During the last decades, a substantial body of research has focused on the role of the two major inositol (Ins) stereoisomers, myoinositol (myoIns) and D-chiroinositol (D-chiroIns), both second messengers of insulin, in insulin-dependent processes, including polycystic ovary syndrome (PCOS). MyoIns has been shown to affect different pathways at both ovarian and non-ovarian level. On the contrary, D-chiroIns alone is unable to exert significant improvements in the ovarian cell functions, as its beneficial effects are mainly limited to the non-ovarian tissue in which it may significantly inhibit the negative cellular consequences of hyperinsulinemia. However, both Ins isomers can be positively associated in the management of PCOS patients in a ratio corresponding to their physiological plasma ratio (40:1). This appears to exert a synergistic effect according to a multi-targeted design. In this respect, new fundamental insights into the biological mechanisms displayed by Ins, as well as clinical trials based on the myoIns + D-chiroIns formulations, have already provided encouraging results.
Highlights
- •
A safe and useful inositol treatment for non-insulin-resistant women with polycystic ovary syndrome (PCOS) is proposed.
- •
Myoinositol and D-chiroinositol are associated at a ratio corresponding to their physiological plasma ratio of 40:1.
- •
The association is safe and effective on PCOS patients at both the systemic and ovarian levels.
Biochemistry and metabolism of inositols
During recent years, studies on inositols (Ins) and their phosphate derivatives such as myoinositol-1-phosphate, myoinositol-bisphosphate, Ins pentakisphosphate have gained momentum .
Ins and its derivatives are natural compounds abundant in fruits and some vegetables, thus representing a family of nine hexahydroxycyclohexane stereoisomers with general formula C 6 H 12 O 6 . Ins are chemically very stable polar molecules with versatile properties. These were originally isolated by J.J. Scherer in 1850 from muscle tissues, and they later appeared to be broadly distributed in mammalian tissues and cells, where they perform important biologic functions. This is particularly true for at least six Ins isomers, myo-inositol (myoIns), scyllo-inositol, epi-inositol, neo-inositol, D-chiroinositol, and muco-inositol .
Even though Ins has been deemed for a while to be an “inactive” molecule , the current evidence demonstrates its significant biological properties. Ins and its derived molecules, besides their “traditional” function as mineral “stores,” participate in regulating a plethora of cellular pathways. Indeed, cell signaling via Ins and its phosphates, in particular via the second messenger myoIns 1,4,5-trisphosphate, comprises a huge field of biology, and thus, participates in the regulation of a plethora of cellular pathways: insulin signal transduction, Ca 2+ flow, cytoskeletal protein assembly, lipid metabolism, modulation of serotoninergic pathways, cell growth and differentiation, oocyte maturation, and fertility .
Moreover, some Ins isomers have been proven to be medically relevant: scyllo-Ins in neurodegenerative diseases, D-chiroinositol (D-chiroIns) in diabetes and myoIns in cancer, metabolic syndrome, and polycystic ovary syndrome (PCOS) . It is therefore appropriate to consider investigating the roles and applications of these “old” simple molecules as “new” pharmacological, pleiotropic agents. Therefore, scientific literature on Ins and its diverse phosphate derivatives is continuously updated in order to cover the widespread array of released studies . The use of Ins, as both a nutraceutical-based preventive strategy and pharmacological support in medical treatments, has been proposed in many diseases, including psychiatric/neurological illnesses , infertility, diabetes, inflammatory processes , and cancer .
MyoIns and D-chiroIns
MyoIns is the most abundant form of Ins in both nature and mammalian cells, with up to 99% of the overall Ins amount. The remaining 1% is represented by the other stereoisomer D-chiroIns. Although having different metabolic functions, both these molecules are mediators of insulin action inside the cell . MyoIns is converted into D-chiroIns by an NAD/NADH epimerase, an insulin-dependent enzyme with tissue-specific expression. Hence, the activity of this enzyme strongly influences different concentration ratios observed between the two molecules in hepatic or adipose cells, or muscle fibers .
MyoIns has defined roles in both somatic and germ cells, as a precursor of phosphoinositides, membrane components, and signaling molecules (reviewed by ) and as a hyperosmotic stress protectant .
