METABOLIC ACTIVATION OF VITAMIN A: RELATIONSHIP TO PHYSIOLOGICAL FUNCTION AND DISEASE

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 1010174
• Project Start Date: Oct 1, 2016
• Project End Date: Sep 30, 2021
• Program Code: [(N/A)]- (N/A)

Recipient Organization
UNIVERSITY OF CALIFORNIA, BERKELEY
(N/A)
BERKELEY,CA 94720

Performing Department
Nutritional Sciences

Non Technical Summary
This project will determine the genes regulated by all-trans-retinoic acid, a metabolite of vitamin A (retinol), which control stem cell conversion into fat and bone cells. This project also will determine how all-trans-retinoic acid regulates the function of brown adipose tissue (BAT). BAT controls thermoregulation and therefore burns calories disproportionately increased relative to other tissues. As such, BAT activity regulated adiposity and obesity. The central theme of this project is to understand the mechanisms whereby all-trans-retinoic acid regulates weight and adiposity to determine whether dysregulation of these genes results in adiposity-related diseases and/or gain of weight and loss of bone during aging.

Animal Health Component

20%

Research Effort Categories

Basic

80%

Applied

20%

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
702	3840	1010	100%

Knowledge Area
702 - Requirements and Function of Nutrients and Other Food Components;

Subject Of Investigation
3840 - Laboratory animals;

Field Of Science
1010 - Nutrition and metabolism;

Keywords

retinol

vitamin a

retinoic acid

adipose

obesity

brown fat

osteoporosis

bone

Goals / Objectives
This project has as its specific goals to: 1) determine the precise mechanism(s) whereby Rdh1 regulates the capacity of brown adipose tissue (BAT) to take up, store and mobilize triacylglycerol (fat). This is important because BAT is extremely proficient at burning fat for thermal regulation. It has been estimated that as few as 50 g of BAT could use up to 20% of basal caloric needs in humans, if maximally stimulated. Thus, the ability of Rdh1 to control BAT function may contribute to weight control in humans, as suggested by the Rdh1-null mouse. 2) determine the precize mechanism(s) whereby Rdh10 regulates conversion of stem cells into adipose and bone. This is important because as humans age they lose bone mass but increase in adipose mass. This may be a function of Rdh10 activity because the Rdh10-null mouse (heterozygote) has increased body fat, adipocyte proliferation in bone marrow, and liver steatosis. These data suggest that Rdh10 regulates stem cell differentiation into bone vs. fat cells, and the process is dysregulated with aging. The outcomes anticipated are discovery of the specific transcription factors regulated by Rdh1 and Rdh10 that regulate body fat and bone mass, and whether loss of these processes with aging lead to the decreased bone mass and increased adiposity of aging.

