Obesity studies Open access Original research BMJ Open Diab Res Care: first published as 10.1136/bmjdrc-2020-002096 on 3 May 2021. Downloaded from Antidiabetic E4orf1 protein prevents hepatic steatosis and reduces markers of aging- related cellular damage in high fat fed older mice Zahra Mostofinejad,1,2 Md Akheruzzaman ,1 Md Abu Bakkar Siddik ,1 Presheet Patkar,1 Nikhil V Dhurandhar,1 Vijay Hegde 1 To cite: Mostofinejad Z, ABSTRACT Akheruzzaman M, Introduction Older age is associated with greater Significance of this study Abu Bakkar Siddik M, et al. prevalence of hyperinsulinemia, type 2 diabetes, and fatty Antidiabetic E4orf1 protein liver disease. These metabolic conditions and aging are What is already known about this subject? prevents hepatic steatosis bidirectionally linked to mitochondrial dysfunction and ► Metabolic disorders such as hyperinsulinemia, type and reduces markers of 2 diabetes and fatty liver disease and aging are bi- aging- related cellular damage telomere attrition. Although effectively addressing these conditions is important for influencing the health and directionally associated with mitochondrial dysfunc- in high fat fed older mice. tion and telomere attrition. BMJ Open Diab Res Care the lifespan, it is particularly challenging in older age. ► Modifying certain biomarkers like deregulated nutri- 2021;9:e002096. doi:10.1136/ We reported that E4orf1, a protein derived from human bmjdrc-2020-002096 adenovirus Ad36, reduces hyperinsulinemia, improves ent sensing, mitochondrial dysfunction, cellular se- glucose clearance, and protects against hepatic steatosis nescence caused by telomere attrition and genomic in younger mice exposed to high fat diet (HFD). Here, we instability are strategies to improve the health span ZM and MA contributed equally. tested if E4orf1 will improve glycemic control, liver fat of an individual. accumulation, mitochondrial integrity, and reduce telomere ► Despite lifestyle modifications and antidiabetic Components of this study agents showing health benefits, there are limitations have been presented at the attrition in older mice. and better therapeutic interventions are needed for American Society of Nutrition Research design and methods We used 9- month- old annual meeting in 2018 and at mice that inducibly expressed E4orf1 in adipose tissue and delaying aging-rela ted risk factors. non-E4orf1 expressing control mice. Mice were maintained Obesity week 2020. What are the new findings? on a 60% (kcal) HFD for 20 weeks and glycemic control In high fat fed older mice, adenoviral protein E4orf1 Received 24 December 2020 was determined by intraperitoneal glucose tolerance test ► significantly improves glycemic control in an insulin Revised 30 March 2021 at week 20. Following 20 weeks of HF- feeding, mice independent manner. http://drc.bmj.com/ Accepted 10 April 2021 were sacrificed and liver tissues collected to determine E4orf1 expression reduces hepatic steatosis and fi- the expression of aging genes using qRT-PCR based RT2 ► brosis despite high fat diet in these mice. Profiler PCR array. Results Compared with the control mice, E4orf1 ► E4orf1 expression also reduces aging- related hepat- significantly improved glycemic control and reduced ic markers of mitochondrial integrity and telomere hepatic steatosis and fibrosis. Additionally, E4orf1 attrition. maintained markers of mitochondrial integrity and How might these results change the focus of on September 26, 2021 by guest. Protected copyright. telomere attrition. research or clinical practice? Conclusion E4orf1 has the potential to improve glycemic ► Given the challenges among the elderly in improv- control in older mice, and the improvement persists ing aging associated comorbidities, use of E4orf1 as even after longer term exposure. E4orf1 expression also © Author(s) (or their a therapeutic target in improving glycemic control, employer(s)) 2021. Re- use maintains mitochondrial integrity and telomere attrition, protecting against hepatic fat accumulation and permitted under CC BY-NC. No thus delaying age- associated diseases. This provides reducing aging- associated cellular damage despite commercial re- use. See rights strong evidence for therapeutic utility of E4orf1 in chronic high fat diet will be clinically desirable. and permissions. Published improving age- associated metabolic and cellular changes by BMJ. that occur with aging in humans. 