Inside the cells, myoIns is present both in free form and as a component of the membrane phosphoinositides. Phosphatidyl-myoIns is the precursor of phosphatidyl-Ins phosphate and phosphatidyl-Ins bisphosphate (PIP 2 ), molecules with important physiological roles . The hydrolysis of PIP 2 by phospholipase C (PLC) produces Ins trisphosphate (Ins-1,4,5P 3 , InsP 3 ), which acts as a second messenger regulating the activities of several hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and insulin . By interaction with the membrane receptors of mitochondria and the endoplasmic reticulum, InsP 3 induces calcium influx into the cytosol, which activates protein kinase C and mediates cellular responses. Other membrane lipids containing Ins (glycosyl-phosphatidyl-Ins) serve as anchors for many membrane proteins.
MyoIns is involved in processes including glucose metabolism, transport, and breakdown , and the regulation of cell proliferation , relevant during development in all its phases : pre- as well as postimplantation embryogenesis and, in the adult, oogenesis and spermatogenesis.
In mammals, a tissue-specific epimerase converts myoIns into D-chiroIns. This reaction is induced by insulin and is particularly sustained in hepatic and muscle cells where it is involved in glycogen synthesis. Differences in myoIns/D-chiroIns distribution are responsible for the distinct functions the two isomers play in the tissues.
D-chiroIns is considered to be a relevant component of the insulin signaling pathway, participating along with galactosamine in a wide array of molecular mechanisms, including stimulation of pyruvate dehydrogenase phosphatase, protein phosphatase 2C, and Ins-phosphate glycan (IPG) that altogether seem to dump the consequences of deregulated glucose metabolism. This role is, however, still debatable, as the serum D-chiroIns levels are increased in insulin-resistant women with preeclampsia and elevated D-chiroIns levels are suspected to contribute to insulin resistance . In rats, D-chiroIns has to be considered an essential nutrient, as it cannot be synthesized endogenously nor be produced from myoIns .
Yet, D-chiroIns is required to ensure proper development. Indeed, mice genetically engineered to develop folate-resistant neural tube defects in utero can be more effectively treated with D-chiroIns than with myoIns . In addition, D-chiroIns is able to prevent and reverse endothelial dysfunction or bone architectural defects in both rats and rabbits .
MyoIns and D-chiroIns
MyoIns is the most abundant form of Ins in both nature and mammalian cells, with up to 99% of the overall Ins amount. The remaining 1% is represented by the other stereoisomer D-chiroIns. Although having different metabolic functions, both these molecules are mediators of insulin action inside the cell . MyoIns is converted into D-chiroIns by an NAD/NADH epimerase, an insulin-dependent enzyme with tissue-specific expression. Hence, the activity of this enzyme strongly influences different concentration ratios observed between the two molecules in hepatic or adipose cells, or muscle fibers .
MyoIns has defined roles in both somatic and germ cells, as a precursor of phosphoinositides, membrane components, and signaling molecules (reviewed by ) and as a hyperosmotic stress protectant .
Inside the cells, myoIns is present both in free form and as a component of the membrane phosphoinositides. Phosphatidyl-myoIns is the precursor of phosphatidyl-Ins phosphate and phosphatidyl-Ins bisphosphate (PIP 2 ), molecules with important physiological roles . The hydrolysis of PIP 2 by phospholipase C (PLC) produces Ins trisphosphate (Ins-1,4,5P 3 , InsP 3 ), which acts as a second messenger regulating the activities of several hormones such as follicle-stimulating hormone (FSH), thyroid-stimulating hormone (TSH), and insulin . By interaction with the membrane receptors of mitochondria and the endoplasmic reticulum, InsP 3 induces calcium influx into the cytosol, which activates protein kinase C and mediates cellular responses. Other membrane lipids containing Ins (glycosyl-phosphatidyl-Ins) serve as anchors for many membrane proteins.
MyoIns is involved in processes including glucose metabolism, transport, and breakdown , and the regulation of cell proliferation , relevant during development in all its phases : pre- as well as postimplantation embryogenesis and, in the adult, oogenesis and spermatogenesis.
In mammals, a tissue-specific epimerase converts myoIns into D-chiroIns. This reaction is induced by insulin and is particularly sustained in hepatic and muscle cells where it is involved in glycogen synthesis. Differences in myoIns/D-chiroIns distribution are responsible for the distinct functions the two isomers play in the tissues.