Project Methods
Procedures Aim 1.1a. Global gene changes in differentiating Rdh1-null mice (KO) vs WT will be determined by RNAseq of stromal vascular (SV) pre-adipocytes from BAT to contrast gene expression differences in pre-brown and mature brown adipocytes. This will provide insight into the regulatory and metabolic genes and will guide further mechanistic and biochemical studies. Of interest are BAT markers, pre-adipocyte markers, and genes associated with RA biosynthesis, metabolism and function, and RA-induced genes. Genes encoding metabolic enzymes, transcription factors, or transporters also will be of interest. Changes in gene expression would be verified by changes in protein expression and/or by biochemical measurements.1b. Biochemical functions of primary brown adipocytes. Cell function will be assessed by responses to the β3-adrenergic agonist isopreterenol in the presence and absence of the PPARγ agonist rosiglitazone. β3-adrenergic agonists drive thermogenic activity in BAT and increase mitochondria number, whereas PPARγ agonists reduce the effects of β3-agonists and promote increased lipid storage, but do not increase mitochondria number. Assessments will include proliferation, UCP1 induction, quantification of lipid droplet accumulation, mitochondrial number, mitochondrial function (oxygen consumption and fatty acid oxidation), and rates of triacylglycerol synthesis, fatty acid oxidation, lipolysis, and fatty acid uptake. Metabolic activity will be assessed with the Seahorse XF24 analyzer using isolated mitochondria and intact cells. Seahorse measures rates of oxygen consumption and proton concentrations. Mitochondrial respiration in BAT will be stimulated with glycerol-3-phosphate. In BAT, mitochondrial GPDH action transfers cytoplasmic reducing equivalents to the mitochondria respiratory chain by oxidizing glycerol-3-phosphate and generating FADH2 to provide electrons to complex III via CoQ. Addition of glycerol-3-phosphate promotes baseline O2 consumption. Oligomycin is then added to inhibit ATP synthesis to measure resting respiration. GDP is then added to inhibit UCP1. The next step adds carbonyl cyanide 4-(trifluoromethoxy) phenylhydrozone (FCCP) to inhibit complex IV and achieve maximal uncoupler-stimulated respiration. Next antimycin A is added to inhibit complex III and all mitochondria respiration. GDP data indicate degree of UCP1 activity, whereas FCCP indicates degree of non-UCP1 catalyzed uncoupled respiration. These data will be complemented by introducing energy producing substrates other than glycerol-3-phosphate (e.g. palmitate, succinate, glucose) to probe function of the TCA cycle and metabolic pathways. Isolated mitochondria will assess function not confounded by cell reactions and substrates that support mitochondria respiration. Respiration in intact cells will complement insight into that obtained with mitochondria. TAG synthesis, FAO and lipolysis will be quantified. Fatty acid uptake will be quantified by using the "Quencher-based LCFA uptake assay".Procedures Aim 2.2.1. Energy use and adiposity. Mice will be fed a HFD (50% fat and 4 IU vitamin A/g). At wk 4 (before onset of the phenotype) and wk 16 (well into the phenotype), blood will be taken to measure adiponectin, insulin, leptin, resistin, TNFα, monocyte chemoattractive protein, and IL6. Serum FFA, glycerol and TAG will be measured. Colonic temperatures will be taken. Mice will be assessed by EchoMRI for changes in adiposity vs. lean body mass. Mice will be evaluated by indirect calorimetry by monitoring 48 hr for: activity; O2 consumption; CO2 production; food and water intake; respiratory exchange ratio, RER (Vco2/Vo2); energy expenditure (kcal/h = [3.815 + 1.232 X RER X Vo2] x 1000); FA oxidation (kcal/h = EE X [1-RER/0.3]). Data treatment involves ANCOVA by plotting lean mass vs. RER to distinguish effects caused by the genotype from effects caused by differences in fat mass. Food and water intake will be recorded. Activity will be based on breaks of infrared beams in the x-axis (total horizontal), breaks of two or more beams in the x-axis (ambulatory) and breaks in the z-axis (rearing). Mice will undergo a GTT (i.p. 1.5 g/kg glucose) after a 6 hr fast and an ITT (0.5 U insulin/kg). Mice will be euthanized at wk 16 and dissected to quantify each fat pad, liver, testis and kidney. Organ weights will be expressed as absolute weights and % total body mass. Histology with H&E staining will be done on BAT and subcutaneous and visceral WAT samples to determine cell sizes (hypertrophy and /or hyperplasia?). Fatty infiltration into liver (TAG content) will be assessed via BODIPY staining.2.2. Adipocyte proliferation in bone marrow. Mice fed a HFD will be used to quantify the earliest onset of adipocyte proliferation in marrow, whether males and females differ, and whether aging exacerbates the phenotype. Distal femurs will be assessed ex vivo at 8, 12, 16 and 24 weeks of age. Femurs will be fixed, decalcified, embedded in paraffin, and sectioned longitudinally (10 μm sections) starting at the coronal face. At least 10 sections, each separated by 10 sections, in the middle of the distal aspect will be H&E stained and quantified for adipocytes from the epiphysis edge of the growth plate inward 1 mm into the metaphysis. Adipocytes will be counted independently by two observers using Image J. Similarly, osteoblasts will be quantified via Alp staining and/or Alp immunohistochemistry, and osteoclasts will be identified as TRAP (tartrate-resistant acid phosphatase) positive cells with a minimum of 3 nuclei. A second study of mice fed a HFD will be done starting at 6-weeks-old and continuing at 8, 12, 16, 24 and 36-weeks-old for total bone mineral density (gm/cm2) and bone mineral content (mg) by DEXA scans. These mice also will be assessed by in vivo µCT to quantify changes in bone formation vs. resorption by generating data on femoral tabeculations and cortical density. Multiple variables will be quantified by µCT, including the ratio trabecular and cortical bone volume/total volume, cortical thickness, cortical porosity, pore volume, etc. µCT also can generate adiposity data that correlates highly (r2 = 0.96) with weights determined by tissue excision and physical weighing, and will serve as an adjunct to the EchoMRI measurements. At the end of this study adipocyte numbers in bone marrow will be quantified by histological assessment in distal femurs to normalize these data with the data of the previous experiment.2.3. Retinoid concentrations. We will quantify serum and tissue atRA and retinol. Samples from mice will include WAT form several depots including subcutaneous and visceral, BAT, skeletal muscle, liver, bone marrow, pancreas, testis, ovary, kidney and serum. Because atRA and other retinoids change in a tissue-specific manner in re-fed mice vs. fasted mice, assays will be done during the fasted (22 hr total) and re-fed (16 hr fasted, 6 hr re-fed) states. We anticipate tissue-specific decreases in atRA in the Rdh10+/- mice, especially in fat pads and bone marrow. We also anticipate differences in atRA between WT and Rdh10+/- mice will depend on whether the mouse has been fasted or fed. For example, in Rdh1-null mice, atRA in BAT is similar between WT and knockout during fasting, but is lower in the knockout after feeding.2.4. Differences in gene expression in vivo. Gene changes (qPCR) and protein differences (western blot) will be assessed in liver, femoral bone marrow, interscapular brown adipose, and in two WAT depots (one each of visceral and subcutaneous). The impact of reducing Rdh10 will be gauged by assaying genes and proteins essential for atRA homeostasis. Genes regulated by atRA in addition also will be assessed. The efficacy of SVF cells to differentiate will be compared between WT and Rdh10+/-

