The G209R mutant mouse as a model for human PCSK1 polyendocrinopathy.

PCSK1 encodes an enzyme required for prohormone maturation into bioactive peptides. A striking number of SNPs and rare mutations in PCSK1 are associated with a range of clinical phenotypes. Infants bearing two copies of a catalytically inactivating mutation, such as G209R, exhibit life-threatening chronic diarrhea and subsequently develop systemic endocrinopathies. Using CRISPR/Cas9 technology, we have engineered a mouse model bearing a G209R missense mutation in exon 6 of the murine Pcsk1 locus. Most pups homozygous for the G209R mutation succumbed by day 2, and surviving pups were severely dwarfed. In homozygous (but not heterozygous) pups, blood glucose levels were significantly lower, accompanied by elevated plasma insulin-like immunoreactivity and accumulation of large quantities of unprocessed proinsulin in the pancreas. Peptide hormone processing was also aberrant in G209R mouse pituitary, with mature ACTH levels markedly reduced in homozygotes, accompanied by a significant accumulation of POMC. We also observed a significant reduction in PC1/3 protein in the brains of G209R homozygous mice by Western blotting, while PC2 levels remained unaffected. Most likely due to the continued presence of PC2, pituitary and brain levels of α-MSH were not impaired. Analysis of intestinal cell types indicated a modest reduction of enteroendocrine cells in G209R homozygotes. We suggest that the G209R Pcsk1 mouse model recapitulates many of the dramatic neonatal deficiencies of human patients with this homozygous mutation.

Mice lacking PC1/3 expression in POMC-expressing cells do not develop obesity.

Pro-opiomelanocortin (POMC) neurons form an integral part of the central melanocortin system regulating food intake and energy expenditure. Genetic and pharmacological studies have revealed that defects in POMC synthesis, processing, and receptor signaling lead to obesity. It is well established that POMC is extensively processed by a series of enzymes, including prohormone convertases PC1/3 and PC2, and that genetic insufficiency of both PC1/3 and POMC is strongly associated with obesity risk. However, whether PC1/3-mediated POMC processing is absolutely tied to body weight regulation is not known. To investigate this question, we generated a Pomc-CreER  T2; Pcsk1  lox/lox mouse model in which Pcsk1 is specifically and temporally knocked out in POMC-expressing cells of adult mice by injecting tamoxifen at eight weeks of age. We then measured the impact of Pcsk1 deletion on POMC cleavage to ACTH and α-MSH, and on body weight. In whole pituitary, POMC cleavage was significantly impacted by the loss of Pcsk1, while hypothalamic POMC-derived peptide levels remained similar in all genotypes. However, intact POMC levels were greatly elevated in Pomc-CreER  T2; Pcsk1  lox/lox mice. Males expressed two-fold greater levels of pituitary PC1/3 protein than females, consistent with their increased POMC cleavage. Past studies show that mice with germline removal of PC1/3 do not develop obesity, while mice expressing mutant PC1/3 forms do develop obesity. We conclude that obesity pathways are not disrupted by PC1/3 loss solely in POMC-expressing cells, further disfavoring the idea that alterations in POMC processing underlie obesity in PCSK1 deficiency.

Mouse Models of Human Proprotein Convertase Insufficiency.

The kexin-like proprotein convertases perform the initial proteolytic cleavages that ultimately generate a variety of different mature peptide and proteins, ranging from brain neuropeptides to endocrine peptide hormones, to structural proteins, among others. In this review, we present a general introduction to proprotein convertase structure and biochemistry, followed by a comprehensive discussion of each member of the kexin-like subfamily of proprotein convertases. We summarize current knowledge of human proprotein convertase insufficiency syndromes, including genome-wide analyses of convertase polymorphisms, and compare these to convertase null and mutant mouse models. These mouse models have illuminated our understanding of the roles specific convertases play in human disease and have led to the identification of convertase-specific substrates; for example, the identification of procorin as a specific PACE4 substrate in the heart. We also discuss the limitations of mouse null models in interpreting human disease, such as differential precursor cleavage due to species-specific sequence differences, and the challenges presented by functional redundancy among convertases in attempting to assign specific cleavages and/or physiological roles. However, in most cases, knockout mouse models have added substantively both to our knowledge of diseases caused by human proprotein convertase insufficiency and to our appreciation of their normal physiological roles, as clearly seen in the case of the furin, proprotein convertase 1/3, and proprotein convertase 5/6 mouse models. The creation of more sophisticated mouse models with tissue- or temporally-restricted expression of specific convertases will improve our understanding of human proprotein convertase insufficiency and potentially provide support for the emerging concept of therapeutic inhibition of convertases.

