E4orf1 improves adipose tissue-specific metabolic risk factors and indicators of cognition function in a mouse model of Alzheimer's disease.

OBJECTIVE: Obesity, impaired glycemic control, and hepatic steatosis often coexist and are risk factors for developing dementia, and Alzheimer's disease (AD). We hypothesized that a therapeutic agent that improves glycemic control and steatosis may attenuate obesity-associated progression of dementia. We previously identified that adenoviral protein E4orf1 improves glycemic control and reduces hepatic steatosis despite obesity in mice. Here, we determined if this metabolic improvement by E4orf1 will ameliorate cognitive decline in a transgenic mouse model of AD. METHODS: Fourteen- to twenty-month-old APP/PS1/E4orf1 and APP/PS1 (control) mice were fed a high-fat diet. Cognition was determined by Morris Water Maze (MWM). Systemic glycemic control and metabolic signaling changes in adipose tissue, liver, and brain were determined. RESULTS: Compared to control, E4orf1 expression significantly improved glucose clearance, reduced endogenous insulin requirement and lowered body-fat, enhanced glucose and lipid metabolism in adipose tissue, and reduced de novo lipogenesis in the liver. In the brain, E4orf1 mice displayed significantly greater expression of genes involved in neurogenesis and amyloid-beta degradation and performed better in MWM testing. CONCLUSION: This study opens-up the possibility of addressing glycemic control and steatosis for attenuating obesity-related cognitive decline. It also underscores the potential of E4orf1 for the purpose, which needs further investigations.

E4orf1 Prevents Progression of Fatty Liver Disease in Mice on High Fat Diet.

Non-alcoholic fatty liver disease (NAFLD) covers a broad spectrum of liver diseases ranging from steatosis to cirrhosis. There are limited data on prevention of hepatic steatosis or its progression to liver disease. Here, we tested if either transgenic (Tg) doxycycline-induced expression in adipose tissue of E4orf1 (E4), an adenoviral protein, or dietary fat restriction attenuated hepatic steatosis or its progression in mice. Twelve to fourteen-week-old TgE4 mice (E4 group) and control mice were exposed to a 60% (Kcal) high fat diet (HFD) for 20 weeks, while another group of mice on HFD for 10 weeks were switched to a chow diet (chow group) for another 10 weeks. Glycemic control was determined at weeks 10 and 20. Tissues were collected for gene and protein analysis at sacrifice. Compared to control, diet reversal significantly reduced body weight in the chow group, whereas E4 expression attenuated weight gain, despite HFD. E4 mice evinced significantly improved glucose clearance, lower endogenous insulin secretion, reduced serum triglycerides, attenuated hepatic steatosis and inflammation. Interestingly, in spite of weight loss and lower liver fat, chow mice showed significant upregulation of hepatic genes involved in lipid metabolism. Despite HFD, E4 prevents hepatic lipid accumulation and progression of hepatic steatosis, while diet reversal maintains hepatic health, but is unable to improve molecular changes.

E4orf1-induced reduction in endogenous insulin level is independent of pancreas endocrine function.

BACKGROUND: Obesity is often associated with hyperinsulinemia due to insulin resistance. In mice models of hyperinsulinemia, adenovirus-derived E4orf1 protein promotes glucose disposal via insulin-independent pathway, and reduces insulin response to glucose load, described as its "Insulin Sparing Action". This is likely because less insulin is needed for disposing glucose in presence of E4orf1, however, there are other potential possibilities. This study determined if E4orf1 reduces insulin response to glucose load because it a) suppresses the ability of pancreatic ß-cells to secret insulin, or b) upregulates glucagon production by the pancreas. METHODS: C57BL/6J wild type (control) and transgenic C57BL/6J (E4orf1) mice that express E4orf1 protein in adipose tissue upon doxycycline feeding, were used. Post-doxycycline feeding, insulin and glucagon secretion in response to glibenclamide or phenylephrine were compared between the two groups. The pancreases were examined for histological changes. RESULTS: In response to glibenclamide, E4orf1 mice secreted more insulin and exhibited lower blood glucose compared to control (47.4 ± 4.4 vs 27.4 ± 3.7 mg/dl, p < 0.003), but showed no difference in glucagon secretion. Post-phenylephrine injection, no differences were observed between the two groups for glucagon or insulin, except E4orf1 mice had a lower blood glucose rise after 10-min of injection compared to the control (39.7 ± 4.7 vs. 58.3 ± 7.5 mg/dl, p < 0.05). E4orf1 mice had significantly larger pancreatic islets and higher number of islets per mm2 tissue area. Neither the size nor the number of islets met the criteria of hypertrophy or hyperplasia. CONCLUSIONS/INTERPRETATION: E4orf1 retains and may enhance the ability of the pancreases to secret insulin in response to insulin secretagogue. Glucagon does not seem to play a role in the Insulin Sparing Action of E4orf1. Overall, the histology studies support better pancreatic islet health in presence of E4orf1, compared to that in control mice. The "insulin-independent" role of E4orf1 has potential therapeutic implications in addressing hyperinsulinemia in obesity.

