Metformin modifies the exercise training effects on risk factors for cardiovascular disease in impaired glucose tolerant adults

Impaired glucose tolerant (IGT) adults are at elevated risk for cardiovascular disease (CVD). Exercise or metformin reduce CVD risk, but the efficacy of combining treatments is unclear.

Objective:

To determine the effects of exercise training plus metformin (EM), compared with each treatment alone, on CVD risk factors in IGT adults.

Design and Methods:

Subjects were assigned to placebo (P), metformin (M), exercise training plus placebo (EP), or EM (8/group). In a double‐blind design, P or 2,000 mg/d of M were administered for 12 weeks and half performed aerobic and resistance training 3 days/week for ～60 min/day at 70% pretraining heart rate peak. Outcomes included adiposity, blood pressure (BP), lipids, and high sensitivity C‐reactive protein (hs‐CRP). Z‐scores were calculated to determine metabolic syndrome severity.

Results:

M and EM, but not EP, decreased body weight compared with P (P < 0.05). M and EP lowered systolic blood pressure by 6% (P < 0.05), diastolic blood pressure by 6% (P < 0.05), and hs‐CRP by 20% (M: trend P = 0.06; EP: P < 0.05) compared with P. Treatments raised high‐density lipoprotein cholesterol (P < 0.05; EM: trend P = 0.06) compared with P and lowered triacyglycerol (P < 0.05) and metabolic syndrome Z‐score compared with baseline (EP; trend P = 0.07 and EM or M; P < 0.05).

Conclusions:

Although exercise and/or metformin improve some CVD risk factors, only training or metformin alone lowered hs‐CRP and BP. Thus, metformin may attenuate the effects of training on some CVD risk factors and metabolic syndrome severity in IGT adults.     

Metabolic Characterization of a Mouse Deficient in All Known Leptin Receptor Isoforms

We have characterized a newly generated mouse model of obesity, a mouse strain deficient in all five previously described leptin receptor isoforms. These transgenic mice, named the db ₃₃₃/db ₃₃₃ mice, were identified from an ENU mutagenesis screen and carry a point mutation in the seventh exon of the db gene encoding the leptin receptor, resulting in a premature stop codon (Y₃₃₃Stop) and gene product that lacks STAT signaling domains. db ₃₃₃/db ₃₃₃ mice have a morbidly obese phenotype, with body weights diverging from wild type as early as 4 weeks of age (P < 0.05). To determine the contribution of the short isoforms of the leptin receptor in this metabolic phenotype, we performed an extensive metabolic characterization of the db ₃₃₃/db ₃₃₃ mouse in relation to the well-characterized db/db mouse lacking only the long form of the leptin receptor. db ₃₃₃/db ₃₃₃ mice have similar endocrine and metabolic parameters as previously described in other leptin receptor transgenic mice including db/db mice that lack only the long isoform of the leptin receptor. However, db ₃₃₃/db ₃₃₃ mice show a subtle trend toward higher body weight and insulin levels, lower oxygen, carbon dioxide production, respiratory exchange ratio (RER), and temperature than db/db mice suggesting the short isoforms may play an additional role in energy homeostasis.     

Excretion and metabolic fate of radiolabeled estradiol and testosterone in the cockatiel (Nymphicus hollandicus)

The metabolism and time courses for clearance of radiolabeled estradiol and testosterone were studied in the female cockatiel (Nymphicus hollandicus) using a simple technique of solubilizing dried fecal/urine matter in an aqueous solution. Carbon ¹⁴ radiolabeled estradiol and testosterone were injected intramuscularly and the urine and fecal matter collected for the pursuant 168 hr. The predominant radiolabel peak was found associated with the aqueous residue of the ether extracted aliquot for both hormones. High-performance liquid chromatographic (HPLC) separation of solubilized fecal/urine material collected during the first sampling interval (0–4 hr after injection) demonstrated that the majority of the excreted radiolabel was in the form of conjugated steroid metabolites in both the estradiol and testosterone injected birds. Subsequent hydrolysis of the aqueous residue of the ether extracted aliquots and HPLC characterized the estrogen and testosterone metabolites as being estrone/estradiol and a variety of androgen based moieties, respectively. By 24 hr postinjection, 79.4 and 79.1% of the original radiolabel was recovered in birds injected with estradiol and testosterone, respectively. These findings demonstrate that steroid hormone excretion in the cockatiel is a rapid and efficient process that is 79% complete by 24 hr and that the primary excretion products are conjugated metabolites. This study also supports the concept that fecal/urine collection is a practical and efficient method of monitoring sex steroid excretion and provides additional evidence that simple solubilization of fecal matter is a sufficient and efficient method for processing feces for subsequent metabolite measurements. Zoo Biol 16:505–518, 1997. © 1997 Wiley-Liss, Inc.     

