Adenovirus 36 and Obesity: An Overview

Abstract

1. Introduction

Obesity is a disease characterized by an excess of fat mass. The incidence of obesity in recent decades has grown dramatically [1].

The prevalence of obesity doubled in adults and tripled in children during the last 20 years, even in countries with traditionally low rate of the disease. In 2014, more than 1.9 billion adults, 18 years old and older, were overweight. Of these, over 600 million were obese. The worldwide prevalence of obesity more than doubled between 1980 and 2014. (World Health Organization (WHO) data; [2]). The risks of diabetes, hypertension and dyslipidaemia increase starting from a body mass index (BMI) of about 21.0 kg/m², thereby reducing life expectancy and greatly increasing the health and social economic burden. Obesity is clearly related to increased mortality, morbidity and disability rates. Overweight and obesity are the fifth leading risk for global deaths. Obesity is now described as a phenomenon in character epidemic [3], and according to the WHO, obesity represents “one of the major Public Health problems of our time”. Obesity is also a risk factor for numerous chronic diseases such as cardiovascular diseases and diabetes, orthopedic problems and mental disorders. The scientific community started to study the obesity epidemic looking at the possible causes with the aim to find effective treatments to reduce morbidity and mortality.

Although obesity is a multifactorial disease caused by the interaction between genetics, metabolism, social, cultural and environmental factors, viral infection has been suggested as a possible cofactor for the development of obesity [4].

Immune response depends by nutritional status and can be easily dysregulated in states of imbalanced nutrition such as obesity. This may predispose obese individuals to increased susceptibility to infection [5,6].

In the hospital setting, obese patients are more likely to develop secondary infections and complications such as sepsis, pneumonia, bacteremia, wound infections, and respiratory infections [7,8]. Obesity has been associated with increased risk of complications due to surgical site infections [9,10,11]. Obese individuals have increased risk of Helicobacter pylori [12] infection and overweight children show a three times greater risk of Neisseria meningitides [13]. Obesity is also a risk factor of severe infection and death caused by the pandemic influenza strain H1N1 [14].

Overall, these observations indicate that excessive adipose tissue expansion predisposes individuals to various infections. On the other hand, new data have been generated in the last few years suggesting infectious agents being the cause of obesity in addition to being more easily hosted in an obese individual. Among the multitude of infectious agents, adenoviruses are the human pathogens that more than others are causatively and correlatively linked with animal and human obesity, respectively, and seem to directly influence the adipose tissue [15,16].

In animals, adenovirus serotype Adv31 and Adv9 correlate with obesity and are adipogenic in animal cells culture [17,18]. An avian adenovirus (SMAM-1) and the human adenovirus type 36 (Adv36) have been associated with obesity [19] and there are reports a suggesting significant role of adenoviruses in the development of human obesity.

Here we provide an overview of the data available on the relationship between adenovirus infection and obesity.

2. Association of Viral Infections with Obesity in Animal Models

Five infectious agents were implicated in contributing to obesity, including canine distemper virus (CDV), rous associated virus (RAV)-7, borna disease virus (BDV) and adenoviruses [19]. Apart from adenoviruses, all the other viruses of this list are associated with brain damage and, in experimental animals, obesity develops via brain involvement or via direct damage of fat tissue. CDV is a paramyxovirus that infects dogs and other wild mammals; it was one of the first infectious agents identified to cause enlarged fat cells and increased body weight in mice [19]. The CDV infected animals showed twofold increase in body weight and a selective, virus-induced disruption of critical brain catecholamine pathways [20]. RAV-7 is one of the most common retroviruses affecting chickens [21]. The infected chickens are smaller than hatchmates and develop obesity, ataxia, lymphoblastoid infiltration of thyroid and pancreas, liver fat accumulation and a frank lipemia. BDV is a nonsegmented negative-stranded RNA virus that may cause obesity in various animals, including rats and chickens. Most studies suggest that BDV causes obesity by inflammation of the hypothalamus. In infected animals, hyperplasia of pancreatic islets, increase in glucose and triglyceride levels is also seen [19].

Adenoviruses are the only infective agents reported to be linked with adiposity in both experimental animal models and naturally infected humans [22].

3. Adenoviruses

Adenoviruses are medium size viruses. All human adenoviral genomes have the same general organization. The genome consists of a single linear, double-stranded DNA molecule with short inverted terminal repeats at each end. A protein, terminal protein, is covalently attached to both the 5′ ends of the genomic DNA. The viral chromosome has five early transcription units (E1A, E1B, E2, E3 and E4) (Figure 1).

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Figure 1

Genomic organization of Adv36: The early proteins (E1A, E1B, E2A, E2B, E3, and E4) are involved in the regulation of replication of DNA. The late proteins (L1–L5), products of the translation of late mRNA, constitute structural capsid proteins.

