Recombinant Bovine Adipogenin (ADIG)

What is Adipogenin (ADIG) and what cellular functions does it perform?

Adipogenin (ADIG) is an evolutionarily conserved microprotein that is highly expressed in adipose tissues and testis. It functions as a critical regulator for lipid droplet formation in adipocytes by directly interacting with seipin to form a rigid complex . ADIG selectively binds to the dodecameric seipin complex, promoting seipin assembly by stabilizing and bridging adjacent seipin subunits .

Functionally, ADIG plays a key role in generating and expanding lipid droplets in adipocytes. In vitro and in vivo studies demonstrate that ADIG deletion results in aberrant lipid droplet morphology during adipogenesis, while overexpression significantly enhances lipid droplet growth . Research has shown that ADIG is transcriptionally controlled by PPARγ, leading to its high expression in adipose tissues .

How is ADIG expression regulated during adipocyte differentiation?

ADIG expression is dynamically regulated during adipogenesis. Northern blot and protein expression analyses reveal that ADIG is strongly induced during adipocyte differentiation in both brown and white adipocytes . This induction occurs concomitantly with the expression of other adipogenic markers such as PPARγ and Perilipin1 .

The regulation of ADIG expression is primarily mediated through PPARγ, a master regulator of adipogenesis. ChIP-seq data demonstrates that PPARγ directly binds to the promoter region of the ADIG gene in adipocytes . Further supporting this regulatory mechanism, treatment with the PPARγ agonist rosiglitazone induces ADIG expression . This transcriptional control explains the high specificity of ADIG expression in adipose tissues.

What is the subcellular localization of ADIG and how does it relate to its function?

ADIG is predominantly localized to the endoplasmic reticulum (ER) in adipocytes. This localization was confirmed using Adig-Apex2 constructs, which showed most Adig-Apex2 signals were expressed on the ER . Additionally, significant Apex2-positive signals were detected at contact sites between the ER and lipid droplets (LDs) .

Co-localization studies in A431 cells demonstrated that ADIG, seipin, and LDs display spatial proximity during LD induction . This subcellular distribution is consistent with ADIG's functional role in LD formation, as the ER-LD contact sites are critical zones for lipid droplet biogenesis. The strategic positioning of ADIG at these interfaces facilitates its interaction with seipin and subsequent regulation of LD formation and expansion.

What expression systems are most effective for producing recombinant bovine ADIG?

For recombinant bovine ADIG production, bacterial expression systems can be employed for basic structural studies, but mammalian expression systems are recommended for functional analyses due to proper folding and post-translational modifications. Based on experimental approaches used in ADIG research, the following methodological considerations are important:

• Bacterial expression (E. coli): Suitable for producing ADIG for structural studies and antibody generation. Tag the recombinant protein with fusion partners like His6, GST, or MBP to facilitate purification and increase solubility.

• Mammalian expression systems: For functional studies, HEK293 or CHO cells would be appropriate to ensure proper protein folding and modifications. These systems are particularly important if studying ADIG-seipin interactions, as demonstrated in studies using HeLa and A431 cells with Adig-FLAG overexpression .

• Insect cell systems: Baculovirus-infected insect cells can be an alternative for high-yield production while maintaining proper folding of mammalian proteins.

For purification, immobilized metal affinity chromatography followed by size exclusion chromatography has proven effective for obtaining pure, properly folded ADIG protein suitable for structural and functional studies.

What are the critical considerations for designing gain-of-function and loss-of-function ADIG experiments?

When designing experiments to study ADIG function, several critical considerations must be addressed:

For gain-of-function studies:

• Expression vector selection: Use adipocyte-specific promoters for targeted expression. Doxycycline-inducible systems have proven effective for controlled ADIG overexpression in vivo, as demonstrated in mouse models (Adig iTG) .

• Delivery methods: For in vitro studies, adenoviral or lentiviral vectors achieve high transduction efficiency in adipocytes. For in vivo studies, AAV vectors with adipocyte-specific promoters provide targeted expression.

• Expression verification: Confirm overexpression through Western blotting and immunofluorescence microscopy. Research has shown that Adig overexpression can significantly increase endogenous seipin expression, which should be monitored .

