Proteasome inhibitors, including curcumin, improve pancreatic ß-cell function and insulin sensitivity in diabetic mice.

BACKGROUND: Type 2 diabetes stems from obesity-associated insulin resistance, and in the genetically susceptible, concomitant pancreatic ß-cell failure can occur, which further exacerbates hyperglycemia. Recent work by our group and others has shown that the natural polyphenol curcumin attenuates the development of insulin resistance and hyperglycemia in mouse models of hyperinsulinemic or compensated type 2 diabetes. Although several potential downstream molecular targets of curcumin exist, it is now recognized to be a direct inhibitor of proteasome activity. We now show that curcumin also prevents ß-cell failure in a mouse model of uncompensated obesity-related insulin resistance (Lepr(db/db) on the Kaliss background). RESULTS: In this instance, dietary supplementation with curcumin prevented hyperglycemia, increased insulin production and lean body mass, and prolonged lifespan. In addition, we show that short-term in vivo treatment with low dosages of two molecularly distinct proteasome inhibitors celastrol and epoxomicin reverse hyperglycemia in mice with ß-cell failure by increasing insulin production and insulin sensitivity. CONCLUSIONS: These studies suggest that proteasome inhibitors may prove useful for patients with diabetes by improving both ß-cell function and relieving insulin resistance.

Obesity in C57BL/6J mice is characterized by adipose tissue hypoxia and cytotoxic T-cell infiltration.

BACKGROUND: Obesity is currently viewed as a state of chronic low-grade inflammation in which there is a pro-inflammatory alteration in the serum adipocytokine profile as well as an infiltration of white adipose tissue by activated macrophages. The etiology of this inflammation, however, is poorly understood. METHODS: Hypothesizing that local hypoxia within expanding white adipose tissue depots may contribute to obesity-related inflammation, we compared body composition, serum inflammatory marker concentrations and the expression of several hypoxia-regulated genes in white adipose tissue derived from lean, dietary-induced obese (DIO) and ob/ob male C57BL/6J mice. We also examined white adipose tissue for the presence of hypoxia using both a pimonidazole-based antibody system and a fiberoptic sensor for real-time pO(2) quantification in vivo. Finally, using cell-specific leukocyte antibodies, we performed immunohistochemistry and flow cytometric analyses to further characterize the cellular nature of adipose inflammation. RESULTS: We determined that obesity in male C57BL/6J mice is associated with increased expression of HIF (hypoxia-inducible factor) isoforms and GLUT-1, and that white adipose tissue hypoxia was present in the obese mice. Immunohistochemistry revealed hypoxic areas to colocalize predominantly with F4/80+ macrophages. Interestingly, CD3+ T cells were present in large numbers within the adipose of both DIO and ob/ob obese mice, and flow cytometry revealed their adipose to possess significantly more CD8+ T cells than their lean cohort. CONCLUSIONS: White adipose hypoxia and cytotoxic T-cell invasion are features of obesity in C57BL/6J mice and are potential contributors to their local and generalized inflammatory state.

Increased expression of hypothalamic leptin receptor and adiponectin accompany resistance to dietary-induced obesity and infertility in female C57BL/6J mice.

BACKGROUND: Obesity is strongly associated with female infertility, but the mechanisms underlying this relationship are largely unknown. METHODS: We investigated the effect of increasing dietary fat percentage upon body mass, hypothalamic neuropeptide gene expression, adipose hormone secretion and fertility in females of the inbred mouse strains C57BL/6J and DBA/2J. To assess the effect of obesity independent of dietary influence, we also compared these parameters in wild-type female C57BL/6J mice to those congenic for the obesogenic mutations ob/ob and A(y)/a. RESULTS: After 24 weeks, rather than exhibiting an obese, leptin-resistant phenotype like their female DBA/2J counterparts, wild-type female C57BL/6J mice remained lean, fertile and manifested increased hypothalamic LEPR-B expression. Although both mutant genotypes were associated with obesity and subfertility, ob/ob mice demonstrated significantly increased hypothalamic LEPR-B expression, whereas A(y)/a mice had a significant reduction. Interestingly, wild-type female C57BL/6J mice were noted to manifest significantly higher and lower levels of adiponectin and tissue plasminogen activator inhibitor-1 (tPAI-1), respectively, than weight-matched wild-type female DBA/2J mice. CONCLUSIONS: We conclude that (1) resistance to the obese-infertile phenotype in female C57BL/6J mice is associated with increased hypothalamic leptin receptor expression and alterations in adipokine levels consistent with decreased adipose tissue inflammation and (2) that long-standing hyperleptinemic obesity in mice is associated with a downregulation of the hypothalamic leptin receptor.

Relaxin positively regulates matrix metalloproteinase expression in human lower uterine segment fibroblasts using a tyrosine kinase signaling pathway.

