Vinpocetine inhibits the differentiation of 3T3-L1 cells and the expression of adipogenesis-associated factors

To confirm the effect of vinpocetine on lipid accumulation, cells were treated with different doses of vinpocetine at 2 days after MDI treatment. Adipogenesis was measured by ORO staining of lipid droplets and microscopic imaging 6 days after MDI treatment. ORO staining and microscopy results showed that treatment with vinpocetine reduced 3T3-L1 cell differentiation in a dose-dependent manner. Specifically, 50 and 100 μg/ml vinpocetine were the most effective doses for inhibiting adipocyte differentiation (Fig. 1a). Vinpocetine also decreased the protein expression levels of PPARγ, C/EBPα, and C/EBPβ (three master regulators of adipogenesis) and FASN and FABP4 (downstream factors of PPARγ) (Fig. 1b). To confirm that the inhibition of adipocyte differentiation was not due to attenuated cell viability, a cell viability assay was performed. The effect of vinpocetine on adipocyte differentiation was not attributable to a decline in cell viability (Fig. 1c).

Fig. 1: Effects of vinpocetine on MDI-induced adipogenesis in 3T3-L1 cells. a 3T3-L1 cells were treated with 0, 5, 10, 25, 50, or 100 μg/ml vinpocetine at 2 days after MDI treatment. Then, Oil Red O staining was performed on day 6 as described in the “Materials and methods”, and images were acquired by microscopy. b Vinpocetine was administered to 3T3-L1 cells at a range of doses, and cell lysates were harvested at 0 and 6 days after inducing differentiation. Protein expression of PPARγ, C/EBPα, C/EBPβ, and downstream factors, such as FABP4 and fatty acid synthase in 3T3-L1 cells was detected by western blotting. β-actin was used as a load control. c Cytotoxicity in 3T3-L1 preadipocytes treated with up to 100 μg/ml vinpocetine was measured by MTT assay (n = 3 for each lane). d 3T3-L1 cells were treated with vinpocetine at 50 or 100 μg/ml, and Oil Red O staining was performed as described in the “Materials and methods”. Cells were differentiated for 6 days. e Lipid accumulation in 3T3-L1 cells was measured using spectrophotometry as described in the “Materials and methods” (n = 3 for each lane). f, g 3T3-L1 cells were treated with 50 or 100 μg/ml vinpocetine at 2 days after MDI treatment, and cell lysates were harvested at days 0, 2, 4, and 6 after inducing the differentiation of the cells. The mRNA expression levels of the genes encoding adipogenesis-associated factors, such as PPARγ, C/EBPα, and C/EBPβ, and downstream factors, such as FABP4 and fatty acid synthase, were detected by RT-PCR analysis. The protein expression of adipogenesis-associated factors and downstream factors was detected by western blotting. β-actin was used as a load control. Data are presented as the mean ± standard error of the mean (SD); ns = not significant; **P < 0.01 for untreated control vs. 50 and 100 μg/ml vinpocetine Full size image

We performed ORO staining of the cells treated with 50 and 100 μg/ml vinpocetine, and the results showed that vinpocetine effectively attenuated MDI-mediated 3T3-L1 cell differentiation (Fig. 1d). Furthermore, vinpocetine-treated adipocytes had 40% less lipid accumulation than did the dimethyl sulfoxide-treated control adipocytes (Fig. 1e). Consistently, the protein and mRNA expression levels of PPARγ, C/EBPα, and C/EBPβ, as well as FABP4 and FASN were markedly reduced in 3T3-L1 cells after vinpocetine treatment (Fig. 1f, g). Collectively, vinpocetine inhibits adipocyte differentiation by decreasing the expression of adipogenesis-associated proteins and genes.

