In Vitro Effects of Pioglitazone on the Expression of Components of Wnt Signaling Pathway and Markers of Bone Mineralization.

Pioglitazone is an insulin-sensitizing thiazolidinedione (TZD) whose use is associated with bone loss. We examined the effects of pioglitazone on components of the Wnt signaling pathway (Wnt1, ß-catenin) and markers of bone mineralization [osteoprotegerin (OPG), bone sialoprotein (BSP), fibroblast growth factor (FGF)23] as well as mineral content in human osteoblast hFOB 1.19 cells. hFOB 1.19 cells were cultured in K12/DMD medium with or without pioglitazone. PPARγ Wnt1, OPG, BSP, or FGF23 mRNA expression was measured using qRT-PCR; ß-catenin, OPG, BSP, or FGF23 using ELISA; and calcium or phosphate content using colorimetry. Treatment with pioglitazone resulted in increased expression of PPARγ mRNA in hFOB 1.19 osteoblasts. Pioglitazone decreased Wnt1 mRNA levels and suppressed components of Wnt signaling pathway as evidenced by a decrease in ß-catenin gene expression and secretion as well as ß-catenin specific activity. The expression and the activity of OPG, BSP, and FGF23 were also reduced by pioglitazone together with total (but not specific) calcium and phosphate content. Pioglitazone affects Wnt1 signaling pathway and mineral matrix regulation components in human osteoblasts.

Thiazolidinediones (TZDs) affect osteoblast viability and biomarkers independently of the TZD effects on aromatase.

Thiazolidinediones (TZDs) are insulin sensitizers used for treatment of diabetes. We have previously reported that TZDs reduce estrogen synthesis by inhibiting aromatase activity in human granulosa cells (HGC). Multiple clinical trials demonstrated that TZDs increase the risk of fractures in postmenopausal women with type 2 diabetes. We studied mouse osteoblasts alone or in a co-culture with HGC to determine whether TZD inhibition of aromatase plays a role in their effects on bone metabolism. Mouse osteoblasts were cultured with and without HGC, and incubated in a medium with or without testosterone, pioglitazone or rosiglitazone. Cell growth, oleic acid uptake, alkaline phosphatase activity, and osteocalcin production were measured. TZDs inhibited estradiol production by up to 84% in HGC/mouse osteoblast co-cultures. TZDs induced mouse osteoblast death and increased oleic acid uptake. TZDs also inhibited alkaline phosphatase activity (58-75%, p<0.046) and osteocalcin production (52-75%, p<0.031). For all the parameters, there were no significant differences between the osteoblast cultures alone and the HCG/osteoblast co-cultures. TZD effects on osteoblast viability, oleic acid uptake, alkaline phosphatase and osteocalcin production are independent of their effects on aromatase.

Differential roles of MAPK-Erk1/2 and MAPK-p38 in insulin or insulin-like growth factor-I (IGF-I) signaling pathways for progesterone production in human ovarian cells.

Insulin and insulin like-growth factor-I (IGF-I) participate in the regulation of ovarian steroidogenesis. In insulin resistant states ovaries remain sensitive to insulin because insulin can activate alternative signaling pathways, such as phosphatidylinositol-3-kinase (PI-3 kinase) and mitogen-activated protein-kinase (MAPK) pathways, as well as insulin receptors and type 1 IGF receptors. We investigated the roles of MAPK-Erk1/2 and MAPK-p38 in insulin and IGF-I signaling pathways for progesterone production in human ovarian cells. Human ovarian cells were cultured in tissue culture medium in the presence of varying concentrations of insulin or IGF-I, with or without PD98059, a specific MAPK-Erk1/2 inhibitor, with or without SB203580, a specific MAPK-p38 inhibitor or with or without a specific PI-3-kinase inhibitor LY294002. Progesterone concentrations were measured using radioimmunoassay. PD98059 alone stimulated progesterone production in a dose-dependent manner by up to 65% (p<0.001). Similarly, LY294002 alone stimulated progesterone production by 13-18% (p<0.005). However, when used together, PD98059 and LY294002 inhibited progesterone production by 17-20% (p<0.001). SB203580 alone inhibited progesterone production by 20-30% (p<0.001). Insulin or IGF-I alone stimulated progesterone production by 40-60% (p<0.001). In insulin studies, PD98059 had no significant effect on progesterone synthesis while SB203580 abolished insulin-induced progesterone production. Either PD98059 or SB203580 abolished IGF-I-induced progesterone production. Both MAPK-Erk1/2 and MAPK-p38 participate in IGF-I-induced signaling pathways for progesterone production, while insulin-induced progesterone production requires MAPK-p38, but not MAPK-Erk1/2. These studies provide further evidence for divergence of insulin and IGF-I signaling pathways for human ovarian cell steroidogenesis.

