The annual reproductive cycle in sheep may reflect a functional remodeling within the GnRH system. Specifically, changes in total synaptic input and association with the polysialylated form of neural cell adhesion molecule have been observed. Whether seasonal changes in a specific subset(s) of GnRH inputs occur or whether glial cells specifically play a role in this remodeling is not clear. We therefore examined GnRH neurons of breeding season (BS) and nonbreeding season (anestrus) ewes and tested the hypotheses that specific (i.e. gamma-aminobutyric acid, catecholamine, neuropeptide Y, or beta-endorphin) inputs to GnRH neurons change seasonally, and concomitant with any changes in neural inputs is a change in glial apposition. Using triple-label immunofluorescent visualization of GnRH, glial acidic fibrillary protein and neuromodulator/neural terminal markers combined with confocal microscopy and optical sectioning techniques, we confirmed that total numbers of neural inputs to GnRH neurons vary with season and demonstrated that specific inputs contribute to these overall changes. Specifically, neuropeptide Y and gamma-aminobutyric acid inputs to GnRH neurons increased during BS and beta-endorphin inputs were greater during either anestrus (GnRH somas) or BS (GnRH dendrites). Associated with the changes in GnRH inputs were seasonal changes in glial apposition, glial acidic fibrillary protein density, and the thickness of glial fibrils. These findings are interpreted to suggest an increase in net stimulatory inputs to GnRH neurons during the BS contributes to the seasonal changes in GnRH neurosecretion and that this increased innervation is perhaps stabilized by glial processes.

gamma-Aminobutyric acid (GABA) inhibits the embryonic migration of GnRH neurons and regulates hypothalamic GnRH release. A subset of GnRH neurons expresses GABA along their migratory route in the nasal compartment before entering the brain, suggesting that GABA produced by GnRH neurons may help regulate the migratory process. To examine this hypothesis and the possibility that persistence of GABA production by GnRH neurons may affect subsequent reproductive function, we generated transgenic mice in which the expression of glutamic acid decarboxylase-67 (GAD-67), a key enzyme in GABA synthesis, is targeted to GnRH neurons under the control of the GnRH gene promoter. On embryonic d 15, when GnRH neurons are still migrating, the transgenic animals had more GnRH neurons in aberrant locations in the cerebral cortex and fewer neurons reaching the hypothalamic-preoptic region, whereas migration into the brain was not affected. Hypothalamic GnRH content in mutant mice was low during the first week of postnatal life, increasing to normal values during infantile development (second week after birth) in the presence of increased pulsatile GnRH release. Consistent with these changes, serum LH and FSH levels were also elevated. Gonadotropin release returned to normal values by the time steroid negative feedback became established (fourth week of life). Ovariectomy at this time demonstrated an enhanced gonadotropin response in transgenic animals. Although the onset of puberty, as assessed by the age at vaginal opening and first ovulation, was not affected in the mutant mice, estrous cyclicity and adult reproductive capacity were disrupted. Mutant mice had reduced litter sizes, increased time intervals between deliveries of litters, and a shorter reproductive life span. Thus, GABA produced within GnRH neurons does not delay GnRH neuronal migration, but instead serves as a developmental cue that increases the positional diversity of these neurons within the basal forebrain. In addition, the results suggest that the timely termination of GABA production within the GnRH neuronal network is a prerequisite for normal reproductive function. The possibility arises that similar abnormalities in GABA homeostasis may contribute to syndromes of hypothalamic amenorrhea/oligomenorrhea in humans.