Publisher Correction: Increased expression and retention of the secretory chaperone proSAAS following cell stress.

Due to an unfortunate mistake during the production process, the last sentence of the second last paragraph of the Discussion section contains an error as the words 'increases' and 'decreased' were transposed.

Increased expression and retention of the secretory chaperone proSAAS following cell stress.

The secretory pathway of neurons and endocrine cells contains a variety of mechanisms designed to combat cellular stress. These include not only the unfolded protein response pathways but also diverse chaperone proteins that collectively work to ensure proteostatic control of secreted and membrane-bound molecules. One of the least studied of these chaperones is the neural- and endocrine-specific molecule known as proSAAS. This small chaperone protein acts as a potent anti-aggregant both in vitro and in cellulo and also represents a cerebrospinal fluid biomarker in Alzheimer's disease. In the present study, we have examined the idea that proSAAS, like other secretory chaperones, might represent a stress-responsive protein. We find that exposure of neural and endocrine cells to the cell stressors tunicamycin and thapsigargin increases cellular proSAAS mRNA and protein in Neuro2A cells. Paradoxically, proSAAS secretion is inhibited by these same drugs. Exposure of Neuro2A cells to low concentrations of the hypoxic stress inducer cobalt chloride, or to sodium arsenite, an oxidative stressor, also increases cellular proSAAS content and reduces its secretion. We conclude that the cellular levels of the small secretory chaperone proSAAS are positively modulated by cell stress.

Reduced Stability and pH-Dependent Activity of a Common Obesity-Linked PCSK1 Polymorphism, N221D.

Common mutations in the human prohormone convertase (PC)1/3 gene (PCKSI) are linked to increased risk of obesity. Previous work has shown that the rs6232 single-nucleotide polymorphism (N221D) results in slightly decreased activity, although whether this decrease underlies obesity risk is not clear. We observed significantly decreased activity of the N221D PC1/3 enzyme at the pH of the trans-Golgi network; at this pH, the mutant enzyme was less stable than wild-type enzyme. Recombinant N221D PC1/3 also showed enhanced susceptibility to heat stress. Enhanced susceptibility to tunicamycin-induced endoplasmic reticulum stress was observed in AtT-20/PC2 cell clones in which murine PC1/3 was replaced by human N221D PC1/3, as compared with wild-type human PC1/3. However, N221D PC1/3-expressing AtT-20/PC2 clones processed proopiomelanocortin to α-MSH similarly to wild-type PC1/3. We also generated a CRISPR-edited mouse line expressing the N221D mutation in the PCKSI gene. When homozygous N221D mice were fed either a standard or a high-fat diet, we found no increase in body weight compared with their wild-type sibling controls. Sexual dimorphism was observed in pituitary ACTH for both genotypes, with females exhibiting lower levels of pituitary ACTH. In contrast, hypothalamic α-MSH content for both genotypes was higher in females compared with males. Hypothalamic corticotropin-like intermediate peptide content was higher in wild-type females compared with wild-type, but not N221D, males. Taken together, these data suggest that the increased obesity risk linked to the N221D allele in humans may be due in part to PC1/3-induced loss of resilience to stressors rather than strictly to decreased enzymatic activity on peptide precursors.

Hindbrain Estrogen Receptor Regulation of Ventromedial Hypothalamic Glycogen Metabolism and Glucoregulatory Transmitter Expression in the Hypoglycemic Female Rat.