Rosiglitazone and pioglitazone inhibit estrogen synthesis in human granulosa cells by interfering with androgen binding to aromatase.

The effects of rosiglitazone or pioglitazone (thiazolidinediones, TZDs) on estrogen production and aromatase activity in human ovarian cells were examined. Human granulosa cells were incubated in the tissue culture medium supplemented with androstenedione or testosterone, with or without insulin, TZDs, or type 1 17ß-hydroxysteroid-dehydrogenase (17ß-HSD) inhibitor. Estrogen concentrations in the conditioned medium, aromatase mRNA and protein expression in the cells and androgen substrate binding to aromatase were measured. With androstenedione as substrate, rosiglitazone or pioglitazone inhibited estrone production by up to 22% (p<0.012) while type 1 17ß-HSD inhibitor enhanced this effect of rosiglitazone or pioglitazone by 37% (p<0.001) and by 67% (p<0.001), respectively. With testosterone as substrate, rosiglitazone or pioglitazone inhibited estradiol production by 32% (p<0.001). With (3)H-testosterone as substrate, rosiglitazone or pioglitazone inhibited the (3)H-tritiated water release by the cultured cells by 45% and 35%, respectively, thus directly demonstrating inhibition of aromatase. Rosiglitazone or pioglitazone, however, had no significant effect on aromatase mRNA or protein expression. Rosiglitazone or pioglitazone inhibited (125)I-androstenedione and (125)I-testosterone binding to aromatase by 38% (p<0.001). It was concluded that rosiglitazone or pioglitazone inhibit estrogen synthesis in human granulosa cells by interfering with androgen binding to aromatase.

Continuous insulin infusion is associated with a reduced post-surgical length of stay, but not with the complication rate, in patients with diabetes mellitus undergoing coronary artery bypass graft.

OBJECTIVE: To establish if glucose management with continuous intravenous insulin infusion (CII) in the early post-operative period after coronary artery bypass graft (CABG) surgery is associated with complication rate and length of hospital stay (LOS) in patients with diabetes mellitus (DM). RESEARCH DESIGN AND METHODS: We reviewed the records of 587 patients with DM who underwent CABG from January 1999 until January 2008; 316 patients were placed on CII, while 271 patients were treated with subcutaneous insulin. We examined patient age, glycated hemoglobin (HgbA1c), 24- and 72-h post-operative average capillary blood glucose (CBG), length of stay (LOS), and the rate of complications. RESULTS: There was no difference in HgbA1c between the groups. Mean CBG values at both 24 h and 72 h remained the same in the CII group (167 mg/dl), while in the non-CII group they were 194 mg/dl and 189 mg/dl, respectively (p<0.001 between the groups). Post-surgical median LOS was 6 days in the CII group and 6.5 days in the non-CII group (p=0.003). Complications occurred at similar rate (in 10% and 11% of patients) in the two groups. CONCLUSIONS: CII is associated with a reduced post-surgical LOS in patients with DM who undergo CABG.

Vitamin D regulates steroidogenesis and insulin-like growth factor binding protein-1 (IGFBP-1) production in human ovarian cells.

