Lipid synthesis is promoted by hypoxic adipocyte-derived exosomes in 3T3-L1 cells

https://doi.org/10.1016/j.bbrc.2014.01.183Get rights and content

Highlights

  • We characterize proteomic profiles of adipocyte-derived exosomes.

  • The protein content of the exosomes under hypoxia is compared with normoxia.

  • Hypoxia increases the total amount of exosomal proteins in adipocytes.

  • Hypoxic adipocyte-released exosomes have enzymes related to de novo lipogenesis.

Abstract

Hypoxia occurs within adipose tissues as a result of adipocyte hypertrophy and is associated with adipocyte dysfunction in obesity. Here, we examined whether hypoxia affects the characteristics of adipocyte-derived exosomes. Exosomes are nanovesicles secreted from most cell types as an information carrier between donor and recipient cells, containing a variety of proteins as well as genetic materials. Cultured differentiated 3T3-L1 adipocytes were exposed to hypoxic conditions and the protein content of the exosomes produced from these cells was compared by quantitative proteomic analysis. A total of 231 proteins were identified in the adipocyte-derived exosomes. Some of these proteins showed altered expression levels under hypoxic conditions. These results were confirmed by immunoblot analysis. Especially, hypoxic adipocyte-released exosomes were enriched in enzymes related to de novo lipogenesis such as acetyl-CoA carboxylase, glucose-6-phosphate dehydrogenase, and fatty acid synthase (FASN). The total amount of proteins secreted from exosomes increased by 3–4-fold under hypoxic conditions. Moreover, hypoxia-derived exosomes promoted lipid accumulation in recipient 3T3-L1 adipocytes, compared with those produced under normoxic conditions. FASN levels were increased in undifferentiated 3T3-L1 cells treated with FASN-containing hypoxic adipocytes-derived exosomes. This is a study to characterize the proteomic profiles of adipocyte-derived exosomes. Exosomal proteins derived from hypoxic adipocytes may affect lipogenic activity in neighboring preadipocytes and adipocytes.

Introduction

Adipose tissues store excess energy in the form of lipids [1], [2]. The tissues are the largest energy reserve in mammals and are capable of accommodating prolonged nutrient excess by altering their mass. However, abnormal or excess accumulation of lipids in adipose tissues causes obesity, which may impair health [3], [4], [5]. Adipose tissue expansion occurs when adipocyte numbers and size increase, which is known as hyperplasia and hypertrophy, respectively [6]. Limiting adipocyte hyperplasia leads to lipid accumulation in existing adipocytes, resulting in hypertrophy. Uptake of exogenous lipids or synthesis of endogenous lipids in the cytosol causes hypertrophy. Smaller adipocytes may be more likely to synthesize fatty acids endogenously (de novo lipogenesis) to begin the lipids accumulation process, while uptake of exogenous fatty acids is more predominant in developing cells [7].

De novo lipogenesis [8] is the process in which non-lipid precursors are converted to fatty acids, and requires acetyl-CoA, which is generated during various metabolic processes. Acetyl-CoA provides the carbon atoms necessary for fatty acid synthesis. It is converted to malonyl-CoA, and the rate-limiting steps in de novo lipogenesis are catalyzed mainly by acetyl-CoA carboxylase (ACC). Successive malonyl-CoA molecules, which serve as a two-carbon donor, are added to acetyl-CoA by the multi-functional enzyme complex, fatty acid synthase (FASN). Glucose-6-phosphate dehydrogenase (G6PD) is a key enzyme that supplies the cellular NADPH required for lipid biosynthesis.

Adipocytes have a limited capacity to accumulate lipid droplets. When adipocytes suffer from lipid overload, hypoxia develops; the reduction in oxygen tension is directly linked to adipocyte dysfunction. To avoid lipid overload and the associated cellular stress in adipose tissues, expression of enzymes related to de novo lipogenesis is reduced [9]. Additionally, adipocytes do not increase in size in a synchronized fashion [10]. Small adipocytes and preadipocytes can act as reservoirs by increasing their storage capacity when larger adipocytes no longer accommodate increased lipid storage. However, how adipocytes without lipid overload are activated to store excess energy remains unknown. Adipocytes communicate with each other and with other tissues [11], but the types of communication between stressed larger adipocytes under hypoxic stress and non-stressed, less hypoxic adipocytes are unknown. Three types of signals are known to control communication between adipocytes [11]: cell-to-cell contact, soluble factors, and exosomes.

