Biochemical and Biophysical Research Communications
Endoplasmic reticulum-mitochondrial interaction mediated by mitofusin-1 or mitofusin-2 is not required for lipid droplet formation or adipocyte differentiation
Section snippets
Background
Triacylglycerol (TG) is a neutral lipid and functions mainly as an energy reserve in eukaryotic organisms that is stored in an organelle known as lipid droplets [1], [2]. Under certain metabolic conditions, such as prolonged low circulating glucose levels, TGs in lipid droplets in adipocytes are broken down to glycerol and free fatty acids, the latter which is used to generate ATP [3]. Lipid droplets are unique in that they are the only organelle with a single phospholipid monolayer that
Cell culture and transfection
Mouse embryonic fibroblasts (wild-type (WT) and Mfn-deficient (Mfn2−/− and Mfn1−/−/Mfn2−/−)) and 3T3-L1 pre-adipocytes (American Type Tissue Culture Collection) were cultured in Dulbecco’s modified Eagle’s medium (DMEM) with 10% fetal bovine serum in a 37 °C incubator with 5% CO2. For some experiments, cells with incubated with 0.5 mM oleate complexed to 0.67% fatty acid-free bovine serum albumin (molar ratio, 4.7:1) for 12 h to stimulate TG synthesis and lipid droplet formation.
For cell
Cells lacking Mfn1 and Mfn2s have altered LD morphology
To determine if ER-mitochondrial interactions mediated by Mfn1 and/or Mfn2 were important for lipid droplet formation, we incubated wild-type (WT) mouse embryonic fibroblasts (MEFs) and MEFs lacking either Mfn2 or both Mfn1 and Mfn2 with 0.5 mM oleate to stimulate lipid droplet formation. In the absence of oleate, cells lacking Mfn2 or both Mfn1 and Mfn2 had lipid droplets similar in both size and number to that of wild-type cells (Fig. 1A and B). However, when TG synthesis was stimulated with
Discussion
In the present study, we investigated the role that the ER-mitochondrial interaction mediated by Mfn2 has in TG storage in lipid droplets. We found that: 1) mouse embryonic fibroblasts lacking Mfn2 have altered lipid droplet morphology, 2) triacylglycerol biosynthesis was not dependent on ER-mitochondrial tethering mediated by Mfn2 and 3) Mfn2 does not have a direct role in adipocyte differentiation.
The increase in lipid droplet size in mitofusin-deficient cells suggested that the TG
Abbreviations
AGPAT1, -acylglycerol 3-phosphate acyltransferase; CoA, coenzyme A; DG, diacylglycerol; DGAT, acyl CoA:diacylglycerol acyltransferase; DMEM, Dulbecco’s modified Eagle’s medium; ER, endoplasmic reticulum; FL-DGAT2, FLAG-tagged DGAT2; GPAT, glycerol 3-phosphate acyltransferase; HRP, horse radish peroxidase; HSP70, heat shock protein 70; MAM, mitochondrial-associated membranes; MEFs, mouse embryonic fibroblasts; NEM, N-ethylmaleimide; PBS, phosphate-buffered saline; TG, triacylglycerol; WT,
Author contributions
PM, HV and PA performed the experiments. SS designed the study and wrote the manuscript. All authors analyzed the results and approved the final version of the manuscript.
Competing interests
The authors declare that they have no competing interests.
Acknowledgements
This work was supported by the Canadian Institutes of Health Research (FRN# 123385) and the Canadian Foundation for Innovation (22785).
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