In addition to increasing insulin sensitivity and adipogenesis, peroxisome proliferator-activated receptor (PPAR)-gamma agonists cause weight gain and hyperphagia. Given the central role of the brain in the control of energy homeostasis, we sought to determine whether PPARgamma is expressed in key brain areas involved in metabolic regulation. Using immunohistochemistry, PPARgamma distribution and its colocalization with neuron-specific protein markers were investigated in rat and mouse brain sections spanning the hypothalamus, the ventral tegmental area, and the nucleus tractus solitarius. In several brain areas, nuclear PPARgamma immunoreactivity was detected in cells that costained for neuronal nuclei, a neuronal marker. In the hypothalamus, PPARgamma immunoreactivity was observed in a majority of neurons in the arcuate (including both agouti related protein and alpha-MSH containing cells) and ventromedial hypothalamic nuclei and was also present in the hypothalamic paraventricular nucleus, the lateral hypothalamic area, and tyrosine hydroxylase-containing neurons in the ventral tegmental area but was not expressed in the nucleus tractus solitarius. To validate and extend these histochemical findings, we generated mice with neuron-specific PPARgamma deletion using nestin cre-LoxP technology. Compared with littermate controls, neuron-specific PPARgamma knockout mice exhibited dramatic reductions of both hypothalamic PPARgamma mRNA levels and PPARgamma immunoreactivity but showed no differences in food intake or body weight over a 4-wk study period. We conclude that: 1) PPARgamma mRNA and protein are expressed in the hypothalamus, 2) neurons are the predominant source of PPARgamma in the central nervous system, although it is likely expressed by nonneuronal cell types as well, and 3) arcuate nucleus neurons that control energy homeostasis and glucose metabolism are among those in which PPARgamma is expressed.

Fibroblast growth factor 21 (FGF21) is a novel metabolic regulator shown to improve glycemic control. However, the molecular and functional mechanisms underlying FGF21-mediated improvements in glycemic control are not completely understood. We examined FGF21 effects on insulin sensitivity and glucose fluxes upon chronic (daily injection for 8 d) and acute (6 h infusion) administration in ob/+ and ob/ob mice. Results show that chronic FGF21 ameliorated fasting hyperglycemia in ob/ob mice via increased glucose disposal and improved hepatic insulin sensitivity. Acute FGF21 suppressed hepatic glucose production, increased liver glycogen, lowered glucagon, and improved glucose clearance in ob/+ mice. These effects were blunted in ob/ob mice. Neither chronic nor acute FGF21 altered skeletal muscle or adipose tissue glucose uptake in either genotype. In conclusion, FGF21 has potent glycemic effects caused by hepatic changes in glucose flux and improved insulin sensitivity. Thus, these studies define mechanisms underlying anti-hyperglycemic actions of FGF21 and support its therapeutic potential.

In the setting of insulin resistance, agonists of peroxisome proliferator-activated receptor (PPAR)-gamma restore insulin action in muscle and promote lipid redistribution. Mice with muscle-specific knockout of PPARgamma (MuPPARgammaKO) develop excess adiposity, despite reduced food intake and normal glucose disposal in muscle. To understand the relation between muscle PPARgamma and lipid accumulation, we studied the fuel energetics of MuPPARgammaKO mice. Compared with controls, MuPPARgammaKO mice exhibited significantly increased ambulatory activity, muscle mitochondrial uncoupling, and respiratory quotient. Fitting with this latter finding, MuPPARgammaKO animals compared with control siblings exhibited a 25% reduction in the uptake of the fatty acid tracer 2-bromo-palmitate (P < 0.05) and a 13% increase in serum nonesterified fatty acids (P = 0.05). These abnormalities were associated with no change in AMP kinase (AMPK) phosphorylation, AMPK activity, or phosphorylation of acetyl-CoA carboxylase in muscle and occurred despite increased expression of fatty acid transport protein 1. Palmitate oxidation was not significantly altered in MuPPARgammaKO mice despite the increased expression of several genes promoting lipid oxidation. These data demonstrate that PPARgamma, even in the absence of exogenous activators, is required for normal rates of fatty acid uptake in oxidative skeletal muscle via mechanisms independent of AMPK and fatty acid transport protein 1. Thus, when PPARgamma activity in muscle is absent or reduced, there will be decreased fatty acid disposal leading to diminished energy utilization and ultimately adiposity.

