D-Chiro-Inositol Regulates Insulin Signaling in Human Adipocytes

Abstract

D-Chiro-Inositol (D-Chiro-Ins) is a secondary messenger in the insulin signaling pathway. D-Chiro-Ins modulates insulin secretion, the mitochondrial respiratory chain, and glycogen storage. Due to these actions D-Chiro-Ins has been proposed to correct defective insulin function in a variety of conditions characterized by metabolic dysfunction, such as polycystic ovary syndrome (PCOS), obesity, gestational diabetes and fat accumulation at menopause. Since it is unclear whether D-Chiro-Ins directly acts on adipocytes, we aimed to study D-Chiro-Ins's actions on adipocyte viability, proliferation, differentiation, and insulin-related protein expression using a human adipocyte cell line derived from Simpson-Golabi-Behmel Syndrome (SGBS) which fully differentiates to mature adipocytes. Throughout differentiation, cells were treated with D-Chiro-Ins, 17β-estradiol (E2) or Insulin. Cell viability and proliferation were not affected by D-Chiro-Ins, then D-Chiro-Ins promoted cell differentiation only during the final days of the process, while E2 enhanced it from the first phases. D-Chiro-Ins stimulated lipid storage and the production of big lipid droplets, thus reducing the content of free fatty acids. We also found that D-Chiro-Ins, either alone or in combination with insulin and E2 increased the expression and activation of insulin receptor substrate-1 (IRS1) and glucose transporter type 4 (GLUT4). In conclusion, this work shows that D-Chiro-Ins plays a direct role in the differentiation and in the function of human adipocytes, where it synergizes with insulin and estrogen through the recruitment of signal transduction pathways involved in lipid and glucose storage. These findings give clear insights to better understand the actions of D-Chiro-Ins on fat metabolism in women in physiology and in a variety of diseases.

Keywords: 17β-estradiol (E2); D-Chiro-Inositol (D-Chiro-Ins); Simpson–Golabi–Behmel syndrome cells (SGBS); insulin pathway; insulin receptor substrate (IRS1).

Figure 1

D-Chiro-Ins does not affect SGBS cells viability. SGBS cells viability was measured by trypan blue assay (A), or MTT assay (B) after 24-hour treatment with increasing concentration of D-Chiro-inositol (D-Chiro-Ins), 17β-estradiol (E2) or combinations of D-Chiro-Ins and E2. All the results represent the media out of three independent experiments, each of them performed in triplicates. Data are expressed as mean ± SEM. One-way ANOVA was performed followed by Dunnett’s multiple comparisons. No significative difference was found between treatments and control after 24-hour treatment.

Figure 2

D-Chiro-Ins does not affect SGBS cells proliferation rate. SGBS cells proliferation was measured by MTT assay for 72-hour treatment with increasing concentration of D-chiro-Inositol (D-Chiro-Ins), 17β-estradiol (E2) or their combinations. All the results are expressed as mean ± SEM, obtained after three independent experiments, each of them performed in triplicates. To analyse the proliferation slope, linear regression analysis was performed; however, the difference between the slopes was not significant (ns: non-significant). In addition, to compare each treatment in the time-response curve, a two-way RM ANOVA was performed followed by Dunnett’s multiple comparisons test; treatments did not affect the cell proliferation rate, however, only time progression was found to affect cell proliferation in a significative way (***p < 0.001 versus every preceding time).

Figure 3

Adipogenic differentiation of SGBS cells. SGBS cells differentiation was according to differentiation protocol. Cells imaging was taken at days 0, 4, 10, 16, 22 and 28 of differentiation. The presence of lipid droplets was assessed at magnification of 20×. Analysis of the different size of lipid droplets (100–499, 500–999 and >1000 nm) was perform with by ImageJ. All data are presented as mean ± SEM after three independent experiments.

