KK Mice Research Articles

Sleep restriction exacerbates cardiac dysfunction in diabetic mice by causing cardiomyocyte death and fibrosis through mitochondrial damage.

Diabetic cardiomyopathy (DCM) is a cardiovascular complication of diabetes mellitus with a poor prognosis and is the leading cause of death in diabetic patients. Sleep deficiency is not only recognized as an important risk factor for the development of type 2 DM, but is also associated with increased morbidity and mortality of cardiovascular disease. The underlying role and mechanisms of sleep restriction (SR) in DCM are far from clear. The KK/Upj-Ay mouse model of T2 DM was used as a study subject, and the small animal ultrasound imaging system was used to detect the function of the heart; immunopathological staining was used to clarify the histo-structural pathological alterations of the heart; and TUNEL staining, qPCR, transmission electron microscopy (TEM), and ELISA kits were used to detect apoptosis, oxidative stress, inflammation, and mitochondrial damage, and related molecular alterations. SR led to a significant increase in mortality, cardiac hypertrophy, necrosis, glycogen deposition and fibrosis further deteriorated in DM KK mice. SR increased cardiomyocyte death in KK mice through the Bax/Bcl2 pathway. In addition to this, SR not only exacerbated the inflammatory response, but also aggravated mitochondrial damage and promoted oxidative stress in KK mice through the PRDM16-PGC-1α pathway. Overall, SR exacerbates structural alterations and dysfunction through inflammation, oxidative stress, and apoptosis in DM KK mice, increasing the risk of death. Clinicians and diabetic patients are prompted to pay attention to sleep habits to avoid accelerating the transition of DCM to heart failure and inducing death due to poor sleep habits.

Abstract Tu010: Matrix metallopeptidase 9 contributes to the beginning of plaque and is a potential biomarker for the early identification of atherosclerosis in asymptomatic patients with diabetes

Aims: To determine the roles of matrix metallopeptidase-9 (MMP9) on human coronary artery smooth muscle cells (HCASMCs) in vitro , early beginning of atherosclerosis in vivo in diabetic mice, and patients with diabetes. Methods: Active hMMP9 ( act-hMMP9) was added to HCASMCs and markers of inflammation were measured. Act-hMMP9 (n=16) or placebo (n=15) was administered to d i abetic KK.Cg- A y /J (KK) mice. Carotid artery inflammation and atherosclerosis measurements were made at 2 and 10 weeks after treatment. An observational study of newly diagnosed drug naïve patients with type 2 diabetes mellitus (T2DM n=234) and healthy matched controls (n=41) was performed and patients had ultrasound of carotid arteries and some had coronary computed tomography angiogram for the assessment of atherosclerosis. Serum MMP9 was measured and its correlation with carotid artery or coronary artery plaques were determined. Results: Act-hMMP9 induced inflammatory response in HCASMCs in vitro , including increased gene and protein expressions of MCP-1, ICAM-1, VCAM-1, and enhanced macrophage adhesion. Exogenous act-hMMP9 increased inflammation and initiated atherosclerosis in KK mice at 2 and 10 weeks: increased vessel wall thickness, lipid accumulation, and Galectin-3+ macrophage infiltration into the carotid arteries. In newly diagnosed T2DM patients, serum MMP9 correlated with carotid artery plaque size with a possible threshold cutoff point. In addition, serum MMP9 correlated with number of mixed plaques and grade of lumen stenosis in coronary arteries of patients with drug naïve T2DM. Conclusions: MMP9 contributes to the initiation of atherosclerosis and may be a potential biomarker for the early identification of atherosclerosis in patients with diabetes

Single-housing-induced islet epigenomic changes are related to polymorphisms in diabetic KK mice.

A lack of social relationships is increasingly recognized as a type 2 diabetes (T2D) risk. To investigate the underlying mechanism, we used male KK mice, an inbred strain with spontaneous diabetes. Given the association between living alone and T2D risk in humans, we divided the non-diabetic mice into singly housed (KK-SH) and group-housed control mice. Around the onset of diabetes in KK-SH mice, we compared H3K27ac ChIP-Seq with RNA-Seq using pancreatic islets derived from each experimental group, revealing a positive correlation between single-housing-induced changes in H3K27ac and gene expression levels. In particular, single-housing-induced H3K27ac decreases revealed a significant association with islet cell functions and GWAS loci for T2D and related diseases, with significant enrichment of binding motifs for transcription factors representative of human diabetes. Although these H3K27ac regions were preferentially localized to a polymorphic genomic background, SNVs and indels did not cause sequence disruption of enriched transcription factor motifs in most of these elements. These results suggest alternative roles of genetic variants in environment-dependent epigenomic changes and provide insights into the complex mode of disease inheritance.

