Recombinant Human ADM2 (ADM2), partial

Which fragments of ADM2 exist and how do they differ in biological activity?

Three primary bioactive fragments of ADM2 have been identified, formed by cleavage at conserved arginine residues:

• ADM2/IMD 1-53: Formed by cleavage at Arg 94-His 95

• ADM2/IMD 1-47: Formed by cleavage at Arg 100-Thr 101

• ADM2/IMD 1-40: Formed by cleavage at Arg 107-Val 108

These fragments exhibit distinct biological activities:

• ADM2/IMD 1-40 demonstrates greater potency in stimulating cAMP generation in vitro and inhibiting food intake in vivo compared to ADM2/IMD 1-47

• ADM2/IMD 1-47 shows stronger hypotensive, heart rate-raising, and gastric-emptying effects than ADM2/IMD 1-40 after intraperitoneal administration

• ADM2/IMD 1-53 produces more potent hypertensive effects following intracerebroventricular injection compared to ADM2/IMD 1-47

Additionally, ADM2 17-47 functions as an antagonist rather than an agonist, inhibiting the effects of ADM2 on trophoblast cell invasion and migration .

Which receptors does ADM2 interact with and what are their relative affinities?

ADM2 interacts with multiple receptors shared with other CGRP family members. The receptors and their relative affinities for ADM2 follow this order:

Despite AM 2 receptor having the highest affinity, most cardiovascular effects of ADM2 are mediated by CGRP receptors and AM 1 receptors. This is because CRLR, RAMP1, and RAMP2 are highly expressed in heart and vascular tissue, while RAMP3 expression is relatively low .

Research also suggests the possible existence of additional, unidentified receptors for ADM2, as ADM2/IMD 17-47 acts as an inverse agonist in the CNS rather than a full antagonist, and combined use of known receptor antagonists only partially blocks ADM2 action in rat spinal cord cells .

What signaling pathways are activated by ADM2 in different tissue contexts?

ADM2 activates multiple signaling pathways in a tissue-dependent manner:

• cAMP signaling pathway: Primary second messenger system activated by ADM2 binding to its receptors

• HIF2α pathway: In adipose tissue, ADM2 markedly increases protein levels and nuclear distribution of HIF2α (but not HIF1α). This activation is mechanistically important, as ADM2-induced increases in ACER2 expression can be reversed by treatment with PT2385, a HIF2α and β dimerization inhibitor

• VEGF signaling pathway: Enriched in ADM2-induced cellular responses

• Wnt signaling pathway: Identified in ADM2-responsive gene sets

The specific downstream effects vary by tissue context. For example, in adipocytes, ADM2 activates HIF2α, which increases ACER2 expression and influences ceramide metabolism, ultimately protecting against NAFLD .

What are the optimal storage and reconstitution conditions for recombinant human ADM2?

To maintain the biological activity of recombinant human ADM2, the following storage conditions are recommended:

• Liquid form: Stable for approximately 6 months at -20°C/-80°C

• Lyophilized form: Stable for approximately 12 months at -20°C/-80°C

• Working aliquots: Store at 4°C for up to one week

For proper reconstitution of lyophilized ADM2:

• Briefly centrifuge the vial before opening to bring contents to the bottom

• Reconstitute in deionized sterile water to a concentration of 0.1-1.0 mg/mL

• Add glycerol to a final concentration of 5-50% (optimally 50%) and prepare aliquots for long-term storage

• Avoid repeated freeze-thaw cycles as they significantly degrade biological activity

What concentrations of ADM2 are most effective for in vitro experiments?

Effective concentrations vary by experimental model and endpoint:

• Neuronal migration studies: 0.5 μM ADM has proven effective in rescuing hypoxia-induced migration deficits in cortical interneurons

• Trophoblast invasion assays: ADM2 significantly enhances the invasion and migration of first-trimester HTR-8SV/neo cells, increasing the invasive index by 2.2-fold compared to controls. This effect can be inhibited by the antagonist ADM2 17-47

• Receptor activation studies: Concentration-dependent effects should be established using dose-response curves, as different fragments (ADM2/IMD 1-53, 1-47, and 1-40) exhibit different potencies in cAMP generation and other receptor-mediated responses

For receptor blockade studies, ADM 22-52 (10 μM) has been used effectively as a specific RAMP2/3 ADM receptor blocker to prevent ADM2-mediated effects .

