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Division of Endocrinology, Department of Medicine (L.P.) and Division of Reproductive Endocrinology, Department of Obstetrics and Gynecology (Z.R.), New York Presbyterian Hospital and Weill Medical College of Cornell University, New York, New York 10021; and Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, Stanford University Medical Center (N.A.C., L.C.G.), Stanford, California 94305
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 Top Abstract I. IntroductionII. Insulin and Insulin...III. IGFs and Their...IV. IGF-Binding Proteins...V. Polycystic Ovary Syndrome...VI. The Insulin-Related Ovarian...VII. Summary and ConclusionsReferences |
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I. Introduction |
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TopAbstractI. IntroductionII. Insulin and Insulin...III. IGFs and Their...IV. IGF-Binding Proteins...V. Polycystic Ovary Syndrome...VI. The Insulin-Related Ovarian...VII. Summary and ConclusionsReferences |
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This field was further expanded by studies of the ovarian production
and ovarian effects of the insulin-like growth factors, IGF-I and
IGF-II, by the discovery of ovarian type I and type II IGF receptors,
and by the discovery of the ovarian production of binding proteins
[IGF-binding proteins (IGFBPs)] for these two growth factors
(13, 14, 15). Thus, in addition to insulin, a role for the structurally
related IGFs in ovarian function has gained recognition. Over the last
decade, a significant amount of information has accumulated about the
role of insulin and IGFs in the ovary at the molecular, cellular, and
clinical levels in a variety of normal and pathological conditions.
Therefore, a need has arisen for a comprehensive review of what we term
the insulin-related ovarian regulatory system. This system consists of
the following components (Table 1
):
insulin; IGF-I and IGF-II; insulin receptor; type I and type II IGF
receptors; IGFBPs 1–6; and IGFBP proteases.
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This article reviews the role of each component of the insulin-related ovarian regulatory system in both normal ovarian physiology and in relevant pathological states, the interactions among the components of this system, and the therapeutic implications of this system for women with abnormal ovarian function.
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II. Insulin and Insulin Receptor |
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TopAbstractI. IntroductionII. Insulin and Insulin...III. IGFs and Their...IV. IGF-Binding Proteins...V. Polycystic Ovary Syndrome...VI. The Insulin-Related Ovarian...VII. Summary and ConclusionsReferences |
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Insulin is a 5900 mol wt polypeptide secreted by the ß-cells of the pancreatic islets of Langerhans. The human insulin gene is located on chromosome 11 (39) and encodes pre-proinsulin, a 110-amino acid single-chain polypeptide that is the precursor of insulin (1). Pre-proinsulin is proteolytically converted to proinsulin, which consists of the A chain, B chain, and C peptide. Proinsulin is homologous with IGF-I and -II and can bind to the insulin receptor with approximately 10% of the affinity of insulin. Insulin is produced after the C-peptide is cleaved from proinsulin by endopeptidases active in the Golgi apparatus and in secretory granules. The endopeptidases preferentially cleave either at the C peptide/B chain junction, between Arg31 and Arg32 (endopeptidase type I), or at the C peptide/A chain junction, between Lys64 and Arg65 (endopeptidase type II). The resulting insulin molecule consists of an A chain (21 amino acids) and a B chain (30 amino acids), with three disulfide bridges: two between the A and the B chains (A7-B7 and A20-B12) and one within the A chain (A6-A11).
The insulin receptor is a heterotetramer consisting of two 
- (135
kDa molecular mass) and two ß- (95 kDa molecular mass)
subunits (2). The gene for the insulin receptor is located on the short
arm of chromosome 19 (43, 44, 45), contains 22 exons, is more than 150 kb
in length, and encodes the proreceptor, a single-chain polypeptide with
a molecular mass of 190 kDa that contains one and one ß-subunit.
The mature 2ß2 heterotetrameric form of
the receptor results from dimerization and several posttranslational
processing steps, including proteolytic cleavage. An isoform of the
receptor lacking 12 amino acids encoded by exon 11 results from
alternative mRNA splicing. Insulin receptors lacking exon 11 may have
biological properties somewhat different from those containing exon 11
(46), although no significant differences in insulin binding and
insulin receptor kinase activity between these two variants were
observed (47).
Insulin receptor -subunits are extracellular structures possessing
cysteine-rich domains that serve as insulin-binding sites. Insulin
receptor ß-subunits have extracellular, transmembrane, and
intracellular domains, the latter containing an ATP-binding site and
several tyrosine autophosphorylation sites. After insulin binds to the
-subunits, the ß-subunits become phosphorylated on tyrosine
residues and acquire kinase activity, initiating a cascade of
intracellular protein phosphorylation (48, 49). The most important
intracellular proteins phosphorylated under the influence of the
insulin-receptor tyrosine kinase are the insulin receptor substrates
(IRS), several of which have been described (50, 51, 52, 53, 54, 55, 56, 57, 58). IRS-1, the first
of these to be discovered (2, 59), has a molecular mass of 131 kDa and
possesses 14 potential tyrosine phosphorylation sites. IRS-1 appears to
be important in insulin receptor function and its variant forms are
sometimes associated with diabetes (60, 61). Mice deficient in IRS-2
develop a syndrome resembling type 2 diabetes (62). Some IRS-1
mutations are associated with insulin resistance and hyperinsulinemia
(63), and codon 972 polymorphism of the IRS-1 gene is associated with
impaired glucose tolerance, PCOS (64), and late onset of type 2
diabetes mellitus (65). IRS-1 binds phosphatidylinositol-3-kinase (PI-3
kinase), a src homology-2 (SH2) domain-containing enzyme,
activation of which is necessary for the initiation of glucose
transport (2, 59, 66, 67, 68, 69). In addition to PI-3 kinase activation,
mitogen-activated protein kinase (MAPK) is also phosphorylated after
insulin receptor binding (2, 49, 59, 70). MAPK activation is thought to
be responsible for the growth-promoting effects of insulin (2). MAPK
can be activated not only by the insulin receptor, but also by other
tyrosine kinase receptors, such as the type I IGF receptor, and
receptors for epidermal growth factor (EGF) and platelet-derived growth
factor (PDGF), as well as G protein-linked receptors (2, 71, 72). The
molecular link between the MAPK cascade and the insulin receptor may be
p21 Ras, a highly conserved protein involved in cell growth that may be
a critical element in growth factor receptor and insulin receptor
tyrosine kinase action (2, 49, 59).