Deletion of insulin receptor in the proximal tubule and fasting augment albumin excretion

The contribution of proximal tubules (PT) to albumin uptake is now well recognized, however, its regulation is understudied area. There are reports suggesting that insulin resistance is associated with the development of albuminuria in nondiabetic individuals. We have previously reported reduced insulin receptor (IR) expression in renal-tubular-epithelial cells, including PT in various models of insulin resistance. However, the effect of a physiological fall in insulin levels and the role for IR in PT in tubular albumin uptake is not clear. To address these gaps in our understanding, we estimated urine excretion and renal uptake of albumin in fasted and fed C57Bl/6 mice injected with fluorescein isothiocyanate (FITC)-albumin (5 µg/mL/kg body weight, intraperitoneal, n = 6 per group). In addition, we compared spot urine analysis from 33 clinically healthy humans after overnight fasting (when insulin levels are lower than in the fed state) and then at 2 hours after 75 g oral glucose challenge (postprandial). Fasted mice had attenuated renal uptake of FITC-albumin and higher excretion in urine, relative to fed mice ( P = 0.04). Moreover, a significant drop in urine albumin-to-creatinine ratio (ACR) and urine albumin concentration (UAC) was observed in the postprandial state in these subjects ( P = 0.001 and P = 0.017, for ACR and UAC, respectively). The drop was negatively associated with postprandial blood glucose levels (ρ = −0.36, P = 0.03 for ΔUAC and ρ = −0.34, P = 0.05 for ΔACR). To test the role of IR in PT, we analyzed 24-hour urine albumin excretion in male mice with targeted deletion of IR from PT (insulin receptor knockout [IRKO]) and their wild-type (WT) littermates ( n = 7 per group). IRKO mice had significantly higher 24-hour urine albumin excretion relative to WT. Moreover, kidneys from KO mice revealed reduced expression of megalin and cubulin proteins in the PT relative to the WT. We also demonstrated insulin (100 nM) induced albumin internalization in human proximal tubule cells (hPT) and this effect of insulin was attenuated in hydroxy-2-naphthalenylmethylphosphonic acid (100 µM), a tyrosine kinase inhibitor, pretreated hPT. Our findings revealed albumin excretion was attenuated by glucose administration to fasting individuals implying a regulatory role for insulin in PT albumin reabsorption. Thus albuminuria associated with insulin resistance/diabetes may relate not only to glomerular dysfunction but also to impairment in insulin-mediated reabsorption.     

Anuran gender identification by fecal steroid analysis

This study tested the hypothesis that steroid hormone metabolites can be measured in anuran feces and their concentrations used to identify the sex of adults. Fecal samples from American toads, Bufo americanus, and boreal toads, B. boreas boreas, were extracted using ethyl acetate, and the concentrations of estradiol, progesterone and testosterone metabolites were measured by enzyme immunoassays with antibodies commonly used to evaluate steroid hormone concentrations in mammalian species. In American toads, mean testosterone metabolite concentrations (P<0.05) between males (224.3±15.5 ng/g feces) and females (80.7±10.6 ng/g), but estradiol and progesterone metabolite concentrations did not. In contrast, estradiol immunoreactivity differed (P<0.05) between male (19.0±1.8 ng/g) and female (48.3±6.3 ng/g) boreal toads. Progesterone and testosterone metabolite concentrations did not differ. Fecal hormone metabolite analysis offers a promising noninvasive approach to gender identification in anuran amphibians. However, the group of metabolites differentiating gender may not be consistent among species. Zoo Biol 0:1–12, 2005. © 2005 Wiley‐Liss, Inc.     