How does ADM2 contribute to cardiovascular homeostasis and potential cardioprotection?

ADM2 exerts multiple cardiovascular effects that contribute to homeostasis and protection:

• Hypotensive effects: ADM2 administration produces vasodilatory actions, though different fragments exhibit varying potencies

• Anti-fibrotic effects: ADM2/IMD 1-53 inhibits myocardial fibrosis directly through down-regulation of TGFβ

• Heart failure modulation: Microinjection of ADM2 into the hypothalamic paraventricular nucleus decreases cardiac sympathetic afferent reflex through AM 1/2 receptors, improving heart failure outcomes

• Biomarker potential: The expression of ADM2, CRLR and RAMP1/2/3 are increased in cardiac tissues in heart failure models. Plasma levels of ADM2 are elevated in heart failure patients, and pre-pro-ADM2/IMD fragments (25-56 and 57-92) may serve as biomarkers for heart failure

This data is summarized in the following table:

How does ADM2 regulate trophoblast invasion and migration during pregnancy?

ADM2 plays a significant role in placental development and function:

• Expression pattern: ADM2 mRNA is expressed in human placenta, and immunoreactive ADM2 is localized in syncytiotrophoblasts, cytotrophoblasts, and endothelial cells throughout human pregnancy

• Enhanced invasion: ADM2 increases the invasive index of first-trimester HTR-8SV/neo trophoblast cells by 2.2-fold compared to controls, an effect that can be inhibited by ADM2 17-47

• Increased migration: In in vitro migration assays, ADM2 accelerates the migration of trophoblast cells toward scratched areas compared to untreated controls

• Physiological relevance: The consistent expression of ADM2 in placental tissues throughout gestation and its effects on trophoblast invasion and migration suggest it plays an important role in placental development and pregnancy maintenance

These findings indicate ADM2 may be crucial for placental implantation and development, with potential implications for understanding and treating pregnancy complications related to abnormal placentation.

How does adipose-specific ADM2 overexpression influence metabolism and NAFLD?

Adipose-specific overexpression of ADM2 has significant metabolic effects, particularly in relation to non-alcoholic fatty liver disease (NAFLD):

• Transgenic model findings: A transgenic mouse model with adipose-specific overexpression of human ADM2 gene (aADM2-tg mice) showed amelioration of NAFLD through promotion of ceramide catabolism

• HIF2α activation: ADM2 overexpression in adipose tissue leads to:

  • Increased protein levels of HIF2α (but not HIF1α) and its target genes in epididymal white adipose tissue

  • Enhanced HIF2α nuclear distribution in adipocytes

  • Induction of ACER2 (alkaline ceramidase 2) expression, which can be reversed by HIF2α inhibition with PT2385

• Mechanistic pathway: The protective effects of ADM2 against NAFLD appear to be mediated through the ADM2 → HIF2α → ACER2 → ceramide catabolism pathway

This research suggests ADM2 could be a potential therapeutic target for NAFLD, acting through modulation of ceramide metabolism in adipose tissue.

What approaches can distinguish between the effects of ADM2 and other CGRP family members?

Distinguishing between ADM2 and other CGRP family members requires specialized methodological approaches:

• Selective antagonists: Use specific receptor antagonists to block particular pathways:

  • ADM2 17-47: Functions as an antagonist of ADM2

  • ADM 22-52: Acts as a specific RAMP2/3 ADM receptor blocker

• Receptor profiling: Exploit differential receptor affinities since ADM2 has a unique receptor affinity profile (AM 2 receptor ≥ CGRP receptor > AM 1 receptor ≥ AMY 1 receptor > AMY 3 receptor)

• Fragment comparison: Different ADM2 fragments (1-53, 1-47, 1-40) have distinct biological activities that can be compared with activities of other CGRP family members

• Genetic models: Use of tissue-specific knockout or overexpression models can help isolate specific peptide effects, as demonstrated with adipose-specific ADM2 overexpression

• Multidimensional analysis: Combine multiple approaches, including receptor binding, functional assays, and genetic manipulations to comprehensively distinguish between family members

What are the current methods for detecting ADM2 fragments in biological samples?