For loss-of-function studies:

• Knockout approaches: Use CRISPR-Cas9 for cell lines or conditional knockout systems for animal models. The Adig flox (Adigf/f) mouse model with adiponectin-rtTA/TRE-Cre has been successful for inducible adipocyte-specific deletion .

• Timing considerations: When studying adipocyte differentiation, the timing of ADIG deletion is crucial. In mouse models, inducing Adig knockout at 5-6 weeks of age allows for examining ADIG function in fully developed adipose tissue while avoiding potential developmental effects .

• Controls and validation: Include proper controls (e.g., Adigf/f mice without Cre expression) and validate knockout efficiency through protein expression analysis .

How should researchers design experiments to study ADIG-seipin interactions?

To effectively study ADIG-seipin interactions, researchers should implement the following experimental approaches:

What methodologies are most effective for studying ADIG's impact on lipid droplet formation?

To effectively study ADIG's impact on lipid droplet formation, researchers should employ a combination of the following methodologies:

How can researchers accurately quantify changes in lipid droplet morphology following ADIG manipulation?

Accurate quantification of lipid droplet morphology changes requires systematic analytical approaches:

• High-content imaging platform: Use automated microscopy systems with multiwell capabilities to acquire standardized images across experimental conditions. This approach enables high-throughput analysis while maintaining consistency.

• Standardized image analysis pipeline: Develop a robust analysis workflow that includes:

  • Cell segmentation to identify individual cells

  • LD detection using intensity thresholds

  • Measurement of parameters for each LD (area, perimeter, circularity)

  • Classification of LDs by size (e.g., small, medium, large, supersized categories)

• Multi-parameter quantification: As demonstrated in ADIG research, analyze multiple metrics including:

  • Total LD area per cell

  • Total number of LDs per cell

  • Size distribution of LDs

  • Percentage of cells with supersized LDs

• Statistical analysis: Apply appropriate statistical tests to determine significance of morphological changes. In Adig KO studies, this approach revealed that loss of Adig decreases the total area and total number of LDs (>0.5 μm²) in individual cells, while increasing the percentage of supersized LDs .

• 3D analysis techniques: For more comprehensive assessment, consider confocal microscopy with 3D reconstruction to capture the complete morphology of LDs within the cellular volume.

What are the key differences in ADIG function between brown and white adipose tissues?

Research has revealed important functional differences in ADIG activity between brown adipose tissue (BAT) and white adipose tissue (WAT):

• Differential effects on adipogenesis:

  • In brown adipocytes, Adig deletion does not significantly affect the adipogenic differentiation rate .

  • In contrast, loss of Adig substantially impairs adipogenesis in white adipocytes .

• Lipid droplet morphology impacts:

  • In BAT, Adig deletion results in smaller LDs with occasional supersized LDs .

  • In WAT, Adig KO cells show either very small or very large LDs, with dramatically decreased total LD area and number .

• Thermogenic capacity:

  • Adig overexpression in BAT significantly enhances thermogenesis during cold exposure, as evidenced by attenuated drops in core body temperature in Adig iTG mice .

  • Conversely, Adig deletion in BAT impairs cold tolerance .

• Tissue remodeling:

  • Long-term Adig overexpression causes substantial remodeling of BAT, with visible shrinkage and whitening of the tissue, suggesting BAT-to-WAT conversion .

  • In WAT, long-term Adig overexpression increases tissue weight and adipocyte size .

How can structural information about the ADIG-seipin complex inform targeted drug design?

The high-resolution structural data of the ADIG-seipin complex offers valuable insights for structure-based drug design approaches:

• Targeting interface residues: The Cryo-EM structure of the seipin/ADIG complex (2.98Å resolution) reveals that ADIG selectively stabilizes and binds to the transmembrane domains of the dodecameric seipin complex . Key evolutionarily conserved amino acid residues identified through AlphaFold3 prediction mediate these interactions . These interface residues represent potential binding sites for small molecules that could either disrupt or enhance the interaction.

• Stabilization of functional complex: Since ADIG promotes seipin assembly by stabilizing and bridging adjacent seipin subunits , compounds that mimic this stabilizing effect could potentially enhance seipin function in conditions where it is impaired.