Progress 10/01/16 to 09/30/21

Outputs
Target Audience:The target audience includesscientists who work in the vitamin A (retinoid)field, scientists who need to understand the actions of vitamin A to perform their own research, medical health professionals who need to understand nutrient functions, and policy makers who govern public health issues. Efforts to reach these audiences include publication of primary scientific reports, review articles, and presentations at scientific meetings. Classroom teaching ofdiverse audiences also communicates the outcome of these studies and the status of the field in general. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This research allowed training of multiplepost-doctoral fellows, graduate students and undergraduates in areas of analytical biochemistry, physiology, molecular biology, biochemistry and nutrition. Trainees were trained in lab techniques,planning of expreiments,data recording and analyses, report writting, and oral presentations. How have the results been disseminated to communities of interest?Information has been disseminated through the scientific literature and by oral and poster reports at scientific meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The work supported showed for the first time the physiological functions of the vitamin A metabolite all-trans-retinoic acid with respect to liver steatosis, bone marrow adipocyte formation, and abnormal increases in white adipose tissue hypertophy due to increased fat storage, 1. We have found that Rdh1 regulates fatty acid uptake in brown adipocytes, promotes lipolysis, enhances Ucp1 protein and is necessary for normal mitochondrial fatty acid oxidation. In the absence of Rdh1 lipids accure in brown adipose tissue because of defects in uptake, lipolysis, Ucp1 expression, and mitochondrial fatty acid oxidation. This causes lowering of the body temperature and excessive storage of fat in white adipose promoting obesity, glucose intolerance and insulin resistance. 2. We enhanced the data that Rdh10 prevents liver steatosis in malemice fed a high-fat diet and arrestes adipocyte formation in female mice whether fed a low-fat or a high-fat diet. This shows that retinoic acid has sex-specific effects. The data suggest that defects in converting retinol into retinoic acid may underly some causes of diabesity.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Immunotherapy Added to Antibiotic Treatment Reduces Relapse of Disease in a Mouse Model of Tuberculosis. Mourik BC, Leenen PJ, de Knegt GJ, Huizinga R, van der Eerden BC, Wang J, Krois CR, Napoli JL, Bakker-Woudenberg IA, de Steenwinkel JE. Am J Respir Cell Mol Biol. 2017 Feb;56(2):233-241. doi: 10.1165/rcmb.2016-0185OC. PMID:27654457 Restoring Retinoic Acid Attenuates Intestinal Inflammation and Tumorigenesis in APCMin/+ Mice. Penny HL, Prestwood TR, Bhattacharya N, Sun F, Kenkel JA, Davidson MG, Shen L, Zuniga LA, Seeley ES, Pai R, Choi O, Tolentino L, Wang J, Napoli JL, Engleman EG. Cancer Immunol Res. 2016 Nov;4(11):917-926. PMID: 27638841 Normalizing Microbiota-Induced Retinoic Acid Deficiency Stimulates Protective CD8(+) T Cell-Mediated Immunity in Colorectal Cancer. Bhattacharya N, Yuan R, Prestwood TR, Penny HL, DiMaio MA, Reticker-Flynn NE, Krois CR, Kenkel JA, Pham TD, Carmi Y, Tolentino L, Choi O, Hulett R, Wang J, Winer DA, Napoli JL, Engleman EG. Immunity. 2016 Sep 20;45(3):641-655. doi: 10.1016/j.immuni.2016.08.008. PMID:27590114 Functions of Intracellular Retinoid Binding-Proteins. Napoli JL. Subcell Biochem. 2016;81:21-76. PMID:27830500 Quantitation of retinaldehyde in small biological samples using ultrahigh-performance liquid chromatography tandem mass spectrometry. Wang J, Yoo HS, Obrochta KM, Huang P, Napoli JL. Anal Biochem. 2015 Sep 1;484:162-8. doi: 10.1016/j.ab.2015.05.016. PMID:26045160

