Obesity in humans can lead to metabolic problems such as glucose intolerance and insulin resistance, which may result from pancreatic islet dysregulation and reduced insulin sensitivity in the liver. LEW.1WR1 (1WR1) rats became more glucose intolerant than LEW/SsNHsd (SsNHsd) rats after 12 weeks on a moderate sucrose diet.1 We hypothesize that the 1WR1 rats develop decreased insulin sensitivity due to impaired islet function and liver responses to insulin. To test this hypothesis we measured blood hormone levels and islet and liver gene expression. The terminal blood insulin (14988+/- 4024 vs. 22703+/-5101 pg/mL; p=0.0085; n=7,7) and glucagon (127.3+/-73.31 vs. 188.6+/-46.87 pg/mL; p=0.0537; n=7,7) were higher in the the 1WR1 rats. Using qRT-PCR, we determined the islets of 1WR1 rats had 3 fold increased insulin (p<0.0001; n=3,3) and glucagon (p<0.0001; n=3,2) relative gene expression. Yet, the β-cell area (22.05+/-6.408 vs. 2.276 +/-1.284mm2; p=0.0016; n=3,4) was significantly reduced in 1WR1 rats. Islet Plin5 expression was upregulated in 1WR1 rats (5.388+/-0.3806 F.C.; p<0.0001; n=3,3) indicating increased lipid droplet production, while Cyclin D (0.5726+/-0.08797 F.C.; p=0.0035; n=3,2) was downregulated indicating decreased cell cycle proliferation. These results indicate that the islets of the 1WR1 rats were insensitive to insulin signaling, which may have been caused by increased lipid droplets and a decrease in compensatory islet area. We also measured the relative expression of insulin-sensitive genes in the liver tissue to determine if there were alterations in liver insulin signaling. Downregulation of Irs-2 (0.5840+/-0.001045 F.C.; p<0.0001; n=7,7) expression was likely caused by the upregulated fat10 gene in 1WR1 rats.2 Fat10 (2315+/-0.01380 F.C.; p<0.0001; n=4,6) expression in the liver was significantly increased. Foxo1 (2.644+/- 0.001211 F.C.; p<0.0001; n=7,7) expression, which is normally reduced by insulin, was upregulated which indicates reduced insulin sensitivity. Upregulated expression of Fgf21 (2.260+/-0.002376 F.C.; p<0.0001; n=6,7), which improves glucose homeostasis, in the liver is why the fasting blood glucose of 1WR1 rats were not significantly different from the SsNHsd rats.1 In conclusion, 1WR1 rats show increasingly impaired metabolism over time. These rats have increased insulin and glucagon levels coupled with liver fat10 overexpression leading to impaired gene regulation of insulin-responsive genes in the liver. These changes synergistically increase susceptibility to pathological obesity and metabolic disease. References: (1) Collins et al., Journal of the Endocrine Society. 2019 3(S1). (2) Ge, Q. et al., Frontiers in Physiology. 2018; 9(1051): 1–16.

Obesity in humans can lead to metabolic problems such as glucose intolerance and insulin resistance, which may result from pancreatic islet dysregulation and reduced insulin sensitivity in the liver. Increased fat10 expression is associated with adiposity, insulin resistance, and inflammation that increases with age as well as type 1 diabetes. The effect of increased fat10 expression on beta cell physiology, however, is not well understood. LEW.1WR1 (1WR1) rats possess a defect in the fat10 promoter which leads to sustained expression of diubiquitin. We suggested previously that fat10 may play a role in increased glucose intolerance, insulin sensitivity, age‐related fat deposition, and changes in beta cell size and density in 1WR1 rats. In that study, we identified through semiquantitative analysis that the 1WR1 rats seemed to have fewer islets compared to the LEW/SsNHsd (SsNHsd) rats. The purpose of this project was to compare normalized beta cell area from these rodents and analyze this data to confirm that the perceived changes in islet number corresponded to differences in beta cell area. We hypothesized that, since the 1WR1 rats had a lower islet number, they would also have lower normalized beta cell area, dysregulated insulin signaling, and an altered cell cycle. Seven week old 1WR1 and SsNHsd rats were given a control diet for 12 weeks. Once the animals were sacrificed, the pancreases were harvested, fixed in formalin, and sent to Histowiz (New York, NY) for immunohistochemical staining against insulin. These stained slides were obtained digitally and opened in ImageJ, which was used to measure beta cell and pancreas area. Comparing these showed that the 1WR1 rats indeed have a lower average normalized beta cell area than SsNHsd rats on a moderate sucrose diet. Using qRT‐PCR, we determined the islets of 1WR1 rats had 3 fold increased insulin and glucagon gene expression. Yet, the β‐cell area (22.05+/‐6.408 vs. 2.276 +/‐1.284mm2; p=0.0016; n=3,4) was significantly reduced in 1WR1 rats. Islet Plin5 expression was upregulated in 1WR1 rats (5.388+/‐0.3806 F.C.; p<0.0001; n=3,3) indicating increased lipid droplet production, while Cyclin D (0.5726+/‐0.08797 F.C.; p=0.0035; n=3,2) was downregulated indicating decreased cell cycle proliferation. These results indicate that the islets of the 1WR1 rats were insensitive to insulin signaling, which may have been caused by increased lipid droplets and a decrease in compensatory islet area. This experiment increases our understanding of the characteristics of the pancreas of LEW.1WR1 rats, which may help us better understand the impact of unregulated fat10 expression on beta cell physiology.

