Basic ScienceAdiponectin homolog novel osmotin protects obesity/diabetes-induced NAFLD by upregulating AdipoRs/PPARα signaling in ob/ob and db/db transgenic mouse models
Introduction
Obesity-associated diabetes-induced nonalcoholic fatty liver disease (NAFLD) is a common cause of chronic liver diseases. NAFLD is representative of the increasing prevalence of metabolic syndrome [1]. NAFLD represents a spectrum of diseases ranging from simple steatosis to steatohepatitis through fibrosis and cirrhosis and is considered to represent the hepatic component of metabolic syndrome, which is related to insulin resistance and other metabolic risk factors such as diabetes mellitus, central abdominal obesity and dyslipidemia [2,3]. NAFLD has become an area of interest in research due to an increasing incidence of obesity-associated diabetes in both children and adults [4]. NAFLD is strongly associated with obesity, insulin resistance conditions such as diabetes mellitus and certain other features of metabolic syndrome such as elevated triglyceride (TG) and low-density lipoprotein (LDL) levels and reduced high-density lipoprotein (HDL) levels. NAFLD is reported to reduce insulin sensitivity in muscle, liver and adipose tissue [5]. Excessive lipids in pancreatic beta cells dysregulate insulin secretion and alter the expression of peroxisome proliferator-activated receptor-alpha (PPAR-α) and glucokinase, ultimately leading to insulin resistance as a result of free fatty acid (FFA)-induced beta cell apoptosis [6]. Conditions such as obesity, diabetes mellitus and hyperlipidemia are the major players that promote NAFLD [4].
Normally, adiponectin levels are high in human serum; however, a decline in the adiponectin level has been observed in obese patients and those with hepatic steatosis [7]. Adiponectin reduces the storage of excess lipids in the liver and provides protection against inflammation and fibrosis due to its insulin-sensitizing property [8]. Established studies have reported that adiponectin and its receptors (AdipoR1/R2) have been associated with various metabolic disorders, such as diabetes, obesity, and cardiovascular and neurodegenerative diseases [[9], [10], [11], [12], [13], [14]]. Moreover, AdipoR1/R2 signaling regulates substrate metabolism through activation of a couple of essential players in cellular energy management including 5, AMP-activated protein kinase (AMPK), sirtuins (SIRT1), and the nuclear receptor PPAR-γ [15,16]. The AdipoR1/R2 regulate age-associated metabolic disorder pathways such as lipid oxidation, glucose uptake and insulin signaling [17]. The osteoanabolic agent GTDF was found to bind to AdipoRs where it enhanced adiponectin signaling and improved the metabolic homeostasis system in rodent diabetes models [18]. Recently, Polyzos et al., reviewed the key role of adipokines in NAFLD and suggested that adipokines approach as noninvasive diagnostic markers of NAFLD and for the therapeutic purpose of NAFLD [19]. Adiponectin is a pleiotropic endogenous adipokine that displays anti-inflammatory and protective activities in various metabolic disorders such as obesity and diabetes [1,20]. Numerous ligands of AdipoRs have been reported, and among them, osmotin is the most accessible and interesting natural plant-derived novel tool and acts as a ligand of AdipoRs. Osmotin, previously known as an antimicrobial protein associated with the plant defense system and a member of the pathogenesis-related protein family 5 (PR-5), is reported to be a structural and functional homolog of the mammalian adiponectin protein [21,22]. Recently, osmotin was reported to possibly act as a homolog of the mammalian hormone adiponectin and induce similar effect in various in vitro and in vivo models [23]. Moreover, we recently reported that osmotin acts via AdipoRs and has neuroprotective properties against metabolic disorders and age-associated neurological disorders such as Alzheimer's disease (AD) [[24], [25], [26]]. Metabolic disorders and age-associated neurological disorders are closely related; AD is even referred to as type 3 diabetes [27]. Further recently a few exciting studies explored the protective effect of osmotin in HUVEC cells and 9Hc2 cells via activation of the AdipoRs [28,29]. Therefore, we hypothesized that osmotin might have beneficial therapeutic effects on the obesity- and diabetes-associated metabolic disorder NAFLD via regulating AdipoR1/R2 and its downstream APPL1/PPAR-α/AMPK/SIRT1 signaling in leptin-deficient ob/ob and db/db mice and that all of these changes in signaling were verified mechanistically in human liver carcinoma HepG2 cells exposed to palmitic acid in vitro.
Section snippets
Osmotin Purification
Osmotin was purified from salt-adapted cultured Nicotiana tabacum cells. The detail methods for the osmotin isolation and purification are described in the supplementary information.
Animal Genotyping, Housing, Ethical Considerations, Grouping and Drug Treatment
The mice used, including wild-type (WT) 7-month-old male (C57BL/6J) mice with a body weight of (22 ± 1.5 g), 7-month-old male leptin-deficient obese (ob/ob) mice (B6.cg.Lepr. <Ob>/J) with a body weight of (49.6 ± 1.5 g), and diabetes (db/db) mice (B6.BKS (D) Lepr, db>/J) with a body weight of (47.4 ± 2.0 g), were
Osmotin Acted via AdipoR1/R2-dependent Mechanism to Reduce Palmitic Acid-induced Toxicity In Vitro
To optimize the palmitic acid in vitro model using human liver carcinoma HepG2 cells, we first treated HepG2 cells with various concentrations of palmitic acid for 12 h. Our results showed that palmitic acid at 100 μM, 200 μM, 500 μM and 1000 μM remarkably decreased HepG2 cell viability (Fig. 1A), while osmotin treatment alone at various concentrations (0.1 μM, 0.2 μM, 0.4 μM, 0.8 μM, and 1 μM) had no toxic effect and improved HepG2 cell viability (Fig. 1B). Further, we co-treated cells with
Discussion
Recently, numerous studies have reported that hepatic adiponectin and AdipoRs signaling play a key and pivotal protective role in the insulin resistance and obesity-associated hepatic diseases e.g. NAFLD [[34], [35], [36], [37], [38], [39]]. Therefore the therapeutic approach to enhance the AdipoR1/R2 targets and its downstream signaling demonstrate a new therapeutic approach against obesity and diabetes-induced NAFLD. Here, our study focused on the effect of the natural plant-derived novel
Conclusion
Taken together, our in vivo and in vitro results suggest that osmotin protects against NAFLD via activation of AdipoRs signaling and APPL1/PPAR-α/AMPK/SIRT1 pathways and reduces body weight, insulin resistance and hepatic glucogenesis, resulting in increased fatty acid oxidation and mitochondrial function. In conclusion, we proposed that supplementation of plant protein osmotin, which has been considered to be a homolog of mammalian adiponectin, is a potential novel therapeutic agent to prevent
Acknowledgements
This research was supported by the Brain Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning (2016M3C7A1904391).
Conflict of Interest
No potential conflicts of interest declared.
Author Contribution
AA designed the experimental work, arranged the data and wrote the manuscript. AA, MWK, AK, MI, and RSU performed western blot analysis. TA, SK, MHJ, NBA, MK, RU and MGJ performed confocal microscopy and in vitro experiments. TA contributed equally in finalizing the whole manuscript. The MOK is a corresponding author, reviewed and approved the manuscript and holds all the responsibilities related to this manuscript. All authors reviewed the manuscript. MOK is a guarantor of this work and, as
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Ahmad and Ali equally contributed this work.