There are more than 50 immunologically distinct serotypes of adenovirus that can cause infections in humans. Serotypes are operationally defined by the ability of the antibodies induced by infection to neutralize that serotype only, and not other adenovirus. The human adenoviruses have been further classified into six subgroups (A–F) based upon hemoagglutination proprieties and genetic relatedness.

Although the epidemiological characteristics of the adenovirus serotype change, all are transmitted by direct contact, fecal-oral transmission, and sometimes by environmental transmission (contaminated water). Most of the adenovirus cause respiratory diseases, but can also cause gastroenteritis, conjunctivitis and cystitis [19,23].

The adenovirus virion contains a 36 kb genome of double-stranded DNA, which is surrounded by a non-enveloped icosahedral protein capsid. The capsid is composed of 252 capsomers: 240 hexons and 12 pentons at the vertices of the icosahedrons. Each penton consists of a penton base and a penton fiber. The fibers consist of a slender shaft with a globular head. They are involved in the process of attachment of the virus particle to the host cell.

Adv2 and Adv5 have been used for most studies of the adenovirus replication cycle. These viruses can be easily grown in the laboratory by infecting permissive cell lines such as HeLa and A549 cells.

Adv5 and Adv37 increase adiposity in animals and have been indicated as possible contributing factors to the rising problem of obesity [24], whereas human adenoviruses Adv2 and Adv31 are not adipogenic in animals. Among the over 50 human adenoviruses available, Adv36 is the most studied also because of its minimal cross-reactivity with other human adenoviruses [25,26,27]. The exact mechanisms leading to the development of obesity through these viruses are unknown. An accurate understanding of the varied etiological factors of obesity (including infecting agents) may lead to cause-specific treatment and successful management of this disease.

4. Adv36 and Obesity

5. Prevalence of Adv36 in Human Obesity

Six human adenoviruses have been studied in relation to obesity [29,30,31,43] and a number of human studies have shown a correlation between antibodies to the Adv36 and obesity (Table 1). Due to the unique amino acid sequences in protruding region of the hexon (loops 1 and 2) of Adv36 [46] there is a low cross-reactivity between Adv36 and other adenovirus in classical serum neutralization assays thus, the information derived from these studies are reliable. Furthermore, the Adv36 genome has a high stability, which is useful for diagnostic purposes and in the development and application of vaccines and therapeutic reagents [47]. Moreover, bioinformatics comparisons with other human adenoviruses identified significant differences, suggesting unique functions of Adv36 possibly linking Adv36 with adipose tissue [40]. In humans, the natural infection with Adv36 is diagnosed by the presence of the viral DNA in adipose tissue [44,48] or by neutralizing antibodies [49].

Analogously to animals, seropositivity to Adv36 is associated with low level of cholesterol and low triglyceride concentrations in serum of obese individuals [30].

Adv36 DNA has been isolated in few cases. Salehian et al. [48] described a patient with massive fat deposits in the thorax and abdomen arguing that the abnormal adipose tissue deposits might be caused by Adv36. Although this case seems in agreement with the increase of adipose tissue found in animals experimentally infected with Adv36, further studies are needed to understand the actual role of Adv36 in abnormal deposits and fat/lipomatosis in humans [48].

The association of natural Adv36 infection with obesity both in adults and children has been described (Table 1).

The first study of Ad36 infection in adult humans in the United States showed that about 30% of obese and 11% of non-obese had been infected, and there was a strong correlation of obesity with infection [49]. Other studies have confirmed these results (Table 1). The first study of Ad36 infection in adult humans in the United States showed that about 30% of obese and 11% of non-obese had been infected, and there was a strong correlation of obesity with infection [42]. Other studies have confirmed these results (Table 1), while few studies did not confirm this finding [50,51,52]. The average Adv36 prevalence ranged from 65% in Italy down to 6% in Belgium/Holland (Table 1). The Adv36 prevalence reported in obese children and non-obese children is 28% and 18%, respectively [53,54,55,56,57,58,59,60,61]. Two meta-analyses explored the association between Adv36 infection and obesity development. The first selected 10 studies. The authors found that Adv36 infection associates with the risk of obesity, but was not associated with abnormal metabolic markers including waist circumstance suggesting that the infection is more associated with accumulation of subcutaneous fat than that of visceral fat [62].

The second meta-analysis included 11 case-control studies, including 2508 obese subjects and 3005 control. The study identified an association between Ad36 infection and a significantly increased risk of obesity development, especially in children [63].

6. Molecular and Cellular Mechanisms Involved in Ad-36-Induced Glucose Uptake

Evidence emerging from animal and human studies suggests that some forms of obesity may actually lead to healthy obesity [24]. Several studies identified one Adv36 gene that mediates glucose disposal through the Ras/PI3K pathway to improve glucose uptake [64]. The glucose uptake increase may contribute to the improved glycemic control found in Adv36-infected animals [34].