Tyrosine kinase activation is believed to be the main signaling mechanism of the insulin receptor (48); it appears to be the earliest postbinding event and is necessary for many, although not all, of insulin’s effects, including transmembrane glucose transport (73, 74). Overexpression of tyrosine kinase-deficient insulin receptors in muscle causes insulin resistance in transgenic animals (75). Tyrosine kinase activity is required in vivo for phosphorylation of IRS-1 and for PI-3 kinase activation (76).
An alternative signaling pathway for the insulin receptor has also been
described. It involves generation of inositolglycan second
messengers at the cell membrane after insulin binding to receptor
-subunits but independently of ß-subunit tyrosine kinase
activation (77). This alternative pathway for receptor signaling may
mediate some of insulin’s effects, including stimulation of ovarian
steroidogenesis (78, 79, 80) (Fig. 1), but
the role of this system in propagating the insulin signal for glucose
transport and other insulin effects has not been fully established.
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Insulin receptor-like proteins are present in lower organisms that do not produce insulin. For example, in certain species of worms, daf-2, a gene similar to that of the insulin receptor, regulates glucose metabolism and longevity (82). Mutation of the insulin receptor in Drosophila leads to small ovaries lacking oocytes, and thus sterility (83). Insulin receptor-like molecules are present in mosquito ovaries (84). The existence of these homologous proteins in insects suggests that the growth and regulatory functions of the insulin/IGF receptor family arose before the divergence of insects and vertebrates more than 600 million years ago (83). Conservation of the insulin receptor over this length of time in a variety of organisms indicates its importance for their survival. Indeed, mice with a genetic knockout of the insulin receptor die in the neonatal period (85).
B. Presence of insulin and insulin receptor in the ovary
Circulating insulin levels in the peripheral blood of normal women
are approximately 10 µU/ml in the fasting state and up to 50 µU/ml
within 1 h after an oral glucose load. In obese women, these
levels are somewhat higher, averaging approximately 15 µU/ml in the
fasting state and up to 60 µU/ml after a glucose load. In
insulin-resistant hyperinsulinemic states such as PCOS or the early
stages of type 2 diabetes mellitus, serum insulin levels range from
20–35 µU/ml in the fasting state to 120–180 µU/ml after a glucose
load (9, 86). In patients with syndromes of extreme insulin resistance,
circulating insulin levels may be as high as 200 µU/ml in the fasting
state and up to 1400–2000 µU/ml after a glucose load (9).
Ovarian follicular fluid (FF) insulin concentrations range from less than 2 µU/ml to 65 µU/ml, with a mean value of approximately 16 µU/ml (87). These do not correlate with plasma insulin or FF estradiol (E2) or androstenedione (A) concentrations, but do correlate directly with those of progesterone (P) (87). Insulin likely reaches FF from the circulation by transudation. To our knowledge, intrafollicular concentrations of insulin have not been reported in women with insulin resistance with or without ovulatory dysfunction.
Both in humans and in animal models, insulin receptors are widely
distributed throughout all ovarian compartments, including granulosa,
thecal, and stromal tissues (3, 11, 12, 88, 89, 90, 91) (Table 2). Ovarian insulin receptors have the
same heterotetrameric 2 ß2 structure as
insulin receptors in other organs. They possess tyrosine kinase
activity (12) and may stimulate the generation of inositolglycans (79).
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Insulin-induced hyperandrogenism is unlikely to result from an action of insulin through its own receptor, however, in disorders in which receptor expression or availability is significantly compromised, such as the type A syndrome of insulin resistance and acanthosis nigricans, caused by insulin receptor mutations, or the type B syndrome, associated with antiinsulin receptor antibodies (6, 7). In the latter two conditions, insulin receptors likely function as inefficiently in the ovary as in other organs, and another receptor, such as the type I IGF receptor, is more likely to mediate the effects of hyperinsulinemia in the ovary (9).
C. Insulin action and the ovary
Numerous actions of insulin on the ovary have been demonstrated
both in vitro (Table 3) and
in vivo (Tables 3 and 4), with
no significant differences between humans and other species (3).
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At this time, there is only limited knowledge about the specific
effects of insulin on ovarian steroidogenic enzymes. A stimulatory
effect of insulin on aromatase has been suggested by some studies of
animal and human ovarian cells in vitro (102, 103, 104, 105), but one
study (106) failed to confirm this finding. 17-Hydroxylase activity
appears to be stimulated by insulin (29, 107, 108, 109), but a recent study
of 28 women with PCOS and 18 normal controls found no correlation
between insulin levels and 17-hydroxyprogesterone (17-OHP) levels after
treatment with GnRH agonist (GnRHa) (110). Insulin increases
P450 side chain cleavage (scc) enzyme mRNA in porcine
granulosa cells (111) and P450scc activity in goldfish
follicles (112). A similar effect could not be demonstrated, however,
in a human ovarian thecal-like tumor line (101). In the latter study,
insulin had no effect on the enzyme activity or mRNA concentration of
17-hydroxylase/17,20-lyase (P450c17) or
3ß-hydroxysteroid dehydrogenase (HSD), but forskolin stimulation of
3ß-HSD mRNA was enhanced by insulin. In human luteinized granulosa
cells, 3ß-HSD expression was found to be stimulated by insulin (106).
b. In vivo studies (Table 4).
