E4orf1 Enhances Glucose Uptake Independent of Proximal Insulin Signaling.

Impaired proximal insulin signaling is often present in diabetes. Hence, approaches to enhance glucose disposal independent of proximal insulin signaling are desirable. Evidence indicates that Adenovirus-derived E4orf1 protein may offer such an approach. This study determined if E4orf1 improves insulin sensitivity and downregulates proximal insulin signaling in vivo and enhances cellular glucose uptake independent of proximal insulin signaling in vitro. High fat fed mice were injected with a retrovirus plasmid expressing E4orf1, or a null vector. E4orf1 significantly improved insulin sensitivity in response to a glucose load. Yet, their proximal insulin signaling in fat depots was impaired, as indicated by reduced tyrosine phosphorylation of insulin receptor (IR), and significantly increased abundance of ectonucleotide pyrophosphatase/phosphodiesterase-1 (ENPP1). In 3T3-L1 pre-adipocytes E4orf1 expression impaired proximal insulin signaling. Whereas, treatment with rosiglitazone reduced ENPP1 abundance. Unaffected by IR-KD (insulin receptor knockdown) with siRNA, E4orf1 significantly up-regulated distal insulin signaling pathway and enhanced cellular glucose uptake. In vivo, E4orf1 impairs proximal insulin signaling in fat depots yet improves glycemic control. This is probably explained by the ability of E4orf1 to promote cellular glucose uptake independent of proximal insulin signaling. E4orf1 may provide a therapeutic template to enhance glucose disposal in the presence of impaired proximal insulin signaling.

Human adenovirus Ad36 and its E4orf1 gene enhance cellular glucose uptake even in the presence of inflammatory cytokines.

BACKGROUND: Aging and obesity are associated with elevated pro-inflammatory cytokines such as monocyte chemoattractant protein (MCP)-1 and tumor necrosis factor (TNF)α, which are linked to insulin resistance. Anti-inflammatory agents have marginal effect in improving insulin resistance. Hence, agents are needed to improve glycemic control despite the inflammation. Ad36, a human adenovirus, increases TNFα and MCP1 mRNA in adipose tissue, yet improves glycemic control in mice. Ad36 via its E4orf1 gene, up-regulates AKT/glucose transporter (Glut)-4 signaling to enhance cellular glucose uptake. OBJECTIVE: Directly test a role of Ad36, or E4orf1 in enhancing cellular glucose uptake in presence of inflammatory cytokines. METHODS: Experiment 1: 3T3-L1 preadipocytes were treated with 0, 10 or 100 ng/mL lipopolysaccharides (LPS), and infected with 0 or 5 plaque forming units (PFU) of Ad36/cell. 3T3-L1 cells that stably and inducibly express E4orf1 or a null vector (pTRE-E4orf1 or pTRE-null cells), were similarly treated with LPS and then with doxycycline, to induce E4orf1. Experiment 2: 3T3L1 preadipocytes were treated with 25 nM MCP1 or 20 nM TNFα for 16 h, followed by infection with 0 or 5 PFU of Ad36/cell. Experiment 3: pTRE-E4orf1 or -null cells were similarly treated with MCP1 or TNFα followed by doxycycline to induce E4orf1. Cellular glucose uptake and cellular signaling were determined 72 h post-Ad36 infection or E4orf1-induction, in continued presence of MCP1 or TNFα. RESULTS: In 3T3-L1 preadipocytes, Ad36, but not E4orf1, increased MCP1 and TNFα mRNA, in presence of LPS stimulation. Ad36 or E4orf1 up-regulated AKT-phosphorylation and Glut4 and increased glucose uptake (P < 0.05) in the presence of MCP1 or TNFα. CONCLUSIONS: Unlike Ad36, E4orf1 does not appear to stimulate inflammatory response. Ad36 and E4orf1 both enhance cellular glucose uptake even in presence of inflammation. Further research is needed to harness this novel and beneficial property of E4orf1 to improve hyperglycemia despite chronic inflammation that is commonly present in aging and obesity.

An adenovirus-derived protein: A novel candidate for anti-diabetic drug development.

