Tesi etd-10242019-195023
Link copiato negli appunti
Tipo di tesi
Corsi integrativi di I livello
Autore
FIDECICCHI, TIZIANA
URN
etd-10242019-195023
Titolo
Effetti del D-chiro-inositolo e dell'estradiolo sui pre-adipociti umani
Struttura
Cl. Sc. Sperimentali - Medicina
Corso di studi
SCIENZE MEDICHE - Laurea Magistrale in Medicina e chirurgia (LM/41)
Commissione
Tutor Prof. PASSINO, CLAUDIO
Relatore Prof. SIMONCINI, TOMMASO
Membro Dott.ssa FRUZZETTI, FRANCA
Membro Prof. COCEANI, FLAVIO
Membro Prof. RECCHIA, FABIO ANASTASIO
Membro Prof. EMDIN, MICHELE
Membro Prof. LIONETTI, VINCENZO
Membro Dott.ssa ANGELONI, DEBORA
Membro Dott. MEOLA, MARIO
Membro Dott.ssa PETRUCCI, ILARIA
Relatore Prof. SIMONCINI, TOMMASO
Membro Dott.ssa FRUZZETTI, FRANCA
Membro Prof. COCEANI, FLAVIO
Membro Prof. RECCHIA, FABIO ANASTASIO
Membro Prof. EMDIN, MICHELE
Membro Prof. LIONETTI, VINCENZO
Membro Dott.ssa ANGELONI, DEBORA
Membro Dott. MEOLA, MARIO
Membro Dott.ssa PETRUCCI, ILARIA
Parole chiave
- D-chiro-inositol
- insulin-resistance
- polycystic ovary syndrome
- preadipocytes
Data inizio appello
10/12/2019;
Disponibilità
completa
Riassunto analitico
Polycystic ovary syndrome (PCOS) is a heterogeneous endocrine disorder that affects about 6-10% of women in reproductive age. It is characterized by hyperandrogenism, menstrual disorders and polycystic ovary morphology.
During PCOS there is a post-binding defect of insulin receptor signaling that causes an inactivation of the IRS1/PI3K/Akt pathway and, consequently, brings to the development of insulin resistance (IR).
In PCOS, also estradiol (E2) levels are altered and they never reach the ovulatory peak, constantly remaining in the follicular phase range. E2 Receptor α (ERα) in adipocytes modulates anti-lipogenesis, insulin sensitivity and glucose tolerance, reduction of body weight and of white adipose tissue. Both E2 and insulin receptor share a common signaling pathway within human cells, converging on the activation of the PI3K/Akt pathway.
Insulin-sensitizing drugs have been proposed to approach PCOS women, such as inositols. D-chiro-inositol (DCI) and myo-inositol (MI) are incorporated in the cell membrane and are second messengers of different receptors, such as the insulin one. There is more and more evidence suggesting that the deficiency of DCI and MI and the altered ratio between them may be one of the bases of IR. No evidence of the direct modulatory effects of DCI on adipocytes has been generated so far, while some clinical studies have shown their ability to improve insulin sensitivity and to ameliorate menstrual cyclicity in women with PCOS, both when administered alone or in combination with other insulin-sensitizing molecules, like α-lipoic acid (ALA).
The first aim of this study was to evaluate the effects of a six months treatment with DCI and ALA on reproductive and metabolic parameters of women with PCOS with or without IR.
The second aim was to evaluate the effects in vitro of DCI and E2 on the differentiation and the proliferation of human adipocytes, with particular attention to the influence of the molecules on the pathway of insulin receptor.
In the clinical study, 40 women with PCOS and 30 controls have been enrolled. Five patients dropped out from the study; hence, 35 women completed the treatment. Among them, 28 women underwent an oral glucose tolerance test (OGTT) before and after the treatment. The menstrual cycle, body mass index (BMI), homeostasis model assessment index (HOMA-I), fasting insulin and insulin secretion in response to an OGTT were evaluated before and after 6 months of treatment. During the observation period, the patients have been asked to not modify their diet and physical activity.
