Reduction of adenovirus 36-induced obesity and inflammation by mulberry extract

Authors


ABSTRACT

Adenovirus 36 (Ad36) is known to be associated with human obesity and to trigger inflammation in murine models. However, to date no clinical drugs for treating virus-induced obesity have been developed. Therefore, in this study, the anti-obesity and anti-inflammation effects of mulberry extract on Ad36 were evaluated in mice. The mulberry extract-fed group showed a reduction in total body weight and in epidermal fat pads. A combination of various mulberry components (1-deoxynojirimycin, kuromanin chloride and resveratrol) and a mulberry extract prevented viral replication by 50% and 70%, respectively, compared with an untreated Ad36-infected group. Moreover, the extract decreased both concentrations of proinflammatory cytokines, such as MCP-1 and TNF-α, and the numbers of infiltrating immune cells and macrophages in epidermal fat pads. In conclusion, dietary mulberry extract might offer an avenue for the development of therapeutic approaches for treating or preventing virus-induced obesity and inflammation-related metabolic diseases.

List of Abbreviations
Ad36

adenovirus 36

CC

callistephin chloride

DNM

1-deoxynojirimycin

KeC

keracyanin chloride

KuC

kuromanin chloride

NF-κB

nuclear factor of κ light polypeptide gene enhancer in B cells

pfu

plaque-forming unit

qRT-PCR

quantitative real-time polymerase chain reaction

Res

resveratrol

Human adenovirus 36 infection has been shown to increase adiposity in both human and animal model systems [1-5]. In addition, we have determined that Ad36 infection also increases inflammation in adipose tissues and maintains obesity states [6]. Therefore, finding a reagent that generally suppresses inflammation and obesity may help to develop a therapeutic option for treating some forms of virus-induced obesity.

Crop diets are recognized to exert many health benefits, including reduction of obesity and inflammation [7-9]. Thus, they have attracted the attention of the pharmaceutical industry. In particular, mulberry and its components have been recognized to have many therapeutic benefits. Extracts of mulberry leaves have been shown to improve blood glucose concentrations and lipid homeostasis and to reduce obesity [7]. Mulberry can also suppress active oxygen and inflammation in adipocytes via its antioxidant effects [9].

Therefore, we examined whether feeding mulberry extract to mice decreases Ad36-induced obesity by reducing concentrations of proinflammatory cytokines and infiltrating macrophages. Mulberries were obtained from the Rural Development Administration in Korea. The procedure for producing an extract from mulberry has been described in a previous paper [10]. Twenty four-week-old female C57BL/6 mice (Orient Bio, Seongnam, Gyeonggi-do, Korea) were allocated to two groups. The mice received oral mulberry extract supplements. Mice (n = 5) in Group 1 were pre-fed a mulberry diet for 4 weeks before viral infection, whereas mice (n = 5) in Group 2 were fed the same experimental diet and simultaneously subjected to viral infection. After infection, all mice were fed with 60 μg of mulberry extract 5 days/week for 12 weeks. Food and water intakes were measured during each feeding period. All procedures involving mice were approved by the institutional review board of the Catholic University of Korea.

Details of materials and methods regarding such matters as animal handling, mulberry extract methods, virus preparation and plaque-forming assay, qRT-PCR, flow cytometry, ELISA, luciferase assay, body weight and lipid contents, and statistical analysis, are provided in the supporting information.

In this study, we used Ad36 as a trigger for inflammation and obesity because Ad36 infection induces chronic inflammation that may contribute to increased adiposity in animals and humans [1-5]. However, there are no currently available specific treatments or vaccines for Ad36-induced obesity. We have here demonstrated that mulberry has the ability to prevent or treat Ad36-induced obesity, as well as Ad36-induced inflammation (Fig. 1). Four weeks after initiating feeding with mulberry extract, mice were infected with Ad36 (4 × 106 pfu per mouse), after which they were fed the mulberry extract continually for 12 weeks (Group 1). Mice in the mulberry pre-fed group showed a 40% decrease in body weight compared with a control Ad36-infected group that received no mulberry extract (P < 0.05; Fig. 1a). In Group 2, Ad36 infection and commencement of experimental feeding occurred simultaneously and the experimental feeding was continued for 12 weeks. The mean body weight of Group 2 mice was similar to that of the Ad36-infected group that did not receive mulberry extract. There was a tendency toward weight reduction; however, this was not statistically significant (Fig. 1a). The weight gain in the epidermal fat pads was decreased by 30% in both groups compared with the control Ad36-infected group (P < 0.05; Fig. 1a). Thus, mulberry extract was effective in reducing both the total body weight and epidermal fat pad weight in Ad36-infected mice, indicating that it has the potential to act as both a preventive medicine and a therapeutic agent for Ad36-induced obesity.