Neural substrates for estrogen regulation of glucose homeostasis remain unclear. Female rat dorsal vagal complex (DVC) A2 noradrenergic neurons are estrogen- and metabolic-sensitive. The ventromedial hypothalamic nucleus (VMN) is a key component of the brain network that governs counter-regulatory responses to insulin-induced hypoglycemia (IIH). Here, the selective estrogen receptor-alpha (ERα) or -beta (ERß) antagonists MPP and PHTPP were administered separately to the caudal fourth ventricle to address the premise that these hindbrain ER variants exert distinctive control of VMN reactivity to IIH in the female sex. Data show that ERα governs hypoglycemic patterns of VMN astrocyte glycogen metabolic enzyme, e.g. glycogen synthase and phosphorylase protein expression, whereas ERß mediates local glycogen breakdown. DVC ERs also regulate VMN neurotransmitter signaling of energy sufficiency [γ-aminobutyric acid] or deficiency [nitric oxide, steroidogenic factor-1] during IIH. Neither hindbrain ER mediates IIH-associated diminution of VMN norepinephrine (NE) content. Both ERs oppose hypoglycemic hyperglucagonemia, while ERß contributes to reduced corticosterone output. Outcomes reveal that input from the female hindbrain to the VMN is critical for energy reserve mobilization, metabolic transmitter signaling, and counter-regulatory hormone secretion during hypoglycemia, and that ERs control those cues. Evidence that VMN NE content is not controlled by hindbrain ERα or -ß implies that these receptors may regulate VMN function via NE-independent mechanisms, or alternatively, that other neurotransmitter signals to the VMN may control local substrate receptivity to NE.

Effects of estradiol on lactoprivic signaling of the hindbrain upon the contraregulatory hormonal response and metabolic neuropeptide synthesis in hypoglycemic female rats.

BACKGROUND: Caudal dorsomedial hindbrain detection of hypoglycemia-associated lactoprivation regulates glucose counter-regulation in male rats. In females, estradiol (E) determines hypothalamic neuroanatomical and molecular foci of hindbrain energy sensor activation. This study investigated the hypothesis that E signal strength governs metabolic neuropeptide and counter-regulatory hormone responses to hindbrain lactoprivic stimuli in hypoglycemic female rats. METHODS: Ovariectomized animals were implanted with E-filled silastic capsules [30 (E-30) or 300 µg (E-300)/mL] to replicate plasma concentrations at estrous cycle nadir versus peak levels. E-30 and E-300 rats were injected with insulin or vehicle following initiation of continuous caudal fourth ventricular L-lactate infusion. RESULTS: Hypoglycemic hypercorticosteronemia was greater in E-30 versus E-300 animals. Glucagon and corticosterone outflow was correspondingly fully or partially reversed by hindbrain lactate infusion. Insulin-injected rats exhibited lactate-reversible augmentation of norepinephrine (NE) accumulation in all preoptic/hypothalamic structures examined, excluding the dorsomedial hypothalamic nucleus (DMH) where hindbrain lactate infusion either suppressed (E-30) or enhanced (E-300) NE content. Expression profiles of hypoglycemia-reactive metabolic neuropeptides were normalized (with greater efficacy in E-300 animals) by lactate infusion. DMH RFamide-related peptide-1 and -3, arcuate neuropeptide Y and kisspeptin, and ventromedial nucleus nitric oxide synthase protein responses to hypoglycemia were E dosage-dependent. CONCLUSIONS: Distinct physiological patterns of E secretion characteristic of the female rat estrous cycle elicit differential corticosterone outflow during hypoglycemia, and establish both common and different hypothalamic metabolic neurotransmitter targets of hindbrain lactate deficit signaling. Outcomes emphasize a need for insight on systems-level organization, interaction, and involvement of E signal strength-sensitive neuropeptides in counter-regulatory functions.

Hindbrain 5'-Adenosine Monophosphate-activated Protein Kinase Mediates Short-term Food Deprivation Inhibition of the Gonadotropin-releasing Hormone-Luteinizing Hormone Axis: Role of Nitric Oxide.

