Calcitonin Receptor Research Articles

Whole-brain mapping of amylin-induced neuronal activity in receptor activity-modifying protein 1/3 knockout mice.

The pancreatic hormone amylin plays a central role in regulating energy homeostasis and glycaemic control by stimulating satiation and reducing food reward, making amylin receptor agonists attractive for the treatment of metabolic diseases. Amylin receptors consist of heterodimerized complexes of the calcitonin receptor and receptor-activity modifying proteins subtype 1-3 (RAMP1-3). Neuronal activation in response to amylin dosing has been well characterized, but only in selected regions expressing high levels of RAMPs. The current study identifies global brain-wide changes in response to amylin and by comparing wild type and RAMP1/3 knockout mice reveals the importance of RAMP1/3 in mediating this response. Amylin dosing resulted in neuronal activation as measured by an increase in c-Fos labelled cells in 20 brain regions, altogether making up the circuitry of neuronal appetite regulation (e.g., area postrema (AP), nucleus of the solitary tract (NTS), parabrachial nucleus (PB), and central amygdala (CEA)). c-Fos response was also detected in distinct nuclei across the brain that typically have not been linked with amylin signalling. In RAMP1/3 knockout amylin induced low-level neuronal activation in seven regions, including the AP, NTS and PB, indicating the existence of RAMP1/3-independent mechanisms of amylin response. Under basal conditions RAMP1/3 knockout mice show reduced neuronal activity in the hippocampal formation as well as reduced hippocampal volume, suggesting a role for RAMP1/3 in hippocampal physiology and maintenance. Altogether these data provide a global map of amylin response in the mouse brain and establishes the significance of RAMP1/3 receptors in relaying this response.

AM833, a Long Acting Amylin Analogue Induces Hypocalcemia in Young Rats via a Calcitonin Receptor Mediated Mechanism

Abstract Amylin is a peptide hormone that is co-secreted with insulin from pancreatic β-cells. It is member of the calcitonin family and activates amylin receptors (AMYRs) in the calcitonin receptor (CTR) family. During recent years, several AMYR agonists have been in clinical development for treatment of diabetes and obesity. In contrast to pramlintide, which is a short acting amylin analogue approved for use in combination with meal-time insulin in patients with diabetes, the more recent development candidates have been dual acting analogues activating both the CTR and the AMYRs. In rodents the CTR plays a significant role in calcium homeostasis, while in humans this has been associated with little long-term effect on the plasma calcium levels (Ganong, 1993; Longo et al., 2011); Rodents are usually chosen as first choice pre-clinical models for in vivo testing and introducing acute and profound hypocalcaemia can lead to general neurological and neuromuscular disturbances, which can be challenging in clinical development programs. AM833 (NNC0174-0833) is a long acting acylated dual acting amylin analogue activating both CTR and AMYRs. When combined with the long acting GLP-1 analogue semaglutide for weight management, both preclinical studies in rat models of obesity and clinical trials in humans with obesity have demonstrated significant weight loss potential. In young rats aged 7–9 weeks, single sc dose of 3–300 nmol/kg AM833 induced a reduction of total and ionized calcium of 40–50% from baseline with lowest level around 12 hours after dosing. A similar response was seen after administration of salmon calcitonin which also activates both CTR and AMYRs. Tachyphylaxis was introduced, with no effect on calcium found after sub chronic daily treatment for 2 weeks. Age played a key role in the sensitivity to calcium lowering with almost no effect found in 11 months old rats. When administering AM1213 (NNC0174-1213), a selective AMYR agonist with little activation of the CTR, no or little calcium lowering of around 10% was seen. These data indicated that the calcium lowering properties are CTR mediated. The induction of hypercalcemia was a rodent specific phenomenon as no calcium lowering outside of normal range was seen in young dogs and rabbits. Clinical data confirmed that CTR induced calcium lowering was not human relevant, but these data showed that understanding physiology and pharmacology in the animal models of investigation as well as human translational relevance is of outmost importance in the progress of an early development program.

