Pharmacological and Genetic Disruption of C-Type Natriuretic Peptide (nppcl) Expression in Zebrafish (Danio rerio) Causes Stunted Growth during Development.

Human patients with mutations within NPPC or NPR2 genes (encoding C-type natriuretic peptide (CNP) and guanylyl cyclase-B (GC-B), respectively) display clinical signs associated with skeletal abnormalities, such as overgrowth or short stature. Mice with induced models of Nppc or Npr2 deletion display profound achondroplasia, dwarfism and early death. Recent pharmacological therapies to treat short stature are utilizing long-acting CNP analogues, but the effects of manipulating CNP expression during development remain unknown. Here, we use Danio rerio (zebrafish) as a model for vertebrate development, employing both pharmacological and reverse genetics approaches to alter expression of genes encoding CNP in zebrafish. Four orthologues of CNP were identified in zebrafish, and spatiotemporal expression profiling confirmed their presence during development. Bioinformatic analyses suggested that nppcl is the most likely the orthologue of mammalian CNP. Exogenous CNP treatment of developing zebrafish embryos resulted in impaired growth characteristics, such as body length, head width and eye diameter. This reduced growth was potentially caused by increased apoptosis following CNP treatment. Expression of endogenous nppcl was downregulated in these CNP-treated embryos, suggesting that negative feedback of the CNP system might influence growth during development. CRISPR knock-down of endogenous nppcl in developing zebrafish embryos also resulted in impaired growth characteristics. Collectively, these data suggest that CNP in zebrafish is crucial for normal embryonic development, specifically with regard to growth.

Efficacy of hypophysectomy for the treatment of hypersomatotropism-induced diabetes mellitus in 68 cats.

BACKGROUND: Hypersomatotropism (HST) is an increasingly recognized endocrinopathy in cats and is mostly described associated with diabetes mellitus (DM). OBJECTIVES: To evaluate the efficacy and safety of transsphenoidal hypophysectomy in treating HST and DM in cats. ANIMALS: Sixty-eight client-owned cats with HST and DM treated by transsphenoidal hypophysectomy. METHODS: Retrospective cohort study. Medical records were reviewed for glycemic control and serum insulin-like growth factor-1 (IGF-1) concentrations. Postoperative complications, death within 4 weeks, and proportion achieving diabetic remission were recorded. Survival times and DM-free intervals were calculated. RESULTS: Fifty-eight cats (85.3%) were alive 4 weeks postoperatively with 10 (15%) postoperative deaths. Complications included hypoglycemia (n = 9), electrolyte imbalance (n = 9), and transient congestive heart failure (n = 5). Fifty-five cats (95% of 58 surviving cats [81% of all cats undergoing surgery]) had improved control of diabetes. Diabetic remission occurred in 41 cats (71% of 58 surviving cats [60% of all cats]) with insulin administration discontinued after a median of 9 days (range, 2-120). Postoperative 4-week serum IGF-1 concentration nadir was significantly lower in cats achieving diabetic remission (median 20 ng/mL [15-708] than those that did not (324 ng/mL [15-1955]; P = .03). All cats received long-term levothyroxine and hydrocortisone PO, alongside desmopressin (conjunctival) in 38 of 53 cats (72%). Recurrence of DM occurred in 5 of 41 cats (12%) after a median of 248 days (range, 84-1232). Median survival time of all cats was 853 days (range, 1-1740). CONCLUSIONS AND CLINICAL IMPORTANCE: Transsphenoidal hypophysectomy is an effective treatment for cats with HST and DM, with a long-term outcome that compares favorably to existing options.

Natriuretic Peptide Expression and Function in GH3 Somatolactotropes and Feline Somatotrope Pituitary Tumours.

