Novel (2E,4E,6Z)-7-(2-alkoxy-3,5-dialkylbenzene)-3-methylocta-2,4,6-trienoic acid retinoid X receptor modulators are active in models of type 2 diabetes.

Previous data have shown that RXR-selective agonists (e.g., 3 and 4) are insulin sensitizers in rodent models of non-insulin-dependent diabetes mellitus (NIDDM). Unfortunately, they also produce dramatic increases in triglycerides and profound suppression of the thyroid hormone axis. Here we describe the design and synthesis of new RXR modulators that retain the insulin-sensitizing activity of RXR agonists but produce substantially reduced side effects. These molecules bind selectively and with high affinity to RXR and, unlike RXR agonists, do not activate RXR homodimers. To further evaluate the antidiabetic activity of these RXR modulators, we have designed a concise and systematic structure-activity relationship around the 2E,4E,6Z-7-aryl-3-methylocta-2,4,6-trienoic acid scaffold. Selected compounds have been evaluated using insulin-resistant rodents (db/db mice) to characterize effects on glucose homeostasis. Our studies demonstrate the effectiveness of RXR modulators in lowering plasma glucose in the db/db mouse model.

Peroxisome proliferator-activated receptor subtype-specific regulation of hepatic and peripheral gene expression in the Zucker diabetic fatty rat.

Fibrates and thiazolidinediones are used clinically to treat hypertriglyceridemia and hyperglycemia, respectively. Fibrates bind to the peroxisome proliferator-activated receptor (PPAR)-alpha, and thiazolidinediones are ligands of PPAR-gamma. These intracellular receptors form heterodimers with retinoid X receptor to modulate gene transcription. To elucidate the target genes regulated by these compounds, we treated Zucker diabetic fatty rats (ZDF) for 15 days with a PPAR-alpha-specific compound, fenofibrate, a PPAR-gamma-specific ligand, rosiglitazone, and a PPAR-alpha/-gamma coagonist, GW2331, and measured the levels of several messenger RNAs (mRNAs) in liver by real-time polymerase chain reaction. All 3 compounds decreased serum glucose and triglyceride levels. Fenofibrate and GW2331 induced expression of acyl-coenzyme A (CoA) oxidase and enoyl-CoA hydratase and reduced apolipoprotein C-III and phosphoenolpyruvate carboxykinase mRNAs. Rosiglitazone modestly increased apolipoprotein C-III mRNA and had no effect on expression of the other 2 genes in the liver but increased the expression of glucose transporter 4 and phosphoenolpyruvate carboxykinase in adipose tissue. We identified a novel target in liver, mitogen-activated phosphokinase phosphatase 1, whose down-regulation by PPAR-alpha agonists may improve insulin sensitivity in that tissue by prolonging insulin responses. The results of these studies suggest that activation of PPAR-alpha as well as PPAR-gamma in therapy for type 2 diabetes will enhance glucose and triglyceride control by combining actions in hepatic and peripheral tissues.

Metabolic effects of rexinoids: tissue-specific regulation of lipoprotein lipase activity.

Hypertriglyceridemia is a frequent complication accompanying the treatment of patients with either retinoids or rexinoids, [retinoid X receptor (RXR)-selective retinoids]. To investigate the cellular and molecular basis for this observation, we have studied the effects of rexinoids on triglyceride metabolism in both normal and diabetic rodents. Administration of a rexinoid such as LG100268 (LG268) to normal or diabetic rats results in a rapid increase in serum triglyceride levels. LG268 has no effect on hepatic triglyceride production but suppresses post-heparin plasma lipoprotein lipase (LPL) activity suggesting that the hypertriglyceridemia results from diminished peripheral processing of plasma very low density lipoproteins particles. Treatment of diabetic rats with rexinoids suppresses skeletal and cardiac muscle but not adipose tissue LPL activity. This effect is independent of changes in LPL mRNA. In C2C12 myocytes, LG268 suppresses the level of cell surface (i.e., heparin-releasable) LPL activity without altering LPL mRNA. This effect is very rapid (t(1/2) = 2 h) and is blocked by the transcriptional inhibitor actinomycin D. These studies demonstrate that RXR ligands can have dramatic effects on the post-translational processing of LPL and suggest that skeletal muscle may be an important target of rexinoid action. In addition, these data underscore that the metabolic consequences of RXR activation are distinct from either retinoic acid receptor or peroxisome proliferator-activated receptor activation.

The inhibitory effect of intracerebroventricularly injected interleukin 1beta on testosterone secretion in the rat: role of steroidogenic acute regulatory protein.