It has not been consistently
demonstrated that insulin stimulates ovarian steroidogenesis in
vivo (113). Several studies have examined the in vivo
effects of insulin on aromatase. In rats with experimental
hyperinsulinemia, an increased estrone (E1) to A ratio was
demonstrated, consistent with a stimulatory effect of insulin on
ovarian or peripheral aromatase (94). In women, an insulin infusion
study has suggested a similar effect (114), and in hyperinsulinemic
women with PCOS, an increased E2/A ratio was seen after
gonadotropin stimulation, compared with normoinsulinemic women with
PCOS (115). Relatively insulin-deficient women with type 2 diabetes
show reduced aromatase activity (116). The increase in circulating A
level observed during insulin infusions in women (117, 118), on the
other hand, suggests that insulin may inhibit aromatase. In short, it
remains unclear whether or how insulin regulates aromatase in
vivo.
The effect of insulin on ovarian androgen production in women has
been extensively studied (Tables 3
and 4). In PCOS, a positive
correlation has been reported between insulin and T or A levels
(119, 120, 121, 122) in several studies, while more recent studies (123, 124, 125, 126, 127)
failed to find such a relationship. In insulin infusion studies that
maintained hyperinsulinemia for several hours, a stimulatory effect of
insulin on ovarian androgen production has not been consistently found.
Stuart and associates (117, 118, 128) demonstrated elevation of A and
dehydroepiandrosterone (DHEA) in normal lean and obese
women and in women with insulin resistance and acanthosis nigricans
during a euglycemic, hyperinsulinemic clamp study. Micic et
al. (129) demonstrated an increase of T in patients with PCOS
during a 4.5-h insulin infusion. On the contrary, Diamond et
al. (130) could demonstrate no change in total or free T or in A
during either insulin or glucose infusion in normal women. Similarly,
Nestler et al. (131) could not demonstrate a rise in T in
normal women during insulin infusion. Dunaif and Graf (114) examined
gonadotropin and sex hormone levels basally and during insulin infusion
in normal and PCOS women. No effect on gonadotropins was demonstrated;
E2 levels rose in response to insulin in normal women. In
PCOS women, A levels increased, but T, free T, and dihydrotestosterone
(DHT) levels declined.
Another group of studies has examined the effects of food intake or oral or intravenous administration of glucose on circulating androgen concentrations. In normal women, Parra et al. (132) found an increase in free T and no change in A after breakfast, but a decline of free T after an oral glucose load. Elkind-Hirsch et al. (133) failed to demonstrate a rise of either T or A during a tolbutamide-enhanced intravenous glucose tolerance test (IVGTT). Smith et al. (134) found a positive correlation between insulin responses and A, T, and DHT levels during oral glucose tolerance testing (OGTT) in hyperandrogenic and normal women, but Tiitinen et al. (135) demonstrated no significant change in T or A in women with PCOS or weight-matched normal controls after an oral glucose load and Tropeano et al. (136) demonstrated a decline of T, A, and DHEA during an OGTT. On occasion, both a stimulatory response and the lack of it have been observed in the same study. For example, Anttila et al. (137) reported a tendency to increased serum T levels during OGTT mainly in a subgroup of PCOS patients with both hyperinsulinemia and elevated LH levels; most PCOS patients, however, showed a decline in T. Fox et al. (138) found that serum androgens declined in PCOS patients during OGTT, but A rose during a 2-h intravenous insulin infusion in obese controls. Since a decline of serum T in the course of a 3- or 4-h OGTT may be attributed to diurnal variations of T, the lack of an increase of T under these conditions argues against a significant acute stimulatory or inhibitory effect of insulin on ovarian androgen production in vivo.
While studies that raise circulating insulin concentration have
produced variable effects on serum androgen levels, studies in which
insulin levels were reduced have consistently demonstrated a decline in
serum androgen levels in insulin-resistant hyperandrogenic women (139, 140) (see Section VI.A). Whether insulin levels are lowered
with diazoxide (30, 141), octreotide (34, 142), metformin (29, 31, 108, 143, 144, 145, 146), troglitazone (35, 36), or through weight loss
(147, 148, 149, 150, 151, 152, 153, 154, 155, 156), a decline in serum androgen levels is usually found and
ovulatory function improves (Table 4). In contrast to the studies in
which insulin levels were elevated acutely for several hours, the
effect of the reduction of circulating insulin can be studied over many
weeks. If insulin-induced stimulation of ovarian steroidogenesis
requires a prolonged exposure to excess circulating insulin, the latter
group of studies is more likely to be able to demonstrate, albeit
indirectly, a stimulatory effect of insulin on
circulating steroids. A confounding factor in some of these studies is
a decline in circulating LH, which may be responsible, at least in
part, for the reduced androgen secretion (157).
In summary, it appears that insulin may have stimulatory or inhibitory effects on ovarian steroidogenic enzymes, but the responses of specific enzymes may vary with cell type and possibly among species. Further studies are needed on the effects of insulin on steroidogenic enzymes in the ovaries both in vitro and in vivo.
2. Interactions with gonadotropins. Acting at the ovarian level, insulin appears to potentiate the steroidogenic response to gonadotropins, both in vitro and in vivo (96, 102, 157, 158, 159, 160, 161, 162, 163). In granulosa cells, this effect may be mediated by an increase in LH receptor number, since insulin in concert with FSH increases ovarian LH-binding capacity (13, 164). In addition, insulin may act on the pituitary to increase gonadotrope sensitivity to GnRH. Evidence for this effect comes both from in vitro studies (165, 166) and indirectly from studies in insulin-resistant patients treated with insulin sensitizers, in whom circulating LH declined concomitantly with insulin (29, 31, 35, 108). On the other hand, in rats with experimental hyperinsulinemia maintained over six 4-day estrous cycles, the response of gonadotropins to GnRH did not differ from that of controls (94). In normally cycling women, increasing body mass index (BMI) did not have an effect on gonadotropin secretion and in women with PCOS BMI and LH levels were inversely related (167, 168, 169), while gonadotropin responsiveness to GnRH did not change after insulin infusion (114). In summary, it remains unclear whether hyperinsulinemia significantly enhances gonadotrope responsiveness to GnRH in vivo, as it does in vitro.