Hindbrain-derived stimuli restrain the gonadotropin-releasing hormone (GnRH)-pituitary luteinizing hormone (LH) reproductive neuroendocrine axis during energy insufficiency. Interruption of food intake, planned or unplanned, is emblematic of modern life. This study investigated the premise that the hindbrain energy sensor 5'-adenosine monophosphate-activated protein kinase (AMPK) inhibits reproductive neuroendocrine function in short term, e.g. 18-h food-deprived (FD) estradiol (E)-implanted ovariectomized female rats. Intra-caudal fourth ventricular administration of the AMPK inhibitor Compound C (Cc) reversed FD-induced inhibition of rostral preoptic (rPO) GnRH protein expression and LH release in animals given E to replicate proestrus (high-E dose-, but not metestrus (low-E dose)-stage plasma steroid levels. FD caused Cc-reversible augmentation or diminution of preoptic norepinephrine (NE) activity in high- versus low-E rats, respectively, and AMPK-independent reductions in hypothalamic NE accumulation in the latter. Nitric oxide (NO) and kisspeptin are key stimulatory signals for the preovulatory LH surge. Here, FD inhibited rPO neuronal nitric oxide synthase protein expression in high-, but not low-E-dosed animals. Lateral ventricular delivery of the NO donor 3-morpholinosydnonimine (SIN-1) reversed inhibitory GnRH and LH responses to FD in high-E rats, and normalized rPO Vglut2, anteroventral periventricular KiSS1, and dorsomedial hypothalamic RFRP-3 mRNA and/or protein profiles. Data show that FD curtails reproductive neuroendocrine outflow by hindbrain AMPK-dependent mechanisms in the presence of peak estrous cycle E levels. Results indicate that neural networks linking this sensor to GnRH neurons likely involve NO signaling, which may function upstream of one or more neurotransmitters identified here by SIN-1-reversible inhibitory responses to FD.

Mu Opioid Receptor Regulation of Gonadotropin-Releasing Hormone-Luteinizing Hormone Axis during Short-Term Food Deprivation: Role of Alpha1-Adrenoreceptor Signaling.

OBJECTIVES: Brain bio-energetic stability is required for optimal gonadal steroid positive-feedback activation of the gonadotropin-releasing hormone-I (GnRH-I)-pituitary luteinizing hormone (LH) neuroendocrine axis. Caudal hindbrain metabolic-sensory noradrenergic neurons counter energy deficiency by curtailing the mid-cycle LH surge. Central mu opioid receptors (mu-R) impose inhibitory effects of diverse physiological stimuli, including stress, on LH. DESIGN/MATERIAL AND METHODS: To address the premise that mu-R attenuate the LH surge due to metabolic stress of food deprivation (FD), this study examined impacts of lateral ventricular administration of the selective mu-R antagonist CTOP on FD-associated patterns of GnRH-I protein expression and LH release in estradiol-primed ovariectomized female rats. RESULTS: FD caused CTOP-reversible reductions in circulating LH and in micropunch-dissected neural tissue GnRH-I and upstream neurotransmitter (kisspeptin)/biosynthetic enzyme (neuronal nitric oxide synthase) protein content. FD up-regulated mu-R protein expression in reproduction-relevant preoptic structures, e.g. anteroventral periventricular (AVPV) and medial preoptic (MPN) nuclei, responses that were abolished by the alpha1-adrenergic receptor (α1A-R) inverse agonist prazosin. CONCLUSIONS: Current data implicate mu-R in FD attenuation of the E positive-feedback - induced LH surge. Results imply that FD-triggered noradrenergic input to the GnRH-I/LH axis acts in part to enhance reproductive neuroendocrine sensitivity to mu-R inhibition. Further studies are needed to characterize the neurochemical phenotype of AVPV and MPN neurons that express α1A- and/or mu-R, and to determine how these cells are organized within regulatory pathways to impose FD restraint of GnRH-1.

Sex Differences and Role of Estradiol in Hypoglycemia-Associated Counter-Regulation.

