These findings indicate that acute lipid and chronic HFD feeding in vivo, as well as acute palmitate and TNF-α exposure in vitro, induce markers of inflammation or ER stress in the hypothalamic appetite-stimulating NPY/AgRP neurons over time, which may contribute to a dramatic alteration in NPY/AgRP content or expression. Acute and chronic HFD feeding in vivo temporally regulates arcuate TNF-α expression with reactive astrocytosis, which suggests a time-dependent neurotrophic or neurotoxic role of lipids.

The distinct lack of cell lines derived from the adult brain is evident. Ciliary neurotrophic factor (CNTF) triggers neurogenesis in primary culture from adult mouse hypothalamus, as detected by bromodeoxyuridine and Ki67 immunostaining. Using SV-40 T-antigen, we immortalized dividing neurons and generated clonal cell lines expressing neuropeptides and receptors involved in neuroendocrine function. We hypothesized that proglucagon-derived peptides may be the mechanistic downstream effectors of CNTF due to documented neuroprotective and proliferative effects. Indeed, proglucagon gene expression was induced by CNTF, and exposure of primary cells to glucagon-like peptide-1 receptor (GLP-1) agonist, exendin-4, induced cell proliferation. Intracerebroventricular injection of CNTF into adult mice caused increased expression of proglucagon peptide in the hypothalamus. Using a specific GLP-1-receptor antagonist, we found that neurogenesis was significantly attenuated and primary culture from GLP-1-receptor-knockout mice lacked CNTF-mediated neuronal proliferation, thus linking the induction of neurogenesis in the hypothalamus to GLP-1-receptor signaling.

Objectives of the study were to determine developmental changes in morphology and expression of androgen receptor (AR) and estrogen receptor (ER)alpha in the body of the rat penis exposed neonatally to diethylstilbestrol (DES). Male pups received DES at a dose of 10 microg per rat on alternate days from Postnatal Day 2 to Postnatal Day 12. Controls received olive oil vehicle only. Tissue samples were collected on Days 18 (prepuberty), 41 (puberty), and 120 (adult) of age. DES-induced abnormalities were evident at 18 days of age and included smaller, lighter, and thinner penis, loss of cavernous spaces and associated smooth muscle cells, and increased deposition of fat cells in the corpora cavernosa penis. Fat cells virtually filled the entire area of the corpora cavernosa at puberty and adulthood. Plasma testosterone (T) was reduced to an undetectable level, while LH was unaltered in all treated groups. AR-positive cells were ubiquitous and their profile (incidence and staining intensity) did not differ between control and treated rats of the respective age groups. Conversely, ERalpha-positive cells were limited to the stroma of corpus spongiosus in all age groups of both control and treated rats, but the expression in treated rats at 18 days was up-regulated in stromal cells of corpora cavernosa, coincident with the presence of morphological abnormalities. Hence, this study reports for the first time DES-induced developmental, morphological abnormalities in the body of the penis and suggests that these abnormalities may have resulted from decreased T and/or overexpression of ERalpha.

We previously reported that diethylstilbestrol (DES) or estradiol valerate (EV) exposure at a dose of 0.10-0.12 mg/kg, or higher, per day, on alternate days, from postnatal days 2-12, resulted in abnormal penis development and infertility (H. O. Goyal et al., 2005, J. Androl. 26, 32-43). The objective of this study was to identify a critical developmental period(s) during which EV exposure results in the observed penile abnormalities. Male pups received EV at a dose of 0.10-0.12 mg/kg on postnatal day(s) 1, 1-3, 4-6, 1-6, 7-12, 13-18, 19-24, or 25-30. Fertility was tested at 102-115 days of age and tissues were examined at 117-137 days. Both penile morphology and fertility were unaltered in rats treated with EV after 12 days of age. Conversely, except in rats treated on postnatal day 1 only, none of the males treated prior to 12 days of age sired pups, and all had abnormal penises, including varying degrees of abnormal accumulation of fat cells and loss of cavernous spaces and smooth muscle cells in the corpora cavernosa penis, which were maximal in the 1-6-day group. Also, the preputial sheath was partially released or its release was delayed, and the weight of the bulbospongiosus muscle was significantly reduced. Plasma testosterone (T) in the 1-6- and 4-6-day groups and intratesticular T in the 4-6-day group were significantly lower. The testosterone surge, characteristic of controls in the first week of life, was suppressed in the 1-3-day group. Estrogen receptor alpha mRNA expression was enhanced in the body of the penis in the 1-3-day group, but not in the 13-18-day group. Hence, EV exposure prior to 12 days of age (as short as 1-3 days postnatal), but not after 12 days of age, results in long-term abnormal penile morphology, characterized by abnormal accumulation of fat cells in the corpora cavernosa penis and, consequently, loss of fertility.