Exposure to disease or injury often results in impaired reproductive activity accompanied by decreased testosterone levels. After immune activation, the cytokine interleukin 1-beta (IL-1beta) circulates in high concentrations, and its exogenous administration evokes many of the sequelae of immune activation. Previously, we have shown that the administration of this cytokine into the cerebral ventricles blunts hCG-stimulated testosterone secretion. This effect, though time-dependent, occurs before significant elevation of interleukin 6 in the peripheral bloodstream, does not depend on adrenal activation, and/or changes in LH concentrations, leading us to hypothesize a direct connection between the brain and testis. To explore this mechanism further, we isolated testicular tissue from rats treated intracerebroventricularly (icv) with vehicle or IL-1beta 30 or 90 min before they were killed. We found that in vivo cytokine treatment blunted ex vivo testosterone secretion in response to hCG, showing that the mechanism is independent of circulating cytokines. Though hCG binding was moderately reduced by icv IL-1beta in these preparations, the extent of this inhibition did not explain our observations. As the first acutely and hormonally regulated step in the biosynthesis of testosterone is the transfer of cholesterol into the inner mitochondrial membrane, which is mediated by steroidogenic acute regulatory (StAR) protein, we hypothesized that the rapid effects of icv IL-1beta on testicular responsiveness to hCG might be due to reduced levels of StAR. We report here that StAR protein was indeed reduced in Leydig cells isolated from rats treated in vivo with IL-1beta. Furthermore, treatment with a water-permeable form of cholesterol that bypasses the requirement for StAR partially restored hCG-stimulated testosterone secretion from testes isolated from rats treated icv with IL-1beta. Taken together, our data indicate that StAR plays a role in the suppression of testicular function evoked by central administration of IL-1beta.

Divergence in the expression of molecular markers of neuronal activation in the parvocellular paraventricular nucleus of the hypothalamus evoked by alcohol administration via different routes.

Immediate early gene (IEG) expression has been routinely used by neuroscientists as an index of neuronal activation. In the case of the hypothalamic-pituitary-adrenal axis, induction of c-fos and/or NGFI-B mRNAs in the parvocellular paraventricular nucleus (pPVN) has been documented after a variety of stimuli that increase adrenocorticotropin (ACTH) in the systemic circulation. However, the functional relationship between expression of IEGs and transcription of the genes for the ACTH secretagogues corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) is not clear. While investigating the neuroendocrine correlates of alcohol administration via different routes (intraperitoneal vs intragastric), we noted a difference in the time course of NGFI-B mRNA expression in the pPVN, despite comparable dynamics in ACTH secretion. By comparing the temporal cascade of transcriptional events in vivo after alcohol injection via either route, we sought to determine functional relationships between IEGs and the induction of CRF and AVP heteronuclear RNAs (hnRNAs). One advantage of our paradigm is the use of the same stimulus (systemic alcohol injection) in which access to the CNS does not differ between the groups to be compared. Intraperitoneal administration of the drug resulted in significant increases in c-fos mRNA, Fos protein, CRF hnRNA, and AVP hnRNA. In contrast, intragastric treatment evoked a brief, modest elevation in c-fos mRNA and Fos protein, increased AVP hnRNA, and caused no detectable change in CRF hnRNA. These data indicate that robust increases in CRF hnRNA are closely linked to full expression of c-fos mRNA and Fos protein. In addition, the expression of NGFI-B after both routes of administration is indicative of cellular activation within the pPVN in parallel with secretion of ACTH.

The neuroendocrine control of clock-timed gonadotropin release in the female Syrian hamster: role of serotonin.

We hypothesized that rhythms in hypothalamic serotonergic activity were permissive to daily and estrous cycle-related rhythms of LH, FSH and prolactin (PRL). In the Syrian hamster, proestrus (PRO) is characterized by a surge of LH, FSH and PRL; diestrus (DIE) by low LH and FSH and a small surge of PRL, while in photoperiod-induced anestrous (PIA) animals there is a surge of LH and FSH and low PRL. Turnover rates of serotonin (5HT) in four brain areas were determined for the three reproductive states at 2-h intervals. Turnover in the preoptic area and arcuate nuclei did not change, indicating that 5HT projections to these regions probably do not control LH, FSH or PRL release. Serotonin turnover in the median eminence (ME) was elevated at 0600 h in PIA females, at 0600 h, 0800 h, and 1400 h on DIE and at 0600 h and 2200 h on PRO. Since the pattern of 5HT turnover in the ME is different during each of the three reproductive states, 5HT in this area is likely not crucial to the control of LH, FSH and PRL. Turnover of 5HT also did not change in the suprachiasmatic nuclei (SCN) of PRO or PIA animals. However, 5HT turnover rates in the SCN were elevated at 1200 h, 2000 h, and 2400 h on DIE. The correlation of high 5HT turnover in the SCN of DIE but not PRO and PIA animals suggested that elevated serotonergic activity in the SCN is part of the mechanism by which the gonadotropin surge is prevented on DIE. To test this, PRO and DIE hamsters were injected with 5HT receptor ligands. Administration of a 5HT agonist attenuated the PRO surge of LH and blocked the surge of PRL. In contrast, administration of two 5HT antagonists failed to elicit a surge of LH in DIE and phenobarbital-blocked PRO females, an indication that other mechanisms also contribute to inhibition of gonadotropin and PRL surges.

