The Thiamine Transporter 2 (THTR2) encoded by SLC19A3 plays an ill-defined role in the maintenance of tissue thiamine, thiamine monophosphate, and thiamine diphosphate (TDP) levels. To evaluate the impact of THTR2 on tissue thiamine status and metabolism, we expressed the human SLC19A3 transgene in the intestine of total body Slc19a3 knockout (KO) mice. Male and female wildtype (WT) and transgenic (TG) mice were fed either 17 mg/kg (1×) or 85 mg/kg (5×) thiamine hydrochloride diet, while KOs were only fed the 5× diet. Thiamine vitamers in plasma, red blood cells, duodenum, brain, liver, kidney, heart, and adipose tissue were measured. Untargeted metabolomics were performed on the brain tissues of groups with equivalent plasma thiamine. KO mice had ~two- and ~three-fold lower plasma and brain thiamine levels than WT on the 5× diet. Circulating vitamers were sensitive to diet and equivalent in TG and WT mice. However, TG had 60% lower thiamine but normal brain TDP levels regardless of diet, with subtle differences in the heart and liver. The loss of THTR2 reduced levels of nucleic acid and amino acid derivatives in the brain. Therefore, mutation or inhibition of THTR2 may alter the brain metabolome and reduce the thiamine reservoir for TDP biosynthesis.

Trimethoprim is predicted to inhibit several thiamine transporters, including the primary thiamine intestinal absorptive transporter, ThTR-2, and the hepatic and renal organic cation transporters, OCT1, OCT2, and MATEs. To investigate the effect of trimethoprim on thiamine absorption, studies were conducted in cells, mice, and healthy volunteers and supported by use of real-world data. In a randomized, crossover clinical study, seven healthy volunteers were given a single oral dose of thiamine or thiamine plus trimethoprim, followed by blood sampling. The thiamine area under the curve (AUC) increased with trimethoprim co-administration (P value = 0.031). Similar results were seen in mice. Trimethoprim appeared to act on thiamine absorption through inhibition of hepatic OCT1 as evidenced from its ability to modulate levels of isobutyrylcarnitine and propionylcarnitine, OCT1 biomarkers identified from metabolomic analyses. Real-world data further supported this finding, showing an association between trimethoprim use and higher levels of triglycerides, LDL cholesterol, and total cholesterol, consistent with OCT1 inhibition (P values: 2.2 × 10^-16 , 5.75 × 10^-7 , and 5.82 × 10^-7 , respectively). These findings suggest that trimethoprim increases plasma levels of thiamine by inhibiting hepatic OCT1. Trimethoprim reduced urinary excretion and clearance of biomarkers for OCT2 and MATEs, consistent with inhibition of renal organic cation transporters. This inhibition did not appear to play a role in the observed increases in thiamine levels. This study highlights the potential for drug-nutrient interactions involving transporters, in addition to transporters' established role in drug-drug interactions.

Objective

Regulation of fatty acid (FA) metabolism is central to adipocyte dysfunction during diet-induced obesity (DIO). Long-chain acyl-CoA synthetase-4 (ACSL4) has been hypothesized to modulate the metabolic fates of polyunsaturated FA (PUFA), including arachidonic acid (AA), but the in vivo actions of ACSL4 are unknown. The purpose of our studies was to determine the in vivo role of adipocyte ACSL4 in regulating obesity-associated adipocyte dysfunction.

Methods

We developed a novel mouse model with adipocyte-specific ablation of ACSL4 (Ad-KO) using loxP Cre recombinase technology. Metabolic phenotyping of Ad-KO mice relative to their floxed littermates (ACSL4^floxed) was performed, including body weight and body composition over time; insulin and glucose tolerance tests; and energy expenditure, activity, and food intake in metabolic cages. Adipocytes were isolated for ex vivo adipocyte oxygen consumption by Clark electrode and lipidomics analysis. In vitro adipocyte analysis including oxygen consumption by Seahorse and real-time PCR analysis were performed to confirm our in vivo findings.

Results

Ad-KO mice were protected against DIO, adipocyte death, and metabolic dysfunction. Adipocytes from Ad-KO mice fed high-fat diet (HFD) had reduced incorporation of AA into phospholipids (PL), free AA, and levels of the AA lipid peroxidation product 4-hydroxynonenal (4-HNE). Additionally, adipocytes from Ad-KO mice fed HFD had reduced p53 activation and increased adipocyte oxygen consumption (OCR), which we demonstrated are direct effects of 4-HNE on adipocytes in vitro.

Conclusion

These studies are the first to elucidate ACSL4's in vivo actions to regulate the incorporation of AA into PL and downstream effects on DIO-associated adipocyte dysfunction. By reducing the incorporation of AA into PL and free fatty acid pools in adipocytes, Ad-KO mice were significantly protected against HFD-induced increases in adipose and liver fat accumulation, adipocyte death, gonadal white adipose tissue (gWAT) inflammation, and insulin resistance (IR). Additionally, deficiency of adipocyte ACSL4 expression in mice fed a HFD resulted in increased gWAT adipocyte OCR and whole body energy expenditure (EE).