Endocrine and Metabolic Responses to Long-Term Monotherapy with the Antiepileptic Drug Valproate in the Normally Cycling Rhesus Monkey

An association between epilepsy and reproductive disturbances with an apparent increase in a polycystic ovarian syndrome (PCOS) has been reported. Whether this association can be attributed to epilepsy itself or is related to antiepileptic drug therapy, in particular valproate (VPA), remains controversial. We studied effects of a long-term VPA treatment on cycling monkeys, postulating that, if VPA monotherapy were to promote abnormal endocrine and metabolic parameters that are characteristic of PCOS, changes in cyclicity would be readily demonstrated. After a 2-month control, a 12- to 15-month VPA monotherapy was initiated in 7 regularly cycling rhesus monkeys. Overall mean levels of VPA were 88.7 ± 4.0 (se) μg/ml. Mean body weight increased progressively during VPA treatment from 8.5 ± 0.5 kg before treatment to 9.6 ± 0.7 kg in the last week of treatment (P < 0.05). Monkeys continued to have regular ovulatory menstrual cycles throughout VPA monotherapy. Length of the cycles was 28 ± 0.58 d in control and 28.4 ± 1.18 d in the last 3 months of VPA treatment. Follicular and luteal lengths and peak preovulatory estradiol and integrated luteal progesterone levels did not differ between control and treatment. Ovaries from VPA-treated monkeys showed histological evidence of ovulation, and none had characteristic features of PCOS. Endocrine PCOS markers, such as increased early follicular LH/FSH ratio and androgen levels were not different in control and VPA treatment cycles. LH and 17-hydroxyprogesterone responses to GnRH agonist challenges and the insulin response to glucose tolerance tests were similar in control and VPA groups. Lipid profiles were not affected by VPA treatment. The data indicate that a 12- to 15-month therapeutic exposure to VPA does not induce cyclic hormonal or morphological ovarian abnormalities or characteristics of the PCOS when administered to nonepileptic normally cycling nonhuman primates

Short-Term Administration of Antivascular Endothelial Growth Factor Antibody in the Late Follicular Phase Delays Follicular Development in the Rhesus Monkey

Indirect evidence in the nonhuman primate and human suggests that angiogenesis and regulators of angiogenesis such as vascular endothelial growth factor (VEGF) may play an active role in cyclic folliculogenesis. Indeed, the follicle selected for maturation and ovulation possesses a denser microvascular network, and VEGF messenger ribonucleic acid and its protein have been identified in granulosa cells of the developing follicle during the mid- and late follicular phases, with a more intense signal in the mature follicle. The objective of this study was to obtain direct evidence in the nonhuman primate for an active role of VEGF in follicular growth and maturation by studying the effect of VEGF-blocking antibodies in this process. After documenting two normal ovulatory cycles, female rhesus monkeys (n = 7) received iv injections of anti-VEGF antibodies (0.5 mg) twice on successive days in the late follicular phase. Three monkeys also received nonspecific goat IgG (0.5 mg) twice on successive days in the late follicular phase. Daily measurements of estradiol, progesterone, LH, and FSH were obtained during the two control cycles, the anti-VEGF treatment and posttreatment cycles, and the IgG treatment cycle. Anti-VEGF antibody administration significantly lengthened the follicular phase in six of seven monkeys to 17.8 ± 1.7 vs. 10.0 ± 0.7 and 9.8 ± 0.6 in control cycles and 10.7 ± 0.3 days (mean ± se) in IgG-treated cycles. The expected late follicular phase rise in estradiol, as documented in the control cycles (day 0, 96.1 ± 6.0; day 1, 125.5 ± 20.0; day 2, 165.5 ± 24.9; day 3, 183.8 ± 11.0 pg/mL), was interrupted by anti-VEGF antibody treatment (99.3 ± 5.0, day 0, preinjection control) to 63.3± 12.2 (day 1), 48.5 ± 8.7 (day 2), and 57.6 ± 9.0 (day 3). Mean FSH levels were significantly increased by day 2 of anti-VEGF antibody treatment. After a variable delay, estradiol concentrations increased to reach a preovulatory peak in all anti-VEGF-treated animals, followed by ovulation, normal luteal function, and a normal posttreatment cycle. The data clearly demonstrate that short-term inhibition of angiogenesis with an anti-VEGF-blocking antibody during the later growth phase of the dominant follicle interferes with normal follicular development. Persistence of estradiol secretion and delayed resumption of its rise also suggest recovery of the follicle. We conclude that the angiogenic regulator VEGF is a crucial component in the process of follicular growth in the primate

Short-Term Administration of Antivascular Endothelial Growth Factor Antibody in the Late Follicular Phase Delays Follicular Development in the Rhesus Monkey