D-chiroIns is considered to be a relevant component of the insulin signaling pathway, participating along with galactosamine in a wide array of molecular mechanisms, including stimulation of pyruvate dehydrogenase phosphatase, protein phosphatase 2C, and Ins-phosphate glycan (IPG) that altogether seem to dump the consequences of deregulated glucose metabolism. This role is, however, still debatable, as the serum D-chiroIns levels are increased in insulin-resistant women with preeclampsia and elevated D-chiroIns levels are suspected to contribute to insulin resistance . In rats, D-chiroIns has to be considered an essential nutrient, as it cannot be synthesized endogenously nor be produced from myoIns .
Yet, D-chiroIns is required to ensure proper development. Indeed, mice genetically engineered to develop folate-resistant neural tube defects in utero can be more effectively treated with D-chiroIns than with myoIns . In addition, D-chiroIns is able to prevent and reverse endothelial dysfunction or bone architectural defects in both rats and rabbits .
Ins in gynecologic and fertility
During the last decades, the gathered data hint for a significant role exerted by Ins in several pathological conditions belonging to gynecology, as well as in fertility-related processes, including oocyte maturation and sexual structure differentiation. This evidence advocates for a relevant role sustained by Ins and its metabolites in human reproduction, as claimed in a seminal paper by Beemster et al. .
Ins and reproductive function: A role in oogenesis and embryogenesis
Ins, and myoIns in particular, represent important molecules for early development (reviewed by ), its serum concentrations in fetuses and newborn infants being several folds higher than in adults . Its administration during pregnancy reduces the risk of gestational diabetes in humans and the occurrence of developmental defects in the fetuses of mutant mice . MyoIns also plays a positive role in reproduction: its concentration in mammalian female reproductive tracts is substantially higher than that of blood serum, thereby suggesting its positive influence on fertility; its levels in the follicular fluid and in blood serum are positively correlated with oocyte quality and pregnancy outcome in humans ; administration of myoIns to women before the onset of hormonal stimulation in in vitro fertilization (IVF) cycles increases oocyte and embryo quality , and reduces the amount of FSH and the days required for proper stimulation, which are parameters directly linked to the chance of pregnancy .
At the ovarian level, myoIns appears to be involved in different functions, being essential to ensure proper oocyte maturation. MyoIns action is related to the role played by InsP 3 on the modulation of intracellular calcium ion concentration in response to LH and FSH action . In oocytes, this mechanism, acting through interaction with the specific receptors (InsP 3 -R1) , plays a central role in the maturation process . Culture medium supplementation with myoIns has been shown to increase meiotic maturation of mouse oocytes with the production of fertile eggs, while the depletion of intracellular stores of myoIns desensitizes Ins-dependent transduction pathways, thus reducing the levels of InsP3 and the proper release of calcium with a negative impact on oocyte maturation . When oocytes matured in the presence of myoIns were fertilized in vitro and transferred to foster mothers, the implantation rate and postimplantation viability of the resulting embryos also increased .
Experiments on farming species suggest a role for myoIns in mammalian preimplantation development, as myoIns supplementation of culture media improves rabbit and bovine blastocyst formation, expansion, and hatching and allows development to term of healthy animals .
The active transport systems allow the uptake of myoIns into the mammalian cells, including oocytes and preimplantation embryos. In the mouse, activity of at least two different membrane transporters is responsible for a progressive increase in myoIns uptake during early development between the one-cell stage and the blastocyst stage . MyoIns is then rapidly incorporated into phosphoinositides . In the zygote, the concentration oscillations of calcium ions, induced by PLC-dependent InsP 3 production, have key roles, from egg activation at fertilization to blastomere divisions . The inclusion of myoIns in human embryo culture media has recently been shown to allow in vitro-produced and cultured mouse embryos to complete the preimplantation development and develop into the expanded blastocyst stage at rates similar to in vivo-developed embryos . This led us to hypothesize that the inclusion of this molecule in the embryo culture media would cause an increase in the number of high-quality embryos produced in human IVF cycles .
