Recombinant Rat Diacylglycerol O-acyltransferase 1 (Dgat1)

What is Recombinant Rat Diacylglycerol O-acyltransferase 1 (DGAT1)?

Recombinant Rat DGAT1 is an enzymatically active form of DGAT1 protein produced through genetic engineering techniques for research purposes. DGAT1 (EC 2.3.1.20) is an integral membrane protein that catalyzes the terminal and only committed step in triacylglycerol synthesis by using diacylglycerol (DAG) and fatty acyl CoA as substrates . The enzyme is also known by several synonyms including ARGP1, DGAT, ARAT, ACAT-related gene product 1, Acyl-CoA retinol O-fatty-acyltransferase, and Diglyceride acyltransferase . As an integral membrane protein, DGAT1 has historically been challenging to purify to homogeneity, which made recombinant protein production particularly valuable for research applications .

What are the key structural characteristics of DGAT1?

DGAT1 is a member of the membrane-bound O-acyltransferase (MBOAT) family. The protein contains multiple transmembrane domains that anchor it to the endoplasmic reticulum membrane where triacylglycerol synthesis occurs. Research indicates that DGAT1 shares regions of similarity with acyl CoA:cholesterol acyltransferase, another enzyme that uses fatty acyl CoA as a substrate . Despite this similarity, experimental evidence confirms that DGAT1 demonstrates high substrate specificity for diacylglycerol, with no detectable acyltransferase activity when alternative substrates like cholesterol or other potential acyl acceptors (including various hydroxycholesterols, vitamins, sterols, and alcohols) are provided .

How is DGAT1 gene expression regulated in different tissues?

DGAT1 gene expression exhibits tissue-specific patterns that correlate with the physiological roles of triacylglycerol in different organs. Expression analyses reveal that DGAT1 mRNA is detectable in all mammalian tissues examined, reflecting the essential role of DGAT1 in cellular glycerolipid metabolism . The highest expression levels are typically found in the small intestine, consistent with DGAT1's proposed critical role in intestinal fat absorption . Significant expression is also observed in adipose tissue, which corresponds with the high levels of DGAT1 activity required for triglyceride storage .

Studies of cell differentiation demonstrate dynamic regulation of DGAT1 expression. During the differentiation of NIH 3T3-L1 cells into adipocytes, DGAT1 mRNA expression increases markedly in parallel with increases in DGAT1 enzymatic activity, correlating with the accumulation of triacylglycerol mass in these cells . This pattern illustrates the close relationship between DGAT1 expression and the cellular capacity for triglyceride synthesis and storage.

What methods are available for measuring DGAT1 enzyme activity?

Several methodological approaches have been developed to measure DGAT1 activity in research settings:

• Fluorescence-based assays: These high-throughput assays monitor the release of CoASH from DGAT1-mediated reactions. For example, the CPM (7-Diethylamino-3-(4-maleimidophenyl)-4-methylcoumarin) assay measures CoASH released during the DGAT1 reaction. CPM reacts with the sulfhydryl group to form a highly fluorescent product detectable at excitation/emission wavelengths of 355/460 nm .

• Radioactive substrate incorporation: Traditional DGAT1 activity assays use either [14C]DAG or [14C]oleoyl CoA as labeled substrates, with subsequent measurement of radioactive triacylglycerol formation . This approach can be performed in membrane fractions from cells expressing DGAT1.

• ELISA-based detection: Commercial ELISA kits allow for the detection and quantification of DGAT1 protein with high sensitivity (e.g., 0.33 ng/mL) across a detection range of 0.781-50 ng/mL in various biological samples including serum, plasma, and tissue homogenates .

What are the implications of DGAT1 inhibition for metabolic disorder research?

DGAT1 inhibition has emerged as a promising therapeutic strategy for metabolic disorders based on multiple lines of evidence from genetic and pharmacological studies. DGAT1 knockout mice display resistance to diet-induced obesity, improved insulin sensitivity, and enhanced glucose homeostasis . These phenotypes suggest that targeted inhibition of DGAT1 could potentially address multiple aspects of metabolic syndrome.

Pharmacological studies using DGAT1 inhibitors have shown several beneficial metabolic effects:

• Obesity prevention: DGAT1 inhibitors protect against diet-induced weight gain in rodent models . T863, a potent DGAT1 inhibitor, demonstrates significant weight reduction effects in diet-induced obese mice .

• Improved insulin sensitivity: Chronic DGAT1 inhibition enhances insulin sensitivity and glucose homeostasis, recapitulating key metabolic benefits observed in genetic knockout models .

• Reduced hepatic steatosis: Pharmacological DGAT1 inhibition decreases fat accumulation in the liver, potentially addressing a key component of metabolic syndrome .

• Delayed fat absorption: DGAT1 inhibitors modify intestinal lipid metabolism, resulting in delayed fat absorption which may contribute to their anti-obesity effects .

