Functional roles of pantothenic acid, riboflavin, thiamine, and choline in adipocyte browning in chemically induced human brown adipocytes – PubMed Black Hawk Supplements

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Brown fat is a therapeutic target for the treatment of obesity-associated metabolic diseases. However, nutritional intervention strategies for increasing the mass and activity of human brown adipocytes have not yet been established. To identify vitamins required for brown adipogenesis and adipocyte browning, chemical compound-induced brown adipocytes (ciBAs) were converted from human dermal fibroblasts under serum-free and vitamin-free conditions. Choline was found to be essential for…
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Functional roles of pantothenic acid, riboflavin, thiamine, and choline in adipocyte browning in chemically induced human brown adipocytes - PubMed

Functional roles of pantothenic acid, riboflavin, thiamine, and choline in adipocyte browning in chemically induced human brown adipocytes

Yukimasa Takeda et al. Sci Rep. .

Abstract

Brown fat is a therapeutic target for the treatment of obesity-associated metabolic diseases. However, nutritional intervention strategies for increasing the mass and activity of human brown adipocytes have not yet been established. To identify vitamins required for brown adipogenesis and adipocyte browning, chemical compound-induced brown adipocytes (ciBAs) were converted from human dermal fibroblasts under serum-free and vitamin-free conditions. Choline was found to be essential for adipogenesis. Additional treatment with pantothenic acid (PA) provided choline-induced immature adipocytes with browning properties and metabolic maturation, including uncoupling protein 1 (UCP1) expression, lipolysis, and mitochondrial respiration. However, treatment with high PA concentrations attenuated these effects along with decreased glycolysis. Transcriptome analysis showed that a low PA concentration activated metabolic genes, including the futile creatine cycle-related thermogenic genes, which was reversed by a high PA concentration. Riboflavin treatment suppressed thermogenic gene expression and increased lipolysis, implying a metabolic pathway different from that of PA. Thiamine treatment slightly activated thermogenic genes along with decreased glycolysis. In summary, our results suggest that specific B vitamins and choline are uniquely involved in the regulation of adipocyte browning via cellular energy metabolism in a concentration-dependent manner.

Keywords: Brown adipocytes; Choline; Pantothenic acid; Riboflavin; Thiamine; Vitamin B.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Figure 1
Figure 1

Identification of vitamins required for the conversion of HDFs to ciBAs. (A) The expression of UCP1 and FABP4 was measured by qRT-PCR analysis in ciBAs converted using the serum-free brown adipogenic medium (SFBAM) in the presence or absence of eight vitamins (V8) included in the basal culture medium throughout the conversion. One-way ANOVA with Tukey’s multiple comparison tests was performed by comparing each value with the one under the condition of SFBAM(+ V8) including RoFB, as indicated by dashed bars in the figures. (B) The protein levels of UCP1, ATGL, and β-Actin were quantified by immunoblotting in the ciBAs. The band intensities were quantified by densitometry using ImageJ software. β-Actin was used as a loading control for normalization. (C) The expression of UCP1 and FABP4 was measured in ciBAs converted by SFBAM excluding each of the eight vitamins. One-way ANOVA with Tukey’s multiple comparison tests was performed by comparing each value with the one under the condition of RoFB and all the vitamins, as indicated by dashed bars in the figures. (D) The protein levels of UCP1 and β-Actin were quantified by immunoblotting in the ciBAs. (E) The expression of UCP1 and FABP4 was measured in ciBAs converted by SFBAM only containing choline and another vitamin. One-way ANOVA with Tukey’s multiple comparison tests was performed by comparing each value with the one under the condition of RoFB and only choline, as indicated by dashed bars in the figures. (F) The protein levels of UCP1 and β-Actin were quantified by immunoblotting in the ciBAs. Data represent mean ± SD (n = 3). One-way ANOVA with Tukey’s multiple comparison tests: * p < 0.05, ** p < 0.01, *** p < 0.001, N.S.; not significant.