Vital nerve cell functions, including maintenance of transmembrane voltage and information transfer, occur at high energy expense. Inadequate provision of the obligate metabolic fuel glucose exposes neurons to risk of dysfunction or injury. Clinical hypoglycemia rarely occurs in nondiabetic individuals but is an unfortunate regular occurrence in patients with type 1 or advanced insulin-treated type 2 diabetes mellitus. Requisite strict glycemic control, involving treatment with insulin, sulfonylureas, or glinides, can cause frequent episodes of iatrogenic hypoglycemia due to defective counter-regulation, including reduced glycemic thresholds and diminished magnitude of motor responses. Multiple components of the body's far-reaching energy balance regulatory network, including the hindbrain dorsal vagal complex, provide dynamic readout of cellular energetic disequilibrium, signals that are utilized by the hypothalamus to shape counterregulatory autonomic, neuroendocrine, and behavioral outflow toward restoration of glucostasis. The ovarian steroid hormone 17ß-estradiol acts on central substrates to preserve nerve cell energy stability brain-wide, thereby providing neuroprotection against bio-energetic insults such as neurodegenerative diseases and acute brain ischemia. The current review highlights recent evidence implicating estrogen in gluco-regulation in females by control of hindbrain metabolic sensor screening and signaling of hypoglycemia-associated neuro-energetic instability. It is anticipated that new understanding of the mechanistic basis of how estradiol influences metabolic sensory input from this critical brain locus to discrete downstream regulatory network substrates will likely reveal viable new molecular targets for therapeutic simulation of hormone actions that promote positive neuronal metabolic state during acute and recurring hypoglycemia.

Rebound Feeding in the Wake of Short-Term Suspension of Food Intake Differs in the Presence of Estrous Cycle Peak versus Nadir Levels of Estradiol.

BACKGROUND: Short-term interruption of feeding is ordinary in modern life but negatively impacts appetite control and body weight. Estradiol (E) imposes long-term inhibitory tonus on food consumption; however, E influence on energy repletion secondary to food deprivation (FD) is unclear. This study investigated the hypothesis that E signal strength regulates hyperphagic responses to FD of varying duration. METHODS: Ovariectomized female rats were implanted with E-containing silastic capsules (30 [E-30] or 300 µg [E-300]/mL) to replicate plasma concentrations at cycle nadir versus peak levels. RESULTS: Data show that food intake was increased equally in E-30 and E-300 rats after 12 hours of food deprivation (FD-12); yet, FD of 18 hours (FD-18) amplified refeeding by E-300 versus E-30. Caudal fourth ventricular administration of the 5'-monophosphate-activated protein kinase (AMPK) inhibitor compound C (Cc) did not modify FD-induced hyperphagia in E-30 (regardless of FD interval) or E-300 animals exposed to FD-12, but diminished refeeding after FD-18 in E-300 rats. Cc-reversible hyperglycemia occurred in refed FD-18 groups. Serum insulin was resistant to FD-12 plus refeeding, but was elevated by AMPK-dependent mechanisms in refed E-300 FD-18 rats; equivalent Cc-insensitive decrements in circulating leptin occurred in all FD groups. CONCLUSION: Current results show that estrous cycle peak, but not baseline, E levels engage hindbrain AMPK signaling to intensify hyperphagia in response to prolongation of FD. Observations of hindbrain AMPK-dependent hyperglycemia, alongside elevated insulin secretion, in refed rats exposed to FD-18 implicate this sensor in insulin resistance mechanisms of glucose partitioning in response to this metabolic imbalance.

Role of hindbrain adenosine 5'-monophosphate-activated protein kinase (AMPK) in hypothalamic AMPK and metabolic neuropeptide adaptation to recurring insulin-induced hypoglycemia in the male rat.