Figure 4

D-Chiro-Ins modulates SGBS cells differentiation. (A) SGBS were treated with D-Chiro-Ins 10⁻⁸ M, E2 10⁻⁹ M, INS 10⁻⁷ M or without treatment (cv, control) during differentiation according to differentiation protocol. Lipid droplets (LD) formation rate was observed with oil red/dapi staining, on days 0, 4, 10, 16, 22 and 28 of the differentiation process. Images representative of three independent experiments taken with immunofluorescence microscope assessed at magnification of 20×. Red: oil red; blue: dapi; bar: 200 nm. (B–D) Analysis of different size of LD produced during the differentiation: 100–499, 500–999 and >1000 nm size. (E) Analysis of lipid accumulation during differentiation, the results were expressed as ratio of the oil red intensity of lipids-droplets and dapi as a nuclear staining dye (oil red/dapi). All the results are presented as mean ± SEM after three independent experiments. Results were analyzed statistically by two-way RM ANOVA. Differences between experimental treatments and control group at the same matching time were analyzed by Dunnett’s multiple comparisons test (**p <0.01, ***p <0.001 versus control at the corresponding time). D-Chiro-Ins, D-chiro-Inositol; E2, 17β-estradiol, INS, insulin.

Figure 5

D-Chiro-Ins modulates the expression and phosphorylation of IRS1 in mature adipocyte. Mature SGBS adipocytes cells were treated with D-Chiro-Ins 10⁻⁸ M, E2 10⁻⁹ M or INS 10⁻⁷ M, and combinations for 24 ho. Cell lysates were immunoblotted with an anti-IRS1 antibody, anti-p-IRS1 antibody, and anti-GAPDH antibody. (A) Panels show representative blot of IRS1, p-IRS1^Tyr941 and GAPDH. The expression level of IRS1 and p-IRS1 was normalized with the protein amount of GAPDH, relative to the control. In the p-IRS1 Tyr⁹⁴¹ blot the band subjected to quantification is the lower, corresponding to 170 kDa. (B–D) Analysis of the relative expression level of IRS1, p-IRS1 and the ratio p-IRS1/IRS1. Values are presented as mean ± SEM of three independent experiments. Results were analyzed statistically by one-way RM ANOVA followed by Dunnett’s multiple comparisons test (*p < 0.05, **p < 0.01 versus control). D-Chiro-Ins, D-chiro-Inositol; E2, 17β-estradiol, INS, insulin; IRS1, insulin receptor substrate 1; p-IRS1, phospho-IRS1; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 6

D-Chiro-Ins does not modulate the expression of Akt. Mature SGBS adipocytes cells were treated with D-Chiro-Ins 10⁻⁸ M, E2 10⁻⁹ M or INS 10⁻⁷ M, and combinations for 24 h. Cell lysates were immunoblotted with an anti-Akt antibody, anti-p-Akt antibody, and anti-GAPDH antibody. (A) Panels show representative blot of Akt, p-Akt^Ser473 and GAPDH. (B–D) Analysis of the relative expression level of Akt, p-Akt and the ratio p-Akt/Akt. The expression level of Akt and p-Akt was normalized with the protein amount of GAPDH, relative to the control. Values are presented as mean ± SEM of three independent experiments. Results were analyzed statistically by one-way RM ANOVA followed by Dunnett’s multiple comparisons test (*p < 0.05, **p < 0.01 versus control). D-Chiro-Ins, D-chiro-Inositol; E2, 17β-estradiol, INS, insulin; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.

Figure 7

D-Chiro-Ins does not modulate the expression of GLUT4. Mature SGBS adipocytes cells were treated with D-Chiro-Ins 10⁻⁸ M, E2 10⁻⁹ M or INS 10⁻⁷ M, and combinations for 24 h. Cell lysates were immunoblotted with an anti-GLUT4 antibody, anti-p-GLUT4 antibody, and anti-GAPDH antibody. (A) Panels show representative blot of GLUT4, p-GLUT4 ^S488 and GAPDH. (B–D) Analysis of the relative expression level of GLUT4, p-GLUT4 and the ratio p-GLUT4/GLUT4; expression that was normalized with GAPDH and relative to the control. Values are presented as mean ± SEM of three independent experiments. Results were analyzed statistically by one-way RM ANOVA followed by Dunnett’s multiple comparisons test (*p < 0.05, **p < 0.001 versus control). D-Chiro-Ins, D-chiro-Inositol; E2, 17β-estradiol, INS, insulin; GLUT4, Glucose Transporter 4; p-GLUT4, phospho-GLUT4; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.