Autophagy is an evolutionarily conserved process that aims to maintain the energy homeostasis of the cell by recycling long-lived proteins and organelles. We have very recently demonstrated that lipophagy, a special form of autophagy, mediates the association of the lipid droplets (LDs) with lysosomes to deliver the lipid cargo within the LDs to lysosomes for degradation in order to release free cholesterol required for steroid synthesis in human ovary and testis. In this chapter, we describe live cell confocal microscopy technique that allows us to monitor real-time cholesterol trafficking and the association of cholesterol-laden LDs with lysosome (lipophagy) in human granulosa cells.

Lipid droplets (LDs) are a neutral lipid storage organelle that is conserved in almost all species. Excessive storage of neutral lipids in LDs is directly associated with many metabolic syndromes. Zebrafish is a better model animal for the study of LD biology due to its transparent embryonic stage compared to other organisms. However, the study of LDs in fish has been difficult due to the lack of specific LD marker proteins and the limitation of purification technology. In this paper, the purification and proteomic analysis of liver LDs of fish including zebrafish and Carassius auratus were performed for the first time. 259 and 267 proteins were identified respectively. Besides most of the identified proteins were reported in previous LD proteomes of mammals, indicating the similarity between mammal and fish LDs. We also identified many unique proteins of liver LDs in fish that are involved in the regulation of LD dynamics. Through morphological and biochemical analysis, we found that the marker protein Plin2 of zebrafish LD was located on LDs in Huh7 cells. These results will facilitate further study of LDs in fish and liver metabolic diseases using fish as a model animal.

Lipid droplets (LDs) are generally considered to be synthesized in the ER and utilized in the cytoplasm. However, LDs have been observed inside nuclei in some cells, although recent research on nuclear LDs has focused on cultured cell lines. To better understand nuclear LDs that occur in vivo, here we examined LDs in primary hepatocytes from mice following depletion of the nuclear envelope protein lamina-associated polypeptide 1 (LAP1). Microscopic image analysis showed that LAP1-depleted hepatocytes contain frequent nuclear LDs, which differ from cytoplasmic LDs in their associated proteins. We found type 1 nucleoplasmic reticula, which are invaginations of the inner nuclear membrane, are often associated with nuclear LDs in these hepatocytes. Furthermore, in vivo depletion of the nuclear envelope proteins lamin A and C from mouse hepatocytes led to severely abnormal nuclear morphology, but significantly fewer nuclear LDs than were observed upon depletion of LAP1. In addition, we show both high-fat diet feeding and fasting of mice increased cytoplasmic lipids in LAP1-depleted hepatocytes but reduced nuclear LDs, demonstrating a relationship of LD formation with nutritional state. Finally, depletion of microsomal triglyceride transfer protein did not change the frequency of nuclear LDs in LAP1-depleted hepatocytes, suggesting that it is not required for the biogenesis of nuclear LDs in these cells. Together, these data show that LAP1-depleted hepatocytes represent an ideal mammalian system to investigate the biogenesis of nuclear LDs and their partitioning between the nucleus and cytoplasm in response to changes in nutritional state and cellular metabolism in vivo.

The purpose of the present study was to investigate molecular compositions of lipid droplets changing in live hepatic cells stimulated with major fatty acids in the human body, i.e., palmitic, stearic, oleic, and linoleic acids. HepG2 cells were used as the model hepatic cells. Morphological changes of lipid droplets were observed by optical microscopy and transmission electron microscopy (TEM) during co-cultivation with fatty acids up to 5 days. The compositional changes in the fatty chains included in the lipid droplets were analyzed via Raman spectroscopy and chemometrics. The growth curves of the cells indicated that palmitic, stearic, and linoleic acids induced cell death in HepG2 cells, but oleic acid did not. Microscopic observations suggested that the rates of fat accumulation were high for oleic and linoleic acids, but low for palmitic and stearic acids. Raman analysis indicated that linoleic fatty chains taken into the cells are modified into oleic fatty chains. These results suggest that the signaling pathway of cell death is independent of fat stimulations. Moreover, these results suggest that hepatic cells have a high affinity for linoleic acid, but linoleic acid induces cell death in these cells. This may be one of the causes of inflammation in nonalcoholic fatty liver disease (NAFLD).

In Saccharomyces cerevisiae, inositol polyphosphate kinase KCS1 but not VIP1 knockout is of great significance for maintaining cell viability, promoting glycolysis metabolism, and inducing mitochondrial damage. The functions of Candida albicans inositol polyphosphate kinases Kcs1 and Vip1 have not yet been studied. In this study, we found that the growth rate of C. albicans vip1Δ/Δ strain in glucose medium was reduced and the upregulation of glycolysis was accompanied by a decrease in mitochondrial activity, resulting in a large accumulation of lipid droplets, along with an increase in cell wall chitin and cell membrane permeability, eventually leading to cell death. Relieving intracellular glycolysis rate or increasing mitochondrial metabolism can reduce lipid droplet accumulation, causing a reduction in chitin content and cell membrane permeability. The growth activity and energy metabolism of the vip1Δ/Δ strains in a non-fermentable carbon source glycerol medium were not different from those of the wild-type strains, indicating that knocking out VIP1 did not cause mitochondria damage. Moreover, C. albicans KCS1 knockout did not affect cell activity and energy metabolism. Thus, in C. albicans, Vip1 is more important than Kcs1 in regulating cell viability and energy metabolism.

