gAcrp30 Human
Description
Biological Functions and Mechanisms
gAcrp30 exerts potent metabolic effects, primarily via activation of AMP-activated protein kinase (AMPK) and regulation of lipid/glucose metabolism.
Key Mechanisms:
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AMPK Activation:
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Receptor Interactions:
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Therapeutic Effects:
Table 1: Comparative Effects of gAcrp30 vs. Full-Length Adiponectin
Table 2: Biochemical Effects of gAcrp30 in Muscle Tissue
Applications and Clinical Relevance
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Metabolic Disorders: gAcrp30 mimetics are explored for treating obesity, insulin resistance, and type 2 diabetes .
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Cancer and Inflammation: Modulates TNF-α and NF-κB pathways, with potential roles in reducing chronic inflammation .
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Exercise Physiology: May enhance fatty acid oxidation during physical activity, improving endurance .
Challenges and Future Directions
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Structural Complexity: Glycosylation and multimerization status influence activity, complicating therapeutic development .
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Delivery Methods: Optimal routes (e.g., systemic vs. tissue-specific) remain under investigation .
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Paralogs and C1q/TNF-α-Related Proteins (CTRPs): Interactions with CTRP family members (e.g., CTRP1–CTRPs7) require further study .
Product Specs
The globular protein gAcrp30 is a product of adiponectin's proteolytic processing. Adiponectin, a highly abundant plasma protein constituting up to 0.05% of total serum protein, is exclusively produced and secreted by adipocytes. Sharing similarities with Adiponectin, gAcrp30 possesses the ability to reduce hyperglycemia and counteract insulin resistance. Moreover, gAcrp30 plays a crucial role in promoting fat loss by signaling muscle tissue to uptake and metabolize free fatty acids. AdipoR1 and AdipoR2, two recently identified signaling receptors, mediate the actions of both adiponectin and gAcrp30.
Recombinant human gAcrp30, produced in E. coli, is a non-glycosylated polypeptide chain comprising 145 amino acids. With a molecular weight of 16.7 kDa, this protein is purified using proprietary chromatographic techniques.
The product is sterile-filtered and lyophilized from a solution containing 10mM sodium phosphate and 0.5mM DTT at a pH of 7.5.
For reconstitution, it is recommended to dissolve gAcrp30 at a concentration of 0.1 mg/ml in a solution of 10mM sodium phosphate and 0.5mM DTT, pH 7.5. This solution can be further diluted in other aqueous solutions as needed.
The purity of the protein is determined to be greater than 95% by SDS-PAGE analysis.
The biological activity of gAcrp30 is assessed based on its ability to suppress the proliferation of mouse M1 cells. The expected ED50 for this inhibitory effect is 669 ng/ml, which translates to a specific activity of 1.5 x 103 units/mg.
Q&A
What experimental models are most suitable for investigating gAcrp30’s effects on fatty acid oxidation?
gAcrp30’s metabolic actions have been characterized using in vitro and in vivo models. For skeletal muscle studies, rat extensor digitorum longus (EDL) and soleus muscles are dissected and incubated in oxygenated Krebs–Henseleit buffer containing gAcrp30 (2.5 μg/mL) to assess acute effects on AMPK activity, acetyl-CoA carboxylase (ACC) phosphorylation, and malonyl CoA levels . In vivo, retroorbital injection of gAcrp30 (75 μg/mouse) in C57BL/6J mice allows evaluation of AMPK activation in gastrocnemius muscle within 15–30 minutes . Cultured C2C12 myotubes are employed for oleate oxidation assays, where gAcrp30 increases fatty acid oxidation by 39% over 90 minutes . Hepatocytes (e.g., Hepa1-6 cells) show no response, highlighting tissue specificity .
How does gAcrp30 enhance insulin-independent glucose uptake in muscle?
gAcrp30 stimulates glucose transport via AMPK-mediated pathways. In EDL muscle incubated with gAcrp30 (2.5 μg/mL), 2-deoxyglucose uptake increases by 50% after 30 minutes, correlating with transient AMPK activation (2-fold rise in α-2 isoform activity) . This effect is absent in soleus muscle, likely due to fiber-type differences in AMPK sensitivity . Methodologically, glucose transport is quantified using radiolabeled 2-deoxyglucose, with insulin-free conditions to isolate gAcrp30’s direct effects. Researchers must account for incubation duration, as AMPK activation peaks at 30 minutes but diminishes by 60 minutes, whereas ACC phosphorylation remains elevated .
What is the evidence for gAcrp30’s role in weight regulation?
