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. 2022 Dec;11(1):213-226.
doi: 10.1080/21623945.2022.2062852.

Dysfunction of insulin-AKT-UCP1 signalling inhibits transdifferentiation of human and mouse white preadipocytes into brown-like adipocytes

Jie Pan  1   2 , Suchart Kothan  2 , Aye Thidar Moe Moe  2 , Kun Huang  1
Affiliations

Dysfunction of insulin-AKT-UCP1 signalling inhibits transdifferentiation of human and mouse white preadipocytes into brown-like adipocytes

Jie Pan et al. Adipocyte. 2022 Dec.

Abstract

The mechanism of insulin signaling on browning of white preadipocytes remains unclear. Human and mouse primary subcutaneous white preadipocytes (hsASCs and WT lean and obese msASCs, respectively) were induced to transdifferentiate into beige adipocytes under conditions of intact or blocked insulin signaling, respectively. Level of phosphoinositide-3-kinase (PI3K) after induction of beige adipocytes under conditions of normal insulin signaling, phosphorylated protein kinase B (pAKT), peroxisome proliferator-activated receptor γ coactivator-1 alpha (PGC-1α), zinc-fifinger transcriptional factor PRD1-BF1-RIZ1 homologous domain-containing protein 16 (PRDM16), uncoupling protein 1 (UCP1), peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer binding protein beta (C/EBPβ) were significantly increased. Conversely, when insulin signaling is incompletely inhibited, the expression of the thermogenic and adipogenic factors is significantly reduced, with obvious impairment of adipogenesis. However, phosphorylation level of adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) and expression level of sirtuin type 1 (SIRT1) had increased. These white preadipocytes from different donors showed similar dynamic change in morphology and molecular levels during the browning. The present data indicate that insulin signaling plays a important role in regulation of browning of hsASCs and msASCs through PI3K-AKT-UCP1 signaling pathway. The insulin-AMPK-SIRT1 pathway was also involved in the adipocytes browning, while its effect is limited.

Keywords: AKT; Insulin signalling; UCP1; browning; white preadipocytes.

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Conflict of interest statement

No potential conflict of interest was reported by the author(s).