Vitamin D Receptor (VDR) is expressed in both animal and human ovarian tissue, however, the role of vitamin D in human ovarian steroidogenesis is unknown. Cultured human ovarian cells were incubated in tissue culture medium supplemented with appropriate substrates, with or without 50 pM-150 pM or 50 nM-150 nM of 1,25-(OH)2D3, and in the presence or absence of insulin. Progesterone, testosterone, estrone, estradiol, and IGFBP-1 concentrations in conditioned tissue culture medium were measured. Vitamin D receptor was present in human ovarian cells. 1,25-(OH)2D3 stimulated progesterone production by 13% (p<0.001), estradiol production by 9% (p<0.02), and estrone production by 21% (p<0.002). Insulin and 1,25-(OH)2D3 acted synergistically to increase estradiol production by 60% (p<0.005). 1,25-(OH)2D3 alone stimulated IGFBP-1 production by 24% (p<0.001), however, in the presence of insulin, 1,25-(OH)2D3 enhanced insulin-induced inhibition of IGFBP-1 production by 13% (p<0.009). Vitamin D stimulates ovarian steroidogenesis and IGFBP-1 production in human ovarian cells likely acting via vitamin D receptor. Insulin and vitamin D synergistically stimulate estradiol production. Vitamin D also enhances inhibitory effect of insulin on IGFBP-1 production.

Vitamin D in diabetes mellitus-a new field of knowledge poised for D-velopment.

This commentary reviews the current state of knowledge regarding the role of vitamin D in the pathogenesis of diabetes mellitus. In type 1 diabetes mellitus or in adult onset latent autoimmune diabetes (LADA), vitamin D exhibits immunomodulatory actions, influencing the activity of lymphocytes and interleukins. In type 2 diabetes mellitus vitamin D appears to act through different mechanisms, affecting insulin secretion and insulin sensitivity through its effects on the beta cells, mediators of inflammation and parathyroid hormone. Much work remains to be done in this new field of knowledge before the role of vitamin D in the pathogenesis of diabetes mellitus is completely understood.

Metabolic and hormonal effects of oral DHEA in premenopausal women with HIV infection: a randomized, prospective, placebo-controlled pilot study.

Women with HIV infection use dehydroepiandrosterone (DHEA) because of its potential effects on mood and energy. We examined the effects of DHEA on the hypothalamic-pituitary-adrenal and gonadal axes and on insulin sensitivity. Fifteen HIV-positive women were randomized to receive placebo (6 subjects) or oral DHEA (9 subjects). ACTH-, CRF-, and GnRH-stimulation tests were performed before and after 8 weeks of treatment. DHEA, DHEA-S, dihydrotestosterone, total testosterone, free testosterone, sex hormone-binding globulin, estrone, estradiol, cortisol, insulin, IGF-1, IGFBP-1, IGFBP-3, and adiponectin in plasma or serum were measured. There was a significant increase in DHEA (p<0.004), DHEA-S (p<0.008), total testosterone (p<0.008), dihydrotestosterone (p<0.004), androstenedione (p<0.04), and estrone (p<0.03) from baseline within the DHEA group but not within the placebo group. There was a significant increase in DHEA (p<0.0006), DHEA-S (p<0.032), total testosterone (p<0.01), and dihydrotestosterone (p<0.005) in the DHEA group compared with the placebo group. Oral DHEA produces significant increases in circulating DHEA, DHEA-S, testosterone, DHT, and, possibly, androstenedione and estrone levels in premenopausal women with HIV infection. In the current pilot study these hormone changes did not affect the pituitary or adrenal axis or insulin/IGF indices. Long-term studies with larger groups of patients are needed to confirm these data and to determine their clinical significance.

On the paradox of insulin-induced hyperandrogenism in insulin-resistant states.

I have reviewed recent studies that shed light on the paradox of insulin-induced ovarian hyperandrogenism in insulin-resistant states. Depending on the circumstances of the particular syndrome, insulin could act on the ovaries of insulin-resistant patients by activating IGF-I receptors, insulin receptors, hybrid insulin/IGF-I receptors, or any combination of these receptors. Further studies will uncover precise mechanisms of insulin-induced ovarian hyperstimulation in specific syndromes of insulin resistance.