Stress exerts profound inhibitory effects on reproductive function by suppressing the pulsatile release of GnRH and therefore LH. Although the mechanisms by which stressors disrupt the hypothalamic GnRH pulse generator remain to be fully elucidated, numerous studies have implicated the amygdala, especially its medial (MeA) and central nuclei (CeA), as key modulators of the neuroendocrine response to stress. In the present study, we investigated the roles of the MeA and CeA in stress-induced suppression of LH pulses. Ovariectomized rats received bilateral ibotenic acid or sham lesions targeting the MeA or CeA; blood samples (25 μl) were taken via chronically implanted cardiac catheters every 5 min for 6 h for the measurement of LH pulses. After 2 h of baseline sampling, the rats were exposed to either: restraint (1 h), insulin-induced hypoglycemia (IIH) (0.3 U/kg, iv), or lipopolysaccharide (LPS) (25 μg/kg, iv) stress. The restraint but not IIH or LPS stress-induced suppression of LH pulses was markedly attenuated by the MeA lesions. In contrast, CeA lesioning attenuated LPS, but not restraint or IIH stress-induced suppression of LH pulses. Moreover, after restraint stress, the number of Fos-positive neurons and the percentage of glutamic acid decarboxylase(67) neurons expressing Fos was significantly greater in the GnRH-rich medial preoptic area (mPOA) of rats with intact, rather than lesioned, MeA. These data indicate that the MeA and CeA play key roles in psychogenic and immunological stress-induced suppression of the GnRH pulse generator, respectively, and the MeA-mediated effect may involve γ-aminobutyric acid ergic signaling within the mPOA.

The epithelial lining of the epididymal duct expresses the androgen receptor (Ar) along its entire length and undergoes rapid and profound degeneration when androgenic support is withdrawn. However, experiments involving orchidectomy with systemic testosterone replacement, and testicular efferent duct ligation, have indicated that structural and functional integrity of the initial segment cannot be maintained by circulating androgen alone, leaving the role of androgen in this epididymal zone unclear. We addressed this question in a mouse model with intact testicular output and selective Ar inactivation in the proximal epididymis by creating double-transgenic males carrying a conditional Ar(loxP) allele and expressing Cre recombinase under the promoter of Rnase10, a gene specifically expressed in proximal epididymis. At 20-25 d of life, on the onset of Rnase10 expression, Ar became selectively inactivated in the principal cells of proximal epididymis, resulting in epithelial hypoplasia and hypotrophy. Upon the subsequent onset of spermiation, epididymal obstruction ensued, with the consequent development of spermatic granulomata, back pressure-induced atrophy of the seminiferous epithelium, orchitis, and fibrosis of the testicular parenchyma. Consistent with these findings, the mice were infertile. When the effect of Ar knockout on gene expression in the proximal epididymis was compared with that of efferent duct ligation and orchidectomy, we identified genes specifically regulated by androgen, testicular efferent fluid, and both. Our findings demonstrate that the development and function of the epididymal initial segment is critically dependent on direct androgen regulation. The phenotype of the produced knockout mouse provides a novel model for obstructive azoospermia.

Neuropeptide Y (NPY), a member of the pancreatic polypeptide family, is an orexigenic hormone. GnRH-1 neurons express NPY receptors. This suggests a direct link between metabolic function and reproduction. However, the effect of NPY on GnRH-1 cells has been variable, dependent on metabolic and reproductive status of the animal. This study circumvents these issues by examining the role of NPY on GnRH-1 neuronal activity in an explant model that is based on the extra-central nervous system origin of GnRH-1 neurons. These prenatal GnRH-1 neurons express many receptors found in GnRH-1 neurons in the brain and use similar transduction pathways. In addition, these GnRH-1 cells exhibit spontaneous and ligand-induced oscillations in intracellular calcium as well as pulsatile calcium-controlled GnRH-1 release. Single-cell PCR determined that prenatal GnRH-1 neurons express the G protein-coupled Y1 receptor (Y1R). To address the influence of NPY on GnRH-1 neuronal activity, calcium imaging was used to monitor individual and population dynamics. NPY treatment, mimicked with Y1R agonist, significantly decreased the number of calcium peaks per minute in GnRH-1 neurons and was prevented by a Y1R antagonist. Pertussis toxin blocked the effect of NPY on GnRH-1 neuronal activity, indicating the coupling of Y1R to inhibitory G protein. The NPY-induced inhibition was independent of the adenylate cyclase pathway but mediated by the activation of G protein-coupled inwardly rectifying potassium channels. These results indicate that at an early developmental stage, GnRH-1 neuronal activity can be directly inhibited by NPY via its Y1R.

