Availability of abundant thiamine determines efficiency of thermogenic activation in human neck area derived adipocytes

Abstract

Objective: Brown/beige adipocytes are characterized by expression of uncoupling protein-1 (UCP1) that enables them to dissipate energy as heat instead of generating ATP. The activation of this process can alleviate obesity as it augments energy expenditure. Human brown adipose tissue is interspersed in distinct anatomical regions including the deep neck area. We found that UCP1 enriched adipocytes differentiated from progenitors of this depot highly expressed ThTr2 transporter of thiamine, the precursor of thiamine pyrophosphate (TPP) cofactor for mitochondrial fuel generating enzymes, and aimed to investigate the importance of thiamine availability during thermogenic activation. Methods: Human adipocytes were differentiated from subcutaneous and deep neck precursors and activated for thermogenesis by the cell permeable dibutyryl-cAMP, which mimics adrenergic stimulation. Proton leak respiration reflecting heat generation was monitored by Seahorse XF analyzer. Direct mitochondrial effect of TPP was observed in flux assay of permeabilized adipocytes. Expression of thermogenic genes and proteins was analyzed by RT-qPCR and immunoblotting, respectively. Results: Inhibition of ThTr2 during thermogenic activation of deep and subcutaneous neck derived adipocytes by cAMP led to decreased proton leak respiration reflecting lower uncoupling activity. In the absence of thiamine, cAMP-induced elevation of proton leak respiration was diminished but restored by thiamine addition reaching highest levels in deep neck adipocytes and at concentrations larger than present in human blood plasma. Addition of TPP to permeabilized adipocytes increased proton leak respiration fueled by one of the TPP-dependent enzymes, pyruvate dehydrogenase. ThTr2 inhibition also hampered cAMP-dependent induction of UCP1, PGC1a and other browning marker genes, along with mitochondrial complex subunits, and thermogenic induction of these genes was potentiated by thiamine in a concentration dependent manner. Conclusion: Our study has revealed the importance of amply supplied thiamine during thermogenic activation in human adipocytes. Thiamine may increase heat generation by providing TPP for TPP-dependent enzymes which lack this cofactor and by potentiating the induction of UCP1 and other thermogenic genes.

Extracellular thiamine concentration influences thermogenic competency of differentiating neck area-derived human adipocytes

“…In our previous study, we found that high expression of ThTrs and high thiamine influx contributed to the effective thermogenic activation of neck area-derived adipocytes [Arianti et al, 2022]. Therefore, we primarily investigated how the expression of the ThTrs is affected by the presence of excess (25 µM or 50 µM) or gradually increasing (40 nM, 200 nM, 1 µM, or 5 µM) concentrations of thiamine during the differentiation of human SC and DN-derived ADIPs and B-ADIPs.…”

“…Cells were collected in TRIzol, total RNA was isolated by chloroform-isopropanol extraction, and RT-qPCR was performed as described previously [Klusóczki et al, 2019;Kristóf et al, 2016;Arianti et al, 2022]. Custom-made gene primers and probes were designed and supplied by Thermo Fisher Scientific (Waltham, MA, USA) as listed in Supplementary Table 1.…”

“…Immunoblotting and densitometry were carried out as described previously [Szatmári-Tóth et al, 2020;Arianti et al, 2022]. Antibodies and working dilutions are listed in Supplementary Table 2.…”

“…OCR and ECAR of adipocytes, which were differentiated as described in 2.3, were measured using an XF96 oximeter (Seahorse Biosciences, North Billerica, MA, USA) according to previously optimized protocols [Klusóczki et al, 2019;Kristóf et al, 2016;Arianti et al, 2022]. When the adipocytes were differentiated in the presence of excess thiamine, cells were pre-incubated for 1 h and treated in Agilent Seahorse XF Base Medium (Agilent Technologies, cat#103334-100), which is optimized for extracellular flux assays and contains thiamine at 8.2 µM concentration.…”

“…Based on the RNAsequencing analysis, 1049 differentially expressed genes (DEGs) were found in the comparison of SC and DN-derived adipocytes. Among these DEGs, we found 21 solute carrier (SLC) transporters and revealed the importance of alanine-serine-cysteine transporter 1 (ASC1, encoded by SLC7A10) [Arianti et al, 2021] and thiamine transporter 2 (ThTr2, encoded by SLC19A3) [Arianti et al, 2022] during thermogenic activation. One of the strongest upregulated SLC transporters in thermogenic DN-derived adipocytes was ThTr2, whose pharmacological inhibition attenuated cAMP-mediated thermogenic activation of human neck area-derived adipocytes [Arianti et al, 2022].…”