Figure 1.

Reduction in body and lipid weights and changes in MCP-1 concentrations in mice infected with Ad36 and treated with mulberry extract.

(a) Left panel: Changes in body weight were measured weekly for 12 weeks from time of infection to time of death (weight gain = mean weight in week 12 − mean weight immediately after Ad36 infection) (Mock, cultured media-infected control group; Ad36, untreated Ad36-infected group) (n = 5 per group; #, P < 0.05 vs untreated Ad36-infected group). Right panel: Twelve weeks after infection, the epidermal fat pads were removed and weighed in all groups. The lipid weights were compared with the Ad36-infected control group (n = 5 per group; *, P < 0.05 vs. Mock, #, P < 0.05 vs untreated Ad36-infected group). (b) mRNA expression of MCP-1 and TNF-α in epidermal fat pads were measured by qRT-PCR 12 weeks after Ad36 infection. Expression in each group was compared with that in the untreated Ad36-infected group. Protein concentrations of MCP-1 and TNF-α in epidermal fat pads were measured using ELISA (n = 5 per group; *, P < 0.05; **, P < 0.01 vs Mock; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 vs untreated Ad36-infected group). (c) Infiltrating immune cells were identified by hematoxylin and eosin staining of sections of the epidermal fat pads (arrows, infiltrating immune cells). (d) Macrophages from the stromal vascular fraction were identified by flow cytometry (stained with an anti-F4/80 antibody) to confirm the presence of infiltrating macrophages. The percentages of macrophages in the treatment groups were compared with those in the Ad36-infected control group. mRNA expression of CD64 (M1 macrophages) and CD206 (M2 macrophages) was measured 12 weeks after Ad36 infection (*, P < 0.05; **, P < 0.01 vs Mock; #, P < 0.05; ##, P < 0.01; ###, P < 0.001 vs untreated Ad36-infected group). (e) 3T3L1 cells were pretreated with 100 ng/mL mulberry extract for 16 hr and infected with Ad36 (4 MOI) (pretreated mulberry). After infection, the cells were treated with 100 ng/mL mulberry extract (treated mulberry). After 24 hr, the cells were harvested for luciferase assay for NF-κB promoter activity (**, P < 0.01 vs Mock; #, P < 0.05 vs untreated Ad36-infected group).

A previous study showed that Ad36-infected mice had increases in proinflammatory cytokine concentrations, including MCP-1 and TNF-α [6]. In this study, mice in both mulberry-fed groups showed significant decreases in amounts of mRNA and protein of proinflammatory cytokines compared with the control Ad36-infected mice (Fig. 1b). Mulberry feeding can reduce MCP-1 concentrations and may therefore reduce immune cell numbers. In particular, macrophages infiltrating into adipose tissue were reduced, as shown in hematoxylin and eosin-stained samples and by flow cytometry (Fig. 1c,d). Macrophages are divided into two classes according to function: M1 macrophages induce the Th1 pathway during inflammation, whereas M2 macrophages reflect an anti-inflammatory state [6]. Ad36 infection without mulberry extract increased the M1 macrophage count and decreased the M2 macrophage count in adipose tissue (Fig. 1d). However, mulberry extract decreased the count and mRNA expression of M1 macrophages by 90% compared with the control Ad36-infected group (Fig. 1d). Previously, we showed that Ad36 infection induces NF-κB signaling and subsequently increases proinflammatory cytokines [6]. In that study, 3T3-L1 cells were pretreated with 100 ng/mL mulberry extract overnight, then infected with Ad36 (MOI 4) and treated with extract for 72 hr. In another group, the cells were infected with Ad36 and treated with extract simultaneously. In the present study, we found that both prior and concomitant treatment of 3T3L1 cells with mulberry extract reduced NF-κB transcription activity by 50% compared with Ad36-infected cells (Fig. 1e). Therefore, mulberry extract can effectively decrease the severity of inflammation by reducing NF-κB activation. It can be used to remedy various types of inflammation-associated disease [11]. Thus, more intensive studies of the medical application of mulberry extract or mulberry components are needed.