In vitro, Adv36 induced adipogenic accumulation commitment, differentiation of adipocytes and increases the cellular glucose uptake [27,44].

In a work by Wang et al. [64], the authors showed that the infection with Adv36 increases glucose uptake, confirming previous data. In vitro experiments on Human Skeletal Muscle (HSKM) cells showed an increase of GLUT1 and GLUT4 gene expression mediated by Ras-activated PI 3-kinase pathway independent from insulin activation (Figure 2).

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Figure 2

Adv36 mediates glucose uptake independently from insulin, adapted from [64]. Adv36 up-regulates the Phosphoinositide 3-kinase (PI3K) signaling via Ras, increasing cellular glucose uptake by glucose trasporters Glut1 and Glut4 despite a down-regulation of the Insulin Receptor Substrate (IRS) signaling.

Considering the crucial role of skeletal muscle in glucose disposal, the ability to modulate its capacity to uptake glucose may have a major impact on systemic glycemic control. The property of Adv36 to improve skeletal muscle glucose uptake in an insulin-signaling independent manner is of great potential interest for the development of new drug targets in insulin resistance diabetes. Subsequently, many studies have focused on the virus E4orf1 gene that up-regulates the PI3K pathway (Figure 2) [42]. The E4orf1 gene is transcribed from the first open reading frame of the early gene of Adv36 and produces a 17 kDa protein (Figure 1) [42]. Adv36 increases cellular glucose uptake via Ras-mediated activation of the phosphatidyl inositol 3-kinase (PI3K), and improves hyperglycemia in mice, without reducing adiposity [65]. In vitro experiments showed that Adv36 significantly increases the absorption of glucose into 3T3-L1 preadipocytes and identified that the E4orf1 is “sufficient” to up-regulate the absorption of glucose [26]. The 3T3-L1 cell line expressing E4orf1 had an increased glucose uptake compared to null vector control cells. Moreover, E4orf1 up-regulated PI3K pathway and increased the Ras-molecule required to stimulates the Adv36-induced glucose uptake. Glucose uptake increases significantly in preadipocytes, adipocytes, or myoblasts derived from 3T3-L1 cells transiently transfected with E4orf1. Thus, the attractive anti-hyperglycemic effect of Adv36 could be mediated by the E4orf1 protein, which may offer a novel ligand to develop hypoglycemic drugs. Cellular uptake of glucose can be improved by up-regulation of Ras signaling in either insulin-dependent or insulin-independent diabetes. In the presence of an intact insulin signal, Ras plays a negligible role in glucose uptake. On the contrary, when the insulin signal is reduced like in obesity or diabetes, the insulin-independent Ras pathway can be useful to improve the glucose disposal. Adv36 increases cellular uptake of glucose by upregulating the Ras/Glut4pathway (Figure 2). These data improved the knowledge on the role of E4orf1 as a mediator of Adv36-induced glucose uptake [26].

The metabolic effects of E4orf1 are encouraging, and may provide new targets for signaling to prevent NAFLD and insulin resistance even in the presence of a high fat diet. There are limitations to these studies because all results are from in vitro experiments and should be verified in vivo. E4orf1 is not a secretory protein, and therefore does not have a cell surface receptor for cell entry. Thus, E4orf1 provides a valuable model to exogenously modulate hepatic glucose and lipid metabolism [66].

In summary, Adv36 may serve two different purposes, on the one hand, to explain the pathophysiology of certain cases of obesity and, on the other hand, to provide a potential weapon for improving insulin resistance, regardless of the consumption of fat.

Collectively, these results can provide a model to develop new agents for the treatment of hyperglycemia associated with obesity, and type 1 or type 2 diabetes [25]. Although the in vivo efficacy and safety of E4orf1 in improving hyperglycemia remain unknown, and an appropriate drug delivery system is required, Adv36 E4orf1 offers a research opportunity to develop new anti-diabetic agents with new conceptually advances for the use of a rather unconventional source, viral proteins, in the anti-diabetic drug development [25].

7. Adenovirus 36 and Immune Response

7.2. Effect of Adv36 on Leptin

Conventionally, obesity can be considered an over accumulation of white adipose tissue (WAT). Although adipocytes occupy the bulk of the volume of WAT, adipose tissue also includes many more cells types, including a diverse population of preadipocytes, macrophages, endothelial cells, fibroblasts and leukocytes [83]. Adipose tissue, traditionally thought as a passive storage for triglycerides and energy, but in the past two decades it has been linked to the production of several hormones, pro-inflammatory chemokines, adipokines and cytokines, including leptin, adiponectin, resistin, visfatin, B-cell activating factor of the Tumor Necrosis Factor (TNF) family, TNF-like weak inducer of apoptosis, a proliferation inducing ligand, TNF-α, omentin and MCP-1 [5]. Based on this concept, adipose tissue has been defined an endocrine organ. In the obese state, secretion of these adipokines is altered in correlation to the increased adipose tissue mass [84,85]. To date, adipokine modulation of immune function by leptin is the best-characterized link between obesity and immune function. Leptin plays a role in many diverse physiological processes but is primarily involved in energy homeostasis and satiety [86,87].