The gonadotropic function of insulin.

We have reviewed the role of insulin in ovarian physiology. Clinical observations and experimental data strongly support the hypothesis that insulin possesses gonadotropic activity, when acting alone or with FSH or LH. This idea is further supported by the recent discovery of insulin in follicular fluid. The idea that insulin has gonadotropic function can explain a variety of clinical observations, which otherwise are difficult to understand. For example, manifestations of ovarian hypofunction (primary amenorrhea, late menarche, anovulation, low pregnancy rate, and early menopause) in IDDM can be understood if it is accepted that insulin is necessary for the ovary to reach its full steroidogenic potential. The idea that insulin affects ovarian steroidogenesis also helps to understand the observation that hyperandrogenism frequently accompanies each of the various insulin-resistant states, regardless of the latter's etiology (e.g. genetic deficiency in the number of insulin receptors, antiinsulin receptor antibodies, obesity, etc.). The explanation for this association is based on the idea that hyperinsulinemia intensifies ovarian steroidogenesis, which manifests clinically as hyperandrogenism. Continuous stimulation of the ovary by insulin over a long period of time possibly produces morphological ovarian changes, such as hyperthecosis or polycystic changes; these changes commonly are observed among women with insulin resistance. The effects of insulin on ovarian cells are mediated possibly through binding of the peptide to its own receptor or to the IGF-1 receptor (the specificity spillover phenomenon). The latter could be an important mechanism in cases of insulin resistance. Potential mechanisms underlying the gonadotropic activity of insulin include direct effects on steroidogenic enzymes, modulation of FSH or LH receptor number, synergism with FSH or LH, or nonspecific enhancement of cell viability. The gonadotropic function of insulin adds yet another note to what has been termed a symphony of insulin action. Further investigation into this new area may yield greater insights not only into normal ovarian physiology, but also into the pathogeneses of such diverse entities as PCO, obesity, diabetes mellitus, and the syndromes of insulin resistance and acanthosis nigricans.

Absence of antibody to human immunodeficiency virus in long-term, socially rehabilitated methadone maintenance patients.

Human immunodeficiency virus (HIV) infection has become widespread among parenteral drug abusers. We measured antibody to HIV and hepatitis B virus markers in 58 long-term, socially rehabilitated methadone-maintained former heroin addicts. None of the 58 had antibody to HIV, but one or more markers of hepatitis B virus infection were seen in 53 (91%). The duration of methadone maintenance was 16.9 +/- 0.5 years, and the median dose of methadone was 60 mg (range, 5 to 100 mg). Before methadone treatment, the patients had abused heroin parenterally for 10.3 +/- 1.7 years, and they had engaged in additional high-risk practices for HIV infection. We conclude that successful outcomes during methadone maintenance treatment are associated with sparing of parenteral drug abusers from HIV infection.

Effects of an intervention by a diabetes team in hospitalized patients with diabetes.

OBJECTIVE: Hospitalized patients with diabetes have a prolonged length of stay in the hospital. We conducted a controlled prospective randomized feasibility study of the effects of a diabetes team (a diabetes nurse educator and an endocrinologist) on the length of stay and other outcomes of hospitalization in these patients. RESEARCH DESIGN AND METHODS: A total of 179 hospitalized patients with diabetes were randomly assigned to receive usual care supplemented with (85 patients) or without (94 control patients) a diabetes team intervention. Outcome measures included the length of stay, blood glucose control, and rates of readmission. RESULTS: For the primary diagnosis of diabetes, the median length of stay was 5.5 days (95% CI 4-8 days) for patients who received diabetes team intervention and 7.5 days (5-11 days) for the control patients (NS). For the secondary diagnosis of diabetes, the median length of stay was 10.0 days (8-13 days) in the intervention group and 10.5 days (8-13 days) in the control group (NS). One month after the team intervention was initiated, 75% of patients in the intervention group were in good glycemic control, compared with 46% in the control group. Readmissions at 3 months after discharge included 13 (15%) patients from the intervention group and 30 (32%) patients in the control group (P = 0.01). CONCLUSIONS: Randomized controlled prospective trials of clinical interventions in hospitalized patients with diabetes are feasible. Diabetes team intervention appears to reduce the hospital length of stay and to improve glycemic control. Team intervention significantly reduces the rate of recurrent hospitalization.