Patients harbouring mutations in genes encoding C-type natriuretic peptide (CNP; NPPC) or its receptor guanylyl cyclase B (GC-B, NPR2) suffer from severe growth phenotypes; loss-of-function mutations cause achondroplasia, whereas gain-of-function mutations cause skeletal overgrowth. Although most of the effects of CNP/GC-B on growth are mediated directly on bone, evidence suggests the natriuretic peptides may also affect anterior pituitary control of growth. Our previous studies described the expression of NPPC and NPR2 in a range of human pituitary tumours, normal human pituitary, and normal fetal human pituitary. However, the natriuretic peptide system in somatotropes has not been extensively explored. Here, we examine the expression and function of the CNP/GC-B system in rat GH3 somatolactotrope cell line and pituitary tumours from a cohort of feline hypersomatotropism (HST; acromegaly) patients. Using multiplex RT-qPCR, all three natriuretic peptides and their receptors were detected in GH3 cells. The expression of Nppc was significantly enhanced following treatment with either 100 nM TRH or 10 µM forskolin, yet only Npr1 expression was sensitive to forskolin stimulation; the effects of forskolin and TRH on Nppc expression were PKA- and MAPK-dependent, respectively. CNP stimulation of GH3 somatolactotropes significantly inhibited Esr1, Insr and Lepr expression, but dramatically enhanced cFos expression at the same time point. Oestrogen treatment significantly enhanced expression of Nppa, Nppc, Npr1, and Npr2 in GH3 somatolactotropes, but inhibited CNP-stimulated cGMP accumulation. Finally, transcripts for all three natriuretic peptides and receptors were expressed in feline pituitary tumours from patients with HST. NPPC expression was negatively correlated with pituitary tumour volume and SSTR5 expression, but positively correlated with D2R and GHR expression. Collectively, these data provide mechanisms that control expression and function of CNP in somatolactotrope cells, and identify putative transcriptional targets for CNP action in somatotropes.

Regulation and Function of C-Type Natriuretic Peptide (CNP) in Gonadotrope-Derived Cell Lines.

C-type natriuretic peptide (CNP) is the most conserved member of the mammalian natriuretic peptide family, and is implicated in the endocrine regulation of growth, metabolism and reproduction. CNP is expressed throughout the body, but is particularly abundant in the central nervous system and anterior pituitary gland. Pituitary gonadotropes are regulated by pulsatile release of gonadotropin releasing hormone (GnRH) from the hypothalamus, to control reproductive function. GnRH and CNP reciprocally regulate their respective signalling pathways in αT3-1 gonadotrope cells, but effects of pulsatile GnRH stimulation on CNP expression has not been explored. Here, we examine the sensitivity of the natriuretic peptide system in LßT2 and αT3-1 gonadotrope cell lines to continuous and pulsatile GnRH stimulation, and investigate putative CNP target genes in gonadotropes. Multiplex RT-qPCR assays confirmed that primary mouse pituitary tissue express Nppc,Npr2 (encoding CNP and guanylyl cyclase B (GC-B), respectively) and Furin (a CNP processing enzyme), but failed to express transcripts for Nppa or Nppb (encoding ANP and BNP, respectively). Pulsatile, but not continuous, GnRH stimulation of LßT2 cells caused significant increases in Nppc and Npr2 expression within 4 h, but failed to alter natriuretic peptide gene expression in αT3-1 cells. CNP enhanced expression of cJun, Egr1, Nr5a1 and Nr0b1, within 8 h in LßT2 cells, but inhibited Nr5a1 expression in αT3-1 cells. Collectively, these data show the gonadotrope natriuretic peptide system is sensitive to pulsatile GnRH signalling, and gonadotrope transcription factors are putative CNP-target genes. Such findings represent additional mechanisms by which CNP may regulate reproductive function.

Pituitary Pathology and Gene Expression in Acromegalic Cats.

The prevalence of GH-secreting pituitary tumors in domestic cats (Felis catus) is 10-fold greater than in humans. The predominant inhibitory receptors of GH-secreting pituitary tumors are somatostatin receptors (SSTRs) and D2 dopamine receptor (DRD2). The expression of these receptors is associated with the response to somatostatin analog and dopamine agonist treatment in human patients with acromegaly. The aim of this study was to describe pathological features of pituitaries from domestic cats with acromegaly, pituitary receptor expression, and investigate correlates with clinical data, including pituitary volume, time since diagnosis of diabetes, insulin requirement, and serum IGF1 concentration. Loss of reticulin structure was identified in 15 of 21 pituitaries, of which 10 of 15 exhibited acinar hyperplasia. SSTR1, SSTR2, SSTR5, and DRD2 mRNA were identified in the feline pituitary whereas SSTR3 and SSTR4 were not. Expression of SSTR1, SSTR2, and SSTR5 was greater in acromegalic cats compared with controls. A negative correlation was identified between DRD2 mRNA expression and pituitary volume. The loss of DRD2 expression should be investigated as a mechanism allowing the development of larger pituitary tumors.

Radiotherapy and pasireotide treatment of a growth hormone producing pituitary tumor in a diabetic dog.