Gender difference in hypothalamic-pituitary-adrenal axis response to alcohol in the rat: activational role of gonadal steroids.

Alcohol administration activates the hypothalamic-pituitary-adrenal (HPA) axis of both male and female rats, with females secreting more adrenocorticotropin (ACTH) and corticosterone than males in response to the same dose of alcohol. Our earlier work suggested that this gender difference arises due to the activational effects of gonadal steroids. In particular, we hypothesized that both androgens and estrogens play a role, with androgens exerting an inhibitory influence while estrogens elevate activity of the HPA. In the present studies, we tested this hypothesis by manipulating steroidal milieu in male rats using surgical castration and chronic implantation of testosterone (T), dihydrotestosterone (DHT), or estradiol (E2). Intact male and female rats were included as controls. Injection of alcohol (3 g/kg b.wt., i.p.) resulted in elevation of blood alcohol levels, ACTH and corticosterone in all groups. However, the amount of ACTH secreted was greater in females and castrated males implanted with E2 than in intact males. In castrated males, regardless of androgen implantation, the ACTH response was intermediate, with mean levels between those of females and males, but not differing significantly from either. In contrast to the ACTH results, significantly higher corticosterone secretion was measured in females and castrated males which did not receive a steroid implant. Since there were no significant differences between groups in blood alcohol levels (BALs), these results are not due to steroid-dependent alterations in alcohol metabolism. Because the ACTH data confirmed an activational effect of E2, we sought to determine whether this steroid regulated levels of corticotropin-releasing factor (CRF) and arginine vasopressin (AVP) mRNAs in the paraventricular nucleus of the hypothalamus (PVN). Four pretreatment groups were studied: intact males, intact females, castrated males, and castrated males implanted with E2. Two weeks after surgery, alcohol or vehicle was administered 3 h before brains were collected. In intact males, alcohol treatment elevated levels of both CRF and AVP mRNAs in the PVN, as previously reported. In contrast, this treatment decreased CRF mRNA in castrated males implanted with E2. In addition, steroid pretreatment alone elevated CRF mRNA levels in castrated males. Although we did not observe E2-dependent increases in CRF or AVP mRNAs, our data do support a complex effect of gonadal steroids on expression of these mRNAs in the PVN.

Prenatal alcohol exposure results in hyperactivity of the hypothalamic-pituitary-adrenal axis of the offspring: modulation by fostering at birth and postnatal handling.

Exposure of fetal rats to alcohol results in permanent hyperactivity of the hypothalamic-pituitary-adrenal (HPA) axis. In contrast, postnatal handling or fostering have been reported to restrain HPA activity. Because of the deleterious consequences of a hyperresponsive HPA axis, we thought that the possibility that postnatal manipulations might be able to reverse the influence of prenatal alcohol treatment deserved investigation. To test this hypothesis, we exposed rat dams to alcohol by inhalation during the second week of gestation. At birth, pups were either fostered or remained with their dam. For the first 3 weeks, litters were handled daily for 15 min or left undisturbed. At 22 days of age, male and female pups were decapitated under basal conditions, after 10 min of mild electro-footshock, or 10 min after footshock had been terminated. As expected, prenatal exposure to alcohol induced increased adrenocorticotropin (ACTH) secretion in response to footshock, and postnatal handling of control pups resulted in a suppression of corticosterone and ACTH release, although changes in this latter hormone did not reach statistical significance. Surprisingly, however, pups exposed to alcohol that were also fostered and handled after birth, showed an ACTH response to footshock stress that was significantly larger than all other groups. This unexpected response may be due to alterations in maternal-pup behaviors and may indicate that these manipulations act on different neuronal substrates within the central HPA of young rats. Further studies are needed to determine whether adrenal regulation is also altered in animals exposed to alcohol prenatally and reared in a similar manner.

Effect of alcohol on the proestrous surge of luteinizing hormone (LH) and the activation of LH-releasing hormone (LHRH) neurons in the female rat.