Antidiabetic E4orf1 protein prevents hepatic steatosis and reduces markers of aging-related cellular damage in high fat fed older mice.

INTRODUCTION: Older age is associated with greater prevalence of hyperinsulinemia, type 2 diabetes, and fatty liver disease. These metabolic conditions and aging are bidirectionally linked to mitochondrial dysfunction and telomere attrition. Although effectively addressing these conditions is important for influencing the health and the lifespan, it is particularly challenging in older age. We reported that E4orf1, a protein derived from human adenovirus Ad36, reduces hyperinsulinemia, improves glucose clearance, and protects against hepatic steatosis in younger mice exposed to high fat diet (HFD). Here, we tested if E4orf1 will improve glycemic control, liver fat accumulation, mitochondrial integrity, and reduce telomere attrition in older mice. RESEARCH DESIGN AND METHODS: We used 9-month-old mice that inducibly expressed E4orf1 in adipose tissue and non-E4orf1 expressing control mice. Mice were maintained on a 60% (kcal) HFD for 20 weeks and glycemic control was determined by intraperitoneal glucose tolerance test at week 20. Following 20 weeks of HF-feeding, mice were sacrificed and liver tissues collected to determine the expression of aging genes using qRT-PCR based RT2 Profiler PCR array. RESULTS: Compared with the control mice, E4orf1 significantly improved glycemic control and reduced hepatic steatosis and fibrosis. Additionally, E4orf1 maintained markers of mitochondrial integrity and telomere attrition. CONCLUSION: E4orf1 has the potential to improve glycemic control in older mice, and the improvement persists even after longer term exposure. E4orf1 expression also maintains mitochondrial integrity and telomere attrition, thus delaying age-associated diseases. This provides strong evidence for therapeutic utility of E4orf1 in improving age-associated metabolic and cellular changes that occur with aging in humans.

Improvement in Glycemic Control in Mice of Different Age Groups.

AIMS AND METHODS: The declining ability to control blood glucose with advancement of age is an important health risk factor and may lead to insulin resistance, type-2-diabetes and Alzheimer's disease. Adenovirus 36(Ad36) improves glycemic control independent of insulin signaling(insulin sparing effect) as evidenced by cell, animal and observational human studies. This property of Ad36 may be useful in correcting aging-related glucose intolerance and related health conditions. Therefore, we determined the effect of Ad36 on glycemic control in older mice, to identify the age group that best responds to Ad36. Six, 12 or 20-month old C57Bl/6 mice on chow diet were each divided into weight-matched groups(mock-infected or Ad36-infected). Body weight was recorded weekly post infection (p.i.) and fasting glucose measured(week 0, 4, 8 and 20 p.i.). Blood glucose and serum insulin were measured during glucose tolerance test(week 0 and 16 p.i.). At week 20 p.i., animals were sacrificed, blood and tissues collected. RESULTS: Mice from all age groups showed improvement in glucose clearance post Ad36 infection, but a more profound effect was observed in 6-month old mice compared with mock-infected mice. Under fed conditions though there was no difference in blood glucose at 20 wk p.i., interestingly, Ad36 reduced serum insulin in age groups old mice, compared with control mice. CONCLUSIONS: These findings suggest Ad36 infected animals improve glycemic control and clear post-prandial gluco00000se increase without increasing insulin secretion in an insulin sparing manner. These beneficial effects provide strong evidence for developing Ad36-based approaches as a novel tool to attenuate age associated glucose intolerance.

E4orf1, an Adeno-viral protein, attenuates renal lipid accumulation in high fat fed mice: A novel approach to reduce a key risk factor for chronic kidney disease.