Indirect evidence in the nonhuman primate and human suggests that angiogenesis and regulators of angiogenesis such as vascular endothelial growth factor (VEGF) may play an active role in cyclic folliculogenesis. Indeed, the follicle selected for maturation and ovulation possesses a denser microvascular network, and VEGF messenger ribonucleic acid and its protein have been identified in granulosa cells of the developing follicle during the mid- and late follicular phases, with a more intense signal in the mature follicle. The objective of this study was to obtain direct evidence in the nonhuman primate for an active role of VEGF in follicular growth and maturation by studying the effect of VEGF-blocking antibodies in this process. After documenting two normal ovulatory cycles, female rhesus monkeys (n = 7) received iv injections of anti-VEGF antibodies (0.5 mg) twice on successive days in the late follicular phase. Three monkeys also received nonspecific goat IgG (0.5 mg) twice on successive days in the late follicular phase. Daily measurements of estradiol, progesterone, LH, and FSH were obtained during the two control cycles, the anti-VEGF treatment and posttreatment cycles, and the IgG treatment cycle. Anti-VEGF antibody administration significantly lengthened the follicular phase in six of seven monkeys to 17.8 ± 1.7 vs. 10.0 ± 0.7 and 9.8 ± 0.6 in control cycles and 10.7 ± 0.3 days (mean ± se) in IgG-treated cycles. The expected late follicular phase rise in estradiol, as documented in the control cycles (day 0, 96.1 ± 6.0; day 1, 125.5 ± 20.0; day 2, 165.5 ± 24.9; day 3, 183.8 ± 11.0 pg/mL), was interrupted by anti-VEGF antibody treatment (99.3 ± 5.0, day 0, preinjection control) to 63.3± 12.2 (day 1), 48.5 ± 8.7 (day 2), and 57.6 ± 9.0 (day 3). Mean FSH levels were significantly increased by day 2 of anti-VEGF antibody treatment. After a variable delay, estradiol concentrations increased to reach a preovulatory peak in all anti-VEGF-treated animals, followed by ovulation, normal luteal function, and a normal posttreatment cycle. The data clearly demonstrate that short-term inhibition of angiogenesis with an anti-VEGF-blocking antibody during the later growth phase of the dominant follicle interferes with normal follicular development. Persistence of estradiol secretion and delayed resumption of its rise also suggest recovery of the follicle. We conclude that the angiogenic regulator VEGF is a crucial component in the process of follicular growth in the primate

Synergistic effect of phytochemicals on cholesterol metabolism and lipid accumulation in HepG2 cells

Abstract Background Crocin (CRO), chlorogenic acid (CGA), geniposide (GEN), and quercetin (QUE) are all natural compounds with anti-obesity properties, in particular, hypolipidemic effects, which have been widely used for the treatment of obesity-related metabolic diseases. However, it is not yet known whether these compounds interact synergistically. Here, we investigated the effects and molecular mechanisms of CRO, CGA, GEN, QUE, and a combination of all four compounds (CCGQ), on lipid accumulation in human hepatoma (HepG2 cells). Methods The optimal concentration of CRO, CGA, GEN, QUE to stimulate HepG2 cells proliferation was determined using MTT assay. HepG2 cells were pretreated with 10 μmol/L simvastatin, 1 μmol/L CRO, 30 μmol/L CGA, 10 μmol/L GEN, 10 μmol/L QUE, and CCGQ (a combination of 1 μmol/L CRO, 30 μmol/L CGA, 10 μmol/L GEN, and 10 μmol/L QUE) for 24 or 48 h. Oil red O staining and extracellular TC and TG levels were detected. The RT-PCR was used to observe on cholesterol metabolism-related gene expression. Immunocytochemistry and western-blot assayed the 3-hydroxy-3-methylglutaryl-coenzyme (HMGCR) protein expression in HepG2 cells. Results Compared to those of control, we demonstrated that treating HepG2 cells for 48 h with CCGQ resulted in a strong synergistic effect, causing a marked decrease in lipid deposition in comparison to individual treatments, in both triglyceride and total cholesterol (CRO, 5.74- and 1.49-folds; CGA, 3.38- and 1.12-folds; GEN, 4.04- and 1.44-folds; QUE, 3.36- and 1.24-folds; simvastatin, 5.49- and 1.83-folds; and CCGQ, 7.75- and 2.20-folds), and Oil red O staining assays. In addition, CCGQ treatment increased ATP-binding cassette transporter (ABCA1), cholesterol 7α-hydroxylase (CYP7A1), and AMP-activated protein kinase 2α (AMPKα2) mRNA expression, while decreasing sterol regulatory element binding protein 2 (SREBP2), and liver X receptor alpha (LXRα) mRNA expression. Notably, CCGQ was more effective in decreasing HMGCR expression than the individual treatments. Conclusion The CCGQ combination has potential, both as a complementary therapy for hyperlipemia, and in preventing further obesity-related complications