Vinpocetine retards adipocyte differentiation by inhibiting adipogenesis-associated cell signaling at an early stage and inducing lipolysis and UCP1 expression at a late stage

We attempted to further investigate whether the inhibitory effect of vinpocetine works at the early or late stage of adipogenesis. The ORO staining results showed that vinpocetine retarded all stages of adipocyte differentiation, including the early stage (day 0 to day 2), the late stage (day 0 to day 6), and after day 6 (Fig. 2a). Consistently, microscopy images showed that vinpocetine hindered both the early and late stages of adipocyte differentiation (Supplementary Figure S2A). Additionally, lipid accumulation was reduced by vinpocetine at both the early and late stages of differentiation (Fig. 2b). Furthermore, we investigated how vinpocetine attenuates adipocyte differentiation and lipid accumulation in terms of mode of function. Cellular signaling molecules, such as ERK, AKT, AMPK, and JAK2-STAT3, are known to regulate lipid formation and adipogenesis32. Therefore, we aimed to survey whether vinpocetine regulates these signaling pathways. Vinpocetine effectively attenuated the phosphorylation of AKT, ERK, and JAK2-STAT3, but not AMPK, at 4 days after MDI induction (Supplementary Figure S2b). We then investigated whether vinpocetine prohibited the phosphorylation of signaling molecules from day 2 to day 3 of adipocyte differentiation (Fig. 2c). These results showed that vinpocetine reduced the phosphorylation of adipogenesis-associated signaling pathways at the early stages of adipocyte differentiation. We also determined that vinpocetine induces the upregulation of cAMP levels and the phosphorylation of lipolysis-associated factors (Fig. 2d, e). We confirmed that the protein expression of UCP1 and PGC-1α, which induces thermogenesis and reduces lipid accumulation24,25, is augmented in vinpocetine-treated adipocytes 4 and 6 days after MDI treatment (Fig. 2f). OCRs were also augmented in vinpocetine-treated adipocytes 5 days after MDI induction (Fig. 2g). Taken together, vinpocetine represses adipocyte differentiation by inhibiting adipogenesis-associated cell signaling at the stages of differentiation and inhibits lipid accumulation by inducing the lipolysis pathway and UCP1 expression.

Fig. 2: Treatment with vinpocetine retards adipocyte differentiation by inhibiting adipogenesis-associated cell signaling at early stages and inducing lipolysis and UCP1 expression at late stages. a 3T3-L1 cells were treated with 50 or 100 μg/ml vinpocetine from day 0, and Oil Red O staining was performed as described in the “Materials and methods” on day 2, day 6, or after day 6 of adipocyte differentiation. b 3T3-L1 cells were treated with 50 or 100 μg/ml vinpocetine, and its effects on the early and late stages of 3T3-L1 differentiation and lipid accumulation were measured using spectrophotometry as described in the “Materials and methods” (n = 3 for each lane). c 3T3-L1 cells were treated with 50 or 100 μg/ml vinpocetine on day 2 after inducing differentiation, and cell lysates were harvested at 0, 1, 6, 12, and 24 h. The phosphorylated forms of AKT, ERK, JAK2, and STAT3 were detected by western blotting. β-actin was used as a load control. d, e 3T3-L1 cells were treated with vinpocetine, and cell lysates were harvested at 6 days after MDI induction. d The cAMP level was measured using a cAMP direct immunoassay kit as described in the “Material and methods” (n = 3 for each lane). e The phosphorylated and total forms of ATGL, PKA, and HSL were detected by western blotting. f 3T3-L1 cells were treated with 100 μg/ml vinpocetine, and cell lysates were harvested at days 4 and 6. The protein expression of UCP1 and PGC-1α was detected by western blotting. β-actin was used as a load control. g OCR was measured in differentiated 3T3-L1 cells in the presence of 2.6 μM oligomycin, 0.5 μM FCCP, and 1 μM rotenone/antimycin A (n = 3 for each lane). Data are presented as the mean ± SD; *P < 0.05; **P < 0.01; and ***P < 0.001 for untreated control vs. 50 and 100 μg/ml vinpocetine Full size image

Vinpocetine-treated mice have reduced body weight, WAT size, and adipogenesis-associated gene expression

Next, we investigated the anti-obesity effects of vinpocetine. Mice were fed an NFD or an HFD (containing 60% of kcal as fat) with or without vinpocetine for 10 weeks. As shown by the photographic data, the amount of gWAT was lessened in vinpocetine-treated mice, especially in the HFD-induced obese mice (Fig. 3a). Vinpocetine-treated mice also had lower body weight in both the NFD-fed and HFD-fed groups than that of the untreated control mice (Fig. 3b). The extent of the reduction in body weight was particularly notable in the HFD-fed mice. However, there were no significant differences in food intake between the two diet groups (Fig. 3c).