Ins in gynecologic diseases
The last decade has witnessed an increasing body of data concerning the usefulness of Ins in the clinical management of PCOS. The PCOS, first described by Stein and Leventhal, is one of the most common female endocrine disorders .
Despite the controversy regarding the differences among the clinical features of PCOS , it is now widely recognized that insulin resistance (IR) plays a significant pathogenetic role in a large subset of patients . IR in PCOS women is primarily ascribed to the defects in post-binding signaling , hence affecting several downstream targets, including modulation of steroidogenic pathways. Insulin directly prompts the ovary theca cells to enhance the synthesis and release of androgens both directly and indirectly, by modulation of carbohydrate levels. High glucose levels in turn inhibit the hepatic synthesis of sex hormone-binding globulin, thereby causing a consequent increase of circulating free-active androgens . As the relationships among disturbances in insulin signaling and PCOS were established, several antidiabetic, insulin-sensitizing drugs, including metformin and thiazolidinediones, have been investigated as effective adjunct in treating PCOS patients. In particular, Metformin reduces glucose serum levels, downregulates ovarian androgen production, and decreases the circulating androgen levels .
However, while Metformin has shown to counteract hyperandrogenism in the short term in obese, insulin-resistant PCOS women ; controversial results have been published about Metformin efficacy in non-obese non-insulin-resistant patients . In addition, Metformin has a detrimental effect on the follicle number and quality .
Moreover, both Metformin and Thiazolidinediones are burdened by relevant adverse events including gastrointestinal symptoms, metabolic complications , fluid retention, body weight increase, coronary artery disease, and bladder cancer . Yet, it is intriguing that the benefits obtained in Metformin-treated PCOS women have been ascribed to a secondary increase in the availability of IPG triggered by Metformin administration , thus providing support to the hypothesis that the insulin-signaling pathways require Ins.
Indeed, when insulin binds to its receptor, two distinct IPGs, incorporating either myoIns or D-chiroIns (D-chiroIns–IPG and myoIns–IPG), are released by the hydrolysis of glycosyl–phosphatidyl-Ins lipids on the outer side of the cell membrane. IPG in turn affects the intracellular metabolic processes by activating the key enzymes controlling the oxidative and non-oxidative glucose metabolism . Even though some differences have been recorded, both D-chiroIns- and myoIns-containing IPG significantly reduce IR and improve glucose metabolism .
Women affected by PCOS show reduced serum levels of D-chiroIns and increased urinary loss of D-chiroIns–IPG . This observation fostered further investigations that eventually ended up in demonstrating that PCOS patients experience a severe deregulation of Ins metabolism, thus enabling the establishment of a clear mechanistic link between IR and Ins deficiency in PCOS patients . Early clinical studies were aimed at verifying the utility of D-chiroIns in PCOS management. Impressive results were indeed obtained, as PCOS patients treated with low doses of D-chiroIns (i.e., 1.2 g/daily) showed reduced levels of lipid biomarkers, increased insulin sensitivity, decreased serum androgen levels, and higher ovulation frequency .
Those effects have been mainly ascribed to a D-chiroIns systemic activity, able to counteract the main consequences of the metabolic syndrome that is frequently associated with PCOS . However, when administered at higher doses, D-chiroIns seems to exert negative effects on ovarian tissues. Indeed, subsequent clinical trials performed with D-chiroIns doses of 2.4 g/daily were unable to confirm previous positive results on PCOS women, eventually suggesting that D-chiroIns may paradoxically worsen the ovarian response in non-insulin-dependent PCOS patients, even if D-chiroIns was effective in normalizing the IR parameters . These results showed that counteracting IR might not be sufficient to reverse the clinical features of PCOS. Indeed, similar disappointing results have been recorded by treating non-insulin-resistant PCOS women with Metformin. The antidiabetic drug improves PCOS clinical signs only in a fraction of PCOS patients but worsens their oocyte quality , thus highlighting the idea that IR is likely not to be the main causative factor in PCOS pathogenesis. In addition, some data indicate that D-chiroIns may exert detrimental effects on ovarian tissues. The release of high levels of D-chiroIns–IPG under insulin stimulation promotes testosterone biosynthesis from the ovarian theca cells, thus leading to increased serum androgen levels .
