Identification and analysis of lipid droplet-related proteome in the adipose tissue of grass carp (Ctenopharyngodon idella) under fed and starved conditions

Highlights

• Lipid droplet protein composition and activity sheds light on fat accumulation.

• Using proteomics, we found 950 lipid droplet proteins in grass carp adipose tissue.

• Short-term starvation did not change carp body weight, condition, or fat content.

• Lipolysis inhibition and fat production protect carp during short-term starvation.

• Autophagy may be the main strategy against short-term energy deprivation.

Abstract

Fat accumulation in the mesenteric adipose tissue is a serious problem in grass carp (Ctenopharyngodon idella) culture. Lipid droplet-related proteins (LDRPs) are involved in the formation, degradation, and biological functions of lipid droplets. In this study, we aimed to provide reference proteomics data to study lipid droplet regulation in fish. We isolated LDRPs from the mesenteric adipose tissue of grass carp (1-year-old) after normal feeding and 7 days of starvation, and identified and analysed them using isobaric tags for relative and absolute quantitation (iTRAQ) technology. Short-term starvation had no significant effect on the body weight, condition factor, visceral index, hepatopancreas index, intraperitoneal fat index, adipose tissue triglyceride content, and adipocyte size of grass carp. Nine hundred and fifty proteins were identified and annotated using the Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) databases; they are involved in a variety of metabolic and signalling pathways, including amino acid, lipid, and carbohydrate metabolism, and the PI3K-Akt signalling pathway. There were 296 differentially expressed proteins (DEPs), with 143 up-regulated and 153 down-regulated proteins. Three proteins involved in triglyceride and fatty acid syntheses and two proteins involved in autophagy were up-regulated, and six proteins involved in lipid catabolism were down-regulated. These results indicate that under short-term starvation, lipid droplets in the adipose tissue of grass carp may maintain their shape by promoting fat production and inhibiting lipolysis, and autophagy may be one of the main strategies for coping with short-term energy deprivation.

Introduction

The grass carp (Ctenopharyngodon idella) is one of the most important freshwater aquaculture species. With a global production of 5.704 million tons in 2018, the grass carp is a source of low-cost, high-value animal protein in both developing and underdeveloped countries (FAO, 2020). In China, the grass carp has the highest production of all freshwater aquaculture species (Fisheries Bureau of Ministry of Agriculture, 2019). Due to its herbivore nature, the grass carp easily accumulates lipids in the abdominal cavity after feeding high-energy commercial diets in the breeding process, leading to a low feed efficiency, poor growth and meat quality, and high mortality (Tian et al., 2015). The lipids are mostly accumulated in the liver and mesenteric adipose tissue, mainly in the form of lipid droplets in hepatocytes and adipocytes (Tian et al., 2015, Tian et al., 2019).

As an energy-storage organelle, lipid droplets can be found in organisms ranging from yeast to humans. Lipid droplets have a neutral core filled with triglycerides and sterols, surrounded by a monolayer of phospholipid membrane decorated by a series of proteins (Olzmann and Carvalho, 2019). These lipid droplet-related proteins (LDRPs) enable lipid droplets to skate along the cytoskeleton, interact with other cellular organelles, and traffic important molecules (Goodman, 2008). They have been implicated in lipid synthesis and degradation, lipid droplet accumulation and fusion, nutrient signalling pathways, and some physiological and pathological processes (Bartz et al., 2007; Cohen, 2018). The activity of LDRPs is easily influenced by the nutritional status of an organism. For example, under sufficient energy conditions, lipid droplets bud from the endoplasmic reticulum or expand by fusion with other droplets, and the process is mediated by triglyceride synthesis enzymes (Kory et al., 2016); whereas, under energy-shortage conditions, lipid droplets are catabolised by lipolytic enzymes (Frühbeck et al., 2014). Therefore, an understanding of LDRPs can shed light on the regulation of the biological functions of lipid droplets.

The study of LDRPs has been greatly facilitated by the development of proteomic technology, which makes the identification of the composition and changing roles of cellular proteins more convenient. A proteome is the general term used for all proteins expressed in a given cell, tissue, or organism. During recent years, with the development of lipid droplet isolation technology (Wiśniewski et al., 2009; Ding et al., 2013; Brasaemle and Wolins, 2016), numerous LDRP databases based on proteomics technology have been successfully constructed for a variety of cells, including fibroblasts, epidermal cells, fat cells, hepatocytes, macrophages, mammalian muscle cells, and even some organisms such as plants, insects, fungi, algae, and bacteria (Beller et al., 2006; Yang et al., 2012; Krahmer et al., 2013; Bersuker and Olzmann, 2017). By analysing these databases, hundreds of functional proteins associated with lipid droplets have been identified. Some of these proteins have been classified as conservative, including lipid metabolism enzymes, membrane transport-related proteins, signalling proteins, and protein degradation-related proteins (Li et al., 2012; Zhang and Liu, 2019). Overall, these studies indicate the potential role of LDRPs in lipid droplet formation, degradation, and biological functions in cells.

Although lipid droplet-related proteomes of many animals have been constructed (Li et al., 2012; Zhang and Liu, 2019), the proteomics approach has been rarely used to study lipid droplet-related proteomes of fish. Studies in other animal species have shown that the composition of LDRPs varies among different species and even among different tissues of the same species. For instance, in mammals, perilipin-1 is expressed only in the lipid droplets of adipocytes, and testicular and adrenal cells (Kimmel and Sztalryd, 2016). In Caenorhabditis elegans, lipid droplets from different tissues contain different hydroxysteroid dehydrogenase (Liu et al., 2018). Therefore, studies on LDRPs should be species or tissue specific.

The construction of a lipid droplet-related proteome of fish can provide an important theoretical basis for understanding the regulation of fat accumulation in fish. In this study, we aimed to provide a reference for studies on lipid droplet-related proteomics in teleost fish. Lipid droplets were extracted from the adipose tissue of grass carps under fed and starved conditions, and then proteome identification and functional annotation were carried out. A preliminary lipid droplet-related proteome library of grass carp adipose tissue was constructed and differentially expressed proteins (DEPs) were screened.