD mouse model using 5/6 subtotal nephrectomy and retinol administration. Active apoptosis, impaired calcium handling, an imbalance between eNOS and oxidative stress and engaged endoplasmic reticulum stress were observed in our model. RNSseq revealed distinct gene expression patterns between HFpEF after CKD and metabolic induced-HFpEF. Furthermore, we revealed the potential mechanism of the pro-HFpEF effect of retinol. Serum accumulation of retinol in CKD prompts myocardial hypertrophy and fibrosis by activating JAK2 and phosphorylating STAT5. Finally, using small molecule inhibitor AC-4-130, we found STAT5 phosphorylation inhibitor may be a potential intervention target for HFpEF after CKD. In conclusion, we provide a novel animal model and a potential drug target for HFpEF intervention in CKD.

Dietary Vitamin A Affects the Function of Incretin-Producing Enteroendocrine Cells in Male Mice Fed a High-Fat Diet.

BACKGROUND: Retinol-binding protein 2 (RBP2) is an intracellular carrier for vitamin A in the absorptive enterocytes. Mice lacking RBP2 (Rbp2-/-) display an unexpected phenotype of obesity, glucose intolerance, and elevated glucose-dependent insulinotropic polypeptide (GIP) levels. GIP and glucagon-like peptide 1 (GLP-1) are incretin hormones secreted by enteroendocrine cells (EECs). We recently demonstrated the presence of RBP2 and other retinoid-related proteins in EECs. OBJECTIVES: Given RBP2's role in intracellular retinoid trafficking, we aimed to evaluate whether dietary vitamin A affects incretin-secreting cell function and gene expression. METHODS: Male Rbp2-/- mice and sex- and age-matched controls (n = 6-9) were fed a high-fat diet (HFD) for 18 wk containing normal (VAN, 4000 IU/kg of diet) or low (VAL, 25% of normal) vitamin A concentrations. Body weight was recorded biweekly. Plasma GIP and GLP-1 levels were obtained fasting and 30 min after an oral fat gavage at week 16. Glucose tolerance tests were also performed. Mice were killed at week 18, and blood and tissue samples were obtained. RESULTS: Rbp2-/- mice displayed greater weight gain on the VAN compared with the VAL diet from week 7 of the intervention (P ≤ 0.01). Stimulated GIP levels were elevated in Rbp2-/- mice compared with their controls fed the VAN diet (P = 0.02), whereas their GIP response was lower when fed the VAL diet (P = 0.03). Although no differences in GLP-1 levels were observed in the VAN diet group, a lower GLP-1 response was seen in Rbp2-/- mice fed the VAL diet (P = 0.02). Changes in incretin gene expression and that of other genes associated with EEC lineage and function were consistent with these observations. Circulating and hepatic retinoid levels revealed no systemic vitamin A deficiency across dietary groups. CONCLUSIONS: Our data support a role for RBP2 and dietary vitamin A in incretin secretion and gene expression in mice fed a HFD.

Retinoic Acid Receptor ß Loss in Hepatocytes Increases Steatosis and Elevates the Integrated Stress Response in Alcohol-Associated Liver Disease.

In alcohol-associated liver disease (ALD), hepatic reductions in vitamin A and perturbations in vitamin A metabolism are common. However, the roles that the vitamin A receptors, termed retinoic acid receptors (RARs), may have in preventing the pathophysiology of ALD remains unclear. Our prior data indicate that a RARß agonist limits the pathology of alcohol-related liver disease. Thus, we generated liver-specific AlbCre-RARß knockout (BKO) mice and compared them to wild type (WT) mice in an early ALD model. Both strains showed similar blood ethanol concentrations and ETOH-metabolizing enzymes. However, the livers of pair-fed-BKO and ETOH-BKO mice developed higher levels of steatosis and triglycerides than pair-fed-WT and ETOH-WT mice. The increased hepatic steatosis observed in the pair-fed-BKO and ETOH-BKO mice was associated with higher lipid synthesis/trafficking transcripts and lower beta-oxidation transcripts. ETOH-BKO mice also exhibited a higher integrated stress response (ISR) signature, including higher transcript and protein levels of ATF4 and its target, 4-EBP1. In human hepatocytes (HepG2) that lack RARß (RARß-KO), ETOH treatments resulted in greater reactive oxygen species compared to their parental cells. Notably, even without ETOH, ATF4 and 4-EBP1 protein levels were higher in the RARß-KO cells than in their parental cells. These 4-EBP1 increases were greatly attenuated in cultured ATF4-deficient and RARß/ATF4-deficient HepG2, suggesting that RARß is a crucial negative regulator of 4-EBP1 through ATF4 in cultured hepatocytes. Here, we identify RARß as a negative regulator of lipid metabolism and cellular stress in ALD.