Leptin acts as a general signal of energy reserves and modulates food intake. Leptin levels increase proportionately to adipose mass resulting in high circulating leptin concentrations in obese individuals [88,89].

The leptin receptor is expressed by B and T lymphocytes and may directly modulate the T and B responses [90]. Leptin seems to exert its effects on immune cells through the JAK /STAT pathway. In peripheral blood mononuclear cells, leptin increases JAK2/3 and STAT3 phosphorylation, which promote proliferation and activation of T lymphocytes upon mitogen stimulation. In terms of infectious disease, the general consensus seems to be that leptin has an anti-inflammatory role, while at the same time serving a protective capacity against infections [91]. Inflammation is used as a localized, protective response to infection and changes in body weight and metabolic state are often associated with acute or chronic inflammatory processes resulting from infection [38]. The inhibition of leptin gene expression by Adv36 infection may increase lipid accumulation and obesity prevalence reported that human cells infected with Adv36 showed greater differentiation and higher levels of lipid accumulation than non-infected control cells [43] (Figure 3). Adv36 infection may increase appetite and food intake by decreasing norepinephrine levels and leptin [92], thereby increasing obesity prevalence (Figure 4).

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Figure 3

Adv36 and leptin. (A) Leptin binding to its receptor (ObRb) activates the associated JAK-2 tyrosine kinase. Leptin inhibits glucose transport through GLUT-2, and activates PI3K. Additionally, Phosphoinositide 3-kinase (PI3K) activation by leptin reduces Cyclic adenosine monophosphate (cAMP) levels and activates the Protein kinase A (PKA) pathway. Leptin can also inhibit the phospholipase C (PLC)/protein kinase C (PKC) pathway; (B) Adv36 inhibits leptin production. The results are decreased insulin release and increased lipid accumulation.

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Figure 4

Proposed mechanisms underlying the effects of Adv36 in infected individuals.

8. Proof-of-Concept of a Vaccine Using UV Inactivated Adv36

Adv36 infection is characterized by greater adiposity and inflammation. To investigate the possibility that a candidate prophylactic vaccine could protect Adv36-induced obesity and inflammation an inactivated Adv36 vaccine was tested [37]. Mice were inoculated with live Adv36, UV-inactivated Adv36, or with medium alone. Live Adv36 increased the size of the epididymal fat pad at four days post-inoculation, whereas UV-inactivated Adv36 prevented it. In another study, mice were immunized with purified and ultraviolet-irradiated virus as candidate vaccine, live Adv36 was injected into mice as a challenge test. Unvaccinated mice (control group) were immunized with phosphate-buffered saline and then challenged with live Adv36. The control group showed 17% greater body weight and 20% more epididymal fats compared with the vaccinated group. Moreover, the vaccinated group had decreased serum levels of pro-inflammatory cytokines, and infiltrated immune cells in fat tissue [93]. Therefore, the vaccine was able to protect against Adv36-increased body weight and fat as well as inflammatory states after challenge. These results could provide proof-of-concept for prophylactic vaccination against virus-induced adiposity [94].

9. Conclusions

Obesity is a multifactorial pathology and the understanding of the different contributing factors is crucial for its efficient management.

Recent studies have shown a possible correlation between obesity and Adv36 viral infections. Animals infected with Adv36 show increase of body weight and physiological changes, increased glucose absorption and decreased secretion of leptin and cholesterol (Figure 4).

The Adv36 infections should therefore be considered as a possible risk factor for obesity and this could be a potential new way to investigate on the worldwide epidemic of obesity. Further research on viruses contributing to obesity is essential. Studying the favorable effect of Adv36 on metabolic consequences of obesity may provide insight to novel treatments that improve glycemic control despite adiposity. Identifying the viral protein responsible for influencing glucose disposal may help in developing novel anti-diabetic therapeutic agents.

Acknowledgments

Author contributions

E.P. drafted the first version of the manuscript, designed review and interpreted data. L.G. supervised and reviewed the manuscript. All authors read and approved the final manuscript, and agreed with the conclusions of the work.

Conflicts of Interest

The authors declare that they have no competing interests of either financial or non-financial nature regarding the work described in the present manuscript and its publication.

References