Acute and chronic exposure of chick embryo to ethanol alters brain neurosteroid levels

Neurosteroids are modulators of neuronal function that may play important role in brain maturation. The aim of the present investigation was to study the effect of prenatal exposure to acute and chronic ethanol on brain progesterone, estradiol, and testosterone concentration on 10th and 15th days following egg incubation. Eggs were exposed to ethanol at 10 % in chronic treatment and 70 % in acute treatment. Progesterone, estradiol, and testosterone were assayed by radioimmunoassay method. It was shown that brain progesterone level was significantly decreased (P?<?0.05) in chronic ethanol group on embryonic day 10, but it was significantly decreased (P?<?0.05) in acute and chronic groups on embryonic day 15. Brain estradiol level was significantly increased (P?<?0.05) in chronic ethanol group on embryonic day 10, and it was decreased (P?<?0.05) in acute and chronic groups of ethanol on embryonic day 15. Brain testosterone was significantly increased (P?<?0.05) in acute and chronic ethanol-exposed groups on embryonic days 10 and 15. Our observations suggest that ethanol may modulate neurosteroid synthesis in the brain.     

Sex differences in aging,life span and spontaneous tumorigenesis in 129/Sv mice neonatally exposed to metformin

The perinatal (prenatal and early neonatal) period is a critical stage for hypothalamic programming of sexual differentiation as well as for the development of energy and metabolic homeostasis. We hypothesized that neonatal treatment with antidiabetic drug biguanide metformin would positively modify regulation of growth hormone – IGF-1 – insulin signaling pathway slowing down aging and improving cancer preventive patterns in rodents. To test this hypothesis male and female 129/Sv mice were s.c. injected with metformin (100 mg/kg) at the 3rd, 5th and 7th days after birth. Metformin-treated males consumed less food and water and their body weight was decreased as compared with control mice practically over their entire lifespan. There were no significant differences in age-related dynamics of food and water consumption in females and they were heavier than controls. The fraction of mice with regular estrous cycles decreased with age and demonstrated a tendency to decrease in the females neonatally treated with metformin. Neonatal exposure to metformin practically failed to change the extent of hormonal and metabolic parameters in blood serum of male and female mice. In males, neonatal metformin treatment significantly increased the mean life span (+20%, P < 0.05) and slightly increased the maximum life span (+3.5%). In females, the mean life span and median in metformin-treated groups were slightly decreased (?9.1% and ?13.8% respectively, P > 0.05) in comparison to controls, whereas mean life span of last 10% survivors and maximum life span were the same as in controls. Almost half (45%) of control male mice and 71.8% male mice neonatally exposed to metformin survived up to 800 d of age, the same age was achieved by 54.3% of mice in control female group and 30% of metformin-treated females (P < 0.03). Thus, neonatal metformin exposure slows down aging and prolongs lifespan in male but not in female mice.     

Induction of sexual behavior in ovariectomized rhesus females with 19-hydroxytestosterone

Eight sexually experienced long-term ovariectomized female rhesus monkeys were given tests of sexual behavior following treatment with 19-hydroxytestosterone (19-OH-T, 1 mg/day for 13 days), and their performance was compared with that following treatment with estradiol benzoate (EB, 10 μg/day for 13 days). Each female was tested for 10 min with each of nine adult males. Blood samples were taken on the last day of treatment with EB, at the end of the intervening 3-month period of no treatment, and on the last day of treatment with 19-OH-T. Blood levels of testosterone and estradiol were quantified by radioimmunoassay. Mean rate of presenting at a distance (proceptive behavior) was significantly higher (P < 0.05) when they were treated with 19-OH-T, but the ratio of presents to male contacts (receptive behavior) was significantly higher (P <0.05) when they were treated with EB. All other components of female sexual behavior were the same. Males displayed fewer annoyance responses (rejecting jerk, P < 0.05) when the females were treated with 19-OH-T than when they were treated with EB. All other male responses occurred with the same frequencies under the two female treatment conditions. Injection of 19-OH-T and EB both resulted in plasma testosterone and estradiol levels higher than those found in the untreated condition. Testosterone levels did not differ under the two treatments (P > 0.05), but estradiol levels were higher under EB treatment than under 19-OH-T (P < 0.05). This study suggests that both testosterone and estradiol are essential for maximum sexual performance and that various components of sexual behavior may be differentially influenced by the ratio of testosterone to estradiol in plasma.     