Proteins of the activator protein-1 family are known to have roles in many physiological processes such as proliferation, apoptosis, and inflammation. However, their role in fat metabolism has yet to be defined in more detail. Here we study the impact of JunB deficiency on the metabolic state of mice. JunB knockout (JunB-KO) mice show markedly decreased weight gain, reduced fat mass, and a low survival rate compared with control mice. If fed a high-fat diet, the weight gain of JunB-KO mice is comparable to control mice and the survival rate improves dramatically. Along with normal expression of adipogenic marker genes in white adipose tissue (WAT) of JunB-KO mice, this suggests that adipogenesis per se is not affected by JunB deficiency. This is supported by in vitro data, because neither JunB-silenced 3T3-L1 cells nor mouse embryonic fibroblasts from JunB-KO mice show a change in adipogenic potential. Interestingly, the key enzymes of lipolysis, adipose triglyceride lipase and hormone-sensitive lipase, were significantly increased in WAT of fasted JunB-KO mice. Concomitantly, the ratio of plasma free fatty acids per gram fat mass was increased, suggesting an elevated lipolytic rate under fasting conditions. Furthermore, up-regulation of TNFα and reduced expression of perilipin indicate that this pathway is also involved in increased lipolytic rate in these mice. Additionally, JunB-KO mice are more insulin sensitive than controls and show up-regulation of lipogenic genes in skeletal muscle, indicating a shuttling of energy substrates from WAT to skeletal muscle. In summary, this study provides valuable insights into the impact of JunB deficiency on the metabolic state of mice.

The insulinoma-associated 2 (Insm2) gene is a member of the Snail/Gfi1/Insm1 transcriptional repressor superfamily. However, little is known about how the expression of human INSM2 or mouse Insm2 in neuroendocrine tissues is regulated. Here we report the expression of INSM2/Insm2 in human fetal pancreas and mouse embryos, as well as adult pancreatic islets, and its regulation by two major islet transcription factors. Mutagenesis and chromatin immunoprecipitation analysis demonstrated that the proximal E-boxes of the mouse Insm2 promoter are direct targets of neurogenin 3 and neurogenic differentiation 1 (NeuroD1). Furthermore, we found that endogenous Insm2 expression was activated in Ngn3/NeuroD1-transduced pancreatic epithelial duct cells. Our results suggest that Insm2 plays an important role in the differentiation cascade of Ngn3/NeuroD1 signaling in pancreatic islets.

The hypothalamic melanocortin system is unique among neuropeptide systems controlling energy homeostasis, in that both anorexigenic proopiomelanocortin (POMC)-derived and orexigenic Agouti related-peptide (AgRP)-derived ligands act at the same receptors, namely melanocortin 3 and 4 receptors (MC3/4R). AgRP clearly acts as a competitive antagonist at MC3R and MC4R but may also have an inverse agonist action at these receptors. The physiological relevance of this remains uncertain. We generated a mouse lacking both POMC and AgRP [double knockout (DKO) mouse]. Phenotyping was performed in the absence and presence of glucocorticoids, and the response to central peptide administration was studied. The phenotype of DKO mice is indistinguishable from that of mice lacking Pomc alone, with both exhibiting highly similar degrees of hyperphagia and increased body length, fat, and lean mass compared with wild-type controls. After a 24-h fast, there was no difference in the refeeding response between Pomc(-/-) and DKO mice. Similarly, corticosterone supplementation caused an equivalent increase in food intake and body weight in both genotypes. Although the central administration of [Nle⁴, d-Phe⁷]-α-MSH to DKO mice caused a decrease in food intake and an increase in brown adipose tissue Ucp1 expression, both of which could be antagonized with the coadministration of AgRP, there was no effect of AgRP alone. These data suggest AgRP acts predominantly as a melanocortin antagonist. If AgRP has significant melanocortin-independent actions, these are of insufficient magnitude in vivo to impact any of the detailed phenotypes we have measured under a wide variety of conditions.