AIMS: Exposure to human adenovirus Ad36 is causatively and correlatively linked with better glycemic control in animals and humans, respectively. Although the anti-hyperglycemic property of Ad36 may offer some therapeutic potential, it is impractical to use an infectious agent for therapeutic benefit. Cell-based studies identified that Ad36 enhances cellular glucose disposal via its E4orf1 protein. Ability to improve glycemic control in vivo is a critical prerequisite for further investigating the therapeutic potential of E4orf1. Therefore, the aim of this study was to determine the ability of E4orf1 to improve glycemic control independent of insulin despite high fat diet. MATERIALS & METHODS: 8-9wk old male C57BL/6J mice fed a high-fat diet (60% kcal) were injected with a retrovirus plasmid expressing E4orf1, or a null vector (Control). Glycemic control was determined by glucose and insulin tolerance test. Islet cell size, amount of insulin and glucagon were determined in formalin-fixed pancreas. Rat insulinoma cell line (832/13) was infected with E4orf1 or control to determine changes in glucose stimulated insulin secretion. Protein from flash frozen adipose tissue depots, liver and muscle was used to determine molecular signaling by western blotting. RESULTS: In multiple experiments, retrovirus-mediated E4orf1 expression in C57BL/6J mice significantly and reproducibly improved glucose excursion following a glucose load despite a high fat diet (60% energy). Importantly, E4orf1 improved glucose clearance without increasing insulin sensitivity, production or secretion, underscoring its insulin-independent effect. E4orf1 modulated molecular signaling in mice tissue, which included greater protein abundance of adiponectin, p-AKT and Glucose transporter Glu4. CONCLUSIONS: This study provides the proof of concept for translational development of E4orf1 as a potential anti-diabetic agent. High fat intake and impaired insulin signaling are often associated with obesity, diabetes and insulin resistance. Hence, the ability of E4orf1 to improve glycemic control despite high fat diet and independent of insulin, is particularly attractive.

Unbiased proteomic profiling strategy for discovery of bacterial effector proteins reveals that Salmonella protein PheA is a host cell cycle regulator.

Salmonella utilizes a type III secretion system to inject bacterial effector proteins into the host cell cytosol. Once in the cytosol, these effectors hijack various biochemical pathways to regulate virulence. Despite the importance of effector proteins, especially for understanding host-pathogen interactions, a potentially large number of effectors are yet to be identified. Here, we demonstrate that unbiased chemical proteomic profiling using off-the-shelf fluorescent probes leads to the discovery of a host cell cycle regulator encoded in the Salmonella genome. Our profiling combined with bioinformatic analysis implicates 29 Salmonella as potential effectors. We follow up on the top candidate, chorismate mutase-P/prehenate dehydratase, PheA, and present evidence that PheA is an effector that mimics E2F7 transcription factor of the host cell and promotes G1/S cell cycle arrest. This validates our strategy and opens opportunities for effector identification in the future.

Adenovirus 36 attenuates weight loss from exercise but improves glycemic control by increasing mitochondrial activity in the liver.

Human adenovirus type 36 (Ad36) as an obesity agent induces adiposity by increasing glucose uptake and promoting chronic inflammation in fat tissues; in contrast, exercise reduces total body fat and inflammation. Our objective was to determine the association between Ad36 and the effects of exercise on inflammation and glycemic control. In the human trials (n = 54), Korean children (aged 12-14 years) exercised for 60 min on three occasions each week for 2 months. We compared the body mass index (BMI) Z-scores before and after exercise. C57BL/6 mice were infected with Ad36 and Ad2 as a control, and these mice exercised for 12 weeks postinfection. After the exercise period, we determined the serum parameters and assessed the presence of inflammation and the mitochondrial function in the organs. Ad36-seropositive children who were subjected to a supervised exercise regimen had high BMI Z-scores whereas Ad36-seronegative children had lower scores. Similarly, Ad36-infected mice were resistant to weight loss and exhibited chronic inflammation of their adipose tissues despite frequent exercise. However, Ad36 combined with exercise reduced the levels of serum glucose, nonesterified fatty acids, total cholesterol, and insulin in virus-infected mice. Interestingly, virus infection increased the mitochondrial function in the liver, as demonstrated by the numbers of mitochondria, cytochrome c oxidase activity, and transcription of key mitochondrial genes. Therefore Ad36 counteracts the weight-loss effect of exercise and maintains the chronic inflammatory state, but glycemic control is improved by exercise synergistically because of increased mitochondrial activity in the liver.