Reproduction is adversely affected by alcohol abuse in humans and laboratory animals. In rats, alcohol exposure suppresses both luteinizing hormone (LH) and sex steroid secretion, although consensus is lacking as to which level of the hypothalamic-pituitary-gonadal (HPG) axis is primarily affected. We tested the hypothesis that acute alcohol treatment inhibits the HPG axis by blunting release of LH-releasing hormone (LHRH) in female rats, by examining the effect of this drug on the central reproductive endocrine event; i.e., the proestrous surge of gonadotropins, which triggers ovulation. In a first series of experiments, we injected alcohol at 8 A.M. and 12 P.M. on proestrus and measured plasma levels of LH, estradiol (E2), and progesterone during the afternoons of proestrus and estrus. Alcohol administration blocked the proestrous surge of LH and ovulation. In subsequent experiments, alcohol inhibited the surge of LHRH (measured by push-pull cannulation) and LHRH neuronal activation (measured by Fos labeling in LHRH neurons). Because alcohol also decreased E2 levels, we reasoned that it might have prevented positive feedback; however, alcohol retained its ability to inhibit the LH surge evoked by E2 implantation in ovariectomized females, disproving this hypothesis. Additionally, alcohol does not act via increased corticosteroid secretion, because alcohol also blocked the proestrous surge in adrenalectomized females. Last, exogenous administration of LHRH to alcohol-blocked animals evoked LH secretion and ovulation, indicating that pituitary and/or ovarian function could be restored by mimicking the hypothalamic signal. Collectively, these data indicate that in female rats, alcohol inhibits the gonadotropin surge primarily by decreasing LHRH secretion.

Role of arginine vasopressin and corticotropin-releasing factor in mediating alcohol-induced adrenocorticotropin and vasopressin secretion in male rats bearing lesions of the paraventricular nuclei.

In male rats, lesions of the paraventricular nucleus (PVN) of the hypothalamus attenuate, but do not abolish, adrenocorticotropin (ACTH) secretion in response to acute alcohol injection. As the PVN is the major source of corticotropin-releasing factor (CRF) in the median eminence, this observation suggests that extra-PVN brain regions, and/or ACTH secretagogues other than CRF (e.g. arginine vasopressin (AVP)), mediate ACTH stimulation by alcohol. This hypothesis was tested by examining the effect of AVP immunoneutralization in PVN-lesioned (PVNx) rats. Removal of endogenous AVP diminished alcohol-evoked ACTH secretion in both sham-operated and PVNx animals, indicating that AVP from outside the PVN partially mediates the hypothalamic-pituitary-adrenal (HPA) axis response to alcohol. This led us to determine whether alcohol might also regulate AVP steady-state gene expression in the supraoptic nucleus (SON) and PVN, and/or CRF mRNA in the PVN and the central nucleus of the amygdala (AMY). In the magnocellular portion of the PVN, sham-operated animals showed significantly increased PVN levels of both CRF and AVP mRNAs 3 h after alcohol. In the SON, alcohol administration tended to decrease AVP gene expression in PVNx rats, while the drug increased AVP mRNA levels in the SON of sham-operated rats. AMY levels of CRF mRNA were unaffected by these manipulations. Finally, since the regulation of alcohol-induced AVP mRNA levels in the SON appeared to depend on the presence of the PVN, we measured peripheral levels of AVP in both sham-operated and PVNx animals after injection of vehicle or alcohol. Although AVP decreased in all groups, alcohol depressed AVP secretion to a greater extent in PVNx animals, suggesting that AVP systems are more sensitive to inhibition in the absence of the PVN. Our results demonstrate that although AVP of PVN origin may participate in regulating the stimulatory effect to AVP on ACTH secretion, AVP from areas other than the PVN also plays a role. Additionally, regulation of both AVP gene expression in the SON and secretion in the systemic circulation are altered in rats bearing lesions of the PVN.

Gender difference in alcohol-evoked hypothalamic-pituitary-adrenal activity in the rat: ontogeny and role of neonatal steroids.

Alcohol administration results in activation of the hypothalamic-pituitary-adrenal (HPA) axis, with female rats secreting more adrenocorticotropin (ACTH) and corticosterone (B) than males in response to the same dose of alcohol. We first examined the ontogeny of the gender difference in HPA responsiveness to alcohol by administering four doses (0, 1, 2, or 3 g/kg body weight) to animals at 21, 41, and 61 days of age (prepubertal, peripubertal, and postpubertal, respectively). We then investigated the organizational role of steroids by manipulating the neonatal steroidal milieu. Rats of both genders were gonadectomized or injected with testosterone propionate within 24 hr of birth and the HPA response to 3 g/kg body weight alcohol was tested in adulthood (postpubertal period). Our data show that the gender difference in HPA responsiveness to alcohol administration arises peripubertally. In addition, HPA response to alcohol is quantitatively smaller in intact male rats than in feminized groups (gonadectomized males and females, intact females) and masculinized female rats. We conclude that the gender difference in HPA response to alcohol observed in postpubertal rats injected with alcohol depends on the activational role of testicular androgens, rather than on their organizational influence.