Figure 2
Figure 2

Effects of choline and pantothenic acid on UCP1 expression and lipid metabolism in ciBAs. (A) The expression of UCP1, CIDEA, and FABP4 was measured by qRT-PCR analysis in ciBAs converted by the serum-free medium in the presence or absence of choline (Ch) and pantothenic acid (PA) throughout the conversion, as indicated. (B) The protein levels of UCP1, ATGL, CEBPA, and β-Actin were quantified by immunoblotting in the ciBAs. (C) The band intensities were quantified by densitometry using ImageJ software. β-Actin was used as a loading control for normalization. (D) Representative images of bright field, lipid droplets stained by Lipi-Red (red), UCP1 expression (green), and merged image in the ciBAs converted in the presence or absence of PA throughout the conversion. The nuclei were visualised by DAPI (blue). Scale bars represent 200 μm. (E) The area of the staining for lipid droplets and UCP1 was quantified by ImageJ software. P values were determined using student’s t-test. (F,G) Glycerol secretion and triglyceride accumulation were measured in ciBAs converted under each condition. Data represent mean ± SD (n = 3). One-way ANOVA with Tukey’s multiple comparison tests: * p < 0.05, ** p < 0.01, *** p < 0.001, N.S.; not significant.

Figure 3
Figure 3

Concentration-dependent effects of PA on UCP1 expression in ciBAs. (A) The expression of UCP1, CIDEA, and FABP4 was measured by qRT-PCR analysis in ciBAs treated with Ch at concentrations from 0.25 to 16 μg/mL throughout the experiments. (B) The expression was measured in ciBAs treated with PA at concentrations from 0.25 to 16 μg/mL. (C) UCP1 and β-Actin proteins were detected by immunoblotting analysis in ciBAs treated with PA at various concentrations. (D) Representative images of Lipi-Red staining (red), UCP1 expression (green), and DAPI (blue) in the ciBAs treated with PA at various concentrations throughout the experiments. The area of the staining for Lipi-Red and UCP1 was quantified by ImageJ software. (E–G) Glycerol secretion, triglyceride accumulation, and glycerol-3-phosphate dehydrogenase 1 (GPDH) activity were measured in ciBAs treated with PA at various concentrations. (H) The phosphorylation of CREB and HSL proteins was quantified by immunoblotting analysis in ciBAs treated with PA at 0.5 μg/mL and 16 μg/mL throughout the experiments. The band intensities were quantified by densitometry using ImageJ software. (I) Cellular mitochondria contents were evaluated by MitoTracker staining in ciBAs treated with PA at various concentrations. Data represent mean ± SD (n = 3). One-way ANOVA with Tukey’s multiple comparison tests: * p < 0.05, ** p < 0.01, *** p < 0.001, N.S.; not significant.

Figure 4
Figure 4

Concentration-dependent effects of PA on mitochondrial respiration and glycolysis. (A) Oxygen consumption rate (OCR) was measured using Seahorse XFe96 extracellular flux analyzer in control fibroblasts, NoC(V7) (grey circles), and ciBAs converted in the absence of PA, RoFB(V7) (black diamonds). Mitochondrial respiration inhibitors, oligomycin, FCCP, and antimycin A/rotenone, were added during the measurement, as indicated. P values were determined using two-way ANOVA. (B) OCR was compared between RoFB(V7) (grey circles) and either RoFB(V7 + PA, 0.5 μg/mL), RoFB(V7 + PA, 4 μg/mL), or RoFB(V7 + PA, 16 μg/mL) (black diamonds). P values were determined using two-way ANOVA. (C,D) OCR corresponding to basal respiration, maximal respiration, ATP production, and proton leak was compared. (E) Extracellular acidification rate (ECAR) was measured in control fibroblasts, NoC(V7) (grey circles), and ciBAs in the absence of PA, RoFB(V7) (black diamonds). Glucose, oligomycin, and 2-deoxyglucose (2-DG) were sequentially added during the measurement, as indicated. P values were determined using two-way ANOVA. (F) ECAR was also compared between the control ciBAs, RoFB(V7) (grey circles), and either RoFB(V7 + PA, 0.5 μg/mL), RoFB(V7 + PA, 4 μg/mL), or RoFB(V7 + PA, 16 μg/mL) (black diamonds). P values were determined using two-way ANOVA. (G) ECAR corresponding to glycolysis and glycolytic capacity was calculated. Data represent mean ± SD (n = 6–8). (H) Lactate secretion into culture supernatants was quantified in control fibroblasts and ciBAs treated with PA at various concentrations throughout the experiments. Data represent mean ± SD (n = 3). (I) Mitochondrial membrane potential (MMP) was evaluated by staining with the fluorescent probe, MT-1 dye. The area of the staining for the dye was quantified by ImageJ software. Data represent mean ± SD (n = 5). One-way ANOVA with Tukey’s multiple comparison tests: * p < 0.05, ** p < 0.01, *** p < 0.001, N.S.; not significant.