Dietary rapamycin supplementation reverses age‐related vascular dysfunction and oxidative stress,while modulating nutrient‐sensing,cell cycle,and senescence pathways

Inhibition of mammalian target of rapamycin, mTOR, extends lifespan and reduces age‐related disease. It is not known what role mTOR plays in the arterial aging phenotype or if mTOR inhibition by dietary rapamycin ameliorates age‐related arterial dysfunction. To explore this, young (3.8 ± 0.6 months) and old (30.3 ± 0.2 months) male B6D2F1 mice were fed a rapamycin supplemented or control diet for 6–8 weeks. Although there were few other notable changes in animal characteristics after rapamycin treatment, we found that glucose tolerance improved in old mice, but was impaired in young mice, after rapamycin supplementation (both P < 0.05). Aging increased mTOR activation in arteries evidenced by elevated S6K phosphorylation (P < 0.01), and this was reversed after rapamycin treatment in old mice (P < 0.05). Aging was also associated with impaired endothelium‐dependent dilation (EDD) in the carotid artery (P < 0.05). Rapamycin improved EDD in old mice (P < 0.05). Superoxide production and NADPH oxidase expression were higher in arteries from old compared to young mice (P < 0.05), and rapamycin normalized these (P < 0.05) to levels not different from young mice. Scavenging superoxide improved carotid artery EDD in untreated (P < 0.05), but not rapamycin‐treated, old mice. While aging increased large artery stiffness evidenced by increased aortic pulse‐wave velocity (PWV) (P < 0.01), rapamycin treatment reduced aortic PWV (P < 0.05) and collagen content (P < 0.05) in old mice. Aortic adenosine monophosphate‐activated protein kinase (AMPK) phosphorylation and expression of the cell cycle‐related proteins PTEN and p27kip were increased with rapamycin treatment in old mice (all P < 0.05). Lastly, aging resulted in augmentation of the arterial senescence marker, p19 (P < 0.05), and this was ameliorated by rapamycin treatment (P < 0.05). These results demonstrate beneficial effects of rapamycin treatment on arterial function in old mice and suggest these improvements are associated with reduced oxidative stress, AMPK activation and increased expression of proteins involved in the control of the cell cycle.     

Steroid metabolism and validation of noninvasive endocrine monitoring in the African wild dog (Lycaon pictus)

The purpose of this study was to validate noninvasive endocrine monitoring techniques for African wild dogs (Lycaon pictus) and to establish physiological validity of these methods by evaluating longitudinal reproductive-endocrine profiles in captive individuals. To determine the primary excretory by-products of ovarian steroid metabolism, [¹⁴C]-progesterone and [³H]-estradiol were co-administered to a female and all excreta were collected for 80 hr postinjection. Radiolabel excretion peaked ≤ 18 hr postinfusion, and progesterone and estradiol metabolites were excreted in almost equivalent proportions in urine (39.7 and 41.1%, respectively) and feces (60.3 and 58.9%, respectively). Most of the urinary metabolites were conjugated (estradiol, 94.3 ± 0.3%; progesterone, 90.4 ± 0.5%), so that immunoassays for pregnanediol-3α-glucuronide (PdG) and estrogen conjugates (EC) were effective for assessing steroid metabolites. Two immunoreactive estrogens (estradiol and estrone) and at least one immunoreactive progesterone metabolite (3α-hydroxy-5α, pregnan-20-one) were detected in feces. Urine and fecal samples were collected (1–3 times per week) for 1.5 yr from one adult female and two adult males to assess longitudinal steroid metabolite excretion. Overall correlation of urinary PdG to matched, same-day fecal progesterone metabolites immunoreactivity was 0.38 (n = 71, P < 0.05). Similarly, urinary EC was correlated (P < 0.05) with same-day fecal estrogen immunoreactivity (r = 0.49, n = 71). During pregnancy and nonpregnant cycles, copulation occurred at the time of peak (or declining) estrogen metabolites and increasing progesterone metabolites concentrations. Estrus duration was 6–9 days and gestation lasted 69 days with parturition occurring coincident with a drop in progesterone metabolites. Males exhibited seasonal trends in fecal testosterone excretion with maximal concentrations from July to September coincident with peak mating activity. Although these limited longitudinal hormone profiles should be interpreted cautiously, noninvasive gonadal steroid monitoring suggests that: (1) both female and male wild dogs may exhibit reproductive seasonality in North America, (2) females are monoestrous, and (3) peak testicular activity occurs between August and October coincident with mating behavior. From a conservation perspective, noninvasive endocrine monitoring techniques should be useful for augmenting captive breeding programs, as well as for developing an improved understanding of the physiological mechanisms underlying reproductive suppression in response to social and ecological pressures. Zoo Biol 16:533–548, 1997. © 1997 Wiley-Liss, Inc.     