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Laboratory mice are standardly housed at around 23 °C, setting them under chronic cold stress. Metabolic changes in the liver in mice housed at thermoneutral, standard and cold temperatures remain unknown. In the present study, we isolated lipid droplets and mitochondria from their livers in a comparative proteomic study aiming to investigate the changes. According to proteomic analysis, mitochondrial tricarboxylic acid cycle (TCA cycle) and retinol metabolism are enhanced, whereas oxidative phosphorylation is not affected obviously under cold conditions, suggesting that liver mitochondria may increase TCA cycle capacity in biosynthetic pathways, as well as retinol metabolism, to help the liver to adapt. Based on proteomic and immunoblotting results, perilipin 5 and major urinary proteins are increased significantly, whereas mitochondrial pyruvate carrier is decreased dramatically under cold conditions, indicating their involvement in liver adaptation.

Neutral lipid storage disease with myopathy (NLSDM) and with ichthyosis (NLSDI) are rare autosomal recessive disorders caused by mutations in the PNPLA2 and in the ABHD5/CGI58 genes, respectively. These genes encode the adipose triglyceride lipase (ATGL) and α-β hydrolase domain 5 (ABHD5) proteins, which play key roles in the function of lipid droplets (LDs). LDs, the main cellular storage sites of triacylglycerols and sterol esters, are highly dynamic organelles. Indeed, LDs are critical for both lipid metabolism and energy homeostasis. Partial or total PNPLA2 or ABHD5/CGI58 knockdown is characteristic of the cells of NLSD patients; thus, these cells are natural models with which one can unravel LD function. In this review we firstly summarize genetic and clinical data collected from NLSD patients, focusing particularly on muscle, skin, heart, and liver damage due to impaired LD function. Then, we discuss how NLSD cells were used to investigate and expand the current structural and functional knowledge of LDs.

Chronic hepatitis B virus (HBV) infection has been reported to be associated with the prevalence of non‑alcoholic fatty liver disease (NAFLD). However, the present study demonstrated that the incidence of fatty liver disease in HBV‑infected subjects (16/152, 10.5%) was not significantly different from in non‑HBV‑infected subjects (292/1,714, 17%), following adjustment for age (odds ratio=0.656; 95% confidence interval=0.379‑1.134; P=0.131). Hepatitis B protein X (HBx) is considered a key regulator in HBV infection and several studies have confirmed that HBx serves a pivotal role in the process of fatty liver disease. In the present study, it was demonstrated that HBx‑expressing cells exhibited increased mitochondrial membrane potential, ATP generation, and endogenous mitochondrial respiration. In addition, higher levels of mitochondrial reactive oxygen species (ROS) were detected in HBx‑expressing cells compared with in control cells. Increased ROS production may contribute to increased lipid droplet formation in HBx‑expressing cells, whereas the removal of ROS with N‑acetylcysteine may decrease the accumulation of lipid droplets in a time‑dependent manner. In conclusion, the present findings indicated that HBV, and perhaps more specifically HBx, was not a protective factor against NAFLD. HBx may function as a risk factor for fatty liver disease, based on the findings of the present functional study; however, further studies are required to clarify the effects of HBx on hepatic steatosis.

Excess adipose fat accumulation, or obesity, is a growing problem worldwide in terms of both the rate of incidence and the severity of obesity-associated metabolic disease. Adipose tissue evolved in animals as a specialized dynamic lipid storage depot: adipose cells synthesize fat (a process called lipogenesis) when energy is plentiful and mobilize stored fat (a process called lipolysis) when energy is needed. When a disruption of lipid homeostasis favors increased fat synthesis and storage with little turnover owing to genetic predisposition, overnutrition or sedentary living, complications such as diabetes and cardiovascular disease are more likely to arise. The vinegar fly Drosophila melanogaster (Diptera: Drosophilidae) is used as a model to better understand the mechanisms governing fat metabolism and distribution. Flies offer a wealth of paradigms with which to study the regulation and physiological effects of fat accumulation. Obese flies accumulate triacylglycerols in the fat body, an organ similar to mammalian adipose tissue, which specializes in lipid storage and catabolism. Discoveries in Drosophila have ranged from endocrine hormones that control obesity to subcellular mechanisms that regulate lipogenesis and lipolysis, many of which are evolutionarily conserved. Furthermore, obese flies exhibit pathophysiological complications, including hyperglycemia, reduced longevity and cardiovascular function - similar to those observed in obese humans. Here, we review some of the salient features of the fly that enable researchers to study the contributions of feeding, absorption, distribution and the metabolism of lipids to systemic physiology.