1Department of Nutritional Sciences, Texas Tech University, Lubbock, Texas, USA shares many chronic metabolic disorders with 2Department of Nutrition and INTRODUCTION obesity and type 2 diabetes (T2D), such as Environmental Toxicology, Aging is a very intricate process that leads to insulin resistance, poor glycemic control, and University of California, Davis, the structural and functional decline of organs chronic low- grade inflammation.2 Advanced California, USA and tissues, marked by chronic systemic age is also associated with ectopic lipid accu- Correspondence to inflammation, oxidative stress, DNA damage, mulation in the liver and can increase the Dr Vijay Hegde; impaired mitochondrial function, cellular prevalence of nonalcoholic fatty liver disease vijay. hegde@ ttu. edu senescence, and tissue dysfunction.1 Aging (NAFLD).3 While the contribution of obesity, BMJ Open Diab Res Care 2021;9:e002096. doi:10.1136/bmjdrc-2020-002096 1 Obesity studies BMJ Open Diab Res Care: first published as 10.1136/bmjdrc-2020-002096 on 3 May 2021. Downloaded from T2D, NAFLD, and nonalcoholic steatohepatitis (NASH) showed that humans who are naturally exposed to Ad36 in the etiology of aging is somewhat ambiguous, given have better glycemic control and, importantly, lower the complexity, the onset of these diseases through meta- hepatic steatosis or NAFLD prevalence.22 23 All these bolic alterations and acceleration of aging could be a features of E4orf1 protein appear to be attractive for vicious cycle.4 the alleviation of several risk factors of aging, which One of the key factors related with cellular aging is telo- may include improved glycemic control and protection mere attrition and damage.5 Several studies have shown against NAFLD but needs further investigation, particu- that patients with T2D have shorter telomeres in white larly in an older mouse model. blood cell and in pancreatic β-cells than non‐T2D indi- In the current study, we determined if E4orf1 can viduals.6 7 Additionally, decline in mitochondrial function improve glycemic control in an insulin independent or an increase in reactive oxygen species (ROS) has also manner in HFD-fed older mice. More specifically, we been implicated in aging.8 Further, it has been shown wanted to test if E4orf1 mediated protection against that telomere dysfunction is associated with a decline in hepatic steatosis can potentially prevent its progression to mitochondrial function.9 Alternatively, hyperglycemia fibrosis and delay aging- associated molecular dynamics. has been shown to induce mitochondrial dysfunction,10 suggesting a bidirectional relationship between T2D and aging associated telomere attrition and mitochondrial RESEARCH DESIGN AND METHODS dysfunction. Animal model and experimental design In view of the complex nature of the aging process, Transgenic C57BL/6J mice expressing Ad36E4orf1 quantitative biomarkers like epigenetic alterations, protein (n=7, M=4, F=3) or non-transgenic littermates deregulated nutrient sensing, mitochondrial dysfunc- (C57BL/6 mice, n=8, M=4, F=4) from our breeding tion, cellular senescence caused by telomere attrition and colony were included in this study. Animals were handled genomic instability predict health span of an individual11 following Texas Tech University Institutional Animal and potentially can be modified. Dietary restriction Care and Use Committee guidelines. All mice were and exercise have been the most studied interventions group- housed, on a 12 hours light/12 hours dark cycle at in improving aging associated risk factors.12 13 However, 25°C, with ad libitum access to chow diet (protein 24%, poor compliance associated with these lifestyle modifica- carbohydrate 60%, and fat 16%) and water. Weekly body tions makes them challenging, especially for the elderly. weight was recorded. After taking baseline measurements Well maintained glycemic control could prevent or delay at 9 months of age, animals were switched to a high fat- the development of multiple age- related disorders and doxycycline diet (HF- dox; 60% kcal from fat, dox 600 might slow the aging process.14 Currently, several antidi- ppm/kg) until their sacrifice (at week 20). Following 20 abetic agents are used for maintaining glycemic control. weeks of HF- dox feeding, blood glucose was measured Common among them is insulin sensitizer metformin, after 4 hours fasting and 15 min after a glucose challenge which also improves health span in mice15 and shows as described previously.17 Serum insulin was also assessed reduced mortality in patients with diabetes compared by ELISA (EMD Millipore, Cat. EZRMI-13K). Mice were http://drc.bmj.com/ 16 with patients with diabetes not receiving metformin. then sacrificed using CO2 asphyxiation and cervical dislo- One of the limitations with efficacy of many antidiabetic cation and cardiac puncture blood was collected. Liver drugs is the requirement of a functional insulin signaling and other peripheral tissues were harvested, frozen pathway, which is often impaired during T2D. Therefore, immediately in liquid nitrogen, and preserved at −80°C therapeutic interventions for delaying aging