Fig. 3: Vinpocetine-treated mice have diminished weight and cell size of gWAT, as well as adipogenesis-associated gene expression. a Vinpocetine was administered to both HFD-fed and NFD-fed mice as described in the “Materials and methods”. b Body weight was measured using a weighing scale every 2 days. c Food intake was measured by weighing the remaining chow. d Gonadal WAT weight was measured after the 10-week experimental diet period. e The adipocyte size in gonadal WAT sections was determined by staining with hematoxylin and eosin (H&E). Size measurements were performed using ImageJ software. f Inguinal WAT weight was measured after the 10-week experimental diet period. g The adipocyte size of inguinal WAT sections was determined by staining with H&E. Size measurements were performed as above. h Gonadal WAT and inguinal WAT were collected, and tissue lysates were prepared as described in the “Materials and methods”. The mRNA expression of FABP4 and FASN was detected by qPCR analysis. β-actin was used as a normalization control. Data are presented as the mean ± SD; *P < 0.05 and ***P < 0.001 for untreated control vs. 10 and 20 mg/kg vinpocetine (unpaired t-tests). ###P < 0.0001 for untreated control vs. 10 and 20 mg/kg vinpocetine (repeated measures ANOVA) Full size image

Moreover, we identified that the vinpocetine-treated mice had a lower gWAT weight in both the NFD-fed and HFD-fed groups (Fig. 3d) and a lower ratio of gWAT weight to body weight than that of the untreated control mice (Supplementary Figure S3). For histological analysis, we performed H&E staining of sectioned gWAT. Reduced adipocyte sizes in gWAT were detected in the vinpocetine-treated NFD-fed and HFD-fed mice (Fig. 3e). We also determined the effect of vinpocetine in iWAT. The vinpocetine-treated mice had lower iWAT weight in both the NFD-fed and HFD-fed groups (Fig. 3f) and a lower ratio of iWAT weight to body weight than that of control mice (Supplementary Figure S4). Moreover, H&E staining data showed that adipocyte size in iWAT in the vinpocetine-treated NFD-fed and HFD-fed mice was reduced (Fig. 3g). Interestingly, the mRNA expression of FABP4 and FASN was reduced in gWAT and iWAT in the vinpocetine-treated HFD-fed mice (Fig. 3h), suggesting that vinpocetine significantly reduces lipid accumulation through the downregulation of FABP4 and FASN in gWAT. Similar to HFD-fed mice, the mRNA expression of FABP4 and FASN was reduced in vinpocetine-treated NFD-fed mice (Supplementary Figure S5). Taken together, vinpocetine reduces the weight of and cell size in both gWAT and iWAT by downregulating adipogenesis-associated gene expression.

Vinpocetine suppresses lipid accumulation in mouse liver tissue

We determined whether vinpocetine ameliorates lipid accumulation in the liver tissues of NFD-fed and HFD-fed mice. We identified that the liver weight of vinpocetine-treated mice was lower than that of the control mice, but there were no significant differences in the liver weight to body weight ratio (Fig. 4a, Supplementary Figure S6). Next, we performed H&E staining of sectioned liver tissues. As expected, lipid accumulation in liver tissues was lessened in vinpocetine-treated NFD-fed and HFD-fed mice (Fig. 4b). The expression of adipogenesis-associated genes, including FABP4 and FASN, was attenuated in vinpocetine-treated liver tissues (Fig. 4c). Taken together, vinpocetine suppresses lipid accumulation in liver tissues.