Progress 10/01/19 to 09/30/20

Outputs
Target Audience:The target audience includes scientists, adminstrators and lay persons interested in the impact of diet, vitamins (esp. vitamin A) on health and disease. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project provided opportunity to train undergraduates and pos-doctoral fellows in metabolic and molecular biology. How have the results been disseminated to communities of interest?Manuscripts are either being written or evaluated through the peer-review process. What do you plan to do during the next reporting period to accomplish the goals?We now focus on the phenotype of the liver-specific Rdh10 KO to determine whether hepatic steatosis is liver autonomous or requires input from other tissues. We have developed and back-crossed the liver-specific Rdh10 KO and have found that the males do not suffer hepatic steatosis, but do have lesions in livers. We are working with a pathologist to characterize these lesions. After establishing the phenotype, we will proceed to evaluate mechanism.

Impacts
What was accomplished under these goals? This year two projects have been completed. 1) We have determined that the Rdh10-ablated mouse (heterozygote) has a sexually dimorphic response in skeletal muscle. Skeletal muscle consumes nearly half the energy of a typical human and thereby restricts adiposity and insulin resistance. The autacoid all-trans-retinoic acid (RA) suppresses adiposity. The retinol dehydrogenase Rdh10 catalyzes the rate-limiting reaction of RA biosynthesis. We studied a heterozygote Rdh10 ablation (Het) to determine the impact of endogenous RA on muscle performance and energy use in vivo. Relative to WT, Het males experienced reduced fatty-acid oxidation, glucose intolerance and insulin resistance, when fed a high-fat diet. RA in the mixed-fiber type gastrocnemius muscle (GM) decreased 38%. TAG content increased 1.7-fold, whereas Glut1 mRNA and glucose decreased >30%. Het male GM had impaired electron transport chain activity, and a 60% reduction in fasting ATP. Running endurance deteriorated 40%. GM fibers increased 5-fold in centralized nuclei--indicating muscle malady or repair. The proportion of oxidative fibers increased, as did expression of the myogenic transcription factors Myog and Myf5. In contrast, Het females increased reliance on fatty acid oxidation, and did not experience glucose intolerance or insulin resistance. Estrogen increased, revealed by a 40% increase in uterine weight. RA decreased only 17% in Het female GM, due to a 1.8-fold increase in the estrogen-induced retinol dehydrogenase, Dhrs9. Het female GM did not amass TAG, increase in oxidative fibers, decrease in Glut1 mRNA or glucose, nor increase in centralized nuclei. Expression of Myog and Myf5 decreased. Electron transport chain activity increased, increasing fasting ATP >3-fold. Running endurance improved 2.2-fold. Thus, endogenous RA modulates insulin resistance through estrogen-related sexual dimorphic effects on skeletal muscle energy use and function. 2) We have determined that the Rdh10-ablated mouse (heterozygote) has profound disfunction of brown adipose tissue metabolism and energy use. The enzymes that catalyze all-trans retinoic acid (RA) biosynthesis and catabolism control its physiological concentrations in tissues. Retinol dehydrogenases (Rdh1, Rdh10 and Dhrs9) catalyze the first and rate-limiting step of atRA biosynthesis. Physiological functions of endogenous RA in regulation of energy balance and adiposity in adulthood have not yet been fully established. Our recent work investigated contributions of Rdh10 to post-natal atRA in white adipose tissue (WAT). We found that deletion of one copy (Rdh10 KO het) decreased tissue RA levels in liver and WAT modestly, leading to metabolic abnormalities in mice fed a high-fat diet (HFD), including increased adiposity, glucose intolerance, insulin resistance, increased liver steatosis in males, and increased adipocytes in female femur marrow. Chronic in vivo treatment with slow-release, low dose RA pellets corrected the metabolic defects. The present study investigates consequences of Rdh10 loss on brown adipose tissue (BAT) function. Rdh10 KO het mice defended their body temperature during acute cold exposure, but had a significant impairment following an overnight fast. During fasting, Rdh10KO het mice fed a low-fat diet have profound decrease in expression of classical BAT markers, including UCP-1 and Cidea. This was accompanied by marked tissue pathology, including accumulation of both non-collagen and collagen fibers type I and III in the extracellular space. Feeding HFD for 3 months exaggerated tissue fibrosis. This had profound consequences on metabolic profile and secretory function of BAT, assayed by metabolic profiling for lipidated amino acids. RNAseq analysis in BAT after fasting revealed transcriptional dysregulation of long non-coding RNA (lncRNA) of several genes associated with skeletal muscle function (Ttn, Myom1, Myom3, Neb, and Tnt1). Our results indicate that loss of Rdh10 affects WAT and BAT differently.