Recent research has focused on tissue-specific DGAT1 inhibition to mitigate potential side effects. For example, intestine-targeted DGAT1 inhibitors like Compound B (A-922500) demonstrate improved obesity and insulin resistance outcomes without the skin aberrations observed with systemic DGAT1 inhibition or genetic knockout . This selective approach may provide a more favorable therapeutic profile for metabolic disorder treatment.

How do DGAT1 knockout models compare to pharmacological inhibition in research applications?

Genetic knockout models and pharmacological inhibition approaches provide complementary but distinct insights into DGAT1 function:

DGAT1 Knockout Models:

• Complete absence of DGAT1 protein throughout development

• Display resistance to diet-induced obesity and enhanced insulin sensitivity

• Exhibit abnormalities in the skin, potentially due to developmental effects

• Congenital DGAT1 deficiency affects maintenance and/or development of the sebaceous gland

• Provide insights into long-term, complete DGAT1 deficiency

Pharmacological Inhibition:

• Can be tissue-specific based on inhibitor distribution patterns

• Allows titration of inhibition levels and timing of intervention

• Can avoid developmental effects by administering in adult animals

• Compound-specific effects may vary based on binding sites and pharmacokinetics

• Enables therapeutic potential assessment for human applications

Interestingly, intestine-targeted DGAT1 inhibitors like Compound B demonstrate improved obesity and insulin resistance outcomes without the skin aberrations seen in knockout mice . This suggests that tissue-specific inhibition may provide metabolic benefits while avoiding certain side effects associated with global DGAT1 deficiency.

What mechanisms explain DGAT1 inhibitors' metabolic effects?

The metabolic benefits of DGAT1 inhibition arise from multiple mechanisms affecting lipid metabolism and energy homeostasis:

• Reduced intestinal lipid absorption: DGAT1 inhibitors decrease chylomicron formation and secretion in the intestine, delaying dietary fat absorption . This effect is particularly prominent with intestine-targeted inhibitors like Compound B .

• Altered lipid signaling: By inhibiting the final step of triglyceride synthesis, DGAT1 inhibitors may alter the intracellular pool of diacylglycerol and other lipid intermediates that function as signaling molecules.

• Enhanced lipid oxidation: Evidence suggests DGAT1 inhibition promotes fatty acid oxidation rather than storage, increasing energy expenditure.

• Reduced ectopic lipid deposition: By limiting triglyceride synthesis, DGAT1 inhibitors decrease lipid accumulation in non-adipose tissues like liver and muscle, potentially improving insulin sensitivity .

• Improved hepatic metabolism: DGAT1 inhibitors reduce hepatic steatosis and may alter hepatic insulin sensitivity and glucose output .

The T863 inhibitor provides a model for understanding these mechanisms. Studies show T863 binds to the oleoyl-CoA binding pocket of DGAT1, directly blocking the catalytic activity required for triglyceride synthesis . This molecular interaction explains the high specificity of certain DGAT1 inhibitors and their ability to selectively target triglyceride synthesis without affecting other lipid metabolic pathways.

What experimental challenges exist in studying DGAT1 activity?

Researchers face several significant challenges when investigating DGAT1 activity:

• Protein purification difficulties: As an integral membrane protein, DGAT1 has historically been challenging to purify to homogeneity while maintaining functional activity . This has complicated structural studies and in vitro biochemical characterizations.

• Assay system limitations: Traditional radioactive assays for DGAT1 activity require specialized facilities and have throughput limitations. While newer fluorescence-based methods improve throughput, they may have different sensitivity or specificity profiles compared to traditional methods .

• Tissue expression variability: DGAT1 expression varies across tissues, necessitating tissue-specific study designs and potentially complicating the interpretation of systemic interventions .

• Redundancy with other enzymes: Other enzymes like DGAT2 catalyze similar reactions, potentially compensating for DGAT1 inhibition in some contexts and complicating phenotypic analyses.

• Species differences: DGAT1 sequence, activity, and regulation may vary between species, requiring careful consideration when translating findings across experimental models.

For recombinant rat DGAT1 specifically, researchers should consider the assay conditions carefully. Typical assay parameters include:

• pH 7.4-7.5 buffer (100 mM Tris/HCl or 50 mM HEPES)

• 200 μM 1,2-diacylglycerol

• 100 μM oleoyl-CoA

• 1% Triton X-100

• 0.1-2 μg of microsomal proteins containing DGAT1

These conditions may require optimization based on specific experimental goals or sample types.

How can researchers distinguish DGAT1 activity from other acyltransferases?

Distinguishing DGAT1 activity from other acyltransferases requires careful experimental design:

What are the optimal conditions for expressing recombinant rat DGAT1?

The expression of functional recombinant rat DGAT1 requires careful consideration of several factors:

• Expression system selection: Insect cell expression systems have been successfully used for DGAT1 expression . These systems can produce high levels of mammalian membrane proteins with appropriate post-translational modifications.

• Membrane fraction isolation: Since DGAT1 is an integral membrane protein, proper isolation of membrane fractions is critical for activity assays. Differential centrifugation techniques are typically employed to isolate microsomes containing DGAT1.

