Lipid Droplet Biogenesis and Maturation

Cytosolic lipid droplets (LDs) are the cellular stores of fat (triacylglycerol) and cholesterol esters, and represent the largest energy reserve in the body. This energy is mobilized during fasting and prolonged exercise by hydrolyzing the triacylglycerol in the LDs, releasing the fatty acids into the blood stream. Storing too much triacylglycerol in the LDs of fat (adipose) tissue results in overweight and obesity, often accompanied by the development of insulin resistance and type 2 diabetes, which are two of the most important metabolic diseases of the 21st century. A key factor is the accumulation of fat in non-adipose tissues, liver and skeletal muscle, resulting in insulin resistance in these organs. The mechanism by which this so-called ectopic fat stored in the LDs of liver and muscle cells results in defects in insulin signaling is subject of intense research but has so far not been resolved. In addition to obesity and diabetes, LDs play an important role in atherosclerosis which develops by accumulation of lipid-laden macrophages, so-called foam cells that contain very large numbers of lipid droplets.

Despite the central role of LDs in lipid homeostasis fundamental questions concerning the mechanism by which lipids are stored in LDs, as well as the mechanism by which lipids are mobilized from LDs, remain unanswered. Deciphering these mechanisms will be a key to understanding how LD dysfunction relates to important diseases including obesity, diabetes, and atherosclerosis, and opens new ways to prevent and treat these health problems.

Recent research shows that LDs are not merely a static neutral lipid storage site, but in fact dynamic and multi-functional organelles. Although many of the molecular players involved in fat storage in LDs and the mobilization of fatty acids from LDs have been identified, the overall mechanisms of lipid droplet formation and breakdown remain obscure because of a fundamental lack of morphological studies. Because of their chemical composition, LDs are very difficult to preserve during sample preparation, and imaging of LDs therefore requires dedicated methods.

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Our aims are

1. To develop new and optimize existing methods for the visualization and compositional analysis of LDs in their cellular context, in mammalian cells and tissues, including human adipose tissue and skeletal muscle biopsies. These methods will include novel non-linear light microscopy techniques such as CARS microscopy that can be used to determine the lipid composition of individual LDs (Figure 1), as well as electron microscopy techniques that allow high resolution 3-dimensional analysis of LDs in their cellular context (Figure 2)
2. To use these optimized technologies to address key fundamental questions concerning the mechanism of LD formation, growth, and breakdown, and to disclose cell morphological parameters linked to the (mal)function of LDs

At the LM level, we collaborate with the University of Amsterdam (Dr Michiel Müller) to image lipid composition and packing of individual lipid droplets using multiplex CARS microscopy  (Figure 1; Rinia et al., 2007, 2008). At the EM level, we have optimized our method for routine high-pressure freezing of mammalian cell monolayers (Jimenez et al., 2006) for the analysis of LDs (Figure 2; D. Groen, W.J.C. Geerts, and K.N.J. Burger, unpublished)

Fig 1. (a) Multiplex CARS analysis of lipid droplets in HeLa cells. (a) optical section of LDs, (b)  Im{χ(3)} spectrum - equivalent to the spontaneous Raman spectrum - of the LD indicated in (a). This spectrum can be used as a fingerprint for the lipid composition of the LD

 

Fig. 2 EM analysis of lipid droplets in HeLa cells. Conventional chemical fixation (A, B).  High pressure freezing followed by freeze substitution (C, D). The surface layer of lipid droplets can be preserved by inclusion of water in the freeze substitution medium (arrow in D)

Some background on Lipid Droplets

Background on CARS microscopy

Link to our EC network on Lipid Droplets

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References

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