The effects of experimental hyperinsulinemia on steroid secretion, ovarian [125I]insulin binding, and ovarian [125I]insulin-like growth-factor I binding in the rat.

We randomized 32 cycling female Sprague-Dawley rats (82 days old) into experimental and control groups (16 animals/group). Hyperinsulinemia was induced and maintained for 22 days in the experimental group with NPH human insulin (Novolin, Squibb-Novo, Princeton, NJ) as previously described. Controls received an identical volume of vehicle. Fifteen minutes before death, each rat received a sc injection of 100 ng synthetic GnRH (Factrel, Ayerst Laboratories, New York, NY). The mean serum insulin level was significantly higher in the insulin-treated group than in the control group (165 +/- 57 vs. 49 +/- 9 microU/ml; P less than 0.05). The mean final weight also was significantly higher in the insulin-treated group (283 +/- 4 vs. 242 +/- 7 g; P less than 0.001). There were no significant differences in mean final serum levels of testosterone, estradiol, estrone, or androstenedione or in GnRH-stimulated serum levels of LH or FSH. The androstenedione to estrone ratio, however, was significantly lower in the insulin-treated group (2.5 +/- 0.3 vs. 3.4 +/- 0.2; P less than 0.01), suggesting that aromatase activity increased with hyperinsulinemia. Specific [125I]insulin binding to ovarian tissue homogenates was lower in the insulin-treated group (1.7 +/- 0.1% vs. 2.6 +/- 0.6%/0.2 mg protein; P greater than 0.05), suggesting that ovarian insulin receptors tended to down-regulate with hyperinsulinemia. Specific [125I]insulin-like growth factor I [( 125I]IGF-I) binding to ovarian tissue homogenates, in contrast, was significantly higher in the insulin-treated group (13.3 +/- 1.4% vs. 7.2 +/- 0.6%/0.2 mg protein; P less than 0.05), suggesting that ovarian IGF receptors up-regulated with hyperinsulinemia. The affinity of neither [125I]insulin binding nor that of [125I]IGF-I binding changed significantly, with the 50% inhibition point remaining between 2.0 and 5.0 ng/ml for each peptide in both groups. We conclude that hyperinsulinemia increases ovarian [125I]IGF-I binding and stimulates aromatase activity in the rat. These phenomena, if also true in women, could be important factors contributing to the ovarian hyperstimulation observed in various hyperinsulinemic states.

Regulation of insulin receptors in the human ovary: in vitro studies.

The association between insulin resistance and ovarian hyperstimulation has led to a hypothesis that insulin stimulates ovarian steroidogenesis. This possible effect of insulin on the ovary could be mediated by either the insulin receptor or the type I insulin-like growth factor (IGF) receptor, both of which have been described in the human ovary. We examined the in vitro regulation of insulin receptors by LH, FSH, multiplication-stimulating activity (MSA), and insulin in ovarian stromal fragments from 24 women. [125I]Insulin binding was measured in the presence and absence of increasing concentrations of insulin, IGF-I, LH, and FSH. Neither LH nor FSH competed with [125I]insulin for binding sites, but preincubation with LH or FSH reduced [125I]insulin binding by 19-38%. Preincubation with MSA reduced [125I]insulin binding by 34-48%. The affinity of the insulin receptors, determined by Scatchard analysis, did not change (Ka = 3.3 X 10(8) mol-1). A concentration of 10 ng/mL insulin in the preincubation medium reduced [125I]insulin binding by 40%, whereas an insulin concentration of 50 or 500 ng/mL completely obliterated specific [125I]insulin binding. [125I]Insulin binding fully recovered, however, 4 h after termination of tissue exposure to insulin. The specificity of [125I] insulin binding was confirmed by studies with IGF-I. We conclude that of the hormones examined, insulin is the most potent regulator of human ovarian insulin receptors in vitro. Down-regulation of insulin receptors by insulin was reversed within 4 h after withdrawal of insulin. MSA, FSH, and LH also down-regulated the number of human ovarian insulin receptors in vitro, but were less potent. These phenomena, if also present in vivo, could be important factors in the regulation of ovarian function by insulin in normal and pathological states.