Affinity Enhancers for Peptide Ligand Binding to the Extracellular Domain of the Amylin Receptor 2

Amylin is a peptide hormone and its activation of amylin receptors controls blood glucose and food intake. Amylin receptor activation is a known drug target for diabetes and has potential for obesity treatment. The amylin receptor consists of the calcitonin receptor (CTR) and the accessary protein called receptor activity-modifying protein (RAMP). CTR is the receptor for peptide hormone calcitonin, whereas the heterodimer of CTR and RAMP gains affinity for peptide hormone amylin. Each of the CTR and RAMP has an extracellular domain (ECD), and CTR ECD is a known binding site for peptide hormones. However, whether RAMP ECD provides a binding site for peptide ligands remains elusive. RAMP has three types in humans and our structural model of the amylin receptor 2 ECD (RAMP2 ECD:CTR ECD complex) suggested the molecular interaction between RAMP2 ECD Glu101 and the C-terminal Tyr residue of an amylin analog AC413. We examined whether this molecular interaction contributes to AC413 binding for amylin receptor 2 ECD and enhances AC413 affinity. In addition, N-glycosylation of the amylin receptor ECD is known to increase the peptide ligand affinity. Despite, which N-glycosite is the most responsible for this effect and the role of the Asn-linked N-acetylglucosamine (GlcNAc) have been unexplored for amylin receptor 2 ECD. Based on our previous reports, we hypothesized that Asn-linked GlcNAc of CTR ECD Asn130 will enhance the peptide ligand affinity for amylin receptor 2 ECD. Here, we established the functional amylin receptor 2 ECD and applied mutagenesis and enzymatic deglycosylation to address the affinity enhancers. We used fluorescence polarization/anisotropy peptide binding assay to measure peptide ligand affinity for purified amylin receptor 2 ECD. Our results showed that the RAMP2 ECD Glu101 to alanine mutation significantly decreased the amylin analog affinity suggesting its potential interaction with the C-terminal Tyr residue of the amylin analog. Using glycan-trimmed CTR ECD, we showed that the Asn-linked GlcNAc residue of CTR ECD Asn130 is the most responsible among N-glycans for enhancing affinity for the amylin receptor 2 ECD. This study provides evidence that both RAMP2 ECD Glu101 and Asn-linked GlcNAc of CTR ECD Asn130 contribute to amylin analog binding as affinity enhancers.

Aloperine improves osteoporosis in ovariectomized mice by inhibiting RANKL-induced NF-κB, ERK and JNK approaches

Presently, postmenopausal osteoporosis mainly caused by excessive activation of in vivo osteoclasts has become a global public health burden. Natural compounds have gradually become the potential drugs for the treatment of postmenopausal osteoporosis. Aloperine is a new alkaloid extracted from the leaves and seeds of sophora bean. The current studies have proved that aloperine has many biological activities, including anti-inflammatory, antiviral and anticancer activities. This study shows that aloperine can inhibit activity and formation of osteoclast mediated by RANKL in a dose-dependent manner without affecting the activity of bone marrow macrophages (BMM). In addition, it is found that aloperine can inhibit the expression of osteoclast specific marker genes, including nuclear factor of activated T cells cytoplasmic 1 (NFATc1), tartrate resistant acid phosphatase (TRAcP), matrix metallopeptidase 9 (MMP9), cathepsin K (Ctsk), V-ATPase d2 and calcitonin receptor. The in vitro experiment of aloperine proved that aloperine can inhibit the degradation of IκBα and the phosphorylation of P65, ERK and JNK. Additionally, aloperine improves bone loss in ovariectomized (OVX) mice by inhibiting osteoclast activity. This project proved that aloperine can affect the formation of osteoclasts by inhibiting RANKL signaling channel, and it is indicated that aloperine has the potential to be developed as a new drug for the prevention and treatment of postmenopausal osteoporosis.