Publications

• Type: Other Status: Published Year Published: 2020 Citation: Retinoid metabolism and functions mediated by retinoid binding-proteins. Napoli JL, Yoo HS.Methods Enzymol. 2020;637:55-75. doi: 10.1016/bs.mie.2020.02.004. Epub 2020 Apr 1.PMID: 32359659 Post-natal all-trans-retinoic acid biosynthesis.Napoli JL. Methods Enzymol. 2020;637:27-54. doi: 10.1016/bs.mie.2020.02.003. PMID: 32359649

Progress 10/01/18 to 09/30/19

Outputs
Target Audience:The target audience include scientists interested in the impact of diet on metabolic health. Changes/Problems:Major problems include difficulties in breeding sufficient knockout mice to determine statistically-significant differences between WT and KOs. What opportunities for training and professional development has the project provided?This project trained multiple undergraduate and post-doctoral fellows in modern nutritional biochemisty in lab techniques and animal husbandry. How have the results been disseminated to communities of interest?Results were published in research journals. What do you plan to do during the next reporting period to accomplish the goals?In the next period, we will: 1) complete characterizing the impact of Rdh10-ablation (heterozygote) on muscle performance and establish the mechanisms of differences between males and females; 2) finish characterizing the liver-specific phenotpe of Rdh10; 3) finish determining the mechanism of the glucocorticoid effect on RA catabolism.