3. Effects on ovarian growth and cyst formation. In a rat
model, a synergistic interaction between LH/hCG and insulin on the
ovary can be demonstrated directly during experimentally induced
hyperinsulinemia, which enhances hCG-induced ovarian growth and cyst
formation (28, 170) (Fig. 2). This
synergistic action of insulin with LH/hCG is seen regardless of
cotreatment with a GnRH antagonist, suggesting that the growth- and
cyst-promoting effects of insulin are exerted directly on the ovary.
Indeed, insulin can stimulate proliferation of both human and rat
theca-interstitial cells in vitro (171, 172, 173). In humans, the
ability of high insulin levels to stimulate ovarian growth in
vivo has been suggested by a case report of a patient with the
type B syndrome of insulin resistance, whose sonographically determined
ovarian volume doubled during a prolonged insulin infusion (174).
Furthermore, in women with PCOS, circulating insulin levels are
correlated with ovarian volume (175, 176), and after gonadotropin
stimulation, the increase in ovarian dimensions observed in
hyperinsulinemic PCOS is greater than in normoinsulinemic PCOS (115).
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5. Effects on IGFBP-1 production. Another protein under the regulatory control of insulin is IGFBP-1. Insulin and BMI are the major determinants of circulating IGFBP-1 levels in both obesity (185, 186, 187) and PCOS (183, 188, 189, 190, 191, 192). Insulin inhibits IGFBP-1 production in the liver (193, 194, 195, 196, 197, 198), thereby reducing circulating IGFBP-1 levels. Insulin also inhibits IGFBP-1 production in ovarian granulosa cells (see Section IV.B), acting through its own receptor (199). A detailed discussion of the role of IGFBPs in ovarian function and their regulation in the ovary is presented in Section IV.D.
6. Ovulation in diabetes mellitus and in states of extreme insulin resistance. Insulin and IGFs have been shown to suppress apoptosis in ovarian follicles, thus reducing rates of their atresia (200, 201). A variety of clinical and experimental observations in patients with type 1 and type 2 diabetes mellitus and states of extreme insulin resistance suggest that insulin may be involved, either directly or indirectly, in the process of ovulation (3, 9, 202).
Insulin deficiency in type 1 diabetes has been associated with disordered ovulation (3, 202). In rats, streptozotocin-induced diabetes is associated with cessation of ovulatory cycles, which can be restored with insulin treatment (203). In mice with alloxan-induced diabetes, a similar reduction in ovulation rate has been reported (204). While the current availability of insulin therapy does not allow observation of a similar phenomenon in human type 1 diabetes, in the preinsulin era, girls who developed diabetes prepubertally failed to enter puberty (3, 4). It is difficult to determine whether it was insulin deficiency itself, the state of chronic diabetic ketoacidosis, the starvation diets used for treatment, or the dramatic weight loss that caused the failure of pubertal development in these girls. In patients with type 1 diabetes treated with insulin, the hypothalamic-pituitary-gonadal axis appears to be relatively hypoactive, mainly because of failure of the GnRH pulse generator (205, 206); low serum sex hormone levels, including low luteal-phase P levels, have been described (207, 208). Even with insulin treatment, up to one third of young women with type 1 diabetes may experience delayed menarche and oligomenorrhea of hypothalamic origin (205).
Hyperinsulinemia resulting from exogenous insulin administration is often present in treated patients with type 1 diabetes. If such patients gain excessive weight, their LH:FSH ratio increases, SHBG levels decrease, and more than 70% develop polycystic ovaries (209); the response of 17-OHP to GnRHa in oligomenorrheic diabetic adolescents is exaggerated, resembling the response reported in insulin-resistant patients with PCOS (29, 108, 210). Some patients with type 2 diabetes have mildly elevated androgen levels or increased androgen responses to GnRH stimulation (116, 202) as well as reduced SHBG levels (211), particularly in the early, hyperinsulinemic stage of the disease (116, 212). It should be noted that hyperinsulinemia in patients with diabetes is relatively mild, compared with that seen in patients with syndromes of extreme insulin resistance, and that significant hyperandrogenism is not characteristic of women with either type 1 or type 2 diabetes (9).
Hyperandrogenism and polycystic ovaries or ovarian hyperthecosis are commonly found in states of extreme insulin resistance (9, 140, 213). These conditions are sometimes caused by mutations of the insulin receptor gene (214, 215, 216) and include the type A syndrome (6), leprechaunism (9, 217, 218), Rabson-Mendenhall syndrome (9, 215), and syndromes characterized by defective insulin receptor signaling (74, 219, 220). Premenopausal patients with the type B syndrome (insulin resistance and acanthosis nigricans associated with the presence of antiinsulin receptor antibodies) also exhibit hyperandrogenism (7, 8).
Although there is evidence that hyperinsulinemia contributes to the development of hyperandrogenism, not all clinical conditions associated with hyperinsulinemia lead to ovarian androgen overproduction. For example, most women with type 1 diabetes, who are often hyperinsulinemic because of exogenous insulin administration but usually do not exhibit significant insulin resistance, do not become hyperandrogenic, but rather exhibit hypothalamic-pituitary-ovarian axis hypofunction. It is not clear why hyperinsulinemia developing in the setting of insulin resistance, rather than any form of hyperinsulinemia, is associated with ovarian hyperandrogenism, particularly since correction of hyperinsulinemia without correction of insulin resistance may improve ovarian function (38, 221, 222, 223).
Dissecting the effects of hyperinsulinemia from those of insulin resistance is difficult (224, 225). One can postulate, however, that because the postbinding insulin receptor pathways may diverge (2, 9, 226), in conditions characterized by hyperinsulinemia without primary insulin resistance all insulin receptor-signaling pathways are significantly down-regulated, whereas when hyperinsulinemia is caused by insulin resistance, only some of these pathways (e.g., glucose transport) may be deficient, while others may be hyperstimulated (9, 227, 228). Thus, if hyperinsulinemia promotes androgen production by activating insulin-signaling pathway(s) distinct from those involved in glucose transport, hyperandrogenism would be more likely to develop in the setting of insulin resistance and compensatory hyperinsulinemia.