• Protein tagging strategies: Addition of epitope tags (such as FLAG) can facilitate detection and purification of recombinant DGAT1 without compromising enzyme activity . When using tagged constructs, researchers should verify that enzyme activity scales with protein expression.

• Detergent selection: For solubilization and activity assays, non-ionic detergents like Triton X-100 (typically at 1% concentration) help maintain DGAT1 in a functional conformation .

• Expression verification: Western blotting with specific antibodies can confirm successful expression, while activity assays using [14C]DAG or [14C]oleoyl CoA substrates verify functional expression .

When expressed in insect cells, recombinant DGAT1 demonstrates activity levels similar to those found in mammalian tissues, significantly above background levels in non-transfected insect cells . This makes insect cell expression systems particularly valuable for biochemical characterization and inhibitor testing.

How should researchers design experiments to study DGAT1's role in metabolic diseases?

When investigating DGAT1's role in metabolic diseases, researchers should consider these experimental design principles:

What techniques are most effective for studying DGAT1 genetic variations?

Investigating genetic variations in DGAT1 requires specialized approaches:

• siRNA technology: For targeted downregulation of DGAT1 genes to study their functional consequences. This approach has been successfully used to downregulate multiple DGAT1 isoforms simultaneously in soybean, revealing their role in determining seed oil and protein composition .

• Transgenic models: Creating transgenic lines with modified DGAT1 expression provides powerful tools for studying long-term physiological effects. For example, trans-acting siRNA technology has been used to downregulate three DGAT1 genes (Glyma.13G106100, Glyma.09G065300, and Glyma.17G053300) simultaneously in soybean .

• Phenotypic analysis: When studying DGAT1 variations, comprehensive phenotyping is essential. In soybean studies, this included measuring:

  • Seed oil concentration (reduced by up to 18 mg/g or 8.3% with DGAT1 downregulation)

  • Seed protein concentration (increased by up to 42 mg/g or 11%)

  • Fatty acid composition (oleic acid levels increased by up to 121 mg/g or 47.3%)

• Functional validation: For novel DGAT1 variants, expression in heterologous systems (like insect cells) followed by activity assays can confirm functional consequences of genetic variations .

• Comparative genomics: Analyzing DGAT1 sequence conservation across species can provide insights into functionally important domains that may be affected by genetic variations.

These approaches collectively enable researchers to understand how genetic variations in DGAT1 affect enzyme function and contribute to phenotypic differences in metabolism and lipid composition.

What are emerging areas of DGAT1 research beyond metabolic disorders?

While DGAT1 research has primarily focused on metabolic disorders, several emerging areas show promise:

• Plant biotechnology: DGAT1 manipulation in plants offers potential for modifying seed composition for agricultural and industrial applications. Research in soybean demonstrates that downregulation of DGAT1 genes can reduce seed oil concentration while increasing protein content, providing a potential strategy for developing soybean varieties with enhanced nutritional profiles .

• Cancer metabolism: The role of altered lipid metabolism in cancer progression has generated interest in DGAT1's potential involvement in cancer cell survival and proliferation.

• Neurodegenerative disorders: As lipid metabolism dysregulation is increasingly recognized in neurodegenerative diseases, DGAT1's role in brain lipid homeostasis represents an understudied area with potential significance.

• Drug delivery systems: DGAT1's role in lipid droplet formation may be leveraged for developing lipid-based drug delivery systems.

• Tissue-specific functions: The identification of intestine-targeted DGAT1 inhibitors opens new possibilities for understanding tissue-specific roles of DGAT1 beyond classic metabolic functions .

These emerging areas highlight the expanding significance of DGAT1 research beyond its established role in metabolic disorders.

What are the current technical limitations in DGAT1 research and potential solutions?

Several technical challenges continue to impact DGAT1 research:

• Structural characterization: Despite its identification over two decades ago, detailed structural information for DGAT1 remains limited due to challenges in purifying and crystallizing this integral membrane protein . Emerging approaches like cryo-electron microscopy may help overcome these barriers.

• Tissue-specific activity measurement: Current methods for measuring DGAT1 activity often require substantial tissue samples and may not capture in situ activity accurately. Development of cell-based reporters or imaging approaches for DGAT1 activity could address this limitation.

• Distinguishing DGAT1 from DGAT2 contributions: Both enzymes catalyze the same reaction, complicating the interpretation of triglyceride synthesis studies. Development of highly specific inhibitors or genetic models with conditional, tissue-specific knockout capabilities would advance research in this area.

• Translation between model systems: Species differences in DGAT1 sequence, regulation, and metabolic context necessitate careful consideration when translating findings between rodent models and human applications. Humanized animal models or human organoids may provide more translatable systems.

• Integration with systems biology: Current approaches often study DGAT1 in isolation rather than as part of complex metabolic networks. Integration of DGAT1 research with systems biology approaches could provide more comprehensive understanding of its role in health and disease.

Addressing these limitations will require interdisciplinary collaboration and continued development of advanced research tools and methodologies.