Impacts
What was accomplished under these goals? This project contributed to showing that homeostasis of the autacoid all-trans-retinoic acid (RA) relies on a complex metabolon that includes retinoid binding-proteins and enzymes from the short-chain dehydrogenase/reductase (SDR), aldehyde dehydrogenase (Aldh), and cytochrome P-450 (Cyp) gene families. Cellular retinoid-binding proteins (BP) chaperone retinol through esterification, conversion into retinal, reduction of retinal, conversion of retinal into RA, and chaperone RA to catabolism. BP also deliver RA to nuclear receptors and mediate non-genomic actions. Holo-Crbp1 and Crbp2 channel retinol via protein-protein interaction to lecithin:retinol acyl transferase (Lrat) for esterification and storage. In contrast, apo-Crbp1 stimulates retinyl ester hydrolysis. Thereby, the ratio apo-Crbp1/holo-Crbp1 directs flux of retinol into and out of storage as retinyl esters. Multiple retinol dehydrogenases (Rdh1, Rdh10, Dhrs9, Rdhe2, Rdhe2s) convert retinol into retinal for RA biosynthesis. Retinal reductases (Dhrs3, Dhrs4, Rdh11, also SDRs) reduce retinal into retinol. Retinal dehydrogenases (Raldh1, 2, 3) convert retinal irreversibly into RA. Cyp 26a1, b1, c1 catalyze catabolism of RA via 4- and 18-hydroxylation. Crabp1 and 2 deliver RA to catabolic enzymes. Additional BP functions include non-genomic actions of Crabp1, delivery of RA to RAR by holo-Crabp2, and stabilization of HuR by apo-Crabp2. In addition to the retinoid-specific BPs, Fabp5 also binds RA and delivers it to Ppar?. RA autoregulates its tissue concentrations by inducing Lrat and Cyp, thus controlling substrate availability for RA biosynthesis and decreasing RA directly. In addition, energy status regulates RA biosynthesis and degradation. Insulin ejects FoxO1 from the nucleus, ending FoxO1 stimulation of RA biosynthesis. In contrast, fasting increases RA biosynthesis, while decreasing RA catabolism. RA exhibits hormesis. Its effects relate to its concentration as an inverted J-shaped curve, transitioning from beneficial in the "goldilocks" zone to toxicity, as concentrations increase. Hormesis has confused understanding post-natal physiological effects of RA in animals fed chow-diets (copious vitamin A) and/or dosed with toxic RA amounts. Knockouts of retinoid enzymes Rdh1, Rdh10, Raldh1 and Crbp1 produce different phenotypes, and often prompt compensation by other retinoid metabolizing enzymes or BP, consistent with non-redundant but partially overlapping functions. This suggests generation of different RA pools, to serve distinct functions, based on specific contributions of retinoid enzymes and BP. Knockouts have an advantage of altering post-natal endogenous RA concentrations, in contrast to pharmacological (or toxic) dosing of RA to a WT animal. This approach more clearly reflects physiological actions of RA. This work showed that the Rdh1 knockout inhibited thermogenesis and enhanced adiposity when mice were fed a low-fat diet by damaging brown adipose mitochondrial, the Rdh10 knockout damaged liver, muscle and bone and enhanced adiposity when mice were fed a high-fat diet. Only females suffered adipocyte formation in bone and only males suffered hepatic steatosis. Males showed decreased muscle work than WT; females showed increased ability to run compared to WT.

Publications

• Type: Journal Articles Status: Published Year Published: 2019 Citation: Quantification of Dehydroepiandrosterone, 17?-Estradiol, Testosterone, and Their Sulfates in Mouse Tissues by LC-MS/MS. Yoo HS, Napoli JL. Anal Chem. 2019 Nov 19;91(22):14624-14630 RDH1 suppresses adiposity by promoting brown adipose adaptation to fasting and re-feeding. Krois CR, Vuckovic MG, Huang P, Zaversnik C, Liu CS, Gibson CE, Wheeler MR, Obrochta KM, Min JH, Herber CB, Thompson AC, Shah ID, Gordon SP, Hellerstein MK, Napoli JL. Cell Mol Life Sci. 2019 Jun;76(12):2425-2447. DGAT1 inhibits retinol-dependent regulatory T cell formation and mediates autoimmune encephalomyelitis. Graham KL, Werner BJ, Moyer KM, Patton AK, Krois CR, Yoo HS, Tverskoy M, LaJevic M, Napoli JL, Sobel RA, Zabel BA, Butcher EC. Proc Natl Acad Sci U S A. 2019 Feb 19;116(8):3126-3135. Examination of Fluconazole-Induced Alopecia in an Animal Model and Human Cohort. Thompson GR 3rd, Krois CR, Affolter VK, Everett AD, Varjonen EK, Sharon VR, Singapuri A, Dennis M, McHardy I, Yoo HS, Fedor DM, Wiederhold NP, Aaron PA, Gelli A, Napoli JL, White SD. Antimicrob Agents Chemother. 2019 Jan 29;63(2). pii: e01384-18

Progress 10/01/17 to 09/30/18

Outputs
Target Audience:The target audience is other scientists by way of the literature and scientific meetings. Results from these studies are also used to inform lectures given to undergraduate and graduate students, and other faculty. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This project trained a graduate student, a post-doctoral fellow and numberous undergraduate students, who are recognized on the publications that the project produced. How have the results been disseminated to communities of interest?The results have been published in the scientific literature and communicated in oral presentations at several scientific meetings. What do you plan to do during the next reporting period to accomplish the goals?We will continue to study the mechanisms underlying the phenotypes of the Rdh1 and Rdh10 knockouts to better understand the mechanisms of RA action in vivo postnatally.