Figure 5
Figure 5

Comparison of the transcriptome in ciBAs treated with PA at low and high concentrations throughout the conversion. (A) Multidimensional scaling analysis graphically indicates the similarity and variability of the transcriptome in the control fibroblast, NoC(V7), ciBAs, RoFB(V7), and ciBAs treated with PA at low and high concentrations, RoFB(V7 + PA, 0.5 μg/mL) and RoFB(V7 + PA, 16 μg/mL). (B) Gene ontology (GO) enrichment analysis was performed in upregulated differentially expressed genes (DEGs) in RoFB(V7 + PA, 0.5 μg/mL) compared with RoFB(V7). The top 10 GO terms are represented in the category of biological process. (C) GO analysis was performed in downregulated DEGs in RoFB(V7 + PA, 16 μg/mL) compared with RoFB(V7 + PA, 0.5 μg/mL). (D) The FPKM values in the RNA-Seq results indicate the transcriptional levels of UCP1, CKMT1A, CKMT1B, CKMT2, CKB, ALPL (TNAP), SLC6A8, and CKM genes. (E) UCP1, CKMT1, CKMT2, CKB, ALPL (TNAP), and SLC6A8 mRNA were measured by qRT-PCR analysis in ciBAs treated with PA as indicated. Data represent mean ± SD (n = 3). One-way ANOVA with Tukey’s multiple comparison tests: *P < 0.05, **P < 0.01, ***P < 0.001, N.S.; not significant.

Figure 6
Figure 6

Effects of riboflavin on thermogenic gene expression and lipolysis in ciBAs. (A) The expression of UCP1, CIDEA, and FABP4 was quantified by qRT-PCR analysis in ciBAs treated with Ch (4 μg/mL) and riboflavin at concentrations from 0.05 to 5 μg/mL throughout the experiments. (B,C) The expression of UCP1, CIDEA, FABP4, CKMT1, CKMT2, CKB, and ALPL was quantified by qRT-PCR analysis in ciBAs treated with riboflavin in the presence of the other vitamins. (D) The fold change of the expression was evaluated in ciBAs treated with the combination of PA and riboflavin at various concentrations, as indicated. (E–H) Glycerol secretion, triglyceride accumulation, lactate secretion, and MMP were measured in ciBAs treated with riboflavin at various concentrations. Data represent mean ± SD (n = 3). One-way ANOVA with Tukey’s multiple comparison tests: * p < 0.05, ** p < 0.01, *** p < 0.001, N.S.; not significant.

Figure 7
Figure 7

Effects of thiamine on thermogenic gene expression and glycolysis in ciBAs. (A) The expression of UCP1, CIDEA, and FABP4 was quantified by qRT-PCR analysis in ciBAs treated with Ch (4 μg/mL) and thiamine at concentrations from 0.5 to 50 μg/mL throughout the experiments. (B,C) The expression of UCP1, CIDEA, FABP4, CKMT1, CKMT2, CKB, and ALPL was quantified by qRT-PCR analysis in ciBAs treated with thiamine in the presence of the other vitamins. (D-G) Glycerol secretion, triglyceride accumulation, lactate secretion, and MMP were measured in ciBAs treated with thiamine at various concentrations. Data represent mean ± SD (n = 3). One-way ANOVA with Tukey’s multiple comparison tests: * p < 0.05, ** p < 0.01, *** p < 0.001, N.S.; not significant.

Figure 8
Figure 8

Schematic illustration of the role of choline (Ch), pantothenic acid (PA), riboflavin, and thiamine in the thermogenic and metabolic functions in ciBAs. (A) Ch is indispensable for adipocyte formation during the conversion of HDFs into ciBAs. The low concentration of PA is sufficient for Ch-induced immature ciBAs to enhance the expression of UCP1 and phosphocreatine (PCr) metabolic genes, lipolysis, triglyceride accumulation, mitochondrial respiration, and MMP. However, treatment with PA at high concentrations represses PA-activated thermogenic expression and metabolic maturation along with reduced glycolysis. (B) Riboflavin treatment activates lipolysis, however, thermogenic gene expression, triglycerides, and MMP are repressed in a dose-dependent manner. (C) Thiamine treatment slightly activates thermogenic gene expression at high concentrations although glycolysis and lipolysis rates are reduced as a consequence.

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Functional roles of pantothenic acid, riboflavin, thiamine, and choline in adipocyte browning in chemically induced human brown adipocytes – PubMed