Fig 2: Weight Loss Restores Tissue Vitamin A Levels and Vitamin A Signaling in Obese Mice.(A) (a) Body weights of (wt) C57/BL6 mice fed either a standard lab chow diet (chow) or high fat diet (HFD) for 4 months, and wt mice that were previously obese and then switched back to a chow diet for 6 weeks (HFDR). (b) Adipose tissue (epididymal) weights in milligrams (mg) of wet weight from mice described in (a). (B) Quantitation of high performance liquid chromatography (HPLC) of (a) liver retinyl-palmitate and all-trans retinol levels in (b) liver, (c) pancreas, and (d) lung from mice described in A. (C) Chromatographic tracings of VA (absorption units (AU)) extracted from livers of (a) wt mice fed a standard lab chow diet, (b) wt mice fed a HFD for 4 months, and (c) wt mice that were previously obese (HFD-fed for 4 months) and then switched back to a chow diet for 6 weeks (HFDR). All-trans retinol (ROL- arrow, ~32-minute retention time) and retinyl-palmitate (RP- arrow, ~55-minute retention time) were detected at a wavelength of 325 nm and identified by a match of retention times of pure retinoid standards. RAc-arrow, retinyl acetate, added control for extraction efficiency. (D) Relative mRNA levels of retinoic acid receptors (a–c); RARa, RARß2, RAR? respectively) and cellular retinol binding protein-1 (Crbp1) (d) in liver, pancreas, and lungs from mice described in A.

Fig 3: Liver Levels of Vitamin A and Cellular Retinol Binding Protein-1 are Reduced with Increasing Obesity.(A) (a) Body weights of (wt) C57/BL6 mice fed either a standard lab chow diet (chow) or high fat diet (HFD) for 1 month (1M), 2 months (2M) or 4 months (4M). (b) Adipose tissue (epididymal) weights in milligrams (mg) of wet weight from mice described in A. (c) Representative images of hematoxylin and eosin stained epididymal adipose tissue depots from mice described in (a). Magnification 200X, Scale Bars = 50 µm. (B) Quantitation of (a) retinyl-palmitate and (b) all-trans retinol levels in livers from mice described in A using of high performance liquid chromatography (HPLC). (C) Relative mRNA levels of (a) retinoic acid receptor ß2 (RARß2), (b) cytochrome P450-member 26a1 (Cyp26a1), and (c) cellular retinol binding protein-1 (Crbp1), in livers from mice described in A. (D) (a–d) Representative images of liver double immunofluorescence stained with antibodies against Crbp1 (red) and LRAT (green) from mice described in A. Crbp1 positive SCs (yellow/orange) marked with white arrows. Magnification 200X, Scale Bars = 50 µm. (E) Quantitation of % percentage of Crbp1 positive stellate cells (SCs) from D. (F) (a–d) Representative images of hematoxylin and eosin (H & E) stained livers from mice described in A. Magnification 200X, Scale Bars = 50 µm. (G) Quantitation of % percentage hepatic steatosis (% hepatocytes with gross macro- or micro-vesicular lipid) in from mice described in A.

Fig 4: HF Diet and Genetic Models of Obesity Have Decreased Tissue Levels of Vitamin A.(A) (a) Body weights of (wt) C57/BL6 mice fed either a standard lab chow diet (chow) or high fat diet (HFD) for 4 months, and 12 week-old ob/ob mice and db/db mice fed a standard lab chow diet. (b) Adipose tissue (epididymal) weight in milligrams (mg) of wet weight from mice described in A. (B) (a) Serum levels of retinol (µM) and (b) serum retinol binding protein-4 (RBP4) (ng/ml) in mice described in A. Quantitation of high performance liquid chromatography (HPLC) of (c) liver retinyl-palmitate and (d) liver all-trans retinol (ROL) levels, and ROL levels in (e) pancreas, (f) kidney, (g) lung, and (h) brain from mice described in A. (C) Relative mRNA levels of retinoic acid receptor ß2 (RARß2) and cellular retinol binding protein-1 (Crbp1) in (a,b) liver, (c,d) pancreas, (e,f) kidneys, and (g,h) lungs from mice described in A. (D) Representative images of liver, pancreas and kidney double immunofluorescence stained with antibodies against Crbp1 (red) and the stellate cell (SC) marker LRAT (green) in (a–d) liver, and Crbp1 (red) and the SC marker Vimentin (green) in (e–h) pancreas and islets (white dotted lines) and (i–l) kidney (glomerulus (GS), white dotted lines) from mice described in A. (D)(a–h) Crbp1 positive SCs (yellow/orange) marked with white arrows. Magnification 200X, Scale Bars = 50 µm. (E–G) Quantitation of % percentage of Crbp1 positive stellate cells (SCs) in (E) liver, (F) Pancreas, and (G) Kidneys from mice described in A.

Fig 5: Vitamin A Levels and Signaling are Reduced in Human Livers with Steatosis.(A) Representative hematoxylin and eosin (a–d), and trichrome (e–h) stained human livers with increasing percentage of fatty liver (0–80% steatosis). Magnification 100X, Scale Bars = 25 µm. (B) Regression analysis of hepatic (a) retinyl-palmitate (b) all-trans retinol levels, and (c) relative hepatic mRNA transcripts of the retinoic acid receptor ß2 (RARß2) and percent steatosis (0–80%) from human livers. All mRNA levels are expressed as relative fold change compared to livers with 0% steatosis. (C) Representative images of double immunofluorescence stained human livers with antibodies against Crbp1 (red) and LRAT (green). Crbp1 positive SCs (yellow/orange) marked with white arrows. Magnification 200X, Scale Bars = 50 µm. (D) Quantitation of percentage of Crbp1 positive stellate cells (SCs) in C. 10–15 random fields per group (2–5 samples per group) were imaged and analyzed for percent SC positive Crbp1 and then pooled within tertiles of steatosis: (0–20%), (40–60%) and (70–80%).