Impacts
What was accomplished under these goals? The Rdh10 knockout was used to determine the physiological function of endogeneous retinoic acid (RA). Pharmacological dosing of RA controls adiposity by inhibiting adipogenesis and inducing fatty acid oxidation. Retinol dehydrogenases (RDH) catalyze the first reaction that activates retinol into RA. In this study, we examined post-natal contributions of Rdh10 to RA biosynthesis and physiological functions of endogenous RA. Embryonic fibroblasts from Rdh10 heterozygote hypomorphs or with a total Rdh10 knockout exhibit decreased RA biosynthesis and escalated adipogenesis. RA or a RAR pan-agonist reversed the phenotype. Eliminating one Rdh10 copy in vivo (heterozygote knock out) yielded a modest decrease in RA of liver and adipose, but increased adiposity in male and female mice fed a high-fat diet, increased liver steatosis, glucose intolerance and insulin resistance in males fed a high-fat diet, and activated bone marrow adipocyte formation in females, regardless of dietary fat. Chronic dosing with low dose RA corrected the metabolic defects. These datra resolve physiological actions of endogenous RA, reveal sex-specific effects of RA in vivo, and establish importance of Rdh10 to metabolic control by RA. The consequences of a modest decrease in tissue RA suggest that impaired retinol activation may contribute to diabesity, and low-dose RA therapy may ameliorate adiposity and its sequelae of glucose intolerance and insulin resistance. In a second project, we examined the aldehyde dehydrogenase Raldh1 (Aldh1a1), which functions as one of three enzymes that converts the retinol metabolite retinal into RA, and one of many proteins that contribute to RA homeostasis. Female Raldh1-ablated mice resist diet-induced obesity (DIO). This phenotype contrasts with ablations of other enzymes and binding-proteins that maintain RA homeostasis, which gain adiposity. The phenotype observed prompted the conclusion that loss of Raldh1 causes an increase in adipose tissue retinal, and therefore, retinal functions independently of RA to prevent DIO. A second deduction proposed that low nM concentrations of RA stimulate adipogenesis, in contrast to higher concentrations. Using peer-reviewed LC/MS/MS assays developed and validated for quantifying tissue RA and retinal, we show that endogenous retinal and RA concentrations in adipose tissues from Raldh1-null mice do not correlate with the phenotype. Moreover, male Raldh1-null mice resist weight gain regardless of dietary fat content. Resistance to weight gain occurs during adolescence in both sexes. We show that RA concentrations as low as 1 nM, i.e. in the sub-physiological range, impair adipogenesis of embryonic fibroblasts from wild-type mice. Embryonic fibroblasts from Raldh1-null mice resist differentiating into adipocytes, but retain ability to generate RA. These fibroblasts remain sensitive to an RA receptor pan-agonist, and are not affected by an RA receptor pan-antagonist. Thus, the data do not support the hypothesis that retinal itself represses weight gain and adipogenesis independently of RA. Instead, the data indicate that Raldh1 functions as a retinal and atRA-independent promoter of adiposity during adolescence, and enhances adiposity through pre-adipocyte cell autonomous actions.

Publications

• Type: Journal Articles Status: Published Year Published: 2018 Citation: Yang D, Vuckovic MG, Smullin CP, Kim M, Lo CP-S, Devericks E, Yoo HS, Tintcheva M, Deng Y, Napoli JL. 2018. Modest Decreases in Endogenous All-trans-Retinoic Acid Produced by a Mouse Rdh10 Heterozygote Provoke Major Abnormalities in Adipogenesis and Lipid Metabolism. Diabetes 67:662673.
• Type: Journal Articles Status: Published Year Published: 2018 Citation: Jin L, Chang C, Pawlik KM, Datta A, Johnson LM, Vu T, Napoli JL, Datta PK. 2018. Serine Threonine Kinase Receptor-Associated Protein Deficiency Impairs Mouse Embryonic Stem Cells Lineage Commitment Through CYP26A1-Mediated Retinoic Acid Homeostasis. Stem Cells 36:13681379.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Yang D, Krois CR, Huang P, Wang J, Min J, Yoo HS, Deng Y, Napoli JL. 2017. Raldh1 promotes adiposity during adolescence independently of retinal signaling. PLoS ONE 12:e0187669
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Napoli JL. 2017. Cellular retinoid binding-proteins, CRBP, CRABP, FABP5: Effects on retinoid metabolism, function and related diseases. Pharmacol Ther 173:1933.
• Type: Journal Articles Status: Published Year Published: 2017 Citation: Mourik BC, Leenen PJM, de Knegt GJ, Huizinga R, van der Eerden BCJ, Wang J, Krois CR, Napoli JL, Bakker-Woudenberg IAJM, de Steenwinkel JEM. 2017. Immunotherapy Added to Antibiotic Treatment Reduces Relapse of Disease in a Mouse Model of Tuberculosis. Am J Respir Cell Mol Biol 56:233241.