Ketoconazole reverses hyperandrogenism in a patient with insulin resistance and acanthosis nigricans.

We administered ketoconazole to a young woman with ovarian hyperandrogenism, insulin resistance, and acanthosis nigricans. While taking ketoconazole, her serum testosterone, androstenedione, dehydroepiandrosterone, and cortisol levels declined, while serum progesterone, 17-hydroxyprogesterone, and 11-deoxycortisol rose. Serum LH, FSH, and estradiol levels were intermittently higher during ketoconazole treatment, although LH and FSH responsiveness to GnRH did not change. Basal and stimulated serum insulin concentrations were high before and during ketoconazole therapy, while fasting glucose levels and glucose disappearance rate constants were normal throughout the study. A dramatic improvement in hirsutism occurred, and menses resumed after a 6-yr hiatus. Adverse drug effects or clinical evidence of adrenal insufficiency were not encountered. These results support a role for ketoconazole in the therapy of ovarian hyperandrogenism.

Insulin-like growth factor II (IGF-II) inhibits insulin-like growth factor binding protein I (IGFBP-1) production in luteinized human granulosa cells with a potency similar to insulin-like growth factor I (IGF-I) and higher than insulin.

Insulin-like growth factor binding protein I (IGFBP-I) is produced by human granulosa cells and may be important in follicular development. Production of IGFBP-I in human granulosa cells is under inhibitory control of insulin and insulin-like growth factor-I (IGF-I). It had not been known if IGF-II affected IGFBP-I production in these cells. We examined the effect of IGF-II on IGFBP-I production in human granulosa cells and compared this effect of IGF-II to similar effects of insulin and IGF-I. Human granulosa cells were obtained during in vitro fertilization and plated at a density of 10(5) cells/mL in McCoy 5A tissue culture medium supplemented with 10% fetal calf serum. After 48 h of preincubation at 37 C, 90% humidity, 5% CO2, the medium was removed, and serum-free medium was added. After 24 h of incubation in the serum-free medium, the medium was removed, cells were washed, and new serum-free medium supplemented with various concentrations of insulin, IGF-I, or IGF-II was added. After 24 h of incubation with insulin, IGF-I, or IGF-II, the medium was removed and frozen at-20 C until assayed for IGFBP-I. IGFBP-I was measured using kits from Diagnostic System Laboratories, Webster, Texas. Concentrations of IGFBP-I in the medium were 4.4 +/- 1.7 ng/mL for control cells; 3.0 +/- 0.8, 3.0 +/- 0.7, 3.1 +/- 0.7, 1.8 +/- 0.5, and 1.1 +/- 0.3 ng/mL for 1, 10, 10(2), 10(3), and 10(5) ng/mL insulin respectively; 1.4 +/- 0.6, 2.1 +/- 0.5, 0.3 +/- 0.1, 0.1 +/- 0.02, and 0.01 +/- 0.02 ng/mL for 0.5, 1, 5, 10, and 10(2) ng/mL of IGF-I, respectively; and 2.3 +/- 2.2, 1.2 +/- 0.8, 0.9 +/- 0.3, 0.7 +/- 0.2, and 0.04 +/- 0.02 ng/mL for 0.5, 1, 5, 10, and 10(2) ng/mL of IGF-II, respectively. Concentration of IGFBP-I in the medium collected from cells incubated with hormones was statistically lower than that for control cells starting at 10(3) ng/mL of insulin (P < 0.05), 0.5 ng/mL of IGF-I (P < 0.05) and 1 ng/mL of IGF-II (P < 0.05). Within the range of hormone concentrations tested, the P-values (independent groups t-test) never reached less than 0.01 levels for insulin, but reached this level at 5 ng/mL for both IGF-I and IGF-II. We conclude that IGF-II inhibits IGFBP-I production in luteinized human granulosa cells in a dose-dependent manner and with potency similar to IGF-I and higher than insulin. Further studies are needed to determine the physiological significance of this and other effects of IGF-II in the human ovary in both normal and pathological states.