Hypothalamic kisspeptin neurons are critical for driving reproductive function, but virtually nothing is known about their endogenous electrophysiological properties and the effects of leptin on their excitability. Therefore, we used the slice preparation from female guinea pigs to study the endogenous conductances and the effects of leptin on kisspeptin neurons. We targeted the arcuate kisspeptin neurons using visualized-patch whole-cell recording and identified kisspeptin neurons using immuocytochemical staining for kisspeptin or single cell RT-PCR. We also harvested dispersed arcuate neurons for analysis of expression of channel transcripts. Kisspeptin neurons exhibited a relatively negative resting membrane potential, and eighty percent of the neurons expressed a pacemaker current (h-current) and a T-type Ca(2+) current. Furthermore, the glutamate receptor agonist N-methyl D-aspartic acid depolarized and induced burst firing in kisspeptin neurons. Leptin activated an inward current that depolarized kisspeptin neurons and increased (burst) firing, but leptin hyperpolarized NPY neurons. Lanthanum, a TRPC-4,-5 channel activator, potentiated the leptin-induced inward current by 170%. The leptin-activated current reversed near -15 mV and was abrogated by the relatively selective TRPC channel blocker 2-APB. The leptin effects were also blocked by a Janus kinase inhibitor, a phosphatidylinositol 3 kinase inhibitor, and a phospholipase Cγ inhibitor. In addition, the majority of these neurons expressed TRPC1 and -5 and phospholipase Cγ1 based on single cell RT-PCR. Therefore, guinea pig kisspeptin neurons express endogenous pacemaker currents, and leptin excites these neurons via activation of TRPC channels. The leptin excitatory effects on kisspeptin neurons may be critical for governing the excitatory drive to GnRH neurons during different nutritional states.

Angiotensin-converting enzyme (ACE)-2 is a homolog of the well-characterized plasma membrane-bound angiotensin-converting enzyme. ACE2 is thought to play a critical role in regulating heart function, and in 2003, ACE2 was identified as a functional receptor for severe acute respiratory syndrome coronavirus. We have recently shown that like ACE, ACE2 undergoes ectodomain shedding and that this shedding event is up-regulated by phorbol esters. In the present study, we used gel shift assays to demonstrate that calmodulin, an intracellular calcium-binding protein implicated in the regulation of other ectodomain shedding events, binds a 16-amino acid synthetic peptide corresponding to residues 762-777 within the cytoplasmic domain of human ACE2, forming a calcium-dependent calmodulin-peptide complex. Furthermore, we have demonstrated that ACE2 expressed in Chinese hamster ovary cells specifically binds to glutathione-S-transferase-calmodulin, but not glutathione-S-transferase alone, in pull-down assays using cell lysates. Finally, to investigate whether calmodulin has any effect on ACE2 ectodomain shedding in cells that endogenously express the enzyme, cells from a human liver cell line (Huh-7) expressing ACE2 were incubated with calmodulin-specific inhibitors, trifluoperazine and calmidazolium. Both trifluoperazine (25 micromol/liter) and calmidazolium, (25 micromol/liter) significantly increased the release of ACE2 into the medium (44.1 +/- 10.8%, P < 0.05, Student's t test; unpaired, two-tailed, and 51.1 +/- 7.4% P < 0.05, one-way ANOVA, respectively;), as analyzed by an ACE2-specific quenched fluorescence substrate assay. We also show that the calmodulin-specific inhibitor-stimulated shedding of ACE2 is independent from phorbol ester-induced shedding. In summary, we have demonstrated that calmodulin is able to bind ACE2 and suggest that the ACE2 ectodomain shedding and/or sheddase(s) activation regulated by calmodulin is independent from the phorbol ester-induced shedding.