Fluoxetine, 17-β estradiol or folic acid combined with intra-lateral septal infusions of neuropeptide Y produced antidepressant-like actions in ovariectomized rats forced to swim

Folic acid is antidepressant, either alone or combined with several antidepressant drugs. However, the antidepressant-like actions of folic acid combined with intra-lateral septal (LSN) infusions of neuropeptide Y (NPY) in the forced swimming test (FST) have not been tested before. Thus, systemic injections of fluoxetine (20.0 mg/kg, P < 0.05; s.c.) or 17-β estradiol (10.0 μg/rat, P < 0.05; s.c.) or oral administrations of folic acid (50.0 mg/kg, P < 0.05; 75.0 mg/kg, P < 0.05) or NPY intra-LSN (3.0 μg, P < 0.05; 3.5 μg, P < 0.05) reduced immobility of ovariectomized Wistar rats. Subthreshold doses of: folic acid (25.0 mg/kg) or 17-β estradiol (5.0 μg/rat, P < 0.05) or fluoxetine (15.0 mg/kg, P < 0.05; s.c.) combined with subthreshold doses of NPY (2.5 μg/rat, P < 0.05; intra-LSN) and these combinations produced antidepressant-like actions; which were canceled by BIBP 3226 (a NPY-Y1 receptor antagonist). It is concluded that folic acid produced antidepressant-like effects probably through the participation of the NPY Y1 receptors found in the lateral septal nuclei.     

Estrogen Reduces 11β‐Hydroxysteroid Dehydrogenase Type 1 in Liver and Visceral,but Not Subcutaneous,Adipose Tissue in Rats

Following menopause, body fat is redistributed from peripheral to central depots. This may be linked to the age related decrease in estrogen levels. We hypothesized that estrogen supplementation could counteract this fat redistribution through tissue‐specific modulation of glucocorticoid exposure. We measured fat depot masses and the expression and activity of the glucocorticoid‐activating enzyme 11β‐hydroxysteroid dehydrogenase type 1 (11βHSD1) in fat and liver of ovariectomized female rats treated with or without 17β‐estradiol. 11βHSD1 converts inert cortisone, or 11‐dehydrocorticosterone in rats into active cortisol and corticosterone. Estradiol‐treated rats gained less weight and had significantly lower visceral adipose tissue weight than nontreated rats (P < 0.01); subcutaneous adipose weight was unaltered. In addition, 11βHSD1 activity/expression was downregulated in liver and visceral, but not subcutaneous, fat of estradiol‐treated rats (P < 0.001 for both). This downregulation altered the balance of 11βHSD1 expression and activity between adipose tissue depots, with higher levels in subcutaneous than visceral adipose tissue of estradiol‐treated animals (P < 0.05 for both), opposite the pattern in ovariectomized rats not treated with estradiol (P < 0.001 for mRNA expression). Thus, estrogen modulates fat distribution, at least in part, through effects on tissue‐specific glucocorticoid metabolism, suggesting that estrogen replacement therapy could influence obesity related morbidity in postmenopausal women.     

The contribution of Kv7 channels to pregnant mouse and human myometrial contractility