7. Interactions of insulin with leptin; leptin-mediated effects on ovulation. New insights into the relationship between weight and ovulation and the role that insulin may play in modifying this relationship emerged with the discovery and characterization of leptin. Leptin is a 16-kDa protein produced by adipose cells (229, 230, 231, 232, 233). Circulating leptin levels are stimulated by estrogen and inhibited by androgens (234, 235, 236) and are directly proportional to adipose tissue mass (236, 237, 238, 239, 240, 241). Leptin regulates body weight by binding to specific receptors in the hypothalamus and thus decreasing food intake (242, 243, 244). Leptin is encoded by the ob gene, which is defective in genetically obese ob/ob mice (229, 231, 237, 245). These animals are also insulin resistant and infertile. Replacement of leptin in ob/ob mice produces weight loss, reverses metabolic abnormalities, and restores ovulation and fertility (246, 247). Db/db mice and Zucker fatty rats have a similar phenotype, which results from a genetic abnormality of the leptin receptor (237, 245, 248). A human kindred with an ob mutation has been described, in which two prepubertal cousins with a frameshift mutation in the ob gene suffer from massive obesity (249). It is not yet known whether they will develop reproductive abnormalities. Similarly, a mutation of the human leptin receptor gene associated with obesity has been reported (250).
A rise in circulating leptin levels is associated with and precedes puberty (251), and higher circulating leptin levels are associated with a younger age at menarche (252, 253), possibly because leptin serves as a signal for the initiation of an early pubertal gonadotropin-secretory pattern (254, 255, 256, 257). A rapid decline of circulating leptin levels is observed during caloric restriction (258) or starvation (244, 259, 260). A decline in leptin may be responsible for the activation of the hypothalamic-pituitary-adrenal axis and the inhibition of the gonadotropic axis observed with stress (261, 262), since these responses can be abolished in animals by leptin administration (233, 263).
Leptin receptors are present in the ovary (264, 265, 266). Their functional
capacity and their role in both normal and abnormal ovarian function
remain to be firmly established since two leptin receptor isoforms
exist, one with a full-length and another with a truncated
intracellular domain (267). While the action of leptin on gonadotropin
secretion is stimulatory, the direct effects of leptin on ovarian
steroidogenesis may be either inhibitory or stimulatory (264, 266, 268). For example, leptin inhibits insulin-induced P and E2
production in bovine granulosa cells (264) and reduces synergism
between FSH and IGF-I on E2 production in rat granulosa
cells (268). On the other hand, leptin appears to stimulate ovarian
17-hydroxylase (265).
Insulin stimulates secretion of leptin by adipocytes (269, 270, 271, 272). In addition, by promoting lipogenesis, insulin may increase adipose tissue mass, thereby further enhancing leptin production. However, there is no apparent acute effect of feeding on leptin levels (260, 273, 274) and no correlation between leptin and insulin sensitivity in vivo (273). Nevertheless, circulating leptin levels rise with acute massive overfeeding over a 12-h period (275).
Leptin inhibits insulin secretion from isolated pancreatic islets in some studies (276, 277), but stimulates insulin secretion in others, either by a direct stimulatory effect on pancreatic ß-cells (278) or because of its inhibitory effect on somatostatin (279). Leptin may affect pancreatic function through the autonomic nervous system (280) and was shown to improve insulin sensitivity in normal rats, reducing glucose and insulin levels (281). When administered intracerebroventricularly, leptin enhanced insulin-stimulated glucose metabolism (282). Leptin has been shown to possess antidiabetic properties in some studies (283, 284), but in other studies it did not affect glucose-stimulated insulin secretion and did not have a significant effect on glucose transport or insulin action in either adipocytes or muscle cells (285, 286). In some circumstances, as, for example, in the setting of obesity, leptin may contribute to the development of insulin resistance and diabetes (287, 288, 289, 290).
The above observations point to a complex relationship among insulin,
leptin, body weight, ovarian steroidogenesis, and ovulation (Fig. 3). If a certain "threshold" level of
leptin is needed to activate the hypothalamic-pituitary-ovarian axis,
then a certain mass of adipose tissue must be present for ovulation to
occur (291). In states characterized by hypoinsulinemia, such as
starvation, weight loss, or untreated type 1 diabetes mellitus,
amenorrhea may develop (292, 293), possibly because of a decline in
circulating leptin (294) and a resultant deactivation of the
hypothalamic-pituitary-ovarian axis (233, 293, 295). Thus, insulin
deficiency may contribute to abnormalities of ovulatory function either
directly, by affecting gonadotropins or the ovaries, or indirectly, by
negatively influencing secretion of leptin. On the other hand, states
characterized by insulin excess may be associated with higher
circulating levels of leptin. Whether such putative leptin excess would
play a role in the development of the hyperandrogenism or anovulation
observed in hyperinsulinemic states remains to be determined.
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). That this phenomenon may also occur
in humans is suggested by the observations of Samoto et al.
(95) and Nagamani and Stuart (296), who demonstrated that in women with
hyperthecosis or PCOS, ovarian type I IGF receptors are up-regulated,
while insulin receptors are down-regulated. Pepper and colleagues (297)
have reported that ovarian [125I]IGF-I binding in a
patient with ovarian hyperthecosis was increased over that found in
normal controls (12, 298). Interestingly, an increase in type I IGF
receptor expression in PCOS may not be limited to the ovaries: a rise
in erythrocyte type I IGF receptors in these patients has also been
reported (299). Further, hyperinsulinemia may increase expression of
hybrid insulin/type I IGF receptors in a variety of insulin target
tissues (300), although this process has not yet been described in the
ovary.