Obesity and hyperlipidemia are independent risk factors of chronic kidney disease (CKD). In mice, diet induced obesity accelerates lipogenesis, lipid accumulation, and injury in kidneys. Expression of adenoviral protein, E4orf1, improves glucose clearance and reduces endogenous insulin secretion to glucose challenge in mice. Therefore, in this pilot study, we examined, if enhanced glycemic control in HFD fed E4orf1 transgenic (E4orf1-Tg) mice, will reduce renal lipogenesis and lipid accumulation. In two separate experiments, E4orf1-Tg mice were fed 60% (kcal) high-fat diet (HFD) supplemented with doxycycline for 10-weeks or 20-weeks along with wild-type (C57BL6/J) or E4orf1-non-transgenic (E4orf1-non-Tg) control mice, respectively. Protein expression of Fatty Acid Synthase (FAS) and Acetyl-CoA Carboxylase (ACC), accumulation of triglyceride (TG) along with mRNA levels of lipid metabolism and injury markers were determined in kidneys. Renal expression of FAS and ACC, and TG content was significantly reduced in E4orf1-Tg mice compared to controls. E4orf1-Tg mice show significant increase in genes involved in mitochondrial fatty acid oxidation and oxidative stress compared to wild-type mice after 10-weeks of HFD. However, mice exposed to 20-weeks of HFD, show no difference in gene expression. E4orf1 expression reduces lipid synthesis and accumulation in kidneys despite HFD, which may be due to attenuation of hyperinsulinemia by E4orf1.

Reducing endogenous insulin is linked with protection against hepatic steatosis in mice.

BACKGROUND: Obesity and type 2 diabetes (T2D) are closely associated with hepatic steatosis (HS), which if untreated can advance to serious liver conditions. Since insulin promotes hepatic lipogenesis, reducing hyperinsulinemia may help in treating HS. E4orf1 is an adenovirus-derived protein that improves glucose clearance independent of insulin, lowers insulin amount required for glucose disposal, and reduces HS. As a next step, we evaluated the mechanism for E4orf1-induced reduction in HS and tested that E4orf1 does not induce hypoglycemia, an important attribute for its application as a potential anti-diabetic agent. METHODS: C57Bl/6J mice that transgenically express E4orf1 in adipose tissue (E4orf-Tg) and wild-type (WT) mice received a chow diet for 6 weeks, followed by a high-fat (HF) diet for additional 10 weeks. Body composition, blood glucose, and serum insulin levels upon glucose load were measured at 0, 6, 7, and 16 weeks. Serum free fatty acid (FFA), triglyceride (TG), and hepatic TG were measured at study termination. We compared histology and the mRNA/protein markers of hepatic and adipose tissue lipid metabolism between the two groups of mice. RESULTS: On chow diet, both groups remained normoglycemic, but E4orf1 expression reduced insulin response. On HF diet, glycemic control in WT deteriorated, whereas E4orf1 significantly enhanced glycemic control, lowered insulin response, reduced hepatic triglycerides, and serum FFA. Overall, a comparison of hepatic mRNA and/or protein expression suggested that E4orf1 expression significantly decreased de novo lipogenesis (DNL) and intracellular lipid transport and increased fat oxidation and TG export. Adipose tissue mRNA and protein markers suggested that E4orf1 expression lowered DNL and increased lipolysis. CONCLUSION: Considering that E4orf1 is not secreted in circulation, we postulate that reduced endogenous insulin in E4orf1 mice indirectly contributes to reduce HS by altering hepatic lipid metabolism, including lipogenesis. This study underscores the possibility of indirectly impacting HS by manipulating adipose tissue metabolism.

Nanoparticle-mediated in vitro delivery of E4orf1 to preadipocytes is a clinically relevant delivery system to improve glucose uptake.