A Tale of Two Peptides: Identifying Targets for Migraine

Calcitonin gene related-peptide (CGRP) and amylin are closely-related peptides that are both reported to be elevated in the plasma of migraine sufferers. CGRP is highly expressed in neural tissues and migraine relevant structures, including the trigeminal ganglia (TG). Amylin is secreted from pancreatic islets in response to food intake, with conflicting reports of expression in the TG. CGRP potently activates two receptors, the CGRP receptor and Amylin 1 (AMY1) receptor, whereas amylin potently activates the AMY1 receptor. CGRP has been proposed to be auto regulated in an autocrine manner in the TG, however CGRP does not colocalise with the CGRP receptor in the TG. The AMY1 receptor is also expressed in the TG, but where it is expressed and whether it is found together with CGRP or amylin is poorly understood. Therefore, our aim was to determine whether amylin is expressed in the TG and the relative distribution of AMY1 receptor components with CGRP or amylin using thoroughly validated antibodies. To study the distribution of CGRP, amylin and the AMY1 receptor component, calcitonin receptor (CTR), in the TG a range of antibodies were validated. Anti-CGRP and amylin antibodies were validated using immunoblotting and rat pancreas histology. Anti-CTR antibodies were validated using transfected HEK293S cells and knock out tissue. Lead antibodies were then applied to mouse, rat and human TG sections with neural markers to investigate their distribution. Anti-amylin antibodies frequently cross-reacted with CGRP, whereas anti-CGRP antibodies displayed no cross-reactivity with amylin. Lead anti-amylin antibodies displayed little or no detection of amylin in rodent and human β-tubulin III-positive neurons, respectively, whereas abundant CGRP-like immunoreactivity was observed across all species. When co-incubated the little amylin-like immunoreactivity observed strongly colocalised with anti-CGRP staining. This was particularly apparently with T4145, the more cross-reactive amylin antibody. The lead anti-CTR antibody, 188/10, displayed robust detection of rodent CTR in transiently transfected HEK293S cells and a marked loss of staining in knock out mouse nucleus accumbens compared to wild type. CTR-like and CGRP-like immunoreactivity frequently colocalised in rat TG sections, primarily in C-fibre neurons, and rarely in NF200-positive A-fibre neurons. These results demonstrate the importance of thoroughly validating antibodies. The data suggest that in the TG, CGRP, rather than amylin, is the endogenous agonist for the AMY1 receptor. Additionally, it suggests that CGRP autocrine signalling occurs via the AMY1 receptor.

Attenuation of CALCR protein expression in stone affected region of kidney biopsy tissue: Case presentation

Calcitonin is one of the most important hormones in human body which regulates calcium homeostasis. It binds with Calcitonin Receptor (CALCR) and reduces serum calcium level by inhibiting bone resorption and proximal renal reabsorption. Alteration in calcium homeostasis promotes diseases like osteoporosis, urolithiasis etc. An interesting finding of reduced CALCR protein expression in a urolithiasis patient motivated us to investigate its level in stone affected region of kidney biopsy tissue and hence in the present study, CALCR expression was checked in urolithiasis patients who had gone through open nephrolithotomy for stone removal. Basic and clinical parameters of the patients were recorded followed by analysis of stone composition, genotypic analysis and studying CALCR protein expression level in the kidney biopsy tissue samples (in the location of stone and an adjacent control area). CALCR protein expression was found to be attenuated in the stone affected region of kidney biopsy tissue as compared to adjacent control tissues (4-6 cm apart from the stone affected region) in all our studied samples (n=5). This case series represents an interesting finding as it is the first time report in the world, regarding expression of CALCR protein in kidney tissues of urolithiasis patients. Keywords: Urolithiasis; CALCR; Protein expression; SNP.

Peptide targeting of lysophosphatidylinositol-sensing GPR55 for osteoclastogenesis tuning