• Modulation of oligomeric state: The finding that seipin can form two unique oligomers (undecamers and dodecamers) with ADIG selectively binding to the dodecameric complex suggests that targeting the determinants of oligomeric state could modulate seipin-ADIG functional outcomes.

• Lipid binding pocket targeting: Since the complex is involved in lipid droplet formation, compounds designed to interact with lipid binding pockets in the complex could modulate lipid transfer and LD formation.

• Allosteric modulation: Identifying allosteric sites in the complex where small molecule binding could influence the conformational dynamics of the complex without directly competing with protein-protein interactions.

What methodological approaches can detect subtle changes in ADIG expression or activity in various disease states?

Detecting subtle changes in ADIG expression or activity in disease states requires sensitive and specific methodological approaches:

• Single-cell RNA sequencing: This technique can reveal cell-specific changes in ADIG expression that might be masked in bulk tissue analysis. This is particularly valuable for heterogeneous tissues like adipose tissue, which contains multiple cell types.

• Phosphoproteomics and other PTM analyses: Since protein activity is often regulated through post-translational modifications (PTMs), mass spectrometry-based approaches to identify changes in ADIG phosphorylation, acetylation, or other modifications can reveal altered activity states.

• Proximity-dependent biotinylation: Using ADIG fused to promiscuous biotin ligases (BioID or TurboID) can help identify changes in the ADIG interactome under different disease conditions, revealing altered functional associations.

• FRET/BRET-based biosensors: Developing biosensors to detect ADIG-seipin interaction in live cells can provide real-time information about complex formation under various conditions.

• Super-resolution microscopy: Techniques such as STORM or PALM can detect nanoscale changes in ADIG localization relative to LDs and the ER that might not be apparent with conventional microscopy.

• Quantitative proteomics with selective reaction monitoring (SRM): This targeted mass spectrometry approach can detect and quantify specific ADIG peptides with high sensitivity, allowing for precise measurement of protein levels across samples.

How might understanding ADIG function inform therapeutic strategies for metabolic disorders?

The molecular and physiological functions of ADIG suggest several potential therapeutic strategies for metabolic disorders:

• Enhancing adipose tissue expandability: ADIG overexpression substantially increases fat mass with enlarged lipid droplets , suggesting that enhancing ADIG function could promote healthy adipose tissue expansion. This might be beneficial in lipodystrophy or in preventing ectopic lipid accumulation in metabolic syndrome.

• Improving thermogenic capacity: ADIG overexpression elevates thermogenesis during cold exposure , indicating that ADIG-targeted therapies might enhance energy expenditure in obesity. The finding that Adig iTG mice showed attenuated drops in core body temperature during cold exposure supports this application .

• Correcting lipid droplet abnormalities: Since ADIG deletion results in aberrant lipid droplet formation , targeting the ADIG-seipin axis could potentially correct LD abnormalities seen in conditions like Berardinelli-Seip congenital lipodystrophy (associated with seipin mutations).

• Enhancing triglyceride clearance: Adig overexpression significantly increased triglyceride uptake from circulation , suggesting ADIG activation could potentially lower circulating triglyceride levels in hypertriglyceridemia.

• Targeting adipose tissue browning: The observation that long-term Adig overexpression causes BAT to significantly shrink and appear to convert to white adipocytes suggests that modulating ADIG function could influence adipose tissue plasticity and browning/whitening processes.

What are common challenges in producing functional recombinant ADIG protein and how can they be overcome?

Researchers working with recombinant ADIG may encounter several technical challenges:

• Protein solubility issues: As a protein that interacts with membrane structures, ADIG may exhibit solubility problems during recombinant expression.

  • Solution: Use fusion tags (SUMO, MBP, GST) to enhance solubility. Optimize buffer conditions with mild detergents (0.1% DDM, CHAPS) or lipid nanodiscs for membrane-associated protein studies.

• Proper folding: Ensuring correct folding of recombinant ADIG is essential for functional studies.

  • Solution: Express protein at lower temperatures (16-20°C) to slow folding. Consider chaperone co-expression systems. Mammalian or insect cell expression systems may provide better folding environments than bacterial systems.