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D. Summary
The role of insulin in the ovary may be summarized as follows: 1)
Insulin receptors are widely distributed throughout all ovarian
compartments. Ovarian insulin receptors have a subunit structure
identical to insulin receptors in other organs, possess tyrosine kinase
activity, and are capable of stimulating the generation of
inositolglycan second messengers. 2) At this time there is no
convincing direct in vivo evidence that hyperinsulinemia
acutely stimulates ovarian steroid production, but there is direct
in vitro evidence and indirect in vivo evidence
for a stimulatory effect of insulin on ovarian steroidogenesis. The
in vitro evidence suggests that the stimulatory effect of
insulin on steroidogenesis is mainly mediated by the insulin receptor
and may involve the inositolglycan pathway. The in vivo
evidence is largely derived from experiments in which a reduction in
circulating insulin levels produces a decline of circulating androgens
and from clinical observations in women with both insulin deficiency
and insulin excess. 3) The effects of insulin on ovulation are complex.
A threshold level of insulin is likely to be required for the normal
function of the hypothalamic-pituitary-ovarian axis, either because of
the direct stimulatory effects of insulin on this axis or because of
the stimulatory effects of insulin on leptin secretion (both direct,
with insulin stimulating adipocyte production of leptin, and indirect,
because of insulin-stimulated lipogenesis). Leptin, in turn,
participates in the initiation of puberty and activation of the
hypothalamic-pituitary-gonadal axis. On the other hand, excessive
circulating insulin, particularly in the setting of insulin resistance,
may enhance ovarian androgen production and thus may contribute to the
development of anovulation. 4) Insulin may amplify its own effects, the
effects of IGFs, and those of gonadotropins by up-regulating type I IGF
receptors and gonadotropin receptors, as well as by inhibiting
production of IGFBP-1, both in the liver and ovary. In the setting of
insulin resistance and hyperinsulinemia, therefore, a cycle of events
that leads to a self-perpetuating amplification of the ovarian effects
of insulin and IGFs can develop (Fig. 5).
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III. IGFs and Their Receptors |
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TopAbstractI. IntroductionII. Insulin and Insulin...III. IGFs and Their...IV. IGF-Binding Proteins...V. Polycystic Ovary Syndrome...VI. The Insulin-Related Ovarian...VII. Summary and ConclusionsReferences |
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2. IGF-II. IGF-II is a 7.5-kDa, 67-amino acid, single-chain polypeptide that is approximately 70% homologous with IGF-I and 50% homologous with proinsulin (14, 309, 310, 311, 312). The human IGF-II gene is located on chromosome 11, contiguous with the insulin gene. Pre-pro-IGF-II, the precursor of IGF-II, is a 22-kDa protein. Inactivation of the IGF-II gene in animals (308, 313) produces growth-deficient but fertile and otherwise normal individuals. IGF-II is highly expressed in fetal tissues and tumors, as well as in normal adult tissues. IGF-II can bind to type I and type II IGF receptors (see below), as well as to the insulin receptor (302, 314).
3. Type I IGF receptor. The type I IGF receptor precursor
protein consists of 1367 amino acids, comprising both the - and
ß-subunits of the receptor. The human type I IGF receptor gene is
located on chromosome 15. The mature type I IGF receptor protein is a
heterotetramer consisting of two - and two ß-subunits and is
highly homologous with the insulin receptor (315, 316). The
cysteine-rich regions of the -subunits of the insulin receptor and
type I IGF receptor are 64–67% homologous, whereas the tyrosine
kinase domains of the ß-subunits are 84% homologous. In addition to
IGF-I, the type I IGF receptor can also bind IGF-II and insulin,
although with somewhat lower affinity. In addition to binding IGF-I,
IGF-II, and insulin, the type I IGF receptor has also been reported to
interact with IGFBPs (317), but the significance of this finding
remains to be determined. Type I IGF receptor postbinding events,
similar to those of the insulin receptor, include tyrosine
phosphorylation of receptor ß-subunits and IRS proteins, interactions
with PI-3 kinase, and activation of MAPK (69, 315, 318, 319). Type I
IGF receptor knockout mice weigh 45% of normal at birth and die
immediately afterward (320). Patients with a deletion of the distal arm
of chromosome 15 lack one copy of the IGF-I receptor gene and exhibit
both intrauterine and postnatal growth restriction (321, 322).
4. Hybrid insulin/type I IGF receptors. Hybrid receptors that
combine an /ß insulin hemireceptor and an /ß type I IGF
hemireceptor have been reported in a variety of tissues, although not
in the ovary (41, 323). These receptors can form in tissues
coexpressing both insulin and type I IGF receptors, theoretically
including the ovary. Hybrid receptors have properties similar to type I
IGF receptors, binding IGF-I with high affinity and insulin with lower
affinity. Interestingly, in situations that are characterized by
insulin receptor down-regulation, the number of hybrid insulin/type I
IGF receptors tends to increase (228).
5. Type II IGF receptor. The type II IGF receptor is identical to the mannose-6-phosphate (Man-6-P) receptor (309, 324, 325, 326). The gene for the type II IGF receptor is located on the long arm of chromosome 6. This receptor targets Man-6-P-containing enzymes from the Golgi apparatus to the lysosomes and also mediates the rapid internalization of IGF-II (309). The receptor is a single-chain polypeptide of approximately 300 kDa with a large extracellular domain containing IGF-II binding sites (325, 327). The cytoplasmic domain is very short and includes tyrosine, threonine, and serine phosphorylation sites. Type II IGF receptor knockout mice exhibit elevated IGF-II levels and die in utero (328, 329). Interestingly, if the IGF-II gene is knocked out at the same time, about 50% of the fetuses survive to birth (328). Type I/type II IGF receptor double-knockout mice differ from normal controls only in their patterns of growth (328). These observations, taken together, suggest that excessive activation of the type I IGF receptor by IGF-II may be lethal in utero.
The type II IGF receptor can be released from the cell membrane into the circulation. This mechanism may be principally responsible for its loss from the cell surface (330, 331, 332, 333). The circulating form of the IGF-II receptor retains its affinity for IGF-II (325, 334) and may participate in the local modulation of organ size in vivo. For example, overexpression of the soluble IGF-II/Man-6-P receptor in transgenic mice can significantly decrease the weight of their alimentary canal (335).