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

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.

Prophylactic and therapeutic vaccines for obesity.

Chronic diseases such as obesity and diabetes are major causes of death and disability throughout the world. Many causes are known to trigger these chronic diseases, and infectious agents such as viruses are also pathological factors. In particular, it is considered that adenovirus 36 infections may be associated with obesity. If this is the case, a vaccine against adenovirus 36 may be a form of prophylaxis to combat obesity. Other types of therapeutic vaccines to combat obesity are also being developed. Recently, hormones such as glucagon-like peptide-1, ghrelin, and peptide YY have been studied as treatments to prevent obesity. This review describes the ongoing development of therapeutic vaccines to treat obesity, and the possibility of using inactivated adenovirus 36 as a vaccine and an anti-obesity agent.

Immunogenicity and safety of H1N1 influenza hemagglutinin protein expressed in a baculovirus system.

Although most influenza vaccines are produced in eggs, new types of vaccines must be developed. In this study, the immunogenicity and safety of a baculovirus-expressed hemagglutinin (HA) of H1N1 influenza virus (Korea/01/2009; designated "HA-Bac-K") was compared with those of a commercially available baculovirus-expressed HA (designated "HA-Bac-C") and an Escherichia coli-expressed HA (designated "HA-E. Coli-K"). HA-Bac-K succeeded in inducing hemagglutination inhibition and neutralization antibodies in mouse and ferret models. The different immunogenicities observed may be attributable to the different expression systems and purification protocols used. Our work suggests that HA expressed in a baculovirus system is an effective and safe candidate influenza vaccine.

IK induced by coxsackievirus B3 infection transiently downregulates expression of MHC class II through increasing cAMP.

Major histocompatibility complex (MHC) class II expression is critical for the presentation of antigens in the immune response to viral infection. Consequently, some viruses regulate the MHC class II-mediated presentation of viral antigens as a mechanism of immune escape. In this study, we found that Coxsackievirus B3 (CVB3) infection transiently increased IK expression, which reduced the expression of MHC class II (I-A/I-E) on splenic B cells. Interestingly, CVB3-induced IK elevated cAMP, a downstream molecule of the G protein-coupled receptors, which inhibited MHC class II presentation on B cells. Transgenic mice expressing truncated IK showed lower expression of MHC class II on B cells than did wild-type mice after CVB3 infection. Taken together, these results imply that IK plays a role in downregulating MHC class II expression on B cells during CVB3 infection through the induction of cAMP.

Black soybean anthocyanins inhibit adipocyte differentiation in 3T3-L1 cells.

Anthocyanins are naturally occurring polyphenolic pigments in plants that have been shown to decrease weight gain and insulin resistance in mice-fed high-fat diets. We investigated the effects of anthocyanins on cell growth, differentiation, and lipolysis in 3T3-L1 cells to test our hypothesis that anthocyanins could reduce adipose tissue mass by acting directly on adipocytes. Anthocyanin extracts from black soybeans were used and composed of 3 of the following major anthocyanins: cyanidine-3-O-glucoside (68.3%), delphinidin-3-O-glucoside (25.2%), and petunidin-3-O-glucoside (6.5%). Treatment with 12.5 and 50 µg/mL of black soybean anthocyanins exhibited inhibitory effects on the proliferation of both preconfluent preadipocytes (P < .01) and maturing postconfluent adipocytes (P < .01). In fully differentiated adipocytes, the number of viable cells was reduced by black soybean anthocyanins (P < .01). Treatment with 50 µg/mL of black soybean anthocyanins slightly increased epinephrine-induced lipolysis but decreased the basal lipolysis of fully differentiated adipocytes (P < .05). Black soybean anthocyanins also reduced lipid accumulation and suppressed the expression of the peroxisome proliferator-activated receptor γ, a major transcription factor for the adipogenic gene (P < .01). These results suggest that black soybean anthocyanins inhibit adipocyte differentiation and basal lipolysis, which may contribute to their antiobesity and antidiabetic properties.