Detection of ADM2 fragments presents several challenges:

• Antibody limitations: Current antibodies against ADM2 cannot distinguish between different cleavage fragments, making specific fragment identification difficult

• Chromatographic approaches: Gel filtration chromatography and HPLC have been used to demonstrate the presence of ADM2/IMD 1-47 in biological samples

• Fragment markers: Pre-pro-ADM2/IMD fragments (25-56 and 57-92) have been identified in human plasma and may serve as more stable markers compared to mature ADM2 fragments

• Technical challenges: Accurate quantification of ADM2 is complicated by:

  • Low plasma levels

  • Short half-life

  • Receptor binding

  • Presence of binding proteins

Research into more specific detection methods is needed, particularly the development of fragment-specific antibodies or mass spectrometry-based approaches that can distinguish between the various ADM2 forms.

What experimental approaches are effective for studying ADM2's role in neuronal function?

Based on research with ADM and ADM2 in neuronal systems, several approaches have proven effective:

• Real-time imaging of neuronal migration: Live-cell imaging to monitor interneuron movement under control and experimental conditions (e.g., hypoxia with/without ADM2 treatment)

• Saltation analysis: Quantifying migration parameters such as number of saltations, saltation length, and directionality to assess ADM2's effects on neuronal migration

• Receptor blockade: Using ADM 22-52 (a specific RAMP2/3 ADM receptor blocker) to verify that saltation rescue by ADM2 is directly linked to ADM2 receptor activation

• Microtubule dynamics assessment: Examining tubulin hyperpolymerization through techniques such as immunoreactivity studies with Glu-tubulin antibody, as demonstrated in ADM research

• Conditional knockout models: Using tissue-specific gene deletion (e.g., brain-specific knockout) to study the effects of ADM2 absence on neuronal development and function

• Single-cell RNA sequencing: Identifying cell type-specific expression of ADM2 and its receptors under various conditions (e.g., hypoxia)

These approaches provide comprehensive insights into ADM2's roles in neuronal systems while addressing the methodological challenges associated with studying this peptide.

What are the major challenges in developing ADM2 as a therapeutic agent?

Despite promising therapeutic potential, several challenges must be addressed:

• Pharmacokinetic limitations:

  • Short half-life in circulation

  • Undefined enzymes responsible for degradation

  • Need for continued delivery systems for sustained effects

• Fragment specificity:

  • Different ADM2 fragments (1-53, 1-47, 1-40) have varying biological activities

  • Unknown relative amounts of fragments in vivo

  • Lack of reliable methods to distinguish between fragments

• Safety concerns:

  • Haemodynamic effects (hypotensive, vasodilatory) may compromise safety

  • Positive inotropic effects might require careful dosing

• Receptor complexity:

  • Multiple receptor subtypes with different affinities

  • Lack of specific antagonists for AM 1 and AM 2 receptors

  • Unexplored actions through AMY 1/3 receptors

• Delivery challenges:

  • Peptide stability and delivery to target tissues

  • Need for specialized formulations or delivery systems

How can contradictory findings regarding ADM2's functions be reconciled?

Contradictory findings in ADM2 research can be reconciled through several approaches:

• Tissue-specific receptor expression: Different tissues express varying levels of CRLR, RAMP1, RAMP2, and RAMP3, affecting ADM2's actions. For example, CRLR, RAMP1, and RAMP2 are highly expressed in heart and vasculature, while RAMP3 is relatively low

• Fragment-specific effects: The different fragments of ADM2 (1-53, 1-47, 1-40) have distinct biological activities. Inconsistent findings may result from different fragments being used across studies

• Context-dependent signaling: ADM2 activates different pathways depending on cellular context. In adipocytes, it activates HIF2α, while in other tissues, different downstream pathways might be activated

• Experimental considerations:

  • Concentration effects (dose-response relationships)

  • Duration of exposure

  • In vitro versus in vivo models

  • Species differences in receptor distribution and signaling

• Methodological standardization: Development of standardized protocols for ADM2 preparation, storage, and administration would help reduce experimental variability

Future research should include comprehensive characterization of ADM2 fragments, receptor distributions, and signaling pathways across different tissues to build a unified understanding of this multifunctional peptide.