• Post-translational modifications: If bovine ADIG requires specific PTMs for function, bacterial expression systems may be inadequate.

  • Solution: Use mammalian expression systems (HEK293, CHO) or insect cells for proteins requiring mammalian-type modifications.

• Protein stability during purification: Maintaining stability throughout purification can be challenging.

  • Solution: Include glycerol (10%) and reducing agents in buffers. Minimize freeze-thaw cycles. Consider purifying protein in complex with interaction partners like seipin fragments to stabilize structure.

• Activity verification: Confirming that recombinant ADIG retains native functional properties.

  • Solution: Develop activity assays based on known functions, such as seipin binding assays or lipid droplet formation assays in reconstituted systems or cell-based assays with ADIG-deficient cells.

How can researchers resolve conflicting data on ADIG function between in vitro and in vivo studies?

When faced with discrepancies between in vitro and in vivo findings regarding ADIG function, researchers should consider the following approaches:

• Systematic comparison of experimental conditions:

  • Analyze differences in cell types, culture conditions, and expression levels between in vitro models and in vivo tissues.

  • Consider whether the timeframe of observations is comparable between systems.

• Cell type-specific responses:

  • As shown in the research, ADIG deletion affects brown and white adipocytes differently . Validate findings across multiple cell types to identify cell-specific responses.

• Conditional genetic models:

  • Use inducible systems (like the Adig iTG and Adig iAKO models ) to control the timing of ADIG manipulation, helping to distinguish between developmental and acute effects.

• Dose-response relationships:

  • Establish dose-response curves in vitro and aim to achieve comparable expression levels to those observed in vivo. In A431 cells, increasing levels of Adig corresponded to increased seipin-GFP fluorescence and enhanced LD formation .

• Ex vivo approaches:

  • Bridge the gap between in vitro and in vivo by using primary cells or tissue explants from experimental animals, maintaining more of the in vivo context while allowing controlled experimental manipulation.

• Physiological context:

  • Consider how physiological challenges might reveal functional aspects not apparent under basal conditions. For example, cold exposure revealed the thermogenic effect of ADIG overexpression in BAT .

What analytical considerations are important when interpreting triglyceride profiles in ADIG research?

Interpreting triglyceride profiles in ADIG research requires careful analytical considerations:

• Comprehensive lipidomic approach:

  • Use untargeted lipidomics to capture the full range of triglyceride species. In BAT from Adig iTG mice, almost all triglyceride species were increased upon Adig overexpression .

  • Analyze both absolute and relative changes in specific lipid species to identify selective effects on particular fatty acid compositions.

• Context-dependent interpretation:

  • Consider tissue-specific differences in lipid metabolism. BAT and WAT have distinct lipid compositions and turnover rates.

  • Factor in nutritional status and environmental conditions (e.g., temperature) when interpreting results. Cold exposure significantly impacts triglyceride metabolism in BAT .

• Temporal dynamics:

  • Assess acute versus chronic effects. Short-term and long-term Adig overexpression had different impacts on BAT triglyceride content and tissue morphology .

  • Consider using pulse-chase labeling approaches to distinguish between effects on triglyceride synthesis, storage, and mobilization.

• Integration with functional outcomes:

  • Correlate triglyceride profiles with functional outcomes like thermogenesis or glucose homeostasis.

  • Triglyceride clearance tests can provide insights into systemic lipid metabolism beyond static measurements .

• Subcellular distribution:

  • Distinguish between cytosolic and lipid droplet-associated triglycerides.

  • Consider microscomal triglyceride transfer protein (MTP) activity and VLDL secretion when interpreting hepatic triglyceride data.

What are the most promising research directions for understanding ADIG's role beyond adipose tissue?

While ADIG is highly expressed in adipose tissues, exploring its functions in other contexts represents an important frontier:

• ADIG in testicular function: Given that ADIG is also highly expressed in testis , investigating its role in testicular lipid metabolism, spermatogenesis, or steroidogenesis could uncover novel functions.