BackgroundThe G-protein-coupled receptor GPR55 has been implicated in multiple biological activities, which has fuelled interest in its functional targeting. Its controversial pharmacology and often species-dependent regulation have impacted upon the potential translation of preclinical data involving GPR55.ResultsWith the aim to identify novel GPR55 regulators, we have investigated lysophosphatidylinositol (LPI)-induced GPR55-mediated signal transduction. The expression system for wild-type and mutated GPR55 was HeLa cells silenced for their endogenous receptor by stable expression of a short-hairpin RNA specific for GPR55 5′-UTR, which allowed definition of the requirement of GPR55 Lys80 for LPI-induced MAPK activation and receptor internalisation. In RAW264.7 macrophages, GPR55 pathways were investigated by Gpr55 silencing using small-interfering RNAs, which demonstrated that LPI increased intracellular Ca2+ levels and induced actin filopodium formation through GPR55 activation. Furthermore, the LPI/GPR55 axis was shown to have an active role in osteoclastogenesis of precursor RAW264.7 cells induced by ‘receptor-activator of nuclear factor kappa-β ligand’ (RANKL). Indeed, this differentiation into mature osteoclasts was associated with a 14-fold increase in Gpr55 mRNA levels. Moreover, GPR55 silencing and antagonism impaired RANKL-induced transcription of the osteoclastogenesis markers: ‘nuclear factor of activated T-cells, cytoplasmic 1′, matrix metalloproteinase-9, cathepsin-K, tartrate-resistant acid phosphatase, and the calcitonin receptor, as evaluated by real-time PCR. Phage display was previously used to identify peptides that bind to GPR55. Here, the GPR55-specific peptide-P1 strongly inhibited osteoclast maturation of RAW264.7 macrophages, confirming its activity as a blocker of GPR55-mediated functions. Although osteoclast syncytium formation was not affected by pharmacological regulation of GPR55, osteoclast activity was dependent on GPR55 signalling, as shown with resorption assays on bone slices, where LPI stimulated and GPR55 antagonists inhibited bone erosion.ConclusionsOur data indicate that GPR55 represents a target for development of novel therapeutic approaches for treatment of pathological conditions caused by osteoclast-exacerbated bone degradation, such as in osteoporosis or during establishment of bone metastases.92B2o-gegw3E_7VEU7qiRTVideo abstract

Paraventricular Calcitonin Receptor-Expressing Neurons Modulate Energy Homeostasis in Male Mice.

The paraventricular nucleus of the hypothalamus (PVH) is a heterogeneous collection of neurons that play important roles in modulating feeding and energy expenditure. Abnormal development or ablation of the PVH results in hyperphagic obesity and defects in energy expenditure whereas selective activation of defined PVH neuronal populations can suppress feeding and may promote energy expenditure. Here, we characterize the contribution of calcitonin receptor-expressing PVH neurons (CalcRPVH) to energy balance control. We used Cre-dependent viral tools delivered stereotaxically to the PVH of CalcR2Acre mice to activate, silence, and trace CalcRPVH neurons and determine their contribution to body weight regulation. Immunohistochemistry of fluorescently-labeled CalcRPVH neurons demonstrates that CalcRPVH neurons are largely distinct from several PVH neuronal populations involved in energy homeostasis; these neurons project to regions of the hindbrain that are implicated in energy balance control, including the nucleus of the solitary tract and the parabrachial nucleus. Acute activation of CalcRPVH neurons suppresses feeding without appreciably augmenting energy expenditure, whereas their silencing leads to obesity that may be due in part due to loss of PVH melanocortin-4 receptor signaling. These data show that CalcRPVH neurons are an essential component of energy balance neurocircuitry and their function is important for body weight maintenance. A thorough understanding of the mechanisms by which CalcRPVH neurons modulate energy balance might identify novel therapeutic targets for the treatment and prevention of obesity.

Eleutherococcus sessiliflorus Inhibits Receptor Activator of Nuclear Factor Kappa-B Ligand (RANKL)-Induced Osteoclast Differentiation and Prevents Ovariectomy (OVX)-Induced Bone Loss