For the in vitro study, Simpson-Golabi-Behmel syndrome (SGBS) cells were used. Pre-adipocyte proliferation was measured by MTS assays after 24, 48 and 72 hours of treatment with increasing concentrations of DCI (10-10-10-7 M), E2 (10-10-10-7 M) and combinations of DCI (10-8 M) + E2 (10-9 or 10-10 M). Preadipocytes underwent growth arrest and subsequent terminal differentiation into adipocytes. During adipocyte differentiation, cells were treated with 10-8 M DCI, 10-9 M E2, 10-7 M Insulin and vehicle (0.01% alcohol); the lipid droplets formation were observed at days 0, 4, 10, 16, 22 and 28 with Oil Red O Staining Kit. Subsequently, differentiated cells were treated with 10-9 M E2, or 10-8 M DCI, or 10-7 M insulin and combinations for 24 hours; protein expression analysis of the insulin receptor substrate 1 (IRS1), Akt, glucose transporter 4 (GLUT4), and the respective phosphorylated proteins was performed by Western Blot.
From this study emerged that women treated with DCI+ALA showed a reduction of BMI (p<0.0001) and menstrual cycle length (p<0.001) after 6 months, with no significant effects on metabolic parameters. Dividing women according to their basal HOMA-IR, women with IR (HOMA-IR>2.5) showed a significant improvement of all metabolic parameters, with a reduction of BMI and no significant improvement of cycle length, while women without IR only showed an improvement of BMI and cycle length.
About the in vitro study, treatment with DCI, E2 and combination of them didn’t affect cell viability and proliferation. Differentiation was significantly promoted by E2, already starting from intermediate phases of differentiation (day 22), while DCI effects were evident only in the late phases (day 28), when a white-like adipose phenotype was displayed. IRS1 expression after treatment of differentiated cells was enhanced by insulin, DCI+insulin and DCI+E2+insulin, while other proteins were not significantly affected by the treatments, even if GLUT4 expression showed a trend to be increased by DCI+E2+insulin.
In conclusion, DCI showed to have insulin-sensitizing effects both on clinic and directly on adipocytes. PCOS women with IR can benefit the most of this insulin-sensitizing effect of the treatment, but it is useful also for other women, because it provides help in the weight loss and in the normalization of menstrual cycle. Moreover, in this pilot study on adipocytes, we also demonstrated for the first time that DCI can affect adipocyte late differentiation and the expression of proteins related to the insulin signaling pathway also in basal conditions, without IR. The insulin-sensitizing effect of DCI on this pathway can be enhanced by the effect of E2. This is an important finding, that leads to deepen the studies on DCI. More studies on adipocytes in different conditions must be conducted, in order to understand in which pathway DCI acts during differentiation and which is its action during IR in the presence of different E2 concentrations: this could lead to understand the possible role of DCI administration not only during PCOS, but also during menopause transition.
During PCOS there is a post-binding defect of insulin receptor signaling that causes an inactivation of the IRS1/PI3K/Akt pathway and, consequently, brings to the development of insulin resistance (IR).
In PCOS, also estradiol (E2) levels are altered and they never reach the ovulatory peak, constantly remaining in the follicular phase range. E2 Receptor α (ERα) in adipocytes modulates anti-lipogenesis, insulin sensitivity and glucose tolerance, reduction of body weight and of white adipose tissue. Both E2 and insulin receptor share a common signaling pathway within human cells, converging on the activation of the PI3K/Akt pathway.
Insulin-sensitizing drugs have been proposed to approach PCOS women, such as inositols. D-chiro-inositol (DCI) and myo-inositol (MI) are incorporated in the cell membrane and are second messengers of different receptors, such as the insulin one. There is more and more evidence suggesting that the deficiency of DCI and MI and the altered ratio between them may be one of the bases of IR. No evidence of the direct modulatory effects of DCI on adipocytes has been generated so far, while some clinical studies have shown their ability to improve insulin sensitivity and to ameliorate menstrual cyclicity in women with PCOS, both when administered alone or in combination with other insulin-sensitizing molecules, like α-lipoic acid (ALA).