• Pathological lipid accumulation in non-adipose tissues: Exploring whether ADIG contributes to ectopic lipid accumulation in conditions like non-alcoholic fatty liver disease (NAFLD), cardiac steatosis, or muscle insulin resistance.

• Metabolic cross-talk: Investigating how adipose-derived ADIG might influence other tissues through endocrine or paracrine mechanisms. The observation that ADIG overexpression enhances triglyceride clearance from circulation suggests potential inter-organ effects.

• ADIG in cancer metabolism: Exploring whether ADIG-mediated lipid droplet formation plays a role in cancer cell metabolism, particularly in cancers known to accumulate lipid droplets as a survival mechanism.

• Developmental biology: Investigating ADIG's potential role in embryonic development or in tissue remodeling during organismal growth, given its effects on cell differentiation and lipid metabolism.

• Comparative biology: Studying ADIG function across species to understand evolutionary conservation and divergence in its roles. The observation that key ADIG residues are evolutionarily conserved suggests important functional constraints.

How might single-cell technologies advance our understanding of ADIG function in heterogeneous tissues?

Single-cell technologies offer powerful approaches to dissect ADIG function in complex tissues:

• Single-cell RNA sequencing (scRNA-seq):

  • Mapping ADIG expression across diverse cell populations within adipose tissue.

  • Identifying cell-specific transcriptional responses to ADIG manipulation.

  • Discovering rare cell populations with unique ADIG expression patterns or responses.

• Single-cell proteomics and CyTOF:

  • Quantifying ADIG protein levels and modifications at single-cell resolution.

  • Correlating ADIG expression with signaling pathway activation markers.

• Spatial transcriptomics and proteomics:

  • Preserving tissue architecture while profiling ADIG expression and function.

  • Identifying spatial patterns and potential regional specializations within adipose depots.

  • Mapping ADIG expression relative to vascular structures or nerve innervation.

• Cellular indexing of transcriptomes and epitopes (CITE-seq):

  • Simultaneously profiling ADIG RNA expression and surface protein markers.

  • Linking ADIG expression to cell identity and functional state.

• Single-cell ATAC-seq:

  • Profiling chromatin accessibility to identify cell-specific regulatory mechanisms controlling ADIG expression.

  • Discovering transcription factor binding sites in the ADIG promoter across different cell types.

These approaches could reveal how ADIG function varies across different adipocyte subtypes and non-adipocyte populations within adipose tissue, potentially uncovering specialized roles in immune cells, vascular cells, or adipocyte progenitors that cannot be detected in bulk tissue analyses.

What emerging technologies might enhance the study of ADIG-seipin interactions and their functional consequences?

Several cutting-edge technologies hold promise for advancing our understanding of ADIG-seipin dynamics:

• Cryo-electron tomography:

  • Visualizing ADIG-seipin complexes in their native cellular environment without extraction.

  • Capturing the three-dimensional organization of these complexes at ER-LD contact sites.

• Live-cell single-molecule imaging:

  • Tracking individual ADIG and seipin molecules in real-time to understand dynamic assembly and disassembly of complexes.

  • Measuring residence times and stoichiometry of components at lipid droplet formation sites.

• Optogenetic and chemogenetic tools:

  • Developing light-activated or small molecule-responsive ADIG variants to control its activity with precise spatial and temporal resolution.

  • Creating rapid perturbation systems to dissect acute versus chronic effects of ADIG-seipin interactions.

• Organoid and tissue-on-chip technologies:

  • Establishing three-dimensional adipose tissue models that recapitulate the cellular complexity of in vivo tissues.

  • Testing ADIG function in physiologically relevant microenvironments with controlled parameters.

• CRISPR-based screening:

  • Performing genome-wide or targeted screens to identify genetic modifiers of ADIG-seipin function.

  • Using base editors or prime editors for precise manipulation of specific ADIG residues identified in structural studies .

• AlphaFold and machine learning approaches:

  • Building on AlphaFold3 predictions to model dynamic interactions and conformational changes in the ADIG-seipin complex.

  • Predicting the effects of mutations or small molecule binding on complex stability and function.

These technologies could help resolve key questions about how ADIG-seipin interactions regulate lipid droplet formation at the molecular level, potentially revealing new therapeutic targets for metabolic disorders.