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Quantitative Mapping of Triacylglycerol Chain Length and Saturation Using Broadband CARS Microscopy.

ABSTRACT: Lipid droplets (LDs), present in many cell types, are highly dynamic organelles that store neutral lipids, primarily triacylglycerols (TAGs). With the discovery of new LD functions (e.g., in immune response, protein clearage, and occurrence with disease), new methods to study LD chemical composition in situ are necessary. We present an approach for in situ, quantitative TAG analysis using label-free, coherent Raman microscopy that allows deciphering LD TAG composition in different biochemically complex samples with submicrometer spatial resolution. Employing a set of standard TAGs, we generate a spectral training matrix capturing the variation caused in Raman-like spectra by TAG backbone, chain length, and number of double bonds per chain, as well as the presence of proteins or other diluting molecules. Comparing our fitting approach to gas chromatography measurements for mixtures of standard TAGs and food oils, we find the root mean-square error for the prediction of TAG chemistry to be 0.69 CH₂ and 0.15 #C=C. When progressing to more complex samples such as oil emulsions and LDs in various eukaryotic cells, we find good agreement with bulk gas chromatography measurements. For differentiated adipocytes, we find a significant increase in the number of double bonds in small LDs (below 2 ?m in diameter) compared to large LDs (above 2 ?m in diameter). Coupled with a relatively limited sample preparation requirement, this approach should enable rapid and accurate TAG LD analysis for a variety of cell biology and technological applications.

SUBMITTER: Paul A

PROVIDER: S-EPMC6588730 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Quantitative Mapping of Triacylglycerol Chain Length and Saturation Using Broadband CARS Microscopy.

Paul Alexandra A Wang Yujen Y Brännmark Cecilia C Kumar Sachin S Bonn Mischa M Parekh Sapun H SH

Biophysical journal 20190511 12

Lipid droplets (LDs), present in many cell types, are highly dynamic organelles that store neutral lipids, primarily triacylglycerols (TAGs). With the discovery of new LD functions (e.g., in immune response, protein clearage, and occurrence with disease), new methods to study LD chemical composition in situ are necessary. We present an approach for in situ, quantitative TAG analysis using label-free, coherent Raman microscopy that allows deciphering LD TAG composition in different biochemically ...[more]

PMID: 31153590

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Project description:Aims/hypothesisIntramyocellular lipid (IMCL) content associates with development of insulin resistance, albeit not in insulin-sensitive endurance-trained athletes (trained). Qualitative and spatial differences in muscle lipid composition may underlie this so-called athlete's paradox. Here we studied triacylglycerol (TAG) composition of individual myocellular lipid droplets (LDs) in trained individuals and individuals with type 2 diabetes mellitus.MethodsTrained ([Formula: see text] 71.0 ± 1.6 ml O2 [kg lean body mass (LBM)]-1 min-1), normoglycaemic (fasting glucose 5.1 ± 0.1 mmol/l) individuals and untrained ([Formula: see text] 36.8 ± 1.5 ml O2 [kg LBM]-1 min-1) individuals with type 2 diabetes (fasting glucose 7.4 ± 0.5 mmol/l), with similar IMCL content (3.5 ± 0.7% vs 2.5 ± 0.3%, p = 0.241), but at opposite ends of the insulin sensitivity spectrum (glucose infusion rate 93.8 ± 6.6 vs 25.7 ± 5.3 μmol [kg LBM]-1 min-1 for trained individuals and those with type 2 diabetes, respectively) were included from our database in the present study. We applied in situ label-free broadband coherent anti-Stokes Raman scattering (CARS) microscopy to sections from skeletal muscle biopsies to measure TAG acyl chain length and saturation of myocellular LDs. This approach uniquely permits examination of individual LDs in their native environment, in a fibre-type-specific manner, taking into account LD size and subcellular location.ResultsDespite a significant difference in insulin sensitivity, we observed remarkably similar acyl chain length and saturation in trained and type 2 diabetic individuals (chain length: 18.12 ± 0.61 vs 18.36 ± 0.43 number of carbons; saturation: 0.37 ± 0.05 vs 0.38 ± 0.06 number of C=C bonds). Longer acyl chains or higher saturation (lower C=C number) could be detected in subpopulations of LDs, i.e. large LDs (chain length: 18.11 ± 0.48 vs 18.63 ± 0.57 carbon number) and subsarcolemmal LDs (saturation: 0.34 ± 0.02 vs 0.36 ± 0.04 C=C number), which are more abundant in individuals with type 2 diabetes.Conclusions/interpretationIn contrast to reports of profound differences in the lipid composition of lipids extracted from skeletal muscle from trained and type 2 diabetic individuals, our in situ, LD-specific approach detected only modest differences in TAG composition in LD subpopulations, which were dependent on LD size and subcellular location. If, and to what extent, these modest differences can impact insulin sensitivity remains to be elucidated. Graphical abstract.

Dataset Information

Quantitative Mapping of Triacylglycerol Chain Length and Saturation Using Broadband CARS Microscopy.

Publications

Quantitative Mapping of Triacylglycerol Chain Length and Saturation Using Broadband CARS Microscopy.

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