The adrenal zona glomerulosa (ZG) secretes aldosterone to regulate sodium balance. Chronic sodium restriction increases aldosterone accompanied by ZG expansion. The ZG is innervated by sympathetic, vasoactive intestinal polypeptide (VIP) and neuropeptide tyrosine (NPY), and sensory, calcitonin gene-related peptide, nerves. It is unclear whether innervation is affected by ZG growth. Therefore, we measured neurite outgrowth in the ZG of adult male rats after dietary sodium manipulation. In response to 1 wk sodium restriction, VIP and NPY fibers elongated in parallel with expansion of the ZG, shown by aldosterone synthase (AS) expression, but calcitonin gene-related peptide fibers were not affected. Sodium repletion resulted in parallel regression in VIP and NPY fiber length and AS expression. These results show that sympathetic, but not sensory, innervation is coordinated with ZG growth. Mediators underlying changes in innervation are unknown; therefore, we characterized a novel gene TMEM35 [termed the unknown factor-1 (TUF1) due to its unknown function] that shows extensive overlap with AS in ZG. After sodium restriction, TUF1 expanded in parallel with the ZG. TUF1 bound the low-affinity neurotrophin receptor, p75NTR, which was expressed in NPY fibers and showed a response similar to TUF1 after sodium manipulation. TUF1- p75NTR binding was competitively displaced by nerve growth factor but not by TUF1 lacking the p75NTR binding motif. Moreover, TUF1 mRNA in rat ZG cells increased after angiotensin II exposure in vitro. Collectively, these findings suggest that TMEM35/TUF1 is a candidate for modulating neurite outgrowth in the ZG after sodium depletion.

The Lin-11, Isl-1, and Mec-3 (LIM) homeodomain transcription factor Isl-1 has been reported to be involved in pituitary development in the early stages of mouse embryogenesis. Our recent studies have shown that Isl-1 is mainly located in the pituitary gonadotropes throughout pituitary development and persists to adulthood. We still do not know the physiological functions of Isl-1 expression and its related mechanisms in the pituitary gland. The aim of the present study was to examine the hypothesis that Isl-1 is involved in regulating pituitary gonadotropin hormone (FSH/LH) production by activating FSHβ and LHβ gene expressions. We have shown that Isl-1 activates FSHβ and LHβ subunit promoters and endogenous gene transcription in LβT2 cells. In addition, Isl-1 overexpression significantly increased FSH synthesis and secretion but not LH. The actions of Isl-1 were not observed when the homeodomain or LIM1 domains are mutated. This demonstrates that Isl-1 induction of FSHβ and LHβ is by both direct and indirect binding of Isl-1 to DNA sequences. Furthermore, Isl-1 expressional level was up-regulated in LβT2 cells after exposure to GnRH, activin, and leptin. However, RNA interference-induced knockdown of Isl-1 significantly reduced the effect of leptin but did not obviously influence the stimulating effects of GnRH and activin on LH and FSH production. In conclusion, the results demonstrate that the LIM-homeodomain transcription factor Isl-1 functions to increase FSHβ/LHβ gene transcription, and mediates the effects of leptin on gonadotropin synthesis.