Daily rhythms of follicle-stimulating hormone in adult anestrous and prepubertal female Turkish hamsters (Mesocricetus brandti).

We tested the hypothesis that daily FSH surges are correlated with the anestrous condition in adult Turkish hamsters. Blood samples were collected from anestrous hamsters on short (12L:12D) and long photoperiods (16L:8D) by cardiac puncture. Both photoperiod-induced anestrous (PIA) and spontaneously anestrous adult Turkish hamsters had a daily rhythm of FSH secretion with maximum hormone concentrations occurring late in the afternoon. We also hypothesized that daily FSH and progesterone surges are correlated with the reproductive response to short photoperiod in prepubertal female Turkish hamsters. Prepubertal hamsters reared on 16L:8D were sampled at 13, 15, 17, 19, and 21 days of age. Only at 19 days was a rhythm of FSH present. Prepubertal female hamsters reared on 12L:12D were sampled at 30, 35, 55, 75, 95, and 115 days of age or until estrous cycles were established. Those hamsters that delayed puberty in response to this short photoperiod had a daily rhythm of FSH and progesterone secretion. Conversely, no daily rhythms were observed in female hamsters that failed to respond to the inhibitory photoperiod with a delay in puberty. These results indicate that the daily rhythm of FSH secretion in the female Turkish hamster is correlated with reproductively inhibited physiological states.

Daily rhythms of luteinizing hormone and follicle-stimulating hormone persist in female hamsters sterilized by neonatal administration of monosodium glutamate.

Monosodium glutamate (MSG) was used to evaluate the importance of the arcuate nucleus of the hypothalamus in the expression of daily gonadotropin rhythms in female golden hamsters. These daily rhythms of luteinizing hormone (LH) and follicle-stimulating hormone (FSH), which also occur in prepubertal females, are characterized by afternoon surges. Neonatal administration of MSG induces degeneration of perikarya in the arcuate nucleus and renders females permanently anovulatory. MSG was injected at 8 days of age; at 21 days, the animals were weaned and sorted by sex into groups of 5-7. Blood samples were obtained at 1300 and 1700 h at 25, 30, 35, 40, 50, 62, and 192 days of age from MSG-sterilized animals. Saline-injected controls were bled at 25 days and after estrous cycles had been initiated (29-37 days of age). In both control and MSG-injected groups, there was an afternoon surge of LH and FSH at 25 days of age. These daily surges persisted in MSG-injected animals. The ovaries of these animals were characterized by an abundant interstitium and arrested follicular development. Progesterone levels of MSG-anovulatory animals also reflected the rhythmicity of LH and FSH, with a significant increase occurring between 1300 and 1700 h. Thus, MSG did not affect the daily circadian-based rhythmicity in gonadotropin secretion even though adult-age animals were infertile. These results suggest that perikarya of the arcuate nucleus affected by MSG are not required for generation of daily LH and FSH rhythms.

The timing of gonadal refractoriness in the female Turkish hamster (Mesocricetus brandti) is not dependent on the timing of gonadal regression.

When prepubertal female Turkish hamsters are reared on 12L:12D, individuals respond reproductively in two ways; the majority exhibit a delay in the onset of regular vaginal estrous cyclicity (first vaginal estrus; FVE), and some individuals exhibit FVE at a time not different from females reared on 16L:8D. Soon, however, these females also become reproductively quiescent. This study addresses the following questions: (1) At what age do these two subpopulations become refractory to 12L:12D? (2) Is the onset of refractoriness in adults reared on 12L:12D dependent on the time when they become anovulatory? All females reared on 12L:12D become refractory at a similar age (169.1 +/- 4.4 days in those that exhibit delayed FVE vs. 165.3 +/- 6.8 days in those that exhibited an early FVE and then became anovulatory; p much greater than 0.05). Also, there is no correlation between the age at which vaginal cycles ceased and the age at which those individuals became refractory (r2 = 0.12; slope not different from 0). On the basis of these data, we propose that in the female Turkish hamster the onset of photorefractoriness is timed, not by the interval of reproductive quiescence, but by a preceding event, perhaps the initiation of short photoperiod exposure or the onset of photosensitivity.