Chronic gAcrp30 administration (daily low-dose injections) in mice fed a high-fat/sucrose diet induces sustainable weight loss without reducing food intake . This is attributed to increased energy expenditure via enhanced muscle fatty acid oxidation. Key data includes:
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Plasma FFA reduction by 20–30% post high-fat meal
Experimental designs should measure respiratory quotient (RQ) and whole-body calorimetry to confirm shifts toward lipid utilization.
How do temporal dynamics of AMPK activation influence experimental interpretations?
gAcrp30’s activation of AMPK is transient, peaking at 30 minutes in vitro and 15–30 minutes in vivo, while downstream ACC phosphorylation and malonyl CoA reduction persist for >60 minutes . This temporal disconnect necessitates careful timing in assays:
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AMPK activity: Measure at 15–30-minute intervals via immunoprecipitation and kinase assays.
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Malonyl CoA: Quantify enzymatically using fatty acid synthase-coupled reactions .
Researchers must avoid conflating acute AMPK activation with sustained metabolic outcomes, as later effects (e.g., glucose uptake) may involve secondary signaling pathways.
Why does full-length Acrp30 fail to replicate gAcrp30’s metabolic effects?
Full-length hexameric Acrp30 (10 μg/mL) does not alter AMPK activity or ACC phosphorylation in EDL muscle, unlike gAcrp30 . This disparity arises from structural differences:
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Proteolytic cleavage exposes gAcrp30’s globular domain, enabling receptor binding absent in full-length oligomers .
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Dose dependency: Full-length Acrp30 requires 10-fold higher concentrations for partial effects .
To resolve this, studies should compare receptor affinity using surface plasmon resonance (SPR) or co-immunoprecipitation with candidate receptors like AdipoR1/R2.
How can tissue-specific responses to gAcrp30 inform therapeutic strategies?
gAcrp30’s effects vary by muscle fiber type:
What methodological pitfalls arise in quantifying gAcrp30’s in vivo effects?
Key challenges include:
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Rapid clearance: gAcrp30’s half-life is short, requiring timed tissue harvesting (e.g., gastrocnemius muscle at 15–30 minutes post-injection) .
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Plasma FFA variability: Use standardized lipid challenges (e.g., Intralipid infusion) to control baseline FFA levels .
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Off-target effects: Include controls for proteolytic degradation products and validate antibodies to distinguish full-length vs. globular Acrp30 .
Methodological Recommendations
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AMPK Activity Assays: Use phospho-specific antibodies (Thr-172) for Western blotting and kinase activity assays with SAMS peptide substrates .
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Malonyl CoA Quantification: Employ enzymatic assays coupled with HPLC to avoid interference from acyl-CoA esters .
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Oleate Oxidation in Cultured Cells: Pre-incubate C2C12 myotubes in differentiation medium for 7 days to ensure myogenic maturity .
Contradictions and Unresolved Issues
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Tissue-specific AMPK activation: Why gAcrp30 activates AMPK in EDL but not soleus muscle remains unclear. Hypotheses include differential expression of AMPK isoforms (α-2 vs. α-1) or regulatory subunits .
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Physiological relevance of cleavage: While gAcrp30 circulates in humans , the protease responsible for Acrp30 processing in vivo is unidentified, complicating translational studies.
Product Science Overview
Adiponectin consists of a 60 amino acid N-terminal collagenous region and a 137 amino acid C-terminal C1q-like globular domain . The globular domain can be cleaved by leukocyte-derived elastase to form globular adiponectin . This globular form is biologically active and has been shown to have various metabolic effects.
Adiponectin plays a crucial role in regulating glucose levels and fatty acid breakdown. It promotes adipocyte differentiation, fatty acid catabolism, and insulin sensitivity . The hormone is known for its anti-inflammatory properties in metabolic contexts, such as obesity and type 2 diabetes, but can have pro-inflammatory effects in non-metabolic disorders like rheumatoid arthritis and inflammatory bowel disease .
Recombinant human globular adiponectin is produced using various expression systems, including E. coli . The recombinant protein is often used in research to study its effects on metabolism and inflammation. It is supplied as a highly purified protein, with purity levels exceeding 90% as determined by SDS-PAGE .
Low levels of adiponectin are associated with obesity, insulin resistance, and type 2 diabetes . Increasing adiponectin levels or enhancing its receptor sensitivity is considered a potential therapeutic strategy for treating these conditions . Adiponectin receptors, AdipoR1 and AdipoR2, mediate the hormone’s effects on glucose uptake and fatty acid oxidation .