Although the type II IGF/Man-6-P receptor is important for IGF-II internalization and degradation, it is unclear whether this receptor actively mediates IGF-II signaling. Examples of such signaling have been reported, including stimulation of G-protein activation and of thymidine incorporation into rat hepatocyte DNA (325, 336, 337, 338). In most instances, however, the metabolic and growth-promoting actions of IGF-II appear to be mediated by the type I IGF receptor (339) or the insulin receptor (314). The type II IGF receptor, however, may mediate signals involved in angiogenesis (340) and other processes. Ligands for the type II IGF receptor, in addition to IGF-II and Man-6-P, include ß-galactosidase and other lysosomal enzymes, proliferin, renin, latent transforming growth factor (TGF)-ß (329), and leukemia-inhibitory factor (341). In the context of these observations, the functions of the type II IGF receptor within the ovary remain to be determined.
B. Expression of IGFs and IGF receptors in the ovary
1. Human and nonhuman primate. Distinctive features of
IGF expression in the primate ovary include the predominance of IGF-II
and its pattern of localization (Table 2). Other molecules that
modulate IGF action, including the IGF receptors, IGFBPs, and IGFBP
proteases, are also differentially expressed in the primate ovary (see
below). While the majority of studies that examined the ovarian
expression of IGFs and that of their receptors were done on human
tissue, ovaries from cycling rhesus monkeys reveal similar expression
patterns of IGF-I, IGF-II, and type I IGF receptor, and there is strong
evidence that IGF-II, aromatase, and IGFBP-4 can be regarded as markers
of the dominant follicle in the rhesus ovary (342).
In the human ovary, IGF peptide expression is follicle stage-specific
and compartmentalized (Table 2). IGF-I mRNA is barely detectable in the
adult ovary and not in the granulosa layer at any stage of follicular
development (88, 89, 343). IGF-II mRNA is expressed in the theca and
perifollicular vessels of all follicles and in the granulosa cells of
some follicles. In small antral follicles, IGF-II mRNA and protein are
detectable in both granulosa and theca (88, 89, 343). In atretic antral
follicles, on the other hand, IGF-II is minimally expressed by the
theca. IGF-II is abundantly expressed and secreted by granulosa cells
of preovulatory follicles as well as by granulosa-luteal cells
harvested during oocyte retrieval after controlled ovarian
hyperstimulation (COH) (88, 90, 344, 345, 346, 347). These findings, plus the
observations that granulosa cells do not express IGF-II prepubertally,
but do so in a subpopulation of adult follicles, and that gonadotropins
regulate IGF-II mRNA expression and secretion in human granulosa-luteal
cells in vitro (344, 345), suggest that ovarian IGF-II gene
expression is regulated by gonadotropins.
Follicular fluid (FF) constituents such as IGF peptides are derived from the circulation as well as from intraovarian production. In normally cycling women, FF IGF-I levels are similar in estrogen-dominant and androgen-dominant follicles and do not correlate with follicular size (348). In contrast, FF IGF-II levels are higher in estrogen- compared with androgen-dominant follicles and correlate positively with follicle size, cycle day, and E2 and negatively with androgen-estrogen (A:E) ratio (348). In normally cycling women, simultaneous measurements of IGF-I, IGF-II, and insulin concentrations in ovarian and peripheral venous blood reveal an ovarian gradient only for IGF-II (349), and serum IGF-I and IGF-II levels in normally cycling women do not vary during the menstrual cycle (348). These data collectively suggest that FF IGF-I originates from serum by transudation and that FF IGF-II derives primarily from local production by the granulosa and possibly by the theca, in addition to some contribution from the circulation. After COH, FF IGF-II levels are about 8 times higher than those of IGF-I, and both IGF-I and IGF-II levels are lower than in serum (350, 351, 352, 353). In contrast to spontaneous cycles, these levels in COH do not correlate with follicle size, oocyte maturity, or FF E2. FF IGF-I and IGF-II levels were noted to rise with increasing cycle day 3 serum FSH, an index of ovarian reserve (354).
Normal circulating levels of IGF-I are not a prerequisite for normal ovarian follicular development in women, as evidenced by cases of ovulation and fertility in individuals with Laron-type dwarfism, which results from GH receptor deficiency (GHRD) (355, 356, 357, 358). Furthermore, a normal follicular response to injected gonadotropins, leading to ovulation and conception, has been reported in women with GHRD, whose serum GH was markedly elevated and both serum and FF IGF-I barely detectable (355, 356). In such subjects, serum IGF-II levels were about 25% of normal (FF IGF-II was not measured). These clinical observations support the conclusion that IGF-I does not play an important role in the ovulatory process in women.
Both type I and type II IGF receptors are found in the human ovary (88, 298, 343, 359). By in situ hybridization, type I IGF receptor mRNA is predominantly expressed by granulosa cells and oocytes, with more intense expression in dominant compared with small antral follicles (88, 343). By this technique, theca and stroma are negative for type I IGF receptors, but stromal receptors with the specificity of the type I IGF receptor have been reported in ligand binding studies (298). Type II IGF receptors are localized to both granulosa and thecal layers, with more intense expression in the granulosa and in dominant, compared with smaller, antral follicles (88). By RT-PCR, both types of receptors were found to be expressed by granulosa, theca, and stroma and to persist upon culture of both granulosa and thecal cells (347).
2. Rodent. In the rat, ovarian IGF-I gene expression and protein production are granulosa specific (360, 361, 362); significantly, IGF-I is selectively expressed in the granulosa of only healthy antral follicles, not in atretic or luteinized follicles or in theca-interstitial cells (342, 360, 363, 364). IGF-II mRNA expression is limited to the thecal compartment and blood vessels (342, 362, 363), but the postnatal decline in ovarian IGF-II content (365) argues against a significant role for this peptide in rat ovarian physiology. While type I IGF receptor mRNA is abundantly expressed in granulosa cells (365), the corresponding protein is detected not only in the granulosa but also in the thecal compartment, regardless of the maturational stage or health status of the follicle (363), suggesting that regulation of the receptor is unlikely to play a major role in follicular maturation (366).