The aim of this study was to evaluate the effects of root bark of Eleutherococcus sessiliflorus (ES) on osteoclast differentiation and function in vitro and in vivo. In vitro, we found that ES significantly inhibited the RANKL-induced formation of TRAP-positive multinucleated osteoclasts and osteoclastic bone resorption without cytotoxic effects. ES markedly downregulated the expression of nuclear factor of activated T cells cytoplasmic 1 (NFATc1); c-Fos; and osteoclast-related marker genes, such as TRAP, osteoclast-associated receptor (OSCAR), matrix metalloproteinase-9 (MMP-9), calcitonin receptor, cathepsin K, the 38 kDa d2 subunit of the vacuolar H+-transporting lysosomal ATPase (Atp6v0d2), dendritic cell-specific transmembrane protein (DC-STAMP), and osteoclast-stimulatory transmembrane protein (OC-STAMP). These effects were achieved by inhibiting the RANKL-mediated activation of MAPK signaling pathway proteins, including p38, ERK, and JNK. In vivo, ES attenuated OVX-induced decrease in bone volume to tissue volume ratio (BV/TV), trabecular thickness (Tb.Th), trabecular number (Tb.N), and bone mineral density, but increased trabecular separation (Tb.Sp) in the femur. Collectively, our findings showed that ES inhibited RANKL-activated osteoclast differentiation in bone marrow macrophages and prevented OVX-mediated bone loss in rats. These findings suggest that ES has the potential to be used as a therapeutic agent for bone-related diseases, such as osteoporosis.

A genetic map of the mouse dorsal vagal complex and its role in obesity.

The brainstem dorsal vagal complex (DVC) is known to regulate energy balance and is the target of appetite-suppressing hormones, such as glucagon-like peptide 1 (GLP-1). Here we provide a comprehensive genetic map of the DVC and identify neuronal populations that control feeding. Combining bulk and single-nucleus gene expression and chromatin profiling of DVC cells, we reveal 25 neuronal populations with unique transcriptional and chromatin accessibility landscapes and peptide receptor expression profiles. GLP-1 receptor (GLP-1R) agonist administration induces gene expression alterations specific to two distinct sets of Glp1r neurons-one population in the area postrema and one in the nucleus of the solitary tract that also expresses calcitonin receptor (Calcr). Transcripts and regions of accessible chromatin near obesity-associated genetic variants are enriched in the area postrema and the nucleus of the solitary tract neurons that express Glp1r and/or Calcr, and activating several of these neuronal populations decreases feeding in rodents. Thus, DVC neuronal populations associated with obesity predisposition suppress feeding and may represent therapeutic targets for obesity.

Proadrenomedullin N-Terminal 20 Peptides (PAMPs) Are Agonists of the Chemokine Scavenger Receptor ACKR3/CXCR7.

Adrenomedullin (ADM) and proadrenomedullin N-terminal 20 peptide (PAMP) are two peptides with vasodilative, bronchodilative, and angiogenic properties, originating from a common precursor, proADM. Previous studies proposed that the atypical chemokine receptor ACKR3 might act as a low-affinity scavenger for ADM, regulating its availability for its cognate receptor calcitonin receptor-like receptor (CLR) in complex with a receptor activity modifying protein (RAMP). In this study, we compared the activation of ACKR3 by ADM and PAMP, as well as other related members of the calcitonin gene-related peptide (CGRP) family. Irrespective of the presence of RAMPs, ADM was the only member of the CGRP family to show moderate activity toward ACKR3. Remarkably, PAMP, and especially further processed PAMP-12, had a stronger potency toward ACKR3 than ADM. Importantly, PAMP-12 induced β-arrestin recruitment and was efficiently internalized by ACKR3 without inducing G protein or ERK signaling in vitro. Our results further extend the panel of endogenous ACKR3 ligands and broaden ACKR3 functions to a regulator of PAMP-12 availability for its primary receptor Mas-related G-protein-coupled receptor member X2 (MrgX2).

AM833 Is a Novel Agonist of Calcitonin Family G Protein-Coupled Receptors: Pharmacological Comparison with Six Selective and Nonselective Agonists.