Progress 10/01/16 to 09/30/17

Outputs
Target Audience:The target audience includes scientists who work in the vitamin A (retinoid) field, scientists who need to understand the actions of vitamin A to perform their own research, medical health professionals who need to understand nutrient functions, and policy makers who govern public health issues. Efforts to reach these audiences include publication of primary scientific reports, review articles, and presentations at scientific meetings. Classroom teaching of diverse audiences also communicates the outcome of these studies and the status of the field in general. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?This research allowed training of multiple post-doctoral fellows, graduate students and undergraduates in areas of analytical biochemistry, physiology, molecular biology, biochemistry and nutrition. Trainees were trained in lab techniques, planning of expreiments, data recording and analyses, report writting, and oral presentations. How have the results been disseminated to communities of interest?Information has been disseminated through the scientific literature and by oral and poster reports at scientific meetings. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? The work supported showed for the first time the physiological functions of the vitamin A metabolite all-trans-retinoic acid with respect to liver steatosis, bone marrow adipocyte formation, and abnormal increases in white adipose tissue hypertophy due to increased fat storage, 1. We have found that Rdh1 regulates fatty acid uptake in brown adipocytes, promotes lipolysis, enhances Ucp1 protein and is necessary for normal mitochondrial fatty acid oxidation. In the absence of Rdh1 lipids accure in brown adipose tissue because of defects in uptake, lipolysis, Ucp1 expression, and mitochondrial fatty acid oxidation. This causes lowering of the body temperature and excessive storage of fat in white adipose promoting obesity, glucose intolerance and insulin resistance. 2. We enhanced the data that Rdh10 prevents liver steatosis in male mice fed a high-fat diet and arrestes adipocyte formation in female mice whether fed a low-fat or a high-fat diet. This shows that retinoic acid has sex-specific effects. The data suggest that defects in converting retinol into retinoic acid may underly some causes of diabesity.

Publications

• Type: Journal Articles Status: Published Year Published: 2017 Citation: Immunotherapy Added to Antibiotic Treatment Reduces Relapse of Disease in a Mouse Model of Tuberculosis. Mourik BC, Leenen PJ, de Knegt GJ, Huizinga R, van der Eerden BC, Wang J, Krois CR, Napoli JL, Bakker-Woudenberg IA, de Steenwinkel JE. Am J Respir Cell Mol Biol. 2017 Feb;56(2):233-241. doi: 10.1165/rcmb.2016-0185OC. PMID:27654457 Restoring Retinoic Acid Attenuates Intestinal Inflammation and Tumorigenesis in APCMin/+ Mice. Penny HL, Prestwood TR, Bhattacharya N, Sun F, Kenkel JA, Davidson MG, Shen L, Zuniga LA, Seeley ES, Pai R, Choi O, Tolentino L, Wang J, Napoli JL, Engleman EG. Cancer Immunol Res. 2016 Nov;4(11):917-926. PMID: 27638841 Normalizing Microbiota-Induced Retinoic Acid Deficiency Stimulates Protective CD8(+) T Cell-Mediated Immunity in Colorectal Cancer. Bhattacharya N, Yuan R, Prestwood TR, Penny HL, DiMaio MA, Reticker-Flynn NE, Krois CR, Kenkel JA, Pham TD, Carmi Y, Tolentino L, Choi O, Hulett R, Wang J, Winer DA, Napoli JL, Engleman EG. Immunity. 2016 Sep 20;45(3):641-655. doi: 10.1016/j.immuni.2016.08.008. PMID:27590114 Functions of Intracellular Retinoid Binding-Proteins. Napoli JL. Subcell Biochem. 2016;81:21-76. PMID:27830500 Quantitation of retinaldehyde in small biological samples using ultrahigh-performance liquid chromatography tandem mass spectrometry. Wang J, Yoo HS, Obrochta KM, Huang P, Napoli JL. Anal Biochem. 2015 Sep 1;484:162-8. doi: 10.1016/j.ab.2015.05.016. PMID:26045160