Non alcoholic Fatty Liver Disease(NAFLD) is a disease usually found alongside diabetes in patients and is known as a manifestation of the metabolic disorder. The disease describes a progression of increasingly malignant conditions affecting the liver starting from benign steatosis. Steatosis can progress to steatohepatitis, damaging cells and encouraging scar tissue formation. The LEW.1WR1(1WR1) rat overexpresses fat10, a gene which plays a role in age‐related inflammation, insulin resistance, adiposity, and hepatocellular carcinoma. This gene has been shown to be essential in maintaining liver cell protein quality control and Mallory Denk body(MDB) formation(Jia, Ji, & French, 2020). 1WR1 rats have been shown to become more glucose intolerant as they age, developing insulin resistance increasing risk of Diabetes and NAFLD. They have also been shown to develop increased body mass while not having significantly different epididymal fat pad deposits and increased fat10 concentration in the liver compared to control animals (Collins, Clopp, Mercado, Gibson, & Love‐Rutledge, 2019). Increasing understanding of fat10’s functions is important for future research in reducing disease susceptibility in NAFLD. For this project we hypothesized that because weight was not being added to the fat pads in the previous study it would be added to the organs; therefore these animals would develop increased liver mass or fatty liver as they age increasing risk of NAFLD. This project studied glucose intolerance, triglyceride levels, liver weight, MDBs, and NAFLD Activity score(NAS) of 1WR1 and WF/NHsd(WF) rat livers. These rats were subjected to a 7% sucrose or normal diet type by Research Diets from 5‐7 weeks of age. At 21 weeks old a 120 minute human like glucose tolerance test was done after a fasting period of 8 hours. Triglyceride concentrations in the blood, liver, and muscles were measured. Sacrifice occurred at 18 weeks of study and the livers were weighed and formalin fixed. They were then sent to Histowiz for slide processing and digitalization. Scoring for MDBs and NAS were done on the resulting slides. The results of the glucose tolerance test confirmed these rats became more glucose intolerant during the study as their glucose levels were unable to return to baseline after 120 minutes. The Triglyceride assay showed significantly higher concentrations of liver and muscle triglyceride levels to the control. The liver mass measurements showed the 1WR1 rats had significantly heavier livers to the control. The MDB scoring showed their presence primarily in the 1WR1 rats. Finally, the results of our NAS scoring indicated that the 1WR1 rats developed steatosis at a higher rate than the control. The steatosis development at a higher rate correlates to a higher risk of steatohepatitis and further malignant forms of NAFLD. Fat10’s roles in age related glucose intolerance and liver function increases risk of development of later malignant NAFLD forms.