OBJECTIVE: Impaired glycemic control is a common comorbidity of obesity. E4orf1(E4), an adenovirus-derived protein, reduces the activity of insulin receptor substrate (IRS), yet activates Akt and promotes the membrane translocation of GLUT4, resulting in better glycemic control in mice. To develop a clinically suitable delivery system, here we constructed and tested liposome nanoparticles (NP), to deliver E4 to preadipocytes. METHODS: Glutathione-S-transferase (GST)-tagged E4 was encapsulated in Rhodamine-phosphatidylethanolamine (PE)-tagged soy-phosphatidylcholine-NP. The NP were characterized. Preadipocytes were treated with free E4, E4 containing NP (E4 NP) or E4-free NP (void NP). RESULTS: For void and E4 NP, the average size was ~150 and 130 nm, PDI was ~0.25 and 0.27, and Zeta potential was -23 and -25, respectively. The average encapsulation efficiency (EE) was ~50%. Cells treated with E4 showed maximum GST expression and Rhodamine signals at 24 h. The presence of E4 in cells was confirmed at 24, 48, and 72 h. At 72 h after exposure, E4 NP significantly decreased pTyr-IRS, yet increased pAkt protein abundance, membrane translocation of GLUT4, and glucose uptake, compared with cells treated with void NP. Free E4 (without NP) had no effect. CONCLUSIONS: NP-mediated delivery of E4 promotes glucose uptake in preadipocytes. The next step is to test the efficacy of this clinically compatible delivery approach in vivo.

E4orf1 protein reduces the need for endogenous insulin.

BACKGROUND: E4orf1 protein derived from adenovirus-36 reduces glucose excursion in mice, and lowers endogenous insulin response, suggesting a reduced need for insulin. We tested if the E4orf1-mediated lowering of insulin response is due to increased tissue sensitivity to insulin, reduced ability to produce or release insulin, or a reduced need for insulin release. METHODS: Experiment 1: hyperinsulinemic-euglycemic clamps (HEC) and glucose tolerance test (GTT) were performed in high fat fed transgenic mice expressing E4orf1 or non-transgenic littermates (n = 12 each), for 4 weeks. Experiments 2, 3, and 4: E4orf1 or null vectors were expressed in rat-pancreatic ß-cell line (INS-1) for 72 h, and cells were exposed to varying levels of glucose. Cell lysates and media were collected. Experiment 5: 3T3L1-preadipocytes that express E4orf1 upon doxycycline induction, or null vector were induced with doxycycline and then exposed to protein transport inhibitor. Supernatant and cell lysate were collected. Experiment 6: 3T3L1-preadipocytes that express E4orf1 upon doxycycline induction, or null vector were co-cultured with INS-1 cells for 24 h. Media was collected. RESULTS: Experiment 1: E4orf1 transgenic mice cleared glucose faster compared to non-transgenic mice during GTT. HEC showed that E4orf1 did not alter tissue sensitivity to exogenous insulin in mice. Experiments 2, 3, and 4: in INS1 cells, E4orf1 did not alter Glut2 abundance or Akt activation, suggesting no reduction in glucose sensing or insulin synthesis, respectively. E4orf1 did not influence glucose-stimulated insulin secretion in media by INS1 cells. Experiment 5: E4orf1 was present in cell lysate, but not in media, indicating it is not a secretory protein. Experiment 6: INS1 cells released less insulin in media when co-cultured in the presence of E4orf1-expressing 3T3-L1 cells. CONCLUSIONS: Our studies support the working hypothesis that the E4orf1-mediated lowering of insulin response is not due to increased tissue sensitivity to insulin, or reduced ability to produce or release insulin, but likely to be due to a reduced need for insulin release.

Potential role of E4orf1 protein in aging-associated impairment in glycemic control.

Aging constitutes a major risk factor for the development of type-2 diabetes (T2D) where glucose tolerance declines with age, resulting in a high prevalence of T2D and impaired glucose tolerance in the elderly population. Currently more than half of the 20 million U.S. adults with T2D are above the age of 60, and the largest increase in T2D prevalence is expected in the elderly. Obesity is a causative factor for T2D associated insulin resistance and hyperglycemia. Furthermore, the aging process is accelerated by hyperglycemia and effective treatment options are limited for the vulnerable aging population. One of the mechanisms contributing to aging associated hyperglycemia is resistance to insulin-mediated glucose disposal. Chronic hyperglycemia also accelerates aging by increasing pro-inflammatory milieu leading to impaired immune function. Although currently available anti-diabetic agents improve glycemic control, they have potential serious side effects in some cases. Therefore, additional and better drugs are urgently needed for treatment of insulin resistance and aging associated health risk factors. This review presents the novel use of a microbial protein, E4orf1 as a potential anti-diabetic agent, which functions independent of insulin and obesity, highlighting the role of unique sources for future drug development.