Exendin-4, a long-acting glucagon-like peptide-1 receptor (GLP-1R) agonist, is a potential regulator of feeding behavior through its ability to inhibit gastric emptying, reduce food intake, and induce satiety. GLP-1R activation by exendin-4 induces anorexia; however, the specific populations of neuropeptidergic neurons activated by exendin-4 within the hypothalamus, the central regulator of energy homeostasis, remain unclear. This study determines whether exendin-4 regulates hypothalamic neuropeptide expression and explores the signaling mechanisms involved. The distribution and quantity of exendin-4-induced c-Fos immunoreactivity were evaluated to determine activation of α-melanocyte-stimulating hormone/proopiomelanocortin, neuropeptide Y, neurotensin (NT), and ghrelin neurons in hypothalamic nuclei during exendin-4-induced anorexia in mice. Additionally, exendin-4 action on NT and ghrelin transcript regulation was examined in immortalized hypothalamic neurons. With anorexia induced by intracerebroventricular exendin-4, α-melanocyte-stimulating hormone/proopiomelanocortin and neuropeptide Y neurons were activated in the arcuate nucleus, with simultaneous activation of NT-expressing neurons in the paraventricular nucleus, and ghrelin-expressing neurons in the arcuate nucleus, paraventricular nucleus, and periventricular hypothalamus, suggesting that neurons in one or more of these areas mediate the anorexic action of exendin-4. In the hypothalamic neuronal cell models, exendin-4 increased cAMP, cAMP response element-binding protein/activating transcription factor-1 and c-Fos activation, and via a protein kinase A-dependent mechanism regulated NT and ghrelin mRNA expression, indicating that these neuropeptides may serve as downstream mediators of exendin-4 action. These findings provide a previously unrecognized link between central GLP-1R activation by exendin-4 and the regulation of hypothalamic NT and ghrelin. Further understanding of this central GLP-1R activation may lead to safe and effective therapeutics for the treatment of metabolic disorders.

Previously, we reported an association between estrogen receptor-a (ERa) upregulation and detrimental effects of neonatal diethylstilbestrol (DES) exposure in the rat penis. The objective of this study was to employ the ERa knockout (ERaKO) mouse model to test the hypothesis that ERa mediates DES effects in the developing penis. ERaKO and wild-type C57BL/6 mice received oil or DES at a dose of 0.2 mg/pup per day (0.1 mg/kg) on alternate days from postnatal days 2 to 12. Fertility was tested at 80-240 days of age and tissues were examined at 96-255 days of age. DES caused malformation of the os penis, significant reductions in penile length, diameter, and weight, accumulation of fat cells in the corpora cavernosa penis, and significant reductions in weight of the bulbospongiosus and levator ani muscles in wild-type mice. Conversely, ERaKO mice treated with DES developed none of the above abnormalities. While nine out of ten male mice sired pups in the wild-type/control group, none did in the wildtype/DES group. ERaKO mice, despite normal penile development, are inherently infertile. Both plasma and intratesticular testosterone levels were unaltered in the DES-treated wild-type or DES-treated ERaKO mice when compared with controls, although testosterone concentration was much higher in the ERaKO mice. Hence, the resistance of ERaKO mice to developing penile abnormalities provides unequivocal evidence of an obligatory role for ERa in mediating the harmful effects of neonatal DES exposure in the developing penis.

Kisspeptin (Kiss) and G-protein-coupled receptor (Gpr)54 have emerged as key regulators of reproduction. 17β-estradiol (E2)-mediated regulation of these neurons is nuclei specific, where anteroventral periventricular (AVPV) Kiss neurons are positively regulated by E2, whereas arcuate nucleus (ARC) neurons are inhibited. We have generated immortalized Kiss cell lines from male and female adult-derived murine hypothalamic primary culture, as well as cell lines from microdissected AVPV and ARC from female Kiss-green fluorescent protein (GFP) mice. All exhibit endogenous Kiss-1 expression, estrogen receptors (ER)s (ERα, ERβ, and Gpr30), as well as known markers of AVPV Kiss neurons in the mHypoA-50 and mHypoA-Kiss/GFP-4, vs markers of ARC Kiss neurons in the mHypoA-55 and the mHypoA-Kiss/GFP-3 lines. There was an increase in Kiss-1 mRNA expression at 24 hours in the AVPV lines and a repression of Kiss-1 mRNA at 4 hours in the ARC lines. An E2-mediated decrease in ERα mRNA expression at 24 hours in the AVPV cell lines was detected, and a significant decrease in Gpr30, ERα, and ERβ mRNA levels at 4 hours in the ARC cell lines was evident. ER agonists and antagonists determined the specific ERs responsible for mediating changes in gene expression. In the AVPV, ERα is required but not ERβ or GPR30, vs the ARC Kiss-expressing cell lines that require GPR30, and either ERα and/or ERβ. We determined cAMP response element-binding protein 1 was necessary for the down-regulation of Kiss-1 mRNA expression using small interfering RNA knockdown in the ARC cell model. These studies elucidate some of the molecular events involved in the differential E2-mediated regulation of unique and specific Kiss neuronal models.

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