Glucose counter-regulatory dysfunction correlates with impaired activation of the hypothalamic metabolic sensor adenosine 5'-monophosphate-activated protein kinase (AMPK). Hypothalamic AMPK is controlled by hindbrain energy status; we examined here whether hindbrain AMPK regulates hypothalamic AMPK and metabolic neurotransmitter maladaptation to recurring insulin-induced hypoglycemia (RIIH). Brain tissue was harvested after single versus serial insulin (I) dosing for Western blot analysis of AMPK, phospho-AMPK (pAMPK), and relevant biosynthetic enzyme/neuropeptide expression in micro-punch dissected arcuate (ARH), ventromedial (VMH), dorsomedial (DMH) nuclei and lateral hypothalamic area (LHA) tissue. The AMPK inhibitor compound c (Cc) or vehicle was administered to the caudal fourth ventricle ahead of antecedent I injections. RIIH caused site-specific elevation (ARH, VMH, LHA) or reduction (DMH) of total AMPK protein versus acute hypoglycemia; Cc respectively exacerbated or attenuated this response in the ARH and VMH. Hindbrain AMPK correspondingly inhibited or stimulated LHA and DMH pAMPK expression during RIIH. RIIH elicited Cc-reversible augmentation of VMH glutamate decarboxylase profiles, but stimulated (ARH pro-opiomelanocortin; LHA orexin-A) or decreased (VMH nitric oxide synthase) other metabolic neurotransmitters without hindbrain sensor involvement. Results demonstrate acclimated up-regulation of total AMPK protein expression in multiple hypothalamic loci during RIIH, and document hindbrain sensor contribution to amplification of this protein profile in the VMH. Concurrent lack of net change in ARH and VMH tissue pAMPK implies adaptive reductions in local sensor activity, which may/may not reflect positive gain in energy state. It remains unclear if 'glucose-excited' VMH GABAergic and/or ARH pro-opiomelanocortin neurons exhibit AMPK habituation to RIIH, and whether diminished sensor activation in these and other mediobasal hypothalamic neurotransmitter populations may contribute to HAAF.

Hindbrain A2 noradrenergic neuron adenosine 5'-monophosphate-activated protein kinase activation, upstream kinase/phosphorylase protein expression, and receptivity to hormone and fuel reporters of short-term food deprivation are regulated by estradiol.

Estradiol (E) mitigates acute and postacute adverse effects of 12 hr-food deprivation (FD) on energy balance. Hindbrain 5'-monophosphate-activated protein kinase (AMPK) regulates hyperphagic and hypothalamic metabolic neuropeptide and norepinephrine responses to FD in an E-dependent manner. Energy-state information from AMPK-expressing hindbrain A2 noradrenergic neurons shapes neural responses to metabolic imbalance. Here we investigate the hypothesis that FD causes divergent changes in A2 AMPK activity in E- vs. oil (O)-implanted ovariectomized female rats, alongside dissimilar adjustments in circulating metabolic fuel (glucose, free fatty acids [FFA]) and energy deficit-sensitive hormone (corticosterone, glucagon, leptin) levels. FD decreased blood glucose in oil (O)- but not E-implanted ovariectomized female rats and elevated and reduced glucagon levels in O and E, respectively. FD decreased circulating leptin in O and E, but increased corticosterone and FFA concentrations in E only. Western blot analysis of laser-microdissected A2 neurons showed that glucocorticoid receptor type II and very-long-chain acyl-CoA synthetase 3 protein profiles were amplified in FD/E vs. FD/O. A2 total AMPK protein was elevated without change in activity in FD/O, whereas FD/E exhibited increased AMPK activation along with decreased upstream phosphatase expression. The catecholamine biosynthetic enzyme dopamine-ß-hydroxylase (DßH) was increased in FD/O but not FD/E A2 cells. The data show discordance between A2 AMPK activation and glycemic responses to FD; sensor activity was refractory to glucose decrements in FD/O but augmented in FD/E despite stabilized glucose and elevated FFA levels. E-dependent amplification of AMPK activity may reflect adaptive conversion to fatty acid oxidation and/or glucocorticoid stimulation. FD augmentation of A2 DßH protein profiles in FD/O but not FD/E animals suggests that FD may correspondingly regulate NE synthesis vs. metabolism/release in the absence vs. presence of E. Mechanisms underlying translation of E-contingent A2 neuron responses to FD into regulatory signaling remain to be determined. © 2016 Wiley Periodicals, Inc.