C19ORF80 Mouse

What is C19ORF80 and what are its alternative names in scientific literature?

C19ORF80 is a gene that encodes the protein ANGPTL8 (Angiopoietin-like protein 8), also known by several other names including lipasin, RIFL, and previously betatrophin. In humans, this protein is encoded by the C19orf80 gene located on chromosome 19, while in mice it is encoded by the Gm6484 gene on chromosome 9 . The protein was discovered in 2012 and has been subject to several name changes as understanding of its function evolved. Notably, the term "betatrophin" was suggested in 2013 based on initial studies proposing a role in pancreatic islet cell proliferation, but this function was later disproven and the original paper retracted in December 2016 . Current scientific consensus favors using ANGPTL8 as the standard nomenclature.

What antibodies are available for detecting mouse ANGPTL8 in experimental settings?

Several antibodies are available for ANGPTL8 detection in mouse research. Based on the search results, researchers can utilize:

The antibody is directed against human recombinant protein fragment corresponding to amino acids 22-198 of human C19ORF80 (NP_061157) produced in E. coli . While this particular antibody is reactive to human ANGPTL8, similar monoclonal antibodies specific for mouse ANGPTL8 are available for mouse model research. When selecting antibodies for mouse studies, researchers should verify species reactivity and validate antibody performance in their specific experimental conditions.

How does ANGPTL8 influence circadian rhythms in mouse models?

ANGPTL8 has been identified as a mediator in food-driven resetting of the hepatic circadian clock in mice. Intraperitoneal (i.p.) injection of recombinant Angptl8 in mice alters diurnal rhythms of locomotor activity . Research demonstrates that ANGPTL8 administration impacts circadian period, though the effects are relatively minor and may be secondary due to other metabolites produced in response to Angptl8 injection that can penetrate the blood-brain barrier .

The relationship between ANGPTL8 and circadian rhythms was investigated using wheel-running activity monitoring. Six-week-old mice placed in standard mouse cages equipped with infrared sensors received daily i.p. injections of 1 μg/kg recombinant Angptl8 or vehicle (PBS) for 24 consecutive days (at ZT2 for 15 days in light-dark cycle and at CT2 for another 9 days when switched to constant darkness) . The circadian period and phase shift in constant darkness were determined using ClockLab analysis software, revealing ANGPTL8's influence on peripheral clock mechanisms, particularly in liver tissue .

What are the controversies surrounding ANGPTL8's role in pancreatic beta cell function?

One of the most significant controversies in ANGPTL8 research was the initial claim that it promoted pancreatic beta cell proliferation, which led to the alternative name "betatrophin." In 2013, researchers from Harvard suggested that ANGPTL8 promoted mouse pancreatic islet cell proliferation, generating considerable excitement about potential therapeutic applications for diabetes .

Current consensus indicates that ANGPTL8's primary functions relate to lipid metabolism rather than beta cell proliferation, though it may still have relevance to diabetes research through other metabolic pathways .

What genetic approaches can be used to study ANGPTL8 function in mice?

Researchers have employed several genetic approaches to study ANGPTL8 function in mice:

• Global knockout mice: ANGPTL8 knockout (KO) mice have been available for over 5 years and display various metabolic phenotypes, including significant decrease in fat mass at 12 weeks of age .

• Liver-specific knockdown: To overcome potential compensatory changes in whole-body knockouts and to specifically study liver-derived ANGPTL8, researchers have developed an adeno-associated virus 8 (AAV8) system harboring ANGPTL8 shRNA for liver-specific knockdown .

• Neutralizing antibody approach: An alternative approach involves quenching circulating ANGPTL8 using Angptl8-neutralized antibodies administered via intraperitoneal injection (50 μg/25g body weight) .

Each approach has specific advantages and limitations. Whole-body knockouts provide insights into systemic functions but may trigger compensatory mechanisms. Liver-specific knockdown offers more precise targeting of hepatic ANGPTL8 functions, while antibody neutralization allows temporal control over protein availability.

What are the optimal protocols for administering recombinant ANGPTL8 in mouse experiments?

Based on the search results, the following protocol has been successfully employed:

• Preparation: Recombinant mouse His-tagged ANGPTL8 protein (commercially available from suppliers like Cloud-Clone Corp.)

• Administration route: Intraperitoneal (i.p.) injection

• Dosage: For circadian rhythm studies, 1 μg/kg body weight has shown efficacy

• Timing: For circadian studies, administration at specific zeitgeber times (e.g., ZT2) is important for consistency and interpretation of results

• Control: Vehicle (PBS) administered using the same volume and route

When designing ANGPTL8 administration experiments, researchers should consider the protein's half-life, potential degradation in storage, and batch-to-batch variation in recombinant protein preparations.

How can researchers effectively monitor ANGPTL8's effects on circadian rhythms?

Researchers can employ multiple complementary approaches to assess ANGPTL8's effects on circadian rhythms:

• Wheel-running activity monitoring: Place mice in cages equipped with infrared sensors to detect locomotor activity. Record wheel revolutions in 6-minute bins using systems like ClockLab (Actimetrics). Analyze circadian period and phase shift in constant darkness using specialized software .

• Real-time monitoring assays: Utilize reporter cell lines such as human Per2::Luc U2OS cells to monitor circadian oscillations in vitro. After treatment with ANGPTL8 or controls, measure bioluminescence using systems like LumiCycle. Analyze period length and amplitude using software such as MATLAB .

• Temporal tissue sampling: Collect tissue samples at regular intervals across the circadian cycle (e.g., every 4 hours for 24 hours) to analyze rhythmic expression of clock genes via RT-qPCR or RNA-seq .