In the seminiferous epithelium of rat testes, preleptotene spermatocytes residing in the basal compartment are transported across the blood-testis barrier (BTB) to enter the adluminal compartment at stage VIII of the epithelial cycle. This process involves redistribution of tight junction (TJ) proteins via reorganization of actin cytoskeleton in Sertoli cells that serves as attachment site for adhesion protein complexes. Ribosomal protein S6 (rpS6), a downstream molecule of mTORC1 (mammalian target of rapamycin complex 1), participates in this process via a yet-to-be defined mechanism. Here, we constructed an rpS6 quadruple phosphomimetic mutant by converting Ser residues at 235, 236, 240, and 244 to Glu via site-directed mutagenesis, making this mutant constitutively active. When this rpS6 mutant was overexpressed in Sertoli cells cultured in vitro with an established TJ barrier mimicking the BTB in vivo, it perturbed the TJ permeability by down-regulating and redistributing TJ proteins at the cell-cell interface. These changes are mediated by a reorganization of actin microfilaments, which was triggered by a redistribution of activated actin-related protein 3 (Arp3) as well as changes in Arp3-neuronal Wiskott-Aldrich Syndrome protein (N-WASP) interaction. This in turn induced reorganization of actin microfilaments, converting them from a "bundled" to an "unbundled/branched" configuration, concomitant with a reduced actin bundling activity, thereby destabilizing the TJ-barrier function. These changes were mediated by Akt (transforming oncogene of v-akt), because an Akt knockdown by RNA interference was able to mimic the phenotypes of rpS6 mutant overexpression at the Sertoli cell BTB. In summary, this study illustrates a mechanism by which mTORC1 signal complex regulates BTB function through rpS6 downstream by modulating actin organization via the Arp2/3 complex, which may be applicable to other tissue barriers.

Hypothalamic inflammation, involving microglia activation in the arcuate nucleus (ARC), is proposed as a novel underlying mechanism in obesity, insulin and leptin resistance. However, whether activated microglia affects ARC neuronal activity, and consequently basal and hormonal-induced food intake, is unknown. We show that lipopolysaccharide, an agonist of the toll-like receptor-4 (TLR4), which we found to be expressed in ARC microglia, inhibited the firing activity of the majority of orexigenic agouti gene-related protein/neuropeptide Y neurons, whereas it increased the activity of the majority of anorexigenic proopiomelanocortin neurons. Lipopolysaccharide effects in agouti gene-related protein/neuropeptide Y (but not in proopiomelanocortin) neurons were occluded by inhibiting microglia function or by blocking TLR4 receptors. Finally, we report that inhibition of hypothalamic microglia altered basal food intake, also preventing central orexigenic responses to ghrelin. Our studies support a major role for a TLR4-mediated microglia signaling pathway in the control of ARC neuronal activity and feeding behavior.

Throughout most of the ovulatory cycle, estrogen negative feedback restrains the GnRH neuronal system. Just before ovulation, however, estrogen negative feedback is removed to permit stimulation of the preovulatory GnRH/LH surge (positive feedback) by the circadian clock in the suprachiasmatic nucleus (SCN). The mammalian ortholog of avian gonadotropin-inhibitory hormone, RFamide-related peptide 3 (RFRP-3), participates in the circadian-timed removal of estrogen negative feedback to permit the LH surge. The present study examined the specific neurochemical means by which the SCN controls RFRP-3 activity and explored whether the RFRP-3 system exhibits time-dependent responsiveness to SCN signaling to precisely time the LH surge. We found that RFRP-3 cells in female Syrian hamsters (Mesocricetus auratus) receive close appositions from SCN-derived vasopressin-ergic and vasoactive intestinal peptide (VIP)-ergic terminal fibers. Central VIP administration markedly suppressed RFRP-3 cellular activity in the evening, but not the morning, relative to saline controls, whereas vasopressin was without effect at either time point. Double-label in situ hybridization for Rfrp-3 and the VIP receptors VPAC1 and VPAC2 revealed that the majority of RFRP-3 cells do not coexpress either receptor in Syrian hamsters or mice, suggesting that SCN VIP-ergic signaling inhibits RFRP-3 cells indirectly. The timing of this VIP-mediated disinhibition is further coordinated via temporally gated responsiveness of RFRP-3 cells to circadian signaling. Together, these findings reveal a novel circadian hierarchy of control coordinating the preovulatory LH surge and ovulation.