Figures

Figure 1.
Figure 1.
Time course of cell grouping and treatment of low temperature. (a), browning inducing for hsASCs, and (b), msASCs under conditions of different temperature treatment. CBAM, complete browning adipogenic medium. Maintenance medium, as adipogenic medium, for sustained induction of the cell browning. G, group. See the methods in the text for details.
Figure 2.
Figure 2.
Dynamic changes of primary hsASCs during browning under different conditions. (a), Dynamic changes in cell morphology. CBAM, cells were induced to browning with complete induction medium at 37°C. CBAM+OSI, treatment with 1 μM OSI-906 while inducing browning, inhibits insulin signalling throughout browning induction. Notably, OSI-906-treated cells exhibited significantly suppressed adipogenesis compared to CBAM-induced cells, i.e. from 72 ± 3.5% in the CBAM group down to 7 ± 1.12%. Red, Nile red staining of lipids. Blue, Hoechst 33,342 nuclear staining. Dn, days of induction. b, Western blot and quantitative protein expression analysis of key molecules in insulin signalling, showing that pAKT is decreased in an OSI-906 dose-dependent manner. Numbers marked above the protein bands represent the final concentration (μM) of OSI-906. hWACs, human white mature adipocytes; mBAT, WT mouse BAT, protein samples used as non-intervention controls for analysis. c, Target gene expression profiles analysed using qPCR, and (d), Western blot analysis of target proteins of browning-related molecules in hsASCs under conditions that intact normal or partially inhibit insulin signalling. After insulin signalling was blocked (CBAM+OSI), adipogenic molecules were significantly downregulated and browning markers were significantly reduced, but SIRT1 was significantly increased compared to cells with intact insulin signalling (CBAM). (e), Quantitative analysis of target protein expression. Compared with the qPCR data, the target molecule expression levels showed a similar dynamic trend. However, in contrast to the down-regulation of the expression levels of these molecules, pAMPK levels and SIRT1 expression were instead increased after insulin signalling was inhibited. *p < 0,05 and **p < 0.01 represent the different levels between groups (n > 7).
Figure 3.
Figure 3.
Browning characteristics of WT msASCs under intact and insulin signalling inhibited situations. (a), dynamic changes of cell morphology. Compared to the cells induced by CBAM, OSI-906 treated cells (CBAM+OSI) showed significantly repressed adipogenesis, from 78 ± 4.5% down to 5 ± 1.5%. Red colour, Nile red staining for lipids. Blue colour, nuclear staining by Hoechst 33,343. Dn, the number of days of induction. (b), Western blotting and protein expression quantification analysis of pAKT, the level of pAKT is decreases in OSI-906 dose dependent manner. Numbers labelled above the protein bands represent final concentration (μM) of OSI-906. The degree of insulin signalling inhibition depended on the dosage of OSI-906. mWACs, WT mouse white mature adipocytes; mBAT, WT mouse BAT, used as controls. (c, d and e), Target gene and protein expression of browning and adipogenic molecules in WT msASCs under conditions of intact and incomplete inhibition of insulin signalling were analysed by qPCR and Western blotting. Beige adipogenesis and browning marker molecules were significantly downregulated after insulin signalling was inhibited (CBAM+OSI), while SIRT1 was significantly increased compared to insulin signalling intact cells (CBAM). In contrast to the down-regulation of these molecules, pAMPK levels and SIRT1 expression instead increased after insulin signalling was inhibited. The dynamic trends of target gene mRNA and protein levels were similar to those seen in hsASCs. **p < 0.01 represent the different levels between groups (n > 7).
Figure 4.
Figure 4.
Dynamic changes of obese db/db msASCs in browning under different conditions. a, morphology of the cells. CBAM, the cells were induced for browning by using a complete inducing medium under 37°C. CBAM+OSI, the msASCs were induced for browning and also treatment the cells with 1 μM OSI-906, showing lipids production and accumulation of the cells was significantly inhibited. Red colour, Nile red staining for lipids. Blue colour, nuclear staining by Hoechst 33,342. Dn, the number of days of induction. b, qPCR analysis of target genes. c, Western blotting, and d, quantification of protein expression of db/db msASCs during browning. mWACs, WT mouse subcutaneous white mature adipocytes. mBAT, WT mouse BAT, as UCP1 positive control, its total protein was loaded 1/3 compared to other samples. Adipogenesis decreased significantly after inhibition of insulin signalling. On the contrary to down-regulation of insulin signalling molecules and browning markers, pAMPK level and SIRT1 expression were increased after insulin signalling was inhibited. These results were similar as those seen both in WT msASCs and hsASCs. *p < 0.05 and **p < 0.01 represent the different levels between groups (n > 7).
Figure 5.
Figure 5.
The dynamic changes of the browning of hsASCs which were exposed at 26°C for different days (see the grouping in Figure 1a). (a), dynamic changes of the cell morphology. G1, group 1, browning induction, and the culture temperature was switched from 37°C to 26°C at D10, for 2 d. G2, group 2, browning induction, and the culture temperature was switched from 37°C to 26°C at D8, for 4 d. G3, group 3, browning induction under 37°C for 12 d. Red colour, Nile red staining for lipids. Blue colour, nuclear staining by Hoechst 33,342. Dn, the number of days of induction. (b), Western blotting and quantification of PGC-1α, and UCP-1 proteins expression of different groups of the cells during browning. Adipogenesis decreased upon the period of low temperature treatment (a, G3> G1> G2), while the tendency of browning of the cells was positively correlated with the period of low temperature treatment (b, G2> G1> G3). hWACs, human subcutaneous mature white adipocytes. mBAT, WT mouse BAT, as UCP1 positive control, its total protein was loaded 1/3 compared to other samples. * p < 0.05 and ** p < 0.01 represent the different level between groups (n > 7).
Figure 6.
Figure 6.
The dynamic changes of the browning of msASCs exposure at 26°C for different days. (a), dynamic changes of the cell morphology. G1, group 1, browning induction, and the culture temperature was switched from 37°C to 26°C at D6, for 1 da. G2, group 2, browning induction, and the culture temperature was switched from 37°C to 26°C at D5, for 2 d. G3, group 3, browning induction, and the culture temperature was switched from 37°C to 26°C at D4, for 3 d. G4, group 4, browning induction under 37°C for 7 d. Adipogenesis decreased during the period of low temperature treatment, and the tendency of induction of cell browning was positively correlated with the days of low temperature treatment, the tendency which was similar as seen in the hsASCs (G3> G2> G1> G4). Red colour, Nile red staining for lipids. Blue colour, nuclear staining by Hoechst 33,342. Dn, the number of days of induction. (b), Western blotting and quantification of PGC-1α and UCP-1 expression during browning. meACs-G4, WT mouse epididymal ASCs, browning under 37°C for 7 d. mWACs, WT mouse subcutaneous white mature adipocytes. mBAT, WT mouse BAT, as UCP1 positive control, and its total protein was loaded 1/3 compared to other samples. * p < 0.05 and ** p < 0.01 represent the different levels between groups (n > 7).
Figure 7.
Figure 7.
A hypothetical model and potential mechanism of the insulin-PI3K-AKT-UCP1 pathway in animal white preadipocyte browning under different conditions. When insulin signalling is intact, white preadipocytes can be induced into beige adipocytes by the stimuli (such as CBAM in this study) including insulin and T3. However, when OSI-906 was used to block the insulin signal incompeletely, both lipogenesis and Browning were inhibited. Cold exposure induces browning of white adipocytes by affecting the expression and function of C/EBPβ and PGC-1α which are downstream molecules of insulin signalling, and/or molecules (such as AMPK and SIRT1) in a signalling pathway crosstalk with insulin signalling. This indicates that the browning of WAC caused by cold exposure may also related to insulin signalling.
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This work was supported in part by grants from the Natural Science Foundation of China (81070685), and Natural Science Foundation of Shandong Province (ZR2018MC013) to JP, and supported by Chiang Mai University, Thailand.
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