An 8-year-old castrated male border terrier dog was diagnosed with acromegaly resulting from a growth hormone secreting pituitary tumor. Sixteen daily fractions of radiation therapy were delivered followed, approximately 1 year later, by administration of pasireotide. The aforementioned treatment was considered effective and should be further evaluated in similar cases.

Radiothérapie et traitement au pasiréotide pour une tumeur pituitaire produisant une hormone de croissance chez un chien diabétique. Un chien Terrier-Border castré âgé de 8 ans a été diagnostiqué avec de l'acromégalie découlant d'une tumeur pituitaire secrétant une hormone de croissance. Seize fractions quotidiennes de radiothérapie ont été administrées et ont été suivies, environ un an plus tard, de l'administration du pasiréotide. Le traitement précédemment mentionné a été considéré efficace et devrait être étudié de plus près dans des cas similaires.(Traduit par Isabelle Vallières).

Time spent with cats is never wasted: Lessons learned from feline acromegalic cardiomyopathy, a naturally occurring animal model of the human disease.

BACKGROUND: In humans, acromegaly due to a pituitary somatotrophic adenoma is a recognized cause of increased left ventricular (LV) mass. Acromegalic cardiomyopathy is incompletely understood, and represents a major cause of morbidity and mortality. We describe the clinical, echocardiographic and histopathologic features of naturally occurring feline acromegalic cardiomyopathy, an emerging disease among domestic cats. METHODS: Cats with confirmed hypersomatotropism (IGF-1>1000ng/ml and pituitary mass; n = 67) were prospectively recruited, as were two control groups: diabetics (IGF-1<800ng/ml; n = 24) and healthy cats without known endocrinopathy or cardiovascular disease (n = 16). Echocardiography was performed in all cases, including after hypersomatotropism treatment where applicable. Additionally, tissue samples from deceased cats with hypersomatotropism, hypertrophic cardiomyopathy and age-matched controls (n = 21 each) were collected and systematically histopathologically reviewed and compared. RESULTS: By echocardiography, cats with hypersomatotropism had a greater maximum LV wall thickness (6.5mm, 4.1-10.1mm) than diabetic (5.9mm, 4.2-9.1mm; Mann Whitney, p<0.001) or control cats (5.2mm, 4.1-6.5mm; Mann Whitney, p<0.001). Left atrial diameter was also greater in cats with hypersomatotropism (16.6mm, 13.0-29.5mm) than in diabetic (15.4mm, 11.2-20.3mm; Mann Whitney, p<0.001) and control cats (14.0mm, 12.6-17.4mm; Mann Whitney, p<0.001). After hypophysectomy and normalization of IGF-1 concentration (n = 20), echocardiographic changes proved mostly reversible. As in humans, histopathology of the feline acromegalic heart was dominated by myocyte hypertrophy with interstitial fibrosis and minimal myofiber disarray. CONCLUSIONS: These results demonstrate cats could be considered a naturally occurring model of acromegalic cardiomyopathy, and as such help elucidate mechanisms driving cardiovascular remodeling in this disease.

Natriuretic peptide activation of extracellular regulated kinase 1/2 (ERK1/2) pathway by particulate guanylyl cyclases in GH3 somatolactotropes.

The natriuretic peptides, Atrial-, B-type and C-type natriuretric peptides (ANP, BNP, CNP), are regulators of many endocrine tissues and exert their effects predominantly through the activation of their specific guanylyl cyclase receptors (GC-A and GC-B) to generate cGMP. Whereas cGMP-independent signalling has been reported in response to natriuretic peptides, this is mediated via either the clearance receptor (Npr-C) or a renal-specific NPR-Bi isoform, which both lack intrinsic guanylyl cyclase activity. Here, we report evidence of GC-B-dependent cGMP-independent signalling in pituitary GH3 cells. Stimulation of GH3 cells with CNP resulted in a rapid and sustained enhancement of ERK1/2 phosphorylation (P-ERK1/2), an effect that was not mimicked by dibutryl-cGMP. Furthermore, CNP-stimulated P-ERK1/2 occurred at concentrations below that required for cGMP accumulation. The effect of CNP on P-ERK1/2 was sensitive to pharmacological blockade of MEK (U0126) and Src kinases (PP2). Silencing of the GC-B1 and GC-B2 splice variants of the GC-B receptor by using targeted short interfering RNAs completely blocked the CNP effects on P-ERK1/2. CNP failed to alter GH3 cell proliferation or cell cycle distribution but caused a concentration-dependent increase in the activity of the human glycoprotein α-subunit promoter (αGSU) in a MEK-dependent manner. Finally, CNP also activated the p38 and JNK MAPK pathways in GH3 cells. These findings reveal an additional mechanism of GC-B signalling and suggest additional biological roles for CNP in its target tissues.