Donohue syndrome (DS) is characterized by severe insulin resistance due to mutations in the insulin receptor (INSR) gene. To identify molecular defects contributing to metabolic dysregulation in DS in the undifferentiated state, we generated mesenchymal progenitor cells (MPCs) from induced pluripotent stem cells derived from a 4-week-old female with DS and a healthy newborn male (control). INSR mRNA and protein were significantly reduced in DS MPC (for β-subunit, 64% and 89% reduction, respectively, P < .05), but IGF1R mRNA and protein did not differ vs control. Insulin-stimulated phosphorylation of INSR or the downstream substrates insulin receptor substrate 1 and protein kinase B did not differ, but ERK phosphorylation tended to be reduced in DS (32% decrease, P = .07). By contrast, IGF-1 and insulin-stimulated insulin-like growth factor 1 (IGF-1) receptor phosphorylation were increased in DS (IGF-1, 8.5- vs 4.5-fold increase; INS, 11- vs 6-fold; P < .05). DS MPC tended to have higher oxygen consumption in both the basal state (87% higher, P =.09) and in response to the uncoupler carbonyl cyanide-p-triflouromethoxyphenylhydrazone (2-fold increase, P =.06). Although mitochondrial DNA or mass did not differ, oxidative phosphorylation protein complexes III and V were increased in DS (by 37% and 6%, respectively; P < .05). Extracellular acidification also tended to increase in DS (91% increase, P = .07), with parallel significant increases in lactate secretion (34% higher at 4 h, P < .05). In summary, DS MPC maintain signaling downstream of the INSR, suggesting that IGF-1R signaling may partly compensate for INSR mutations. However, alterations in receptor expression and pathway-specific defects in insulin signaling, even in undifferentiated cells, can alter cellular oxidative metabolism, potentially via transcriptional mechanisms.

Thyroid hormones are released from thyroglobulin (Tg) in lysosomes, which are impaired in infantile/nephropathic cystinosis. Cystinosis is a lysosomal cystine storage disease due to defective cystine exporter, cystinosin. Cystinotic children develop subclinical and then overt hypothyroidism. Why hypothyroidism is the most frequent and earliest endocrine complication of cystinosis is unknown. We here defined early alterations in Ctns(-/-) mice thyroid and identified subcellular and molecular mechanisms. At 9 months, T4 and T3 plasma levels were normal and TSH was moderately increased (∼4-fold). By histology, hyperplasia and hypertrophy of most follicles preceded colloid exhaustion. Increased immunolabeling for thyrocyte proliferation and apoptotic shedding indicated accelerated cell turnover. Electron microscopy revealed endoplasmic reticulum (ER) dilation, apical lamellipodia indicating macropinocytic colloid uptake, and lysosomal cystine crystals. Tg accumulation in dilated ER contrasted with mRNA down-regulation. Increased expression of ER chaperones, glucose-regulated protein of 78 kDa and protein disulfide isomerase, associated with alternative X-box binding protein-1 splicing, revealed unfolded protein response (UPR) activation by ER stress. Decreased Tg mRNA and ER stress suggested reduced Tg synthesis. Coordinated increase of UPR markers, activating transcription factor-4 and C/EBP homologous protein, linked ER stress to apoptosis. Hormonogenic cathepsins were not altered, but lysosome-associated membrane protein-1 immunolabeling disclosed enlarged vesicles containing iodo-Tg and impaired lysosomal fusion. Isopycnic fractionation showed iodo-Tg accumulation in denser lysosomes, suggesting defective lysosomal processing and hormone release. In conclusion, Ctns(-/-) mice showed the following alterations: 1) compensated primary hypothyroidism and accelerated thyrocyte turnover; 2) impaired Tg production linked to ER stress/UPR response; and 3) altered endolysosomal trafficking and iodo-Tg processing. The Ctns(-/-) thyroid is useful to study disease progression and evaluate novel therapies.