Fig. 4: Vinpocetine retards lipid accumulation in mouse liver tissues and BAT. Vinpocetine was intraperitoneally injected into HFD mice, as described in the “Materials and methods”. a Both NFD-fed and HFD-fed mice were sacrificed after the 10-week experimental diet period. Then, the liver weight of these mice was measured. b H&E staining of liver sections was performed in each indicated group treated with or without vinpocetine for 10 weeks as described in the “Materials and methods”. c Liver tissues were collected, and tissue lysates were prepared as described in the “Materials and methods”. The mRNA expression of FABP4 and FASN was detected by qPCR analysis. β-actin was used as a normalization control. d Both NFD-fed and HFD-fed mice were sacrificed after the 10-week experimental diet period. Then, the BAT weight was measured using a weighing scale. e H&E staining of BAT sections from mice was performed in the indicated group treated with or without vinpocetine for 10 weeks as described in the “Materials and methods.” f BAT was collected, and tissue lysate was prepared as described in the “Materials and methods” The protein expression of UCP1 and PGC-1α was detected by western blotting. β-actin was used as a load control. g BAT was collected, and tissue lysate was prepared as described in the “Materials and methods”. The mRNA expression of UCP1 was detected by qPCR analysis. β-actin was used as a normalization control. Data are presented as the mean ± SD (n = 4 for NFD-fed mice and n = 8 for HFD-fed mice); *P < 0.05, **P < 0.01, and ***P < 0.001 for untreated control vs. 10 and 20 mg/kg vinpocetine Full size image

Vinpocetine induces BAT activation

Similar to its effect in liver tissue, vinpocetine reduced the weight of BAT in HFD-fed mice to a greater degree than that in untreated control HFD-fed mice, whereas it did not affect BAT weight in NFD-fed mice (Fig. 4d). For histological analysis, we performed H&E staining of sectioned BAT tissues. The results indicated that the lipid ratio was reduced in BAT sections from HFD-fed mice (Fig. 4e). However, there were no differences in the ratio of BAT weight to body weight in HFD-fed mice (Supplementary Figure S7).

We confirmed the protein expression of UCP1 and PGC-1α (Fig. 4f) and also analyzed the mRNA expression of UCP1 in BAT (Fig. 4g). Interestingly, vinpocetine significantly increased the protein and mRNA expression of UCP1 in NFD-fed and HFD-fed mice. Taken together, the results indicate that vinpocetine inhibits lipid accumulation by upregulating the expression of UCP1.

Vinpocetine ameliorates metabolic parameters related to hyperlipidemia, liver function, and glucose homeostasis

We determined whether vinpocetine might ameliorate metabolic parameters, such as the levels of TG, FFA as a blood fat, and ALT as a biomarker for liver health in the serum of the experimental mice. As expected, vinpocetine diminished the levels of TG, FAA, and ALT in both the NFD-fed and HFD-fed mice (Fig. 5a). Considering the effects of vinpocetine on glucose homeostasis, we performed GTT assays for 120 min. Clearance of blood glucose was significantly faster in vinpocetine-treated mice than in control mice (Fig. 5b). Insulin sensitivity was analyzed by ITT. There were no differences in blood glucose levels between the vinpocetine-treated NFD-fed mice and the untreated control NFD-fed mice, but the blood glucose level was lower in the vinpocetine-treated HFD-fed mice than in the control HFD-fed mice (Fig. 5c). These data indicate that vinpocetine improves hyperlipidemia, liver function, and glucose homeostasis in HFD-induced obese mice.

Fig. 5: Vinpocetine improves serum metabolic parameters and glucose homeostasis. a Serum analyses of TG, FFA, and ALT were performed as described in the “Materials and methods”. b For glucose tolerance tests, 1 g/kg glucose was intraperitoneally injected into both the NFD-fed and HFD-fed mice. c For insulin tolerance tests, 1 U/kg insulin was intraperitoneally administered into both the NFD-fed and HFD-fed mice. The blood glucose level was measured at different time points as described in the “Material and methods”. Data are presented as the mean ± SD (n = 4 for NFD-fed mice and n = 8 for HFD-fed mice) *P < 0.05; **P < 0.01; and ***P < 0.001 for untreated control vs. 10 and 20 mg/kg vinpocetine Full size image

Vinpocetine hampers the secretion of adipokines in vitro and in vivo

Because we initially identified that vinpocetine inhibited the activation of the STAT3 pathway (Fig. 2d), we hypothesized that vinpocetine might downregulate cytokines, such as IL-6, IL-10, and IFN-α, in 3T3-L1 cells and in the WAT of HFD-fed mice. The mRNA expression of cytokine-encoding genes, including IL-6, IL-10, and IFN-α, was markedly diminished by vinpocetine in 3T3-L1 cells (Fig. 6a) and in the WAT of HFD-fed mice (Fig. 6b). These data show that vinpocetine inhibits the activation of adipokines both in vitro and in vivo.