Mulberry extract clearly reduced viral titers in the adipocytes of mice and treated cells (Fig. 2). Viral titers in the epidermal fat pads were measured to test the anti-viral effect of mulberry extract. Ad36 (4 × 106 pfu/mouse) was injected into mice in Group 1. Because a previous report has shown that Ad36 can be grown in epidermal fat pads and that the viral titer in fat pads is at its highest 3 days after infection [6], viral titers were analyzed by qRT-PCR 3 days after infection. The titers were calculated by comparison with a standard curve for Ad36. Viral titers in the mulberry-fed group were 60% less than in the control Ad36-infected group (P < 0.05; Fig. 2a). The anti-viral effects of both mulberry components and mulberry extract were also examined in vitro. Mulberry extract (1–100 ng/mL) and various mulberry components (5 μg/mL) such as DNM, KuC, Res, callistephin chloride and keracyanin chloride [9], were assessed in 3T3L1 cells. These cells were infected with Ad36 (4 MOI) and then treated simultaneously with the extract or components. Compared with untreated Ad36-infected cells, mulberry extract decreased viral titers by 80%, whereas DNM, KuC and Res reduced them by 50–60% (P < 0.01; Fig. 2b). Therefore, mulberry extract and some mulberry components have anti-viral effects both in vivo and in vitro. Thus, the inhibition of viral replication by mulberry may be the mechanism by which it prevents Ad36-induced obesity and inflammation. In fact, these data are similar to those of previous reports showing that mulberry seed has an antiviral effect against murine norovirus-1 [12] and that mulberry leaf extract has an antiviral effect against hepatitis B and C viruses [13]. Because these studies used mulberry seed and leaf, it is not surprising that mulberry extract also has antiviral effects against Ad36 infection. Furthermore, although the detailed mechanisms have yet to be revealed, liver steatosis improves in humans with previous Ad36 infection who receive a tailored nutritional interventional treatment [14, 15]. Thus, it is possible that mulberry extract may synergistically affect liver function. Mulberry extract contains many unknown components. It is therefore important to identify the functional components when developing drugs based on mulberry. In this study, we found an inverse correlation between Ad36-induced adiposity/inflammation and consumption of mulberry extract. Further studies are needed to identify the mechanism(s) responsible for these effects and the appropriate dose and injection route.

Figure 2.

Prevention of Ad36 growth using mulberry extract.

(a) Mice were pre-fed with mulberry extract for 4 weeks, infected with Ad36 (4 × 106 pfu/mouse) by intraperitoneal injection and then killed 3 days after infection (n = 5 per group). Expression of the Ad36 E1A viral gene was measured in epidermal fat pads and compared with that of the untreated Ad36-infected group by qRT-PCR (***, P < 0.001 vs Mock; #, P < 0.05 vs untreated Ad36-infected group). (b) 3T3L1 cells were treated with 1–100 ng/mL of mulberry extract and mulberry components (5 μg/mL of DNM, KuC, Res, CC and KeC for 16 hr. After soaking overnight, the cells were infected with 4 MOI of Ad36. mRNA expression of the Ad36 E1A viral gene was measured and compared with that of the untreated Ad36-infected group (ND, not detected; *, P < 0.05 vs Mock; #, P < 0.05 vs Ad36-infected group).

Taken together, mulberry extract has a high potential for decreasing both body weight and inflammation and may therefore be developed into a therapeutic agent for Ad36-induced obesity.

ACKNOWLEDGMENTS

This work was supported by a grant from the Gyeonggi-do Regional Research Center of the Catholic University of Korea and by the Basic Science Research Program of the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2012R1A1A2039819).

DISCLOSURE

None of the authors has any conflict of interest associated with this study.

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