Premature birth accounts for approximately 75% of neonatal mortality and morbidity in the developed world. Despite this, methods for identifying and treating women at risk of preterm labour are limited and many women still present in preterm labour requiring tocolytic therapy to suppress uterine contractility. The aim of this study was to assess the utility of Kv7 channel activators as potential uterine smooth muscle (myometrium) relaxants in tissues from pregnant mice and women. Myometrium was obtained from early and late pregnant mice and from lipopolysaccharide (LPS)‐injected mice (day 15 of gestation; model of infection in pregnancy). Human myometrium was obtained at the time of Caesarean section from women at term (38–41 weeks). RT‐PCR/qRT‐PCR detected KCNQ and KCNE expression in mouse and human myometrium. In mice, there was a global suppression of all KCNQ isoforms, except KCNQ3, in early pregnancy (n= 6, P < 0.001 versus late pregnant); expression subsequently increased in late pregnancy (n= 6). KCNE isoforms were also gestationally regulated (P < 0.05). KCNQ and KCNE isoform expression was slightly down‐regulated in myometrium from LPS‐treated‐mice versus controls (P < 0.05, n= 3–4). XE991 (10 μM, Kv7 inhibitor) significantly increased spontaneous myometrial contractions in vitro in both human and mouse myometrial tissues (P < 0.05) and retigabine/flupirtine (20 μM, Kv7 channel activators) caused profound myometrial relaxation (P < 0.05). In summary, Kv7 activators suppressed myometrial contraction and KCNQ gene expression was sustained throughout gestation, particularly at term. Consequently, activation of the encoded channels represents a novel mechanism for treatment of preterm labour.     

Crosstalk between AMPK activation and angiotensin II‐induced hypertrophy in cardiomyocytes: the role of mitochondria

AMP‐kinase (AMPK) activation reduces cardiac hypertrophy, although underlying molecular mechanisms remain unclear. In this study, we elucidated the anti‐hypertrophic action of metformin, specifically, the role of the AMPK/eNOS/p53 pathway. H9c2 rat cardiomyocytes were treated with angiotensin II (AngII) for 24 hrs in the presence or absence of metformin (AMPK agonist), losartan [AngII type 1 receptor (AT1R) blocker], Nω‐nitro‐L‐arginine methyl ester (L‐NAME, pan‐NOS inhibitor), splitomicin (SIRT1 inhibitor) or pifithrin‐α (p53 inhibitor). Results showed that treatment with metformin significantly attenuated AngII‐induced cell hypertrophy and death. Metformin attenuated AngII‐induced activation (cleavage) of caspase 3, Bcl‐2 down‐regulation and p53 up‐regulation. It also reduced AngII‐induced AT1R up‐regulation by 30% (P < 0.05) and enhanced AMPK phosphorylation by 99% (P < 0.01) and P‐eNOS levels by 3.3‐fold (P < 0.01). Likewise, losartan reduced AT1R up‐regulation and enhanced AMPK phosphorylation by 54% (P < 0.05). The AMPK inhibitor, compound C, prevented AT1R down‐regulation, indicating that metformin mediated its effects via AMPK activation. Beneficial effects of metformin and losartan converged on mitochondria that demonstrated high membrane potential (Δψ_m) and low permeability transition pore opening. Thus, this study demonstrates that the anti‐hypertrophic effects of metformin are associated with AMPK‐induced AT1R down‐regulation and prevention of mitochondrial dysfunction through the SIRT1/eNOS/p53 pathway.     

锈毛草莓积雪草合剂对STZ小鼠降血糖作用研究

采用75%乙醇萃取锈毛草莓及积雪草,制备成合剂,通过STZ诱导建立糖尿病小鼠模型,随机分为模型组、低剂量组及高剂量组,分别以蒸馏水、4及8 g·kg~(-1)锈毛草莓积雪草合剂灌胃,1次·d~(-1),持续4周,另设正常组作为对照。探究绣毛草莓积雪草合剂对糖尿病小鼠的降血糖效果及作用机制。结果表明:(1)不同剂量的锈毛草莓积雪草合剂均能显著降低糖尿小鼠血糖值,提高糖耐量并减轻其多食、多饮、多尿的症状。(2)低剂量组小鼠血清TC、TG含量较模型组有显著提高(P0.01或P0.05),肝脏SOD、CAT活性显著提高(P0.05),MDA含量明显降低。(3)低剂量黄毛草莓积雪草合剂效果优于二甲双胍,毒性实验显示锈毛草莓积雪草合剂实属无毒。该研究结果表明锈毛草莓积雪草合剂具有降血糖作用,同时能够降低糖尿病小鼠的血脂水平,其作用机制可能与提高肝脏抗氧化能力有关。     

Steroid production,cell proliferation,and apoptosis in cultured bovine antral and mural granulosa cells: Development of an in vitro model to study estradiol production