Studying Cat (Felis catus) Diabetes: Beware of the Acromegalic Imposter.

Naturally occurring diabetes mellitus (DM) is common in domestic cats (Felis catus). It has been proposed as a model for human Type 2 DM given many shared features. Small case studies demonstrate feline DM also occurs as a result of insulin resistance due to a somatotrophinoma. The current study estimates the prevalence of hypersomatotropism or acromegaly in the largest cohort of diabetic cats to date, evaluates clinical presentation and ease of recognition. Diabetic cats were screened for hypersomatotropism using serum total insulin-like growth factor-1 (IGF-1; radioimmunoassay), followed by further evaluation of a subset of cases with suggestive IGF-1 (>1000 ng/ml) through pituitary imaging and/ or histopathology. Clinicians indicated pre-test suspicion for hypersomatotropism. In total 1221 diabetic cats were screened; 319 (26.1%) demonstrated a serum IGF-1>1000 ng/ml (95% confidence interval: 23.6-28.6%). Of these cats a subset of 63 (20%) underwent pituitary imaging and 56/63 (89%) had a pituitary tumour on computed tomography; an additional three on magnetic resonance imaging and one on necropsy. These data suggest a positive predictive value of serum IGF-1 for hypersomatotropism of 95% (95% confidence interval: 90-100%), thus suggesting the overall hypersomatotropism prevalence among UK diabetic cats to be 24.8% (95% confidence interval: 21.2-28.6%). Only 24% of clinicians indicated a strong pre-test suspicion; most hypersomatotropism cats did not display typical phenotypical acromegaly signs. The current data suggest hypersomatotropism screening should be considered when studying diabetic cats and opportunities exist for comparative acromegaly research, especially in light of the many detected communalities with the human disease.

Computed tomographic signs of acromegaly in 68 diabetic cats with hypersomatotropism.

In order to describe the signs of acromegaly in cats, a case-control study was done based on computed tomography (CT) scans of the heads of 68 cats with hypersomatotropism and 36 control cats. All cats with a diagnosis of hypersomatotropism had diabetes mellitus, serum insulin-like growth factor-1 >1000 ng/ml and a pituitary mass. Measurements of bones and soft tissues were done by two independent observers without knowledge of the diagnosis. Pituitary masses were identified in CT images of 64 (94%) cats with hypersomatotropism. Analysis of variance found a moderate effect of gender on the size of bones and a large effect of hypersomatotropism on the size of bones and thickness of soft tissues. In cats with hypersomatotropism the frontal and parietal bones were, on average, 0.8 mm thicker (P <0.001); the distance between the zygomatic arches was, on average, 5.4 mm greater (P <0.001); and the mandibular rami were, on average, 1.1 mm thicker (P <0.001) than in control cats. The skin and subcutis dorsal to the frontal bone were, on average, 0.4 mm thicker (P = 0.001); lateral to the zygomatic arch were, on average, 0.7 mm thicker (P <0.001); and ventral to the mandibular rami were, on average, 1.1 mm thicker (P = 0.002) in cats with hypersomatotropism than in control cats. The cross-sectional area of the nasopharynx was, on average, 11.1 mm(2) smaller in cats with hypersomatotropism than in control cats (P = 0.02). Prognathia inferior and signs of temporomandibular joint malformation were both observed more frequently in cats with hypersomatotropism than in control cats (P = 0.03). Overall, differences between affected and unaffected cats were small. Recognising feline acromegaly on the basis of facial features is difficult.

Screening diabetic cats for hypersomatotropism: performance of an enzyme-linked immunosorbent assay for insulin-like growth factor 1.