Obesity and associated comorbidities are a major health burden, and novel therapeutics to help treat obesity are urgently needed. There is increasing evidence that targeting the amylin receptors (AMYRs), heterodimers of the calcitonin G protein-coupled receptor (CTR) and receptor activity-modifying proteins, improves weight control and has the potential to act additively with other treatments such as glucagon-like peptide-1 receptor agonists. Recent data indicate that AMYR agonists, which can also independently activate the CTR, may have improved efficacy for treating obesity, even though selective activation of CTRs is not efficacious. AM833 (cagrilintide) is a novel lipidated amylin analog that is undergoing clinical trials as a nonselective AMYR and CTR agonist. In the current study, we have investigated the pharmacology of AM833 across 25 endpoints and compared this peptide with AMYR selective and nonselective lipidated analogs (AM1213 and AM1784), and the clinically used peptide agonists pramlintide (AMYR selective) and salmon CT (nonselective). We also profiled human CT and rat amylin as prototypical selective agonists of CTR and AMYRs, respectively. Our results demonstrate that AM833 has a unique pharmacological profile across diverse measures of receptor binding, activation, and regulation. SIGNIFICANCE STATEMENT: AM833 is a novel nonselective agonist of calcitonin family receptors that has demonstrated efficacy for the treatment of obesity in phase 2 clinical trials. This study demonstrates that AM833 has a unique pharmacological profile across diverse measures of receptor binding, activation, and regulation when compared with other selective and nonselective calcitonin receptor and amylin receptor agonists. The present data provide mechanistic insight into the actions of AM833.

Discovery of a First-In-Class Small Molecule Antagonist against the Adrenomedullin-2 Receptor: Structure-Activity Relationships and Optimization.

Class B G-protein-coupled receptors (GPCRs) remain an underexploited target for drug development. The calcitonin receptor (CTR) family is particularly challenging, as its receptors are heteromers comprising two distinct components: the calcitonin receptor-like receptor (CLR) or calcitonin receptor (CTR) together with one of three accessory proteins known as receptor activity-modifying proteins (RAMPs). CLR/RAMP1 forms a CGRP receptor, CLR/RAMP2 forms an adrenomedullin-1 (AM1) receptor, and CLR/RAMP3 forms an adrenomedullin-2 (AM2) receptor. The CTR/RAMP complexes form three distinct amylin receptors. While the selective blockade of AM2 receptors would be therapeutically valuable, inhibition of AM1 receptors would cause clinically unacceptable increased blood pressure. We report here a systematic study of structure–activity relationships that has led to the development of first-in-class AM2 receptor antagonists. These compounds exhibit therapeutically valuable properties with 1000-fold selectivity over the AM1 receptor. These results highlight the therapeutic potential of AM2 antagonists.

Rosavin suppresses osteoclastogenesis in vivo and in vitro by blocking the nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) signaling pathways.

BackgroundBone homeostasis is mediated by osteoblast-related bone formation and osteoclast-related resorption. The imbalance of bone homeostasis due to excessive osteoclastogenesis or reduced osteogenesis can result in various disorders, such as postmenopausal osteoporosis (PMOP). The receptor activator of nuclear factor-κB ligand (RANKL)-induced nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) and mitogen-activated protein kinase (MAPK) pathways are essential in osteoclastogenesis. In this study, we aimed to investigate the effects of rosavin, an alkylbenzene diglycoside compound from the traditional Chinese medicine Rhodiola Rosea L, on RANKL-induced osteoclastogenesis in vitro and in vivo.MethodsThe effects of rosavin on osteoclastogenesis were assessed by TRAP staining of bone marrow monocyte cells (BMMCs) and RAW 264.7 cells. The effects of rosavin on osteogenesis were determined using alkaline phosphatase (ALP) and alizarin red staining, as well as real-time quantitative reverse transcription polymerase chain reaction. Actin ring formation and bone formation experiments were performed to evaluate osteoclast function. Western blotting was carried out to determine the expression of osteoclastogenesis-related genes, and the activation of the NF-κB and MAPK pathways was evaluated by performing western blotting and immunofluorescence staining. Ovariectomized mice were used to explore the effect of rosavin on bone loss.ResultsRosavin could inhibit osteoclastogenesis, suppress the function of osteoclasts, and decrease the expression of osteoclast differentiation-related genes, including tartrate-resistant acid phosphatase (TRAP), cathepsin K, matrix metalloproteinase-9 (MMP-9), calcitonin receptor (CTR), TNF receptor-associated factor 6 (TRAF-6), receptor activator of nuclear factor-κB (RANK), and colony-stimulating factor-1 receptor (c-fms). Rosavin inhibited RANKL-induced phosphorylation of p65 and inhibitory subunit of NF-κB alpha (IκBα), and suppressed p65 nuclear translocation. Rosavin was also found to inhibit the phosphorylation of extracellular-signal-regulated kinase (ERK), p38, and c-Jun N-terminal kinase (JNK). Furthermore, rosavin promoted osteogenesis in bone marrow mesenchymal stem cells (BMSCs). In vivo experiments showed that treatment with rosavin could alleviate ovariectomy-induced osteoporosis in mice.ConclusionsOur results indicated that rosavin suppressed RANKL-induced osteoclastogenesis in vivo and in vitro by blocking the NF-κB and MAPK pathways. Rosavin treatment is a potential therapy for the clinical treatment of osteoclastogenesis-related disorders.