Specific insulin binding sites in human ovary.

The strong association between hyperinsulinemic states of insulin resistance and ovarian hyperandrogenism has led to the suggestion that insulin might directly influence the function of the ovary. To assess this possibility, we have attempted to directly measure insulin receptors in the ovarian stroma of three patients who were operated upon for polycystic ovarian disease. 125I-insulin binding was easily detectable in fragments of ovarian stroma in each case. Specific binding was totally inhibited by pre-treatment with serum containing specific anti-insulin receptor autoantibodies (B-2). These data are consistent with the hypothesis that insulin can directly influence ovarian function. Further studies of insulin receptors and insulin action in human ovarian tissue could lead to a better understanding of the link between insulin resistant states and ovarian hyperfunction.

Pneumocystis carinii infection of the thyroid in a hypothyroid patient with AIDS: diagnosis by fine needle aspiration biopsy.

A 49-yr-old homosexual man with acquired immunodeficiency syndrome presented with a left-sided neck mass. He was found to have a firm goiter. He was clinically euthyroid, but had laboratory evidence of primary hypothyroidism. Radioactive iodine scan of the thyroid showed homogeneous uptake over an enlarged right lobe and absence of uptake over the left lobe. Two fine needle aspiration biopsies of the thyroid revealed the presence of Pneumocystis carinii (P. carinii) organisms on the Gomori's methenamine silver strain. After courses of iv and oral therapy with trimethoprim-sulfamethoxazole, a third fine needle aspiration biopsy failed to reveal any organisms. A repeated radioactive iodine scan of the thyroid showed return of uptake over the left lobe. Thyroid function tests normalized with levothyroxine, and the goiter decreased in size. To our knowledge, this is the first report of hypothyroidism associated with P. carinii infection of the thyroid. P. carinii infection should be considered in the differential diagnosis of human immunodeficiency virus infected individuals presenting with cold thyroid nodules. Fine needle aspiration biopsy is a valuable tool in assessing these patients.

Insulin receptor mediates inhibitory effect of insulin, but not of insulin-like growth factor (IGF)-I, on IGF binding protein 1 (IGFBP-1) production in human granulosa cells.