Adenovirus 36 as an obesity agent maintains the obesity state by increasing MCP-1 and inducing inflammation.

BACKGROUND: Although it is well known that adenovirus 36 (Ad36) is associated with obesity in humans as well as in animals, the detailed cellular mechanism is unclear. METHODS: Wild-type (WT) mice and monocyte chemoattractant protein-1 knockout (MCP-1(-/-)) mice were infected with Ad36, and their weights and inflammatory status were measured. Macrophage infiltration was examined in their reproductive fat pads and in a coculture system. The correlation between Ad36 antibody presence and MCP-1 levels was tested in human samples. RESULTS: We have shown that Ad36 infection stimulated an inflammatory state by increasing the level of MCP-1 through the activation of nuclear factor κB, which in turn induced the infiltration of macrophages into adipocytes. This induced inflammation resulted in viral obesity, which caused chronic inflammation and affected lipid metabolism. In contrast to WT mice, MCP-1(-/-) mice were protected from Ad36-induced inflammation and obesity. The MCP-1 levels in Ad36-antibody-positive human group were higher than those in the antibody-negative group. CONCLUSIONS: These findings support the proposition that virus-induced inflammation is the cellular mechanism underlying Ad36-induced obesity. These results also suggest that MCP-1 plays a critical role in Ad36-induced obesity and that MCP-1 may be a therapeutic target in preventing virus-induced obesity.

Systemic analysis of a novel coxsackievirus gene delivery system in a mouse model.

In order to systemically investigate the possibility of using coxsackievirus B3 (CVB3) to deliver foreign genes in vivo, a recombinant strain of CVB3 encoding the renilla gene (CVB3- renilla) was constructed. The recombinant CVB3 resulted in extensive and transient expression of the renilla protein within mouse organs, especially the pancreas. The level of expression was generally dependent upon the viral titer present. Moreover, the CVB3-renilla strain was completely attenuated. Interestingly, the recombinant CVB3 vector was expressed much more strongly in mouse organs than was a comparable adenoviral vector. The CVB3-renilla strain did not express the renilla gene in mice with pre-existing coxsackievirus-specific neutralizing antibodies, but direct organ-specific administration of the virus during openperitoneum surgery was able to circumvent this immunity. This coxsackievirus vector may represent a useful means for delivering and expressing foreign genes in mouse models in an acute and extensive fashion.

Recombinant lentivirus-delivered short hairpin RNAs targeted to conserved coxsackievirus sequences protect against viral myocarditis and improve survival rate in an animal model.

Coxsackieviruses are important human pathogens that induce myocarditis and pancreatitis. However, there are no vaccines or therapeutic reagents for their clinical treatment. Although RNA interference (RNAi)-based approaches to the prevention of viral production have been developed recently, limitations to the in vivo delivery systems and variations in the viral target sequences still hamper the strategy. In this study, to overcome these limitations, we have constructed recombinant lentivirus-delivered short hairpin RNAs (shRNAs) against sequences in highly conserved cis-acting replication element (CRE) within the 2C protein of coxsackievirus B3 (CVB3), designated MET-2C. A recombinant lentivirus, designated Met-2C lenti, was constructed that contains the MET-2C sequence, which acts as a shRNA. Met-2C lenti clearly reduced viral production in CVB3-infected cells in vitro. Moreover, the mice injected intraperitoneally with Met-2C lenti had significant reductions in viral titers, viral myocarditis, and proinflammatory cytokines after challenge with CVB3, compared with those in GFP lenti infected control mice. Moreover, Met-2C lenti improved survival rate compared with that of the GFP lenti infected control group. Therefore, Met-2C lenti is potentially a clinical therapeutic agent for the treatment of viral myocarditis.