Brown and beige adipocytes dissipate energy by uncoupling protein 1 (UCP1)-dependent andindependent thermogenesis, which may be utilized to develop treatments against obesity. We have found that mRNA and protein expression of the alanine-serine-cysteine transporter-1 (ASC-1) was induced during adipocyte differentiation of human brown-prone deep neck and beigecompetent subcutaneous neck progenitors, and SGBS preadipocytes. cAMP stimulation of differentiated adipocytes led to elevated uptake of serine, cysteine, and glycine, in parallel with increased oxygen consumption, augmented UCP1-dependent proton leak, increased creatine-driven substrate cycle coupled respiration, and upregulation of thermogenesis marker genes and several respiratory complex subunits; these outcomes were impeded in the presence of the specific ASC-1 inhibitor, BMS-466442. Our data suggest that ASC-1-dependent consumption of serine, cysteine, and glycine is required for efficient thermogenic stimulation of human adipocytes.

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.

White adipocytes store lipids, have a large lipid droplet and few mitochondria. Brown and beige adipocytes, which produce heat, are characterized by high expression of uncoupling protein (UCP) 1, multilocular lipid droplets, and large amounts of mitochondria. The rs1421085 T-to-C single-nucleotide polymorphism (SNP) of the human FTO gene interrupts a conserved motif for ARID5B repressor, resulting in adipocyte type shift from beige to white. We obtained abdominal subcutaneous adipose tissue from donors carrying FTO rs1421085 TT (risk-free) or CC (obesity-risk) genotypes, isolated and differentiated their preadipocytes into beige adipocytes (driven by the PPARγ agonist rosiglitazone for 14 days), and activated them with dibutyryl-cAMP for 4 hours. Then, either the same culture conditions were applied for additional 14 days (active beige adipocytes) or it was replaced by a white differentiation medium (inactive beige adipocytes). White adipocytes were differentiated by their medium for 28 days. RNA-sequencing was performed to investigate the gene expression pattern of adipocytes carrying different FTO alleles and found that active beige adipocytes had higher brown adipocyte content and browning capacity compared to white or inactive beige ones when the cells were obtained from risk-free TT but not from obesity-risk CC genotype carriers. Active beige adipocytes carrying FTO CC had lower thermogenic gene (e.g., UCP1, PM20D1, CIDEA) expression and thermogenesis measured by proton leak respiration as compared to TT carriers. In addition, active beige adipocytes with CC alleles exerted lower expression of ASC1 neutral amino acid transporter (encoded by SLC7A10) and less consumption of Ala, Ser, Cys, and Gly as compared to risk-free carriers. We did not observe any influence of the FTO rs1421085 SNP on white and inactive beige adipocytes highlighting its exclusive and critical effect when adipocytes were activated for thermogenesis.

Free radicals contribute to the pathophysiology of degenerative diseases which increase mortality globally, including mortality in Indonesia. Amomum compactum Soland. Ex Maton fruit from the Zingiberaceae family, also known as Java cardamom, contains secondary metabolites that have high antioxidant activities. The antioxidant activity of the methanol extract of Java cardamom fruit correlates with its flavonoid and phenolic compound contents, which can be affected by different methods and durations of extraction. This study aimed to measure and compare the effects of extraction methods and durations on total flavonoid and phenolic contents (TFCs and TPCs) and subsequent antioxidant activities by the 2,2′-diphenyl-1-picrylhydrazyl (DPPH) radical, ferric reducing antioxidant power (FRAP), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), and cupric ion reducing antioxidant capacity (CUPRAC) assays. Methanol extracts of Java cardamom were produced by continuous shaking (CSE), microwave-assisted (MAE), or ultrasonic-assisted extractions (UAE) for three different durations. CSE for 360 min resulted in the highest TFCs (3.202 mg Quercetin Equivalent/g dry weight), while the highest TPCs (1.263 mg Gallic Acid Equivalent/g dry weight) were obtained by MAE for 3 min. Out of the investigated methods, MAE for 3 min resulted in the highest antioxidant activity results for the extracts. We conclude that the polyphenolic antioxidant yield of Java cardamom depends on two parameters: the method and the duration of extraction.

Brown adipose tissue (BAT) dissipates energy in the form of heat majorly via the mitochondrial uncoupling protein 1 (UCP1). The activation of BAT, which is enriched in the neck area and contains brown and beige adipocytes in humans, was considered as a potential therapeutic target to treat obesity. Therefore, finding novel agents that can stimulate the differentiation and recruitment of brown or beige thermogenic adipocytes are important subjects for investigation. The current study investigated how the availability of extracellular thiamine (vitamin B1), an essential cofactor of mitochondrial enzyme complexes that catalyze key steps in the catabolism of nutrients, affects the expression of thermogenic marker genes and proteins and subsequent functional parameters during ex vivo adipocyte differentiation. Therefore, we differentiated primary human adipogenic progenitors that were cultivated from subcutaneous (SC) or deep neck (DN) adipose tissues in the presence of gradually increasing thiamine concentrations during their 14 day long differentiation program. Higher thiamine levels resulted in increased expression of thiamine transporter 1 and 2 both at mRNA and protein levels in human neck area-derived adipocytes. Gradually increasing concentrations of thiamine led to increased basal, cAMP-stimulated, and proton-leak respiration along with elevated mitochondrial biogenesis of the differentiated adipocytes. The extracellular thiamine availability during adipogenesis determined the expression levels of UCP1, PGC1a, CKMT2, and other browning-related genes and proteins in primary SC and DN-derived adipocytes in a concentration-dependent manner. Providing abundant amounts of thiamine further increased the thermogenic competency of the adipocytes. Our study raises the possibility of a novel strategy with long-term thiamine supplementation, which can enhance the thermogenic competency of differentiating neck area-derived adipocytes for preventing or combating obesity.

No abstract

Introduction: White adipocytes store lipids, have a large lipid droplet and few mitochondria. Brown and beige adipocytes, which produce heat, are characterized by high expression of uncoupling protein (UCP) 1, multilocular lipid droplets, and large amounts of mitochondria. The rs1421085 T-to-C single-nucleotide polymorphism (SNP) of the human FTO gene interrupts a conserved motif for ARID5B repressor, resulting in adipocyte type shift from beige to white.Methods: We obtained abdominal subcutaneous adipose tissue from donors carrying FTO rs1421085 TT (risk-free) or CC (obesity-risk) genotypes, isolated and differentiated their preadipocytes into beige adipocytes (driven by the PPARγ agonist rosiglitazone for 14 days), and activated them with dibutyryl-cAMP for 4 hours. Then, either the same culture conditions were applied for additional 14 days (active beige adipocytes) or it was replaced by a white differentiation medium (inactive beige adipocytes). White adipocytes were differentiated by their medium for 28 days.Results and Discussion: RNA-sequencing was performed to investigate the gene expression pattern of adipocytes carrying different FTO alleles and found that active beige adipocytes had higher brown adipocyte content and browning capacity compared to white or inactive beige ones when the cells were obtained from risk-free TT but not from obesity-risk CC genotype carriers. Active beige adipocytes carrying FTO CC had lower thermogenic gene (e.g., UCP1, PM20D1, CIDEA) expression and thermogenesis measured by proton leak respiration as compared to TT carriers. In addition, active beige adipocytes with CC alleles exerted lower expression of ASC-1 neutral amino acid transporter (encoded by SLC7A10) and less consumption of Ala, Ser, Cys, and Gly as compared to risk-free carriers. We did not observe any influence of the FTO rs1421085 SNP on white and inactive beige adipocytes highlighting its exclusive and critical effect when adipocytes were activated for thermogenesis.