Despite the importance of relaxin to normal parturition in various species and its potential as an etiological agent in preterm delivery in women, knowledge regarding the mechanisms by which relaxin alters cervical connective tissue is extremely limited. An established in vitro model for human pregnancy cervix, human lower uterine segment fibroblasts, was used to determine the effects of relaxin as well as those of progesterone on the expression of matrix metalloproteinases and tissue inhibitor of metalloproteinase-1. The results demonstrate that relaxin is a positive regulator of matrix metalloproteinase expression, as it stimulates the expression of procollagenase protein and mRNA levels, stimulates prostromelysin-1 protein and mRNA levels, and inhibits tissue inhibitor of metalloproteinase-1 protein expression. Stimulation of procollagenase and prostromelysin-1 expression by relaxin does not involve phorbol-12-myristate-13-acetate- sensitive PKCs. Relaxin-stimulated tyrosine phosphorylation of the putative receptor and inhibition by a receptor tyrosine kinase inhibitor suggest that the relaxin receptor is probably a tyrosine kinase receptor. Inhibition of c-Raf protein expression using an antisense oligonucleotide inhibits relaxin regulation of matrix metalloproteinase and tissue inhibitor of metalloproteinase-1, suggesting that a signaling pathway involving c-Raf kinase mediates relaxin action.

Human follistatin-related protein: a structural homologue of follistatin with nuclear localization.

Follistatin-related protein is a recently discovered glycoprotein that is highly homologous in both primary sequence and exon/intron domain structure to the activin-binding protein, follistatin. We explored their potential for functional redundancy by investigating the relative affinities and kinetics of their interactions with activin, bone morphogenic protein-6, and bone morphogenic protein-7 and by exploring their expression and distribution in human tissues and cells. Follistatin and follistatin-related protein mRNA were ubiquitous by Northern analyses, although their sites of peak distribution differed, with follistatin-related protein and follistatin predominating in the placenta and ovary, respectively. Follistatin-related protein, like follistatin, preferentially bound activin with high affinity and in an essentially irreversible fashion. Although follistatin-related protein, like follistatin, possesses a signal sequence and no known nuclear localization signals, its secretion was undetectable in most cell lines by RIA. Intriguingly, follistatin-related protein was identified as a nuclear protein in human granulosa cells and all human cell lines tested. Furthermore, Western analyses of CHO cells transfected with human follistatin-related protein revealed this protein to reside within the insoluble nuclear protein fraction. We conclude that despite its remarkably high level of similarity to follistatin with regard to structure and activin binding kinetics, follistatin-related protein is a nuclear as well as a secretory protein that may perform distinct intracellular actions.

Follistatin-related protein (FSRP): a new member of the follistatin gene family.

The identification and characterization of follistatin related protein (FSRP) suggests that the follistatin (FS) gene family may actually contain two sub-families. The first includes FS and FSRP by virtue of their high degree of structural homology and comparable activin-binding activity, while the second sub-family contains extracellular matrix proteins that possess one or more 10-cysteine FS domains, but do not bind activin or related TGF-beta family members. Characterization of FSRP indicates that it binds activin with similar affinity and selectivity as FS, but does not bind heparin. Furthermore, although FSRP inhibits activin-mediated gene transcription in heterologous assays, FSRP is much less active than FS in the rat pituitary bioassay. When overexpressed in transgenic mice, FSRP may lead to interruption of follicular development and fertility in females but appears to have only a modest effect on males. These results suggest that FSRP is a structural, but not necessarily a functional homologue of FS.

Critical ovarian hyperstimulation syndrome in a coasted in-vitro fertilization patient.

We report an instance of critical ovarian hyperstimulation syndrome in a highly responsive in-vitro fertilization patient despite the preventive measure of a 4 day 'coast' interval during which no gonadotrophins were administered while gonadotrophin-releasing hormone agonist therapy continued until serum oestradiol concentrations fell below 3000 pg/ml.

Effects of aging and gonadal failure on the hypothalamic-pituitary axis in women.