Screening diabetic cats for feline hypersomatotropism (HS) is currently dependent on using a radioimmunoassay (RIA) for measurement of growth hormone or insulin-like growth factor 1 (IGF-1), both of which require radioactivity, are costly and have limited availability. Performance of an enzyme-linked immunosorbent assay (ELISA) using anti-human IGF-1 antibodies was assessed. Total IGF-1 was determined in diabetic cat samples across a wide range of IGF-concentrations using a previously validated RIA (serum: 92 cats; plasma: 31 cats). Repeat IGF-1 measurement was then performed using the ELISA-system. Mean IGF-1 recovery after serial dilution proved satisfactory with a correlation coefficient of 0.96 (serum) and 0.97 (plasma). Appropriate precision was established [intra-assay coefficient of variation (CV) 9.5 ± 2% (serum) and 13.6 ± 7% (plasma); inter-assay CV 11.4 ± 4% (serum) and 7.6 ± 6% (plasma)] and significant effect of hyperlipidaemia, haemoglobinaemia, bilirubinaemia and storage was excluded, with the exception of an increase in serum IGF-1 when left at room temperature for more than 24 h. ELISA concentrations correlated significantly with RIA concentrations (serum Pearson r(2): 0.75; plasma: 0.83, P <0.001). Receiver operating characteristics analysis showed an area under the curve of 0.99 (serum) and 0.96 (plasma), and indicated high diagnostic accuracy for categorising a diabetic cat correctly as suspicious for HS at a serum IGF-1 cut-off of 997 ng/ml (sensitivity, 100%; specificity, 88.1%). The current study is the first to validate an easy-to-use and economical IGF-1 ELISA for the screening for HS among diabetic cats, which is important given the suspected significant prevalence of HS-induced diabetes mellitus.

Organohalogenated contaminants in domestic cats' plasma in relation to spontaneous acromegaly and type 2 diabetes mellitus: a clue for endocrine disruption in humans?

It was recently hypothesized that pets may serve as sentinels to explore human exposure to organohalogenated chemicals (OHCs) via indoor environments and adverse health effects. The current study investigates OHCs contamination in domestic cats suffering from diabetes mellitus (DM), particularly DM induced by acromegaly and a form of DM akin to human type 2 DM (T2DM). Plasma from three groups of domestic cats was analyzed: acromegaly induced DM, T2DM and age matched control cats without DM. Analytes targeted included organochlorine pesticides, polychlorinated biphenyls (PCBs), and polybrominated diphenyl ethers (PBDEs), together with their hydroxylated (HO-) metabolites. Similar PCB profiles were measured in cat plasma compared to humans, while the PBDE profile (dominated by BDE-99 (48%-55%) and BDE-47 (19%-25%)), the PCB and PBDE metabolite profiles were different in cat plasma than found in humans. Significantly higher OHC concentrations were recorded in plasma of acromegalic cats compared to the other two groups. Group differences in the PCBs/HO-PCBs ratios suggest that acromegalic cats have a lower capacity to metabolize persistent OHCs, like PCBs, than diabetic cats or cats without an endocrinopathy. As pituitary tumorigenesis in animals can be induced by estrogens, and PCBs may act as xenoestrogens, further investigation into whether there could be a causative link with the induction of feline acromegaly is warranted. Interestingly, BDE-47/BDE-99 ratios in cats were similar to the ratios in house dust. The results of this study suggest that domestic cats may represent a good model to assess human exposure to chemicals present in indoor dust.

Feline acromegaly: an essential differential diagnosis for the difficult diabetic.

PRACTICAL RELEVANCE: Clinicians who deal with diabetic cats can have mixed experiences. Some patients are 'textbook cases', responding very well to insulin administration; others prove to be more challenging. Recent studies have shown a significant proportion of problem diabetic cats to have underlying acromegaly (hypersomatotropism). Recognising this syndrome in these cats will be key to successfully managing the concurrent diabetes. PATIENT GROUP: Just like the 'normal' (non-acromegalic) diabetic cat, the acromegalic diabetic cat tends to be a middle-aged to older male neutered domestic short hair. However, with increasing case experience, this signalment may change. Most patients are insulin resistant, although this may not be the initial presenting sign. No breed predispositions have been recognised to date. CLINICAL CHALLENGES: There is no single diagnostic test for feline acromegaly - a confident diagnosis relies on a combination of clinical signs, feline growth hormone and insulin-like growth factor 1 levels, and intracranial imaging. Additionally, the ideal treatment protocol has yet to be established. Currently, radiotherapy is considered by many to be the best treatment; however, costs, the need for multiple anaesthetics, and the often delayed and unpredictable treatment response represent serious limitations of this modality. Previously, medical treatment has proven unsuccessful. Recent studies provide some evidence in favour of, and some against, the use of newer long-acting somatostatin analogue preparations in a proportion of acromegalic cats. EVIDENCE BASE: Two recent studies have revealed a relatively high prevalence of acromegaly among diabetic cats. One also specifically assessed the value of hormonal tests, computed tomography and magnetic resonance imaging during the diagnostic process.