Calcitonin Gene-Related Peptide Protects Against Cardiovascular Dysfunction Independently of Nitric Oxide In Vivo.

[Figure: see text].

From the Literature

From the Literature

The calcitonin receptor regulates osteocyte lacunae acidity during lactation in mice.

The physiological role of calcitonin, and its receptor, the CTR (or Calcr), has long been debated. We previously provided the first evidence for a physiological role of the CTR to limit maternal bone loss during lactation in mice by a direct action on osteocytes to inhibit osteocytic osteolysis. We now extend these findings to show that CTR gene expression is upregulated two- to three-fold in whole bone of control mice at the end of pregnancy (E18) and lactation (P21) compared to virgin controls. This was associated with an increase in osteoclast activity evidenced by increases in osteoclast surface/bone surface and Dcstamp gene expression. To investigate the mechanism by which the CTR inhibits osteocytic osteolysis, in vivo acidification of the osteocyte lacunae during lactation (P14 days) was assessed using a pH indicator dye. A lower pH was observed in the osteocyte lacunae of lactating Global-CTRKOs compared to controls and was associated with an increase in the gene expression of ATPase H+ transporting V0 subunit D2 (Atp6v0d2) in whole bone of Global-CTRKOs at the end of lacation (P21). To determine whether the CTR is required for the replacement of mineral within the lacunae post-lactation, lacunar area was determined 3 weeks post-weaning. Comparison of the largest 20% of lacunae by area did not differ between Global-CTRKOs and controls post-lactation. These results provide evidence for CTR activation to inhibit osteocytic osteolysis during lactation being mediated by regulating the acidity of the lacunae microenvironment, whilst the CTR is dispensable for replacement of bone mineral within lacunae by osteocytes post-lactation.

Amylin and Calcitonin: Potential Therapeutic Strategies to Reduce Body Weight and Liver Fat.

The hormones amylin and calcitonin interact with receptors within the same family to exert their effects on the human organism. Calcitonin, derived from thyroid C cells, is known for its inhibitory effect on osteoclasts. Calcitonin of mammalian origin promotes insulin sensitivity, while the more potent calcitonin extracted from salmon additionally inhibits gastric emptying, promotes gallbladder relaxation, increases energy expenditure and induces satiety as well as weight loss. Amylin, derived from pancreatic beta cells, regulates plasma glucose by delaying gastric emptying after meal ingestion, and modulates glucagon secretion and central satiety signals in the brain. Thus, both hormones seem to have metabolic effects of relevance in the context of non-alcoholic fatty liver disease (NAFLD) and other metabolic diseases. In rats, studies with dual amylin and calcitonin receptor agonists have demonstrated robust body weight loss, improved glucose tolerance and a decreased deposition of fat in liver tissue beyond what is observed after a body weight loss. The translational aspects of these preclinical data currently remain unknown. Here, we describe the physiology, pathophysiology, and pharmacological effects of amylin and calcitonin and review preclinical and clinical findings alluding to the future potential of amylin and calcitonin-based drugs for the treatment of obesity and NAFLD.