The patterns of IGF-I, IGF-II, and type I IGF receptor expression are essentially the same in rat and mouse ovary (342, 364, 367). IGF-I expression increases at the secondary preantral stage and is abundant in healthy follicles through the preovulatory stage. Type I IGF receptor is expressed constitutively, regardless of follicular developmental stage or health (367). These findings lay the groundwork for studies of ovarian function in transgenic mouse models with deletions of these components (368).
3. Livestock species. Porcine granulosa cells in culture
secrete abundant immunoreactive IGF-I, which is increased by FSH, cAMP,
GH, EGF, and TGF-. IGF-I is abundant in porcine FF, especially in
large follicles. Its levels increase in response to PMSG and/or GH
treatment (369, 370, 371). This finding suggests that gonadotropin and GH
action on the granulosa cells of the developing porcine follicle is
mediated in part by local induction of IGF-I. IGF-II in the porcine
ovary is expressed mainly in the theca and is not under gonadotropin or
GH regulation (15, 370, 372). FF IGF-II levels decline in response to
GH (370, 372, 373). In the sheep ovary, at least four localization
studies of IGF-I expression have been published, with divergent
findings (374, 375, 376, 377). IGF-II is localized to the theca, and its levels
in FF are 4-fold greater than those of IGF-I (377, 378). In the cow,
IGF-I is produced by the ovary (379, 380), and its levels in FF
increased with increasing E2 concentrations and increasing
follicle diameter in some (379, 381, 382, 383, 384), but not all (385, 386, 387),
studies. IGF-II is exclusively expressed in the theca, with greater
expression in dominant follicles, compared with subordinate or
nonrecruited ones (388).
C. Role of IGFs in ovulatory function and steroidogenesis (Table 5)
1. Human. Studies of the effects of IGFs on human granulosa
and thecal cells in vitro have primarily employed IGF-I,
although as discussed above, the predominant endogenous locally
produced ligand in vivo is IGF-II. IGF actions on the ovary
include augmentation of DNA synthesis and steroidogenesis. IGF-I
stimulates DNA synthesis and basal E2 secretion in
granulosa and granulosa-luteal cells and inhibits IGFBP-1 production
(199, 389, 390, 391, 392, 393, 394, 395, 396). It also synergizes with gonadotropins in augmenting
E2 and P production (393, 397, 398, 399, 400). Several studies have
been conducted recently of the effects of IGF-II on human ovarian
cellular constituents. IGF-II stimulates basal P and E2
secretion by human granulosa-luteal cells (353, 401). It also
stimulates aromatization of androgen precursors (402) and inhibits
IGFBP-1 (396) and IGFBP-2 (403) production by these cells. The effect
of IGF-II on estradiol production is most pronounced if the cells are
preincubated with insulin (402), possibly due to insulin-induced
up-regulation of type I IGF receptors, formation of hybrid
insulin/IGF-I receptors, or inhibition of IGFBP-1 production. IGF-II
also stimulates granulosa-luteal cell DNA synthesis and proliferation
in vitro (401, 404). In granulosa cells from both
unstimulated and gonadotropin-stimulated preovulatory follicles, IGF-I,
both alone and in synergy with gonadotropins, stimulates P450 aromatase
mRNA expression and activity (405).
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2. Rodent. IGF-I actions in rat granulosa and theca have been
extensively reviewed (14, 23, 409, 410). IGF-I acts as a
co-gonadotropin with FSH to stimulate granulosa cells to produce
E2 and P, and with LH to stimulate thecal androgen
production. IGF-I stimulates LH receptor expression in granulosa and
theca (13, 411, 412) and may be required for FSH receptor expression in
granulosa (368); it also stimulates granulosa cell production of
inhibin -subunit and augments the stimulation of this response by
FSH (413, 414, 415). Stimulation of inhibin- expression in rat granulosa
by FSH requires activation of protein tyrosine kinases by endogenously
produced IGF-I, suggesting that IGF-I signaling is obligatory for this
response (415). IGF-I also stimulates DNA synthesis in granulosa and
theca-interstitial cells (171, 416).
In addition to its role in differentiation and proliferation of granulosa and theca, IGF-I also plays an important role in granulosa survival, since it can inhibit apoptosis (201). Granulosa cell apoptosis, associated with regular cleavage of nuclear DNA by endonuclease, is associated with follicular atresia (417). In vitro, this process is suppressed by IGF-I and gonadotropins and enhanced by the presence of IGFBPs (200). In the human ovary apoptosis is characteristic of androgen- but not estrogen-dominant follicles (418), but regulation of apoptosis by IGFs has not yet been demonstrated in human ovarian follicles or cellular components, as it has in the rat (201). To our knowledge, there are no studies examining specific effects of IGF-II in rodent ovaries.
3. Livestock species. In the sow, similar effects of IGFs on granulosa and thecal cell function have been reported as in humans and rodents (419, 420, 421). IGF-I stimulates granulosa cell proliferation and synergizes with FSH in granulosa cell differentiation (419). IGF-II enhances the delivery of cholesterol to the P450 scc enzyme complex and enhances the functional activity of this first committed step in P biosynthesis (421). In sheep, IGF-I stimulates granulosa cells from small follicles to proliferate and those from larger follicles to produce P (422), an effect likely mediated through the type I IGF receptor (423). In the cow, IGF-I stimulates granulosa and thecal cell proliferation and steroidogenesis (379, 380, 424).
D. Summary
Although both IGF-I and IGF-II have been shown in vitro
to have multiple ovarian effects in various species, IGF-II appears to
be the predominant ovarian IGF in the human. The IGF-II gene is
expressed in the human ovary, and the effects of IGF-II appear to be
similar to those of IGF-I. The metabolic and growth-related effects of
IGF peptides appear to be mediated under most circumstances by type I
IGF receptors, which are present in all human ovarian compartments.
Their numbers appear to be increased under the influence of insulin, as
discussed in Section II.C. Type I IGF receptors may mediate
the effects of insulin in the ovary in extreme insulin-resistant states
with severe hyperinsulinemia. Clarification of the presence and the
role of hybrid insulin/type I IGF receptors in the human ovary awaits
further studies.