Exosomes are small 50–150 μm membrane vesicles secreted from most cell types [12]; they play an important role as information carriers between donor and recipient cells. Exosomes contain a wide variety of cytosolic contents as well as membranous components from donor cells, including genetic materials, lipids, and proteins, which determine the types of information carried [13], [14]. Exosome content is thought to reflect the conditions surrounding the donor cells [15]. Exosomes could fuse with and transfer their internal contents into the cytosol of recipient cells [14]. Upon interacting with exosomes and receiving the internal contents, recipient cells undergo morphological and physiological changes, including cancer metastasis, angiogenesis, and cell differentiation [16], [17], [18], [19]. Adipocytes also secrete exosomes [20]; however, the characteristics of adipocyte-derived exosomes are poorly understood, particularly under pathological conditions.

In this study, we first conducted quantitative proteomic analysis in 3T3-L1 adipocyte-derived exosomes. We demonstrated that multiple enzymes related to de novo lipogenesis were enriched in exosomes secreted under hypoxic conditions. These exosomes may promote lipid accumulation by transferring lipogenic enzymes into recipient cells.

Section snippets

Reagents, cell lines, and animals

Detailed material information can be found in the data supplement.

Exosome purification

Donor cells (3T3-L1 cells or HEK 293T cells) were cultured in DMEM (4500 mg/L glucose) supplemented with 10% exosomes-depleted fetal bovine serum (FBS). Exosomes were depleted of FBS by 12 h ultracentrifugation at 100,000g, 4 °C. Exosomes were prepared from cell supernatants using sequential centrifugation and filtration steps. Briefly, cell supernatants were diluted in an equal volume of phosphate-buffered saline (PBS) and

Serum exosomes are increased in obese animals

To examine whether obesity affects serum exosomes, serum exosomes from leptin-deficient (ob/ob) obesity mice and wild-type (WT) mice were isolated. Exosomes from ob/ob mouse serum contained more protein amount than WT mouse serum (Fig. 1A). Notably, the amount of exosomal protein in ob/ob mice was similar to that in WT mice after compensating for body weight (Fig. 1B), indicating that the increase of serum exosomes in ob/ob mice was due to increased body weight.

Hypoxia enhances exosome secretion in 3T3-L1 adipocytes

Next, using 3T3-L1 cells as a

Discussion

Cellular stress conditions are reflected in the content of cell-derived exosomes [15]. Exosomes contain genetic materials and protein from the cell of origin, and thus depend on the stresses of the donor cells at the time of exosome biogenesis. Exosomes modulate the physiological functions of recipient cells through the transfer of RNA and proteins [14]. Exosomes exposed to some stress have been suggested to generally induce tolerance against further stresses in recipient cells [23]. Although

Conflict of interest and funding

The authors declare no conflict of interest.

Acknowledgments

This study was supported in part by Grant-in-Aid for Scientific Research (24591101) from the Ministry of Education, Science, Sports and Culture, and Hoansha Foundation.

References (33)

  • K.L. Levert et al.

    A biotin analog inhibits acetyl-CoA carboxylase activity and adipogenesis

    J. Biol. Chem.

    (2002)
  • H. van den Bosch

    Phosphoglyceride metabolism

    Annu. Rev. Biochem.

    (1974)
  • E.D. Rosen et al.

    Adipocytes as regulators of energy balance and glucose homeostasis

    Nature

    (2006)
  • A.H. Mokdad et al.

    Prevalence of obesity, diabetes, and obesity-related health risk factors, 2001

    JAMA

    (2003)
  • L.F. Van Gaal et al.

    Mechanisms linking obesity with cardiovascular disease

    Nature

    (2006)
  • S.E. Kahn et al.

    Mechanisms linking obesity to insulin resistance and type 2 diabetes

    Nature

    (2006)
  • Cited by (109)

    • Tissue differences in the exosomal/small extracellular vesicle proteome and their potential as indicators of altered tissue metabolism

      2022, Cell Reports
      Citation Excerpt :

      Among the proteins in this second cluster, several enzymes involved in carbohydrate metabolism (phosphoglycerate kinase 1 [PGK1], pyruvate kinase isozymes M1/M2 [PKM], and lactate dehydrogenase B [LDHB]) or the synthesis of fatty acids (ACLY and FASN) were identified. Previous studies have shown that EVs can transfer enzymes from one cell type to another and promote the activity of metabolic pathway in the target cell (Gabriel et al., 2013; Sano et al., 2014; Yoshida et al., 2019; Zhang et al., 2019), and our data suggest that this may be especially important for sEVs from endothelial cells and adipocytes. Identification of proteins that could specifically mark the tissue of origin of circulating sEVs could be useful in the management and diagnosis of diseases associated with altered exosomal/sEV profiles, such as various types of cancer, Alzheimer’s disease, or metabolic diseases (Eguchi et al., 2016; Logozzi et al., 2017; Mariscal et al., 2016; Rajendran et al., 2006; Saman et al., 2012; Skog et al., 2008).

    View all citing articles on Scopus
    View full text