Diabetes prevention is a large topic of research that is focused on nutritional and exercise‐based interventions with some genetic predisposition based interventions. FAT10 is a ubiquitin‐like protein and Type 1 Diabetes (T1D) susceptibility gene that may play a role in age‐related inflammation, adiposity, cancer, and kidney disease. The young T1D susceptible, LEW.1WR1 rat overexpresses FAT10 and has higher fasting concentrations of blood insulin. It is unclear how the overexpression of FAT10 effects directly affects insulin sensitivity, secretion, or production. It is also unclear if the initial insulin sensitivity of this animal model plays a role in disease susceptibility. Therefore we hypothesized that older LEW.1WR1 rats will have markers of increased insulin resistance like increased insulin and beta cell mass due to the poor regulation of FAT10 expression in this model. The objective of this study was to characterize insulin sensitivity, insulin levels, and characterize the relative beta cell size of adult LEW.1WR1 rats with or without dietary stress to understand if FAT10 expression plays a role in the regulation of inflammation and aging‐induced insulin resistance. To test this hypothesis we used LEW.1WR1 and LEW.SsNHsd rat and monitored weight gain and glucose tolerance over the course of 12 weeks on a high fat or control diet; we also isolated pancreata and used immunohistochemistry staining for insulin to analyze islet size and insulin staining. The LEW.ssNHsd rat served as a control rat because it does not have the alteration in the FAT10 promoter region. Glucose tolerance tests were performed prior to starting the diet as a baseline, and after 10 weeks on the diet to determine the effects of diet and aging on the rat groups in regards to Insulin Sensitivity. We observed that in the course of 10 weeks the control LEW.1WR1 rats became significantly more glucose intolerant and had increased insulin levels. The control LEW.1WR1 rats also gained weight at a rate similar to the high‐fat diet, a diet that is traditionally used to induce insulin resistance and glucose intolerance. This data suggests that FAT10 may be playing a role in age‐related adiposity, glucose intolerance, insulin sensitivity, and beta‐cell changes in the LEW.1WR1 rat. This study begins to lay the groundwork for understanding how alterations in FAT10 and metabolism increase T1D susceptibility.Support or Funding InformationThe University of Alabama in HuntsvilleLouis Stokes Alliance for Minority Participation (NSF)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

FAT10 is an ubiquitin-like protein and Type 1 Diabetes (T1D) susceptibility gene that may play a role in age-related inflammation, adiposity, and cancer risk. The LEW.1WR1(1WR1) rat overexpresses FAT10 and has higher fasting concentrations of blood insulin and triglyceride during the induction of T1D. FAT10 is hypothesized to play a role in the expression of adipogenic genes; however, it is unclear if overexpression of FAT10 directly affects insulin sensitivity and glucose metabolism. We hypothesize that young adult 1WR1 rats will have increased glucose intolerance due to the poor regulation of insulin secretion which over time leads to reduced insulin sensitivity and increased adiposity. To test this hypothesis, we monitored glucose and insulin tolerance by glucose (GTT) or insulin tolerance (ITT) test and weight gain throughout 12 weeks on high fat and high sucrose (HFD) or moderate sucrose (MS) diet. The LEW/ssNHsd (SsNHsd) rat serves as a control rat. All experiments contain seven rats per group. We observed that at four weeks, the HFD diet rat groups had increased but non-significant fasting blood glucose levels during an ITT (109 +/- 4 mg/dL 1WR1-MS; 113 +/- 5 mg/dL SsNHsd-MS; 132 +/- 6 mg/dL 1WR1 HFD; or 131 +/- 13 mg/dL SsNHsd-HFD.) At ten weeks, the 1WR1-MS rats were more glucose intolerant than the control SsNHsd-MS rat (311.7 +/- 64 mg/dL vs. 194 +/- 53 mg/dL; p=0.09.) We compared the eight week GTT of the 1WR1-MS rats to a GTT before starting the diets (zero weeks) and the thirty minute time points are significantly different for the 1WR1-MS after 8 weeks on the MS diet compared to both the 1WR1-MS and SsNHsd-MS prior to the MS diet ( 164 +/- 27 mg/dL zero weeks 1WR1; 167 +/- 26 mg/dL zero weeks SsNHsd; 311 +/-64 mg/dL eight weeks 1WR1-MS p< 0.0001). 1WR1 rats on both diets also gained weight at similar rates, yet the total abdominal fat mass of the 1WR1-MS rats was not increased compared to the SsNHsd-MS (17.43 +/- 3.48 g 1WR1-MS vs. 16.42 +/- 2.73 g SsNHsd-MS p=0.94 and 24.64 +/- 4.27 g 1WR1-HFD p=0.004) and 1WR1 rat epididymal fat was not significantly different between the two diets compared to the significant difference in SsNHsd rats groups (5.85+/-1.01 1WR1-MS vs. 7.27+/- 1.42 1WR1-HFD p=0.07; 4.63 +/- 0.81 vs. 6.77 +/- 0.65 p=0.0052.) This data suggests that metabolic shifts are occurring in the 1WR1 rat, due to either increased sucrose intake or aging. These shifts reflected the increased glucose intolerance after eight weeks and the changes in abdominal fat pads relative to body mass. This study lays the groundwork for characterizing how alterations in FAT10 increase metabolic dysfunction through aging, glucose metabolism, insulin secretion/signaling, and lipid metabolism/catabolism. Unless otherwise noted, all abstracts presented at ENDO are embargoed until the date and time of presentation. For oral presentations, the abstracts are embargoed until the session begins. Abstracts presented at a news conference are embargoed until the date and time of the news confere...