This study was undertaken to characterize the relationship between changes in steroid production, cell cycle activity (ie, cell proliferation) and apoptosis in antral and mural bovine granulosa cells cultured in vitro. This was done to select conditions promoting optimal estradiol production by bovine granulosa cells cultured in completely defined conditions. In the first experiment, antral granulosa cells were cultured over the entire 4 days of the culture period in the presence of either 0, 2, or 10 ng/ml of FSH (chronic conditions) or were maintained under minimal FSH support (0.5 ng/ml FSH) for the first 3 days of culture and then were challenged over the fourth day of culture with either 0, 2, or 10 ng/ml FSH (challenged conditions). Compared with cells exposed to constant FSH levels (chronic conditions), the FSH-induced production of estradiol was higher (P < 0.006) and that of progesterone was lower (P < 0.02) over the last 24 h of culture, when antral granulosa cells were maintained under minimal FSH support during the first 3 days of culture (challenged conditions). In the second experiment, dynamics of estradiol and progesterone productions, conversion of [¹⁴C]androstenedione into subsequent steroid metabolites, DNA content, cell cycle activity, and apoptosis (as assessed by flow cytometry) of antral and mural granulosa cells over the first 3 days of culture under minimal FSH support and in response to a challenge with FSH during the last 24 h of culture were evaluated. Estradiol production as well as the conversion of androstenedione into testosterone and estradiol were greater (P< 0.01) in antral than in mural granulosa cells cultured under challenged conditions. A higher proportion of mural than antral granulosa cells were in the proliferative state at the end of culture (P < 0.03). This may be related to the decreased ability of mural cells to produce estradiol. FSH suppressed (P < 0.05) the spontaneous onset of apoptosis in both cell types. These results suggest that functional differences between these two cell compartments need to be considered in studying bovine granulosa cells in vitro. Because of their large (400 to 600%) FSH-induced estradiol production, antral granulosa cells cultured under challenged conditions provide a model that can be used to examine substances for their ability to alter estradiol production and apoptosis in bovine granulosa cells. Mol. Reprod. Dev. 50:170–177, 1998. © 1998 Wiley-Liss, Inc.     

Energy metabolism and protein balance in growing rats fed different levels of dietary fibre and protein

A study was performed to investigate the effect of different levels of dietary fibre (DF) and dietary protein on visceral organ size, digestibility, nitrogen balance and energy metabolism in rats. Thirty-six male Wistar rats, initial body weight about 76 g, were used in a factorial design consisting of three levels of DF (low, 100 g/kg DM; medium, 250 g/kg DM and high, 290 g/kg DM) and two levels of dietary protein (low, 120 g/kg DM and high, 223 g/kg DM). The added fibre source was soybean hulls and Danish fish meal was used as sole source of dietary protein. Measurements of gas-exchange were done on six rats (one group) while urine and faeces were collected individually. The ratio of food/empty body gain increased (P < 0.05) with increasing DF and decreasing levels of dietary protein. The weight of the digestive tract was larger (P < 0.05) in rats fed the high fibre diet than in those fed the low fibre diet. The digestibility of nutrients and energy decreased linearly with increasing level of soybean fibre (P < 0.05). An increased intake of DF was associated with a concomitant loss of protein and energy to faeces. The microbial degradation of NSP and other unabsorbed carbohydrates caused considerably changes in N metabolism of the colon. In rats fed the low protein diets increased levels of DF decreased N excretion in urine and increased N excretion in faeces, while the ratio of retained/digested protein remained constant. When rats were fed the high protein diet protein retention dropped in response to DF both absolute and relative to digested amount, indicating that energy intake could be a limiting factor. Heat production as a percentage of metabolizable energy (HP/ME) was higher (P < 0.05) in rats fed the low protein diet than in rats fed the high protein diet, but no significant difference was found among DF levels.     