Insulin-like growth factor binding proteins (IGFBPs) may participate in regulating ovarian function by modifying effects of insulin-like growth factors (IGFs) or by directly affecting ovarian steroidogenesis in both normal and pathological circumstances. The latter include hyperinsulinemic insulin resistant states, such as polycystic ovary syndrome. We examined regulation of IGFBP-1 production in human granulosa cells by insulin and IGF-I. The cells were obtained during in vitro fertilization, plated in McCoy-5A tissue culture medium supplemented with 10% fetal calf serum (10(5) cells/0.5 mL), and incubated at 37 C, 90% humidity, 5% CO2 for 48 h. After additional 24 h incubation without fetal calf serum, 1, 10, or 100 ng/mL of insulin or IGF-I were added with or without 2 h preincubation with 10 micrograms/mL monoclonal anti insulin receptor antibody IR-47-9. After 48 h incubation with insulin or IGF-I, the medium was collected and IGFBP-1 and progesterone concentrations were measured, using kits from Diagnostic Systems Laboratories, Webster, TX. Progesterone concentration ranged between 50-100 ng/mL/10(5) cells, without consistent stimulatory effect of either insulin or IGF-I. Control cells produced 7.0 +/- 1.7 ng/mL of IGFBP-1. Incubation with 1 or 10 ng/mL of insulin resulted in culture medium IGFBP-1 concentrations of 7.1 +/- 1.3 ng/mL and 5.4 +/- 0.7 ng/mL, respectively (P = NS). Incubation with 100 ng/mL of insulin reduced IGFBP-1 culture medium concentration to 1.6 +/- 0.3 ng/mL (P < 0.01, compared with controls). 1, 10, and 100 ng/mL of IGF-I inhibited IGFBP-1 concentrations in the conditioned culture medium to 1.3 +/- 0.3 ng/mL, 0.4 +/- 0.1 ng/mL and 0.3 +/- 0.1 ng/mL, respectively (P < 0.01, compared with controls). Preincubation with antiinsulin receptor antibody IR-47-9 alleviated inhibitory effect of insulin, but not of IGF-I on IGFBP-1 production. After preincubation with IR-47-9, IGFBP-1 culture medium concentrations were 5.9 +/- 0.8 ng/mL, 4.9 +/- 1.2 ng/mL, and 4.8 +/- 1.3 ng/mL for 1, 10, and 100 ng/mL of insulin, respectively. The latter number was significantly higher than IGFBP-1 concentration in the medium collected from cells incubated with 100 ng/mL of insulin without IR-47-9 (1.6 +/- 0.3 ng/mL, P < 0.01) and not significantly different from the control cells. For cells preincubated with IR-47-9 and then incubated with 1, 10, or 100 ng/mL of IGF-I, the IGFBP-1 conditioned culture medium concentrations were 1.7 +/- 0.1 ng/mL, 0.5 +/- 0.2 ng/mL, and 0.3 +/- 0.1 ng/mL, respectively. None of these were significantly different from the IGFBP-1 concentrations in the medium collected from cells incubated with the respective concentrations of IGF-I without preincubation with IR-47-9. We conclude that 1) both insulin and IGF-I inhibit IGFBP-1 production by cultured human granulosa cells; 2) IGF-I is a more potent inhibitor of IGFBP-1 production than insulin; 3) in the range of hormone concentrations tested, insulin exerts its inhibitory effect on IGFBP-1 production via insulin receptor, while IGF-I appears to exert its effect via another receptor.

Distribution and characterization of insulin and insulin-like growth factor I receptors in normal human ovary.

Insulin-resistant hyperinsulinemic states are now widely known to be associated with ovarian hyperandrogenism, and this is thought to be due to an action of insulin on the ovary. However, the identity of the receptor that is responsible for insulin action in these patients, whose insulin receptors on classical target tissues are severely impaired, is unclear. We now report the presence of insulin receptors in stromal and follicular compartments as well as in granulosa cells obtained from normal ovaries. After 15-h incubations at 4 C with [125I]insulin and tissue fragments, specific insulin binding was 6-19% and 7-13%/mg protein (n = 8) to stroma and theca, respectively. Granulosa cells obtained in the course of in vitro fertilization were separated from red cells on a Percoll gradient; specific insulin binding ranged from 9-15%/10(6) cells. Insulin binding was characterized by sensitive insulin competition (half-maximal, 10 ng/ml), appropriately shifted proinsulin competition (20 times to the right), and complete inhibition by specific anti-insulin receptor antibodies (B-2). An antibody to the insulin-like growth factor I (IGF-I) receptor (alpha IR-3) that inhibits IGF-I binding to IGF-I receptors in other cell systems had no effect on insulin binding. Further proof that this binding is to classic insulin receptors was obtained from measurement of insulin-stimulated receptor autophosphorylation. When insulin receptors from stroma were extracted with Triton X-100 and incubated with [gamma-32P]ATP and Mn, insulin increased the incorporation of 32P into the beta-subunit of the receptor 5-fold. In parallel studies with [125I-]IGF-I and specific blocking antibodies to its receptor, no detectable IGF-I binding to stroma or follicles was found. We conclude that specific high affinity insulin receptors possessing tyrosine kinase activity are widely distributed in normal human ovary. IGF-I receptors in normal ovary are either absent or present at very low density. Binding of insulin to its own receptor (as opposed to IGF-I receptors) appears to be the most likely first step in the stimulation of ovarian steroidogenesis by insulin in normal ovaries and possibly in insulin-resistant states as well.