Twenty-five years of research about adipogenic adenoviruses: A systematic review.

Infectious etiology is implicated in chronic diseases such as gastric ulcer or atherosclerosis. However, "infection" is a recent term in the field of obesity. Since the first report in 1982 of obesity due to infection, several microbes have been linked to obesity. Among the adipogenic microbes, avian adenovirus SMAM-1 and human adenovirus Ad36 have been studied most extensively for the past 25 years. Here, we present a systematic review of literature about SMAM-1 and Ad36. Reports from North America, Europe, and Asia reveal strong evidence that Ad36 causes obesity in animals and paradoxically improves glycemic control, and in vitro data provides mechanistic explanation. Considering that experimental Ad36 infection of humans is unlikely, its causative role in human obesity or glycemic control has not been demonstrated unequivocally. Nonetheless, most, but not all, observational studies in children and adults link Ad36 infection to obesity and improvement in glycemic control. The E4orf1 gene of Ad36 was identified as responsible for better glycemic control. Overall, 25 years have considerably advanced knowledge about the role of infection in obesity. Potential translational benefits include the development of vaccines to prevent Ad36-induced obesity and drug development based on the E4orf1 protein to improve glycemic control.

E4orf1: A protein for enhancing glucose uptake despite impaired proximal insulin signaling.

BACKGROUND: Type 2 diabetes is often linked with impaired proximal insulin signaling. Hence, a therapeutic agent that enhances cellular glucose uptake without requiring proximal insulin signaling would be desirable for improving glycemic control. The E4orf1 peptide (E4) derived from human adenovirus 36 (Ad36) promotes cellular glucose uptake in vitro and in vivo, independent of insulin. E4 bypasses a part of insulin signaling to upregulate cellular glucose uptake. We tested the hypothesis that E4 requires the distal but not proximal insulin signaling to enhance cellular glucose disposal. METHODS: 3T3-L1 preadipocytes inducibly expressing E4 or a null vector (NV) were treated with inhibitor of insulin receptor (S961), inhibitor of insulin like growth factor-1receptor (IGF-1R) (Picropodophyllin, PPP), PPP+S961, or phosphatidyl inositol-3 kinase (PI3K) inhibitor (Wortmannin, WM). We used PPP and S961 to block the proximal insulin signaling, or WM to block the distal insulin signaling. Cells were exposed to 0 or 100nM insulin. RESULTS: As expected, when the proximal or distal insulin signaling was blocked in NV cells, insulin could not enhance pAKT protein abundance, Glut4 translocation, or glucose uptake. Whereas, E4 cells significantly increased pAKT abundance, Glut4 translocation and glucose uptake independent of the presence of insulin or proximal insulin signaling. Enhanced glucose disposal in E4 cells was completely abrogated when the distal insulin signaling was blocked. CONCLUSIONS: E4 bypasses the proximal insulin signaling but uses the distal insulin signaling to activate pAkt and in turn Glut4 translocation to improve cellular glucose uptake. E4 offers a promising template to improve glycemic control when the proximal insulin signaling is impaired.

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.

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.

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.

E4orf1 induction in adipose tissue promotes insulin-independent signaling in the adipocyte.

BACKGROUND/PURPOSE: Type 2 diabetes remains a worldwide epidemic with major pathophysiological changes as a result of chronic insulin resistance. Insulin regulates numerous biochemical pathways related to carbohydrate and lipid metabolism. METHODS: We have generated a novel mouse model that allows us to constitutively activate, in an inducible fashion, the distal branch of the insulin signaling transduction pathway specifically in adipocytes. RESULTS: Using the adenoviral 36 E4orf1 protein, we chronically stimulate locally the Ras-ERK-MAPK signaling pathway. At the whole body level, this leads to reduced body-weight gain under a high fat diet challenge. Despite overlapping glucose tolerance curves, there is a reduced requirement for insulin action under these conditions. The mice further exhibit reduced circulating adiponectin levels that ultimately lead to impaired lipid clearance, and inflamed and fibrotic white adipose tissues. Nevertheless, they are protected from diet-induced hepatic steatosis. As we observe constitutively elevated p-Akt levels in the adipocytes, even under conditions of low insulin levels, this pinpoints enhanced Ras-ERK-MAPK signaling in transgenic adipocytes as a potential alternative route to bypass proximal insulin signaling events. CONCLUSION: We conclude that E4orf1 expression in the adipocyte leads to enhanced baseline activation of the distal insulin signaling node, yet impaired insulin receptor stimulation in the presence of insulin, with important implications for the regulation of adiponectin secretion. The resulting systemic phenotype is complex, yet highlights the powerful nature of manipulating selective branches of the insulin signaling network within the adipocyte.