Culture of human preovulatory granulosa cells: Effect of extracellular matrix on steroidogenesis

Summary— Human luteal granulosa cells, harvested from preovulatory follicles during in vitro fertilization attempts, were cultured in a serum-precoated substratum (‘serum cells’) or on a collagen matrix (‘collagen cells’). Concerning the ‘serum cell’ model, E2 secretion was very low in the absence of androgen; when androstenedione was added to the culture medium, cells secreted 180 ± 52 pmol/ml/24 h of estradiol, 440 ± 78 pmol/ml/24 h of testosterone and lower quantities of estrone and estriol. Follicle stimulating hormone induced a significant increase in estradiol and estriol, while the secretion of the other steroids was not altered. The secretion of progesterone was 3.15 ± 1 nmol/ml/24 h and significantly enhanced by luteinizing hormone (+ 95%; P < 0.01). The secretions of 17α-hydroxyprogesterone and 20α-dihydroprogesterone were low and not modified by luteinizing hormone. ‘Collagen cells’, in basal conditions, showed an increased secretion of estradiol (+ 50%, P < 0.05), became rounded and were less responsive to gonadotropins when compared with ‘serum cells’. Thus, the use of a collagen matrix, similarly to gonadotropins, stimulated granulosa cell steroidogenesis in relation to modifications of cell shape. The higher responsiveness of serum cells to gonadotropins makes this model more suitable for physiological and pharmacological studies than the collagen one.     

Impact of diabetic polyneuropathy and cardiovascular autonomic neuropathy on the excretion of urinary 8-epi-PGF2α and its metabolites (2, 3-dinor and 2, 3-dinor-5, 6-dihydro)

The objective of this study was to establish if diabetes in the presence of polyneuropathy (PN) and/or cardiovascular autonomic neuropathy (CAN) is associated with alterations in the amounts of 8-epi-PGF_2α (IP) and its metabolites including 2, 3-dinor-8-epi-PGF_2α (dinor-IP) and 2, 3-dinor-5, 6 dihydro-8-epi-PGF_2α (dinor-dihydro-IP) in urine. Mass spectrometric separation showed that excretion of IP was similar in the PN+/CAN ? and PN+/CAN+ groups but higher than in the PN ? /CAN ? group (n = 103, 22 and 60, respectively; P < 0.05). By contrast, excretion of dinor-IP or dinor-dihydro-IP were similar in the PN ? /CAN ? and PN+/CAN ? groups but higher than in PN+/CAN+ group. Correlations were obtained between IP and dinor-IP or dinor-dihydro-IP (r = 0.30; P < 0.001 and r = 0.31; P < 0.001, respectively). A significant association was also observed between dinor-IP and dinor-dihydro-IP (r = 0.48; P < 0.001). In conclusion, these biomarkers should prove useful in studies evaluating the impact of therapeutic drugs or antioxidant interventions aimed at delaying the onset of diabetic complications.     

Activin βA subunit,follistatin and follistatin-like 3 are expressed in the endometrium of ovariectomized rats and regulated by estrogen replacement

Activin A is a growth factor expressed in the endometrium, where it modulates tissue remodeling and enhances decidualization. The effects of activin A are counteracted by two binding proteins, namely follistatin and follistatin-like 3 (FSTL3). We have evaluated the effects of estrogen and progestin on the endometrial expression of activin βA subunit, follistatin and FSTL3 in ovariectomized rats. Adult female Wistar rats (n = 21) were ovariectomized and received one week later a single dose of estradiol benzoate (1.5 mg/kg body weight, i.m. injection), either alone (n = 7) or associated with depot medroxyprogesterone acetate (3 mg/kg body weight, i.m. injection, n = 7), or oil vehicle (control group, n = 7). One week later, activin βA subunit mRNA levels had increased significantly in the uteri of rats treated with estradiol alone (7.4 fold increase over controls, P < 0.05) and to the same extent in rats receiving estradiol plus medroxyprogesterone (6.1 fold increase over controls, P < 0.05). This was accompanied by increase of βA subunit immunostaining in estradiol and estroprogestin treated rats, which was noted only in the surface endometrial epithelium. Follistatin mRNA expression, conversely, showed a significant decrease in the groups treated with estrogen alone and estrogen plus progestin (P < 0.05), and follistatin immunostaining in the glandular epithelium was weaker in estradiol and estroprogestin-treated rats compared to controls. FSTL3 expression was similar in the 3 groups. In conclusion, the expression of activin βA subunit increases and that of follistatin decreases following estrogen replacement in the endometrium of ovariectomized rats, and these effects are not further altered by the addition of progestin. Presented, in part, as a poster at the 55nd Annual Meeting of the Society for Gynecologic Investigation, San Diego, CA, March 2008.