The dual amylin and calcitonin receptor agonist KBP-089 and the GLP-1 receptor agonist liraglutide act complimentarily on body weight reduction and metabolic profile

BackgroundWeight loss therapy is becoming more and more important, and two classes of molecules, namely amylin receptor and GLP-1 receptor agonists, have shown promise in this regard. Interestingly, these molecules have several overlapping pharmacological effects, such as suppression of gastric emptying, reduction of glucagon secretion and weight loss in common; however, they also have distinct effects on prandial insulin secretion. Hence, a combination of these two mechanisms is of significant interest.MethodsIn this study, we investigated the add-on potential of the dual amylin and calcitonin receptor agonist (DACRA) KBP-089 in combination with the GLP-1 receptor agonist liraglutide as obesity treatment in high-fat diet (HFD) fed rats.ResultsIncreasing doses of KBP-089 and liraglutide alone and in combination were studied with respect to their effects on body weight, food intake and glucose metabolism during a 9-week intervention study conducted in HFD rats. Further, the gastric emptying rate during an oral glucose tolerance was assessed. Treatment with KBP-089 and liraglutide dose-dependently lowered body weight 15% (at 2.5 μg/kg/day) and 7% (at 400 μg/kg/day) in HFD rats, respectively, while the combination resulted in a 21% body weight reduction, which was mirrored by reduction in fat depot sizes. Gastric emptying and glucose metabolism were improved, primarily by KBP-089, although liraglutide led to a reduction in fasting plasma glucagon.ConclusionDACRAs complement GLP-1 on food intake, body weight, and glucose tolerance indicating the potential for an add-on therapy.

Treatment with a dual amylin and calcitonin receptor agonist improves metabolic health in an old, obese, and ovariectomized rat model.

Objectives:Menopause is often characterized by detrimental metabolic changes, such as obesity, insulin resistance, and impaired glucose tolerance, often requiring treatment. KeyBioscience Peptides (KBPs) are Dual Amylin and Calcitonin Receptor Agonists which have shown promising metabolic effects in rats. The objective of this study was to investigate the in vivo effect of KBP on the metabolic health in a model driven by unhealthy diet, age, and menopause.Methods:Female Sprague Dawley rats were fed a high-fat diet (HFD) for 3 months before the initiation of the study. At 6 months of age the rats were randomized into groups (n = 12) and subjected to ovariectomy surgery and treatment with KBP: (1) Lean-Sham, (2) HFD-Sham, (3) Lean-OVX, (4) HFD-OVX, (5) HFD-OVX-KBP (10 μg/kg/d), (6) HFD-OVX-KBP (20 μg/kg/d), (7) HFD-OVX-EE2 (30 μg/d 17a-ethynylestradiol). Body weight, food intake, oral glucose tolerance tests (OGTTs), subcutaneous fat, visceral fat, liver weight, and uterus weight were assessed during the 6-month study. Statistical analyses were conducted by one-way ANOVA with Tukey post-hoc test for multiple comparisons.Results:Combination of OVX and HFD led to significant induction of obesity (31% weight increase, P < 0.001) and insulin resistance (13% increase in tAUCglucose during OGTT P < 0.01) compared with the relevant control groups (P < 0.05), and this could be completely rescued by EE2 therapy confirming the model system (P < 0.05).Treatment of OVX-HFD rats with KBP for 26 weeks led to a significant reduction in body weight (13%, P < 0.001) in the high dose and 9% (P < 0.01) in the low dose, with corresponding improvements in fat depot sizes, all compared with HFD-OVX controls. As expected, food intake was suppressed, albeit mainly in the first 2 weeks of treatment, resulting in a reduction of overall caloric intake by 6.5% (P < 0.01) and 12.5% (P < 0.001) in the low and high doses respectively. Furthermore, treatment with KBP reduced the weight of visceral and subcutaneous fat tissues. Finally, KBP treatment significantly improved glucose tolerance, assessed using OGTTs at weeks 8, 16, and 24.Conclusions:The data presented here clearly indicate a positive and sustained effect of KBP treatment on body weight loss, fat depot size, and improved glucose tolerance, illustrating the potential of KBPs as treatments for metabolic complications of overweight and menopause.