The first aim of this study was to evaluate the effects of a six months treatment with DCI and ALA on reproductive and metabolic parameters of women with PCOS with or without IR.
The second aim was to evaluate the effects in vitro of DCI and E2 on the differentiation and the proliferation of human adipocytes, with particular attention to the influence of the molecules on the pathway of insulin receptor.
In the clinical study, 40 women with PCOS and 30 controls have been enrolled. Five patients dropped out from the study; hence, 35 women completed the treatment. Among them, 28 women underwent an oral glucose tolerance test (OGTT) before and after the treatment. The menstrual cycle, body mass index (BMI), homeostasis model assessment index (HOMA-I), fasting insulin and insulin secretion in response to an OGTT were evaluated before and after 6 months of treatment. During the observation period, the patients have been asked to not modify their diet and physical activity.
For the in vitro study, Simpson-Golabi-Behmel syndrome (SGBS) cells were used. Pre-adipocyte proliferation was measured by MTS assays after 24, 48 and 72 hours of treatment with increasing concentrations of DCI (10-10-10-7 M), E2 (10-10-10-7 M) and combinations of DCI (10-8 M) + E2 (10-9 or 10-10 M). Preadipocytes underwent growth arrest and subsequent terminal differentiation into adipocytes. During adipocyte differentiation, cells were treated with 10-8 M DCI, 10-9 M E2, 10-7 M Insulin and vehicle (0.01% alcohol); the lipid droplets formation were observed at days 0, 4, 10, 16, 22 and 28 with Oil Red O Staining Kit. Subsequently, differentiated cells were treated with 10-9 M E2, or 10-8 M DCI, or 10-7 M insulin and combinations for 24 hours; protein expression analysis of the insulin receptor substrate 1 (IRS1), Akt, glucose transporter 4 (GLUT4), and the respective phosphorylated proteins was performed by Western Blot.
From this study emerged that women treated with DCI+ALA showed a reduction of BMI (p<0.0001) and menstrual cycle length (p<0.001) after 6 months, with no significant effects on metabolic parameters. Dividing women according to their basal HOMA-IR, women with IR (HOMA-IR>2.5) showed a significant improvement of all metabolic parameters, with a reduction of BMI and no significant improvement of cycle length, while women without IR only showed an improvement of BMI and cycle length.
About the in vitro study, treatment with DCI, E2 and combination of them didn’t affect cell viability and proliferation. Differentiation was significantly promoted by E2, already starting from intermediate phases of differentiation (day 22), while DCI effects were evident only in the late phases (day 28), when a white-like adipose phenotype was displayed. IRS1 expression after treatment of differentiated cells was enhanced by insulin, DCI+insulin and DCI+E2+insulin, while other proteins were not significantly affected by the treatments, even if GLUT4 expression showed a trend to be increased by DCI+E2+insulin.
In conclusion, DCI showed to have insulin-sensitizing effects both on clinic and directly on adipocytes. PCOS women with IR can benefit the most of this insulin-sensitizing effect of the treatment, but it is useful also for other women, because it provides help in the weight loss and in the normalization of menstrual cycle. Moreover, in this pilot study on adipocytes, we also demonstrated for the first time that DCI can affect adipocyte late differentiation and the expression of proteins related to the insulin signaling pathway also in basal conditions, without IR. The insulin-sensitizing effect of DCI on this pathway can be enhanced by the effect of E2. This is an important finding, that leads to deepen the studies on DCI. More studies on adipocytes in different conditions must be conducted, in order to understand in which pathway DCI acts during differentiation and which is its action during IR in the presence of different E2 concentrations: this could lead to understand the possible role of DCI administration not only during PCOS, but also during menopause transition.
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