Glia are critical for the neuropathology of complex I deficiency in Drosophila.

Mitochondrial electron transport chain (ETC) disorders cause severe neurological disease, typically in the context of fatal encephalomyelopathies. Neuronal cell autonomous energy deficiency due to reduced mitochondrial adenosine triphosphate production is currently the leading hypothesis to explain the neurotoxicity in ETC disorders. To define the mechanisms underlying neuropathology in ETC disorders, we have modeled the most common type of ETC disorder, complex I deficiency, in Drosophila. Our model recapitulates important clinical features of the disease including neuronal loss, mitochondrial enlargement, motor dysfunction and early death. Using cell-type specific gene knockdown, we find that both neurons and glia contribute to the disease phenotype and that glia play a critical non-cell autonomous role in the development of neuronal toxicity. Our results open up an unexpected avenue of research, and could lead to the development of new treatment strategies.

Doxycycline-regulated 3T3-L1 preadipocyte cell line with inducible, stable expression of adenoviral E4orf1 gene: a cell model to study insulin-independent glucose disposal.

Impaired glycemic control and excessive adiposity are major risk factors for Type 2 Diabetes mellitus. In rodent models, Ad36, a human adenovirus, improves glycemic control, independent of dietary fat intake or adiposity. It is impractical to use Ad36 for therapeutic action. Instead, we identified that E4orf1 protein of Ad36, mediates its anti-hyperglycemic action independent of insulin signaling. To further evaluate the therapeutic potential of E4orf1 to improve glycemic control, we established a stable 3T3-L1 cell system in which E4orf1 expression can be regulated. The development and characterization of this cell line is described here. Full-length adenoviral-36 E4orf1 cDNA obtained by PCR was cloned into a tetracycline responsive element containing vector (pTRE-Tight-E4orf1). Upon screening dozens of pTRE-Tight-E4orf1 clones, we identified the one with the highest expression of E4orf1 in response to doxycycline treatment. Furthermore, using this inducible system we characterized the ability of E4orf1 to improve glucose disposal in a time dependent manner. This stable cell line offers a valuable resource to carefully study the novel signaling pathways E4orf1 uses to enhance cellular glucose disposal independent of insulin.

E4orf1 improves lipid and glucose metabolism in hepatocytes: a template to improve steatosis & hyperglycemia.

Hepatic steatosis often accompanies obesity and insulin resistance. The cornerstones of steatosis treatment include reducing body weight and dietary fat intake, which are marginally successful over the long term. Ad36, a human adenovirus, may offer a template to overcome these limitations. In vitro and in vivo studies collectively indicate that via its E4orf1 protein, Ad36 improves hyperglycemia, and attenuates hepatic steatosis, despite a high fat diet and without weight loss. Considering that hepatic insulin sensitivity, or the synthesis, oxidation, or export of fatty acid by hepatocytes are the key determinant of hepatic lipid storage, we determined the role of E4orf1 protein in modulating these physiological pathways. For this study, HepG2 cells, or mouse primary hepatocytes were transfected with E4orf1 or the null vector. Glucose output by hepatocytes was determined under gluconeogenic conditions (cAMP and dexamethasone, or glucagon exposure). Also, de-novo lipogenesis, palmitate oxidation, and lipid export as determined by apoB secretion were measured 48 h post transfection. Results show that compared to null vector transfected cells, E4orf1 significantly reduced glucose output in basal and gluconeogenic conditions. E4orf1 reduced de-novo lipogenesis by about 35%, increased complete fatty acid oxidation 2-fold (p<0.0001), and apoB secretion 1.5 fold(p<0.003). Response of key signaling molecules to E4orf1 transfection was in agreement with these findings. Thus, E4orf1 offers a valuable template to exogenously modulate hepatic glucose and lipid metabolism. Elucidating the underlying molecular mechanism may help develop therapeutic approaches for treating diabetes or non-alcoholic fatty liver disease(NAFLD).