In addition, D-chiroIns may significantly hinder myoIns uptake in mammalian cells, thus leading to an imbalance in their respective ratio within the ovary . As discussed earlier, myoIns is in fact the most abundant Ins isomer within the ovary, accounting for about 99% of all Ins , and is converted to D-chiroIns through an insulin-induced, NAD-dependent epimerase. A decreased epimerase activity has been observed in human muscle tissue of type 2 diabetics and in other non-ovarian tissues in IR patients . Yet, as the ovarian tissue never develops insulin insensitivity, higher circulating insulin levels are likely to induce a prominent and paradoxical increase in D-chiroIns concentration within the ovary. An increased conversion from myoIns to D-chiroIns would in turn significantly reduce the myoIns levels within the ovarian cells. Specifically, a significant increase in the epimerase activity in the theca cells of ovaries of PCOS women has been found to be associated with a significant reduction in the myoIns/D-chiroIns ratio . Indeed, a significant decrease in the myoIns/D-chiroIns ratio has been documented in the follicular fluid of PCOS patients . Consequently, any further increase in D-chiroIns is likely detrimental to ovarian function, thus providing a mechanistic explanation to the so-called “D-chiroIns ovarian paradox.” Furthermore, D-chiroIns has been proven to be largely ineffective in non-hyperinsulinemic PCOS women, as the majority of these patients do not respond to D-chiroIns therapy .
On the contrary, the usefulness of myoIns supplementation in PCOS has been assessed by several reports. In mice, myoIns improves glucose tolerance by increasing insulin sensitivity and potentiates insulin activity . Precisely, myoIns promotes the translocation of glucose transporter 4 to the plasma membrane, therefore lowering plasma glucose and insulin levels .
MyoIns supplementation in the PCOS patients counteracts the main features of the associated metabolic syndrome and improves several ovarian functions: oocyte quality, frequency of ovulation, increased pregnancy rate, and reduced number of FSH treatment required to trigger ovulation. These results have been assessed by both pilot and randomized studies .
It is worth mentioning that the frequency of both ovulation and pregnancy rate increased by myoIns treatment . It thus appears that myoIns improves both ovarian function and systemic features associated with PCOS: deregulation of glucose metabolism, clinical signs of hyperandrogenism, lipid metabolism .
Facchinetti et al. reviewed and analyzed six randomized controlled trials focused on myoIns supplementation in PCOS patients and provided strong evidence for a higher myoIns effectiveness (with a dosage of 2–4 g/day for 12–16 weeks) with respect to conventional therapy or treatments based on D-chiroIns alone. Yet, a potential bias is still represented by the fact that other studies lack proper randomization or are flawed by statistical weakness . Moreover, we still await a compelling investigation on Ins-based mechanisms within the ovary, even if increasing evidence is presented.
Functional difference among D-chiroIns and myoIns may help in understanding why different myoIns/D-chiroIns ratios are actively preserved in fat, muscle, liver, and ovarian tissues, via a tuned regulation of epimerase activity. While D-chiroIns effects are mainly restricted to insulin signaling transduction, myoIns has demonstrated to exert other noticeable activities (see Fig. 1 ), by influencing cytoskeleton remodeling (Bizzarri M, unpublished observations), steroidogenesis, and oocyte maturation . Furthermore, myoIns participates in modulating LH/FSH activity and GnRH agonist-mediated LH inhibition . Both aromatase and 3α-hydroxysteroid dehydrogenase activities are modulated by D-chiroIns and myoIns in a subtle diverse fashion . Moreover, a relative scarcity of myoIns has been hypothesized from an epidemiological point of view to impair fertility and ovary function . Specifically, myoIns has been deemed to significantly modulate steroidogenesis by acting through an insulin-independent pathway that involves cytoskeleton rearrangements . The Ins-based treatment of PCOS mainly relies on the insulin mimicking effect of IPG. However, it is currently recognized that during the development of PCOS, hyperinsulinism or IR act as a second hit, worsening the follicular arrest either through amplification of the intraovarian hyperandrogenism or through dysregulation of granulosa cells . Given that intraovarian deregulation of androgens is currently considered as the main cause for follicular arrest in PCOS, the insulin-independent antiandrogenic effect displayed by myoIns deserves to be investigated thoroughly.