• Reporter gene assays: Utilize luciferase reporters driven by clock gene promoters (e.g., Per1 promoter) to assess ANGPTL8's effects on transcriptional activity. Transfect reporters into appropriate cell lines, treat with ANGPTL8, and measure luciferase activity using dual luciferase systems .

What cell culture systems are most appropriate for in vitro studies of ANGPTL8?

Several cell culture systems have proven effective for in vitro studies of ANGPTL8:

For synchronization experiments, researchers typically treat cells with ANGPTL8 (40 nM) or positive controls like 50% horse serum for 2 hours . For circadian monitoring, the medium is subsequently replaced with recording medium containing luciferin, and bioluminescence is recorded over several days .

How should researchers analyze circadian data from ANGPTL8 experiments?

Analysis of circadian data from ANGPTL8 experiments requires specialized approaches:

• Period and phase analysis: For wheel-running activity data, use software like ClockLab analysis to determine circadian period and phase shifts. Baseline fluctuations should be fitted to polynomial curves and subtracted from raw data .

• Amplitude measurement: Calculate amplitude from raw bioluminescence data (typically from 48 to 120 hours after synchronization) using software like MATLAB .

• Gene expression rhythmicity: For RT-qPCR data of clock gene expression, normalize to housekeeping genes (e.g., 36B4) and analyze rhythmicity using cosinor analysis or similar methods .

• Statistical considerations: When comparing period, phase, or amplitude between experimental groups, use appropriate statistical tests accounting for the circular nature of circadian data when relevant.

These analytical approaches allow for robust quantification of ANGPTL8's effects on circadian parameters and help distinguish direct effects from secondary consequences.

What are the key molecular readouts for assessing ANGPTL8 activity in mouse models?

When evaluating ANGPTL8 activity in mouse models, researchers should consider multiple molecular readouts:

• Clock gene expression: Measure mRNA levels of core clock genes (e.g., Per1, Per2, Bmal1, Clock) via RT-qPCR to assess effects on the molecular clock machinery .

• Protein localization: Use immunocytochemistry to analyze subcellular localization of clock proteins like Per1 following ANGPTL8 treatment .

• Promoter activity: Assess transcriptional effects using luciferase reporters driven by relevant promoters (e.g., Per1 promoter) .

• Protein-protein interactions: Investigate interactions between ANGPTL8 and potential receptors like PirB using colocalization studies .

• Metabolic parameters: Given ANGPTL8's roles in metabolism, measure relevant metabolic parameters such as triglyceride levels, which may be affected by ANGPTL8's activity .

How can conflicting findings about ANGPTL8 function be reconciled?

Conflicting findings about ANGPTL8 function, as exemplified by the beta cell proliferation controversy, can be reconciled through systematic approaches:

• Model system differences: Consider whether discrepancies arise from differences in model systems (e.g., global knockout vs. tissue-specific knockdown). In the case of ANGPTL8, whole-body knockout mice manifest various metabolic phenotypes that could indirectly affect research endpoints .

• Temporal factors: Evaluate whether timing of intervention (acute vs. chronic) or developmental timing contributes to discrepancies.

• Dose-response relationships: Examine whether conflicting results reflect different doses of ANGPTL8, as the protein may have distinct effects at different concentrations.

• Secondary vs. primary effects: Determine whether observed effects are direct consequences of ANGPTL8 activity or secondary to other changes. For example, changes in adipose tissue in ANGPTL8 KO mice lead to alterations in circulating adipokine levels, potentially impacting liver clock homeostasis indirectly .

• Methodological validation: Ensure that key findings are validated using complementary approaches. The retracted beta cell proliferation claim highlights the importance of rigorous validation .

What are promising research areas for understanding ANGPTL8's physiological roles?

Several promising research directions emerge from current understanding of ANGPTL8:

• Circadian metabolism integration: Further explore how ANGPTL8 integrates feeding cues with peripheral clocks, particularly in liver and adipose tissue .

• Receptor identification and signaling: While some evidence suggests PirB as a potential receptor, complete elucidation of ANGPTL8's signaling mechanisms remains an important research goal .

• Therapeutic applications in metabolic disorders: Despite the setback regarding beta cell proliferation, ANGPTL8's roles in lipid metabolism suggest potential therapeutic applications. Inhibition of ANGPTL8 represents a possible therapeutic strategy for hypertriglyceridemia .

• Central nervous system effects: The search results note that physiological roles of ANGPTL8 in the central nervous system remain unknown, presenting an understudied area for future research .

• Interaction with other ANGPTL family members: Understanding how ANGPTL8 functions in concert with related proteins like ANGPTL3 and ANGPTL4, particularly given their structural similarities .

What technological advances would enhance ANGPTL8 research?

Advancing ANGPTL8 research would benefit from several technological developments:

• Conditional and inducible genetic models: Development of more sophisticated mouse models with tissue-specific and temporally controlled ANGPTL8 expression or deletion .

• High-resolution structural data: Experimental determination of ANGPTL8's three-dimensional structure would enhance understanding of its function and interactions .

• Improved in vivo imaging: Methods to visualize ANGPTL8 activity and trafficking in real-time within living organisms would provide dynamic insights into its function.

• Single-cell approaches: Application of single-cell transcriptomics and proteomics to understand cell-type specific responses to ANGPTL8.

• Pathway-specific perturbation tools: Development of tools to selectively modulate specific downstream pathways activated by ANGPTL8 would help dissect its multifaceted functions.