Endotoxin stimulates leptin in the human and nonhuman primate

Leptin, which plays a key role in regulating energy homeostasis, may also modulate the inflammatory response. An inflammatory challenge with endotoxin has been shown to stimulate leptin release in the rodent. This finding has not been reproduced in humans or in nonhuman primates, although leptin levels have been reported to increase in septic patients. We have therefore examined the effects of endotoxin injection on plasma leptin levels in nine ovariectomized monkeys and four postmenopausal women. In an initial study in five monkeys, mean leptin levels did not increase during the first 5 h after endotoxin treatment, but did increase significantly from 6.4 +/- 2.1 ng/ml at baseline to 12.3 +/- 4.4 ng/ml at 24 h (P = 0.043). In a second study, a significant increase in leptin over time was noted after endotoxin treatment (P &lt; 0.001); leptin release during the 16- to 24-h period after endotoxin injection was 48% higher than during the control period (P = 0.043). A similar stimulatory effect of endotoxin on leptin was observed when monkeys received estradiol replacement. In a third study, repeated injections of endotoxin over a 3-d period stimulated IL-6, ACTH, cortisol, and leptin release (P &lt; 0.001). Leptin increased during the first day of treatment in all animals, but only monkeys with baseline plasma leptin levels greater than 10 ng/ml exhibited a sustained increase in leptin throughout the 3-d period. There was a significant correlation (r = 0.81; P = 0.008) between the mean baseline leptin level and the percent increase in leptin over baseline on the last day of treatment. In the human subjects, plasma leptin concentrations did not change significantly during the 7-h period after endotoxin injection. However, leptin increased in all four women from a mean baseline of 8.34 +/- 3.1 to 13.1 +/- 4.3 ng/ml 24 h after endotoxin (P = 0.038). In summary, endotoxin stimulates the release of leptin into peripheral blood in the human and nonhuman primate, but the time course is different from that reported in the rodent. These results are consistent with previous reports of increased blood leptin levels in patients with sepsis. The significance of these findings and the potential role of leptin in modulating the response to inflammation in the human require further study

Astressin B, a Nonselective Corticotropin-Releasing Hormone Receptor Antagonist, Prevents the Inhibitory Effect of Ghrelin on Luteinizing Hormone Pulse Frequency in the Ovariectomized Rhesus Monkey

Administration of ghrelin, a key peptide in the regulation of energy homeostasis, has been shown to decrease LH pulse frequency while concomitantly elevating cortisol levels. Because increased endogenous CRH release in stress is associated with an inhibition of reproductive function, we have tested here whether the pulsatile LH decrease after ghrelin may reflect an activated hypothalamic-pituitary-adrenal axis and be prevented by a CRH antagonist. After a 3-h baseline LH pulse frequency monitoring, five adult ovariectomized rhesus monkeys received a 5-h saline (protocol 1) or ghrelin (100-μg bolus followed by 100 μg/h, protocol 2) infusion. In protocols 3 and 4, animals were given astressin B, a nonspecific CRH receptor antagonist (0.45 mg/kg im) 90 min before ghrelin or saline infusion. Blood samples were taken every 15 min for LH measurements, whereas cortisol and GH were measured every 45 min. Mean LH pulse frequency during the 5-h ghrelin infusion was significantly lower than in all other treatments (P < 0.05) and when compared with the baseline period (P < 0.05). Pretreatment with astressin B prevented the decrease. Ghrelin stimulated cortisol and GH secretion, whereas astressin B pretreatment prevented the cortisol, but not the GH, release. Our data indicate that CRH release mediates the inhibitory effect of ghrelin on LH pulse frequency and suggest that the inhibitory impact of an insufficient energy balance on reproductive function may in part be mediated by the hypothalamic-pituitary-adrenal axis

Effects of Estradiol on Cerebrospinal Fluid Levels of Agouti-Related Protein in Ovariectomized Rhesus Monkeys

Hypothalamic proopiomelanocortin (POMC)-derived MSH peptides and the melanocortin receptor antagonist, agouti-related protein (AgRP), interact to regulate energy balance. Both POMC and AgRP neurons express estrogen receptors, but little is known about estrogen regulation of the melanocortin system in the primate. We have therefore examined the effects of physiological doses of estradiol (E2) on POMC and AgRP in lumbar cerebrospinal fluid (CSF) of ovariectomized monkeys. POMC prohormone was measured by ELISA. AgRP was measured by RIA (sensitive for the more biologically active C-terminal AgRP83-132 but also detects full-length AgRP) and by ELISA (measures primarily full length AgRP). In the first experiment, 14 animals were studied before and after 3 wk of E2. CSF POMC did not change, but AgRP(RIA) decreased from 7.9 ± 1.2 to 4.7 ± 1.2 fmol/ml after E2 (P = 0.03) and the POMC/AgRP(RIA) ratio increased from 4.2 ± 0.89 to 6.8 ± 1.04 (P = 0.04). AgRP(ELISA) did not change, but the ratio of AgRP(RIA) compared with AgRP(ELISA) was reduced after E2 (P = 0.02). In the second experiment, 11 animals were studied after 6 wk of E2, and similar changes were noted. The degree of AgRP(RIA) suppression with E2 was inversely related to body mass index (r = 0.569; P = 0.03). These results show for the first time that E2 suppresses AgRP(C-terminal) in CSF, increases the POMC to AgRP ratio, and may decrease AgRP processing, thus leading to increased melanocortin signaling. Furthermore, obesity was associated with resistance to the suppressive effects of E2 on AgRP, analogous to what is seen with obesity and leptin resistance