OBJECTIVE: Our aim was to determine the effect of aging on the hypothalamic-pituitary-gonadal axis function. STUDY DESIGN: We studied 9 women aged 25 to 40 years with well-defined idiopathic premature ovarian failure and compared them with 8 women aged 51 to 70 years who had age-appropriate menopause. All women underwent 24 hours of frequent blood sampling every 10 minutes before and after replacement with transdermal estradiol targeted to achieve serum concentrations of approximately 100 pg/ml. RESULTS: In the absence of estrogen exposure, women with premature ovarian failure demonstrated a greater 24-hour mean luteinizing hormone concentration compared with that in the older women with age-appropriate menopause (32.3+/-4.3 mlU/ml vs 19.2+/-2.4 mlU/ml, p=0.0001). Despite the lesser luteinizing hormone serum levels in the older group, the luteinizing hormone pulse frequency per 24 hours was similar (22.1+/-3.0 pulses per 24 hours in prematurely menopausal women vs 21.9+/-2.5 pulses per 24 hours in the older postmenopausal women, p=0.94). When exposed to estrogen, mean luteinizing hormone concentrations decreased to 11.6+/-2.7 mlU/ml in prematurely menopausal women versus 4.4+/-1.0 mlU/ml in older postmenopausal women, p=0.017. Both groups had suppressed mean luteinizing hormone secretion compared with their paired, non-estradiol-exposed studies, p=0.0001. Frequency of luteinizing hormone pulsations was reduced to 16.5+/-3.5 pulses per 24 hours in prematurely menopausal women exposed to estradiol (p < 0.0058, compared with non-estradiol-exposed women). Further reduction was observed in older postmenopausal women (11.5+/-1.1 pulses per 24 hours, p=0.0001, compared with nonestradiol exposure, and p=0.0125, vs prematurely menopausal, estradiol-exposed women). Pulse amplitude was suppressed in both prematurely menopausal women (5.6+/-0.5 mlU/ml to 2.3+/-0.5 mlU/ml, p=0.0001) and older postmenopausal women (3.6+/-0.4 mlU/ml to 2.3+/-0.6 mlU/ml p=0.04) in the presence of estradiol. Although luteinizing hormone pulse amplitudes were greater in the women with premature menopause in the absence of estradiol (p=0.0028) compared with those in older postmenopausal women, pulse amplitudes became similar in the presence of estradiol. Parallel changes in mean follicle-stimulating hormone were observed. Women with premature ovarian failure had a mean follicle-stimulating hormone level of 71.1+/-9.4 mlU/ml that was suppressed to 18.0+/-4.1 mlU/ml after estradiol exposure (p=0.0001); values in older postmenopausal women were 45.9+/-6.0 and 10.3+/-2.0, respectively (p=0.0001). Although the women with premature ovarian failure secreted more follicle-stimulating hormone in the absence and presence of estradiol, only the former situation was statistically significant (p=0.0008 and p=0.23, respectively). CONCLUSIONS: These data suggest that there is an age-related decrease in gonadotropin secretion that may be hypothalamic or pituitary in origin. There is less luteinizing hormone secreted in women older than age 50. There is greater suppression of luteinizing hormone and follicle-stimulating hormone secretion by estradiol in aged women. Thus these data indicate that postmenopausal hormone changes involve central hypothalamic-pituitary alterations, as well as ovarian changes.

"Coasting" does not adversely affect cycle outcome in a subset of highly responsive in vitro fertilization patients.

OBJECTIVE: To study the effect of postponing hCG administration while continuing daily GnRH agonist therapy ("coasting") on highly responsive patients undergoing IVF-ET. DESIGN: Retrospective analysis. SETTING: University-affiliated Center for Fertility and Reproductive Medicine. PATIENT(S): Patients undergoing IVF-ET from March 1995 to March 1997. INTERVENTION(S): Three groups of IVF-ET patients were compared to explore the effect of coasting on cycle outcome: a group of highly responsive coasted patients, a group of equally responsive noncoasted patients, and an age-matched normally responsive control group. Two groups of coasted patients were also compared to assess the effect of E2 levels at the time that they met the follicular criteria for hCG administration. Last, the effect of varying coast duration was examined by regression analysis. MAIN OUTCOME MEASURE(S): Patient characteristics, outcome parameters, and incidence of ovarian hyperstimulation syndrome (OHSS). RESULT(S): Coasting had no detrimental effect on cycle outcome in the subset studied. Regression analysis, however, suggests an inverse relationship between coast duration and the number of mature oocytes retrieved as well as the clinical pregnancy rate. CONCLUSION(S): Coasting in the studied subset of IVF patients did not adversely affect cycle outcome parameters or the incidence of OHSS, but prolonged coasting intervals may impair IVF cycle outcome.

Delay of gonadotropin stimulation in patients receiving gonadotropin-releasing hormone agonist (GnRH-a) therapy permits increased clinic efficiency and may enhance in vitro fertilization (IVF) pregnancy rates.

OBJECTIVE: To promote an even temporal distribution of patients starting IVF cycles at our center, patients undergoing GnRH agonist (GnRH-a) suppression frequently delay the start of gonadotropin stimulation. Our objective was to analyze the effect that the delay of initiation of gonadotropin stimulation has on outcome parameters in this population. DESIGN: Retrospective analysis. SETTING: A tertiary referral reproductive medicine unit. PATIENT(S): Patients undergoing IVF cycles on long GnRH-a protocols. INTERVENTION(S): Patients were treated with either a "standard-dose" or "low-dose" leuprolide acetate protocol initiated in the mid-luteal phase. MAIN OUTCOME MEASURE(S): Delay time, clinical pregnancy rate, ongoing pregnancy rate, cancellation rate. RESULT(S): Analysis of the overall group revealed associations between stimulation delay and decreases in stimulation duration and the number of gonadotropin ampules administered. Weighted linear regression analyzes revealed statistically positive relationships between delay time and both clinical pregnancy rates and ongoing pregnancy rates, despite a positive relationship between delay time and cancellation rates. Analysis of the standard-dose and low-dose subgroups revealed that the enhancement of pregnancy rates was attributable primarily to patients in the standard-dose protocol. CONCLUSION(S): Delay of gonadotropin stimulation while patients are receiving GnRH-a therapy allows for increased clinic efficiency. There appears to be an enhancement of clinical and ongoing pregnancy rates for the standard-dose leuprolide acetate protocol that is associated with stimulation delay.