Oct1 deficiency results in significantly reduced liver TG content. (A) Liver weight/body weight ratios of Oct1^+/+, Oct1^−/−, Oct1^+/+,ob/ob, and Oct1^{−/−,ob/ob} mice (n = 10 per genotype for lean mice and n = 6 per genotype for ob/ob mice). (B) Quantification of TG amounts in the livers from mice with four genotypes shown in A. (C) Representative Oil red O-stained (ORO) (Upper) and H&E-stained (Lower) sections of livers. (Scale bar, 50 μm.)

Oct1^−/− mouse livers exhibit changes in enzymes related to steatosis. (A) Representative Western blots of key regulatory enzymes, ACC, AMPK, and LKB1 involved in lipid metabolism. (B) Quantification of Western blotting.

Metabolomic study in stably transfected cells reveals that thiamine is a principal substrate of OCT1/Oct1. (A) Relative abundance of 2-methylbutyrylcarnitine and (B) thiamine in HEK293-EV and HEK293-OCT1 from metabolomic studies (n = 6 cell lines per group). (C) Time course of uptake of [³H]thiamine in HEK293-EV and HEK293-hOCT1 cells. (D) Kinetics of [³H]thiamine uptake by human OCT1, mouse Oct1, and mouse Slc19a2 transporters. Kinetics parameters are summarized in SI Appendix, Table S4.

Characterization of the role of Oct1 on thiamine disposition in vivo and its hepatic location. (A) Level of thiamine derivatives: thiamine monophosphate (TMP) and thiamine pyrophosphate (TPP) in Oct1^+/+ and Oct1^−/− mouse liver extracts. (B) Total [³H]thiamine radioactivity in plasma from Oct1^+/+ and Oct1^−/− mice after administration of an i.v. dose of [³H]thiamine. (C) Total [³H]thiamine radioactivity in various tissues from Oct1^+/+ and Oct1^−/− mice 5 min after administration of an i.v. dose of [³H]thiamine. Data were normalized to WT in the corresponding tissues, which were set at 100%. (D) mRNA expression levels of various thiamine transporters in livers from WT mice. The expression level was normalized to the Slc19a3 set at 1. (E) Central vein is the primary expression location of Oct1 in mouse liver. (Upper) X-Gal detection of enzymatic lacZ activity (panel 1) and immunofluorescence staining of β-Gal in liver sections from Oct1^−/− mice (panel 2). (Lower) Immunofluorescence staining of liver sections of the central vein marker glutamine synthetase (GS, red, panel 3), β-Gal (green, panel 4), and merged (panel 5) from Oct1^−/− mice. (White scale bar, 50 μm; red scale bar, 25 μm.) (F) Immunofluorescence staining of the key glycolysis enzyme pyruvate dehydrogenase (PDH), which is primarily localized in the vicinity of the central vein. (Left) GS, red; (Center) PDH, green; (Right) overlap, yellow. (Scale bar, 50 μm.) (G) Representative livers (Left) and TG levels (Right) from WT mice fed thiamine-deficient (TD) diet or normal diet (n = 6 for each diet) for 3 wk. All data are mean ± SEM *P < 0.05, **P < 0.01, ***P < 0.001.

Human OCT1 transgenic mice fed a high-fat diet show significantly higher TG concentrations and enlarged liver sizes. (A) Location of transgenic hOCT1 in liver sections from Oct1^+/+, Oct1^{−/−,OCT1tg}, and Oct1^+/+,OCT1tg mice. Yellow arrows indicate the sporadic expression of hOCT1. Red arrows indicate that hOCT1 is expressed around the central vein. (Lower Right) Magnification 20×, compared with other panels (10×). (White scale bar, 50 μm; red scale bar, 25 μm.) (B) Representative MRI of livers from mice. The heat map from blue to red represents the fat content from low to high. MRI parameters are summarized in SI Appendix, Table S5. (Scale bar, 4 mm.)

The effect of metformin, Oct1, and thiamine deficiency on OCT1-mediated thiamine disposition and liver energy status. (A) The inhibitory effect of metformin and phenformin on OCT1-mediated [³H]thiamine uptake (IC₅₀: metformin, 1.4 mM; phenformin, 0.07 mM). (B) Thiamine and metformin uptake by nonsynonymous human OCT1 genetic polymorphisms. OCT1-ref refers to the reference OCT1 without any of the nonsynonymous polymorphisms. (C) [³H]thiamine accumulation in various tissues from Oct1^+/+ mice 5 min after i.v. treatment with [³H]thiamine (2 mg/kg) alone (white bars) or [³H]thiamine (2 mg/kg) plus metformin (50 mg/kg) (black bars). (D) Plasma levels of thiamine and its metabolites in wild-type C57BL/6J mice treated with metformin (100 mg/kg, i.p.) or saline for 7 d. *P < 0.05, **P < 0.01 compared with the mice treated with saline; n = 3 (saline) and n = 5 (metformin). (E) Quantifications of P-ACC (using ELISA, see SI Appendix) and AMP/ATP ratio in liver cell lysates from mice that were fed a normal diet or a thiamine-deficient diet for 7 d. (F) Adenine nucleotide ratios (AMP/ATP and ADP/ATP) in livers from Oct1^+/+ and Oct1^−/− mice. All data are mean ± SEM *P < 0.05, **P < 0.01, ***P < 0.001. (G) Cartoon illustration of the physiological role of OCT1 in thiamine disposition in the liver. Reduced hepatic thiamine and consequently lower TPP levels occur as a result of deletion of OCT1 or potentially from metformin treatment. The reduced ATP production as a result of decreasing PDH-TPP and OGDH-TPP activity may further activate AMPK and ACC, triggering β-oxidation to increase the shortfall of energy in the hepatocytes. Metformin treatment may mimic this effect. Red arrows represent the pathways; black up arrow represents increased levels or activities; black down arrow represents reduced levels or activities.