Matrix metallopeptidase 9 contributes to the beginning of plaque and is a potential biomarker for the early identification of atherosclerosis in asymptomatic patients with diabetes.

To determine the roles of matrix metallopeptidase-9 (MMP9) on human coronary artery smooth muscle cells (HCASMCs) in vitro, early beginning of atherosclerosis in vivo in diabetic mice, and drug naïve patients with diabetes. Active human MMP9 (act-hMMP9) was added to HCASMCs and the expressions of MCP-1, ICAM-1, and VCAM-1 were measured. Act-hMMP9 (n=16) or placebo (n=15) was administered to diabetic KK.Cg-Ay/J (KK) mice. Carotid artery inflammation and atherosclerosis measurements were made at 2 and 10 weeks after treatment. An observational study of newly diagnosed drug naïve patients with type 2 diabetes mellitus (T2DM n=234) and healthy matched controls (n=41) was performed and patients had ultrasound of carotid arteries and some had coronary computed tomography angiogram for the assessment of atherosclerosis. Serum MMP9 was measured and its correlation with carotid artery or coronary artery plaques was determined. In vitro, act-hMMP9 increased gene and protein expressions of MCP-1, ICAM-1, VCAM-1, and enhanced macrophage adhesion. Exogenous act-hMMP9 increased inflammation and initiated atherosclerosis in KK mice at 2 and 10 weeks: increased vessel wall thickness, lipid accumulation, and Galectin-3+ macrophage infiltration into the carotid arteries. In newly diagnosed T2DM patients, serum MMP9 correlated with carotid artery plaque size with a possible threshold cutoff point. In addition, serum MMP9 correlated with number of mixed plaques and grade of lumen stenosis in coronary arteries of patients with drug naïve T2DM. MMP9 may contribute to the initiation of atherosclerosis and may be a potential biomarker for the early identification of atherosclerosis in patients with diabetes. https://clinicaltrials.gov, identifier NCT04424706.

Fish oil consumption prevents hepatic lipid accumulation induced by high-cholesterol feeding in obese KK mice.

Fish oil (FO) is rich in the n-3 polyunsaturated fatty acids. It has been demonstrated that FO intake possesses lipid-lowering properties. Conversely, a high-cholesterol (CH) diet promotes lipid accumulation in the liver and induces fatty liver. This study investigated the effects of FO feeding on hepatic lipid accumulation induced by high-cholesterol feeding in KK mice. All experimental diets had a fat energy ratio of 25%, the SO group had all fat sources as safflower oil (SO), the 12.5 FO group had half of the SO replaced with FO, and the 25 FO group had all of the SO replaced with FO, each with or without 2 weight % (wt%) cholesterol (SO/CH, 12.5 FO/CH, and 25 FO/CH groups, respectively), for 8 weeks. The hepatic triglyceride and total cholesterol levels were significantly lower in the 25 FO/CH group than in the SO/CH group. The hepatic mRNAs of fatty acid synthesis-related genes were downregulated by the FO feeding groups. In view of importance to establish the benefit of FO for preventing severe NAFLD, our results suggest that FO intake prevents excessive hepatic fat accumulation induced by a high-cholesterol diet in obese KK mice through the inhibition of fatty acid synthesis.

Fish oil-derived n-3 polyunsaturated fatty acids downregulate aquaporin 9 protein expression of liver and white adipose tissues in diabetic KK mice and 3T3-L1 adipocytes

Aquaporin 9 (AQP9) is an integral membrane protein that facilitates glycerol transport in hepatocytes and adipocytes. Glycerol is necessary as a substrate for gluconeogenesis in the physiological fasted state, suggesting that inhibiting AQP9 function may be beneficial for treating type 2 diabetes associated with fasting hyperglycemia. The n-3 polyunsaturated fatty acids (PUFAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are rich in fish oil and lower the risk of metabolic syndrome; however, the effects of EPA and DHA on AQP9 expression in obese and type 2 diabetes are unclear. The KK mouse is an animal model of obesity and type 2 diabetes because of the polymorphisms on leptin receptor gene, which results in a part of cause for obese and diabetic conditions. In this study, we determined the effect of fish oil-derived n-3 PUFA on AQP9 protein expression in the liver and white adipose tissue (WAT) of KK mice and mouse 3T3-L1 adipocytes. The expression of AQP9 protein in the liver, epididymal WAT, and inguinal WAT were markedly decreased following fish oil administration. We also demonstrated that n-3 PUFAs, such as DHA, and to a lesser extent EPA, downregulated AQP9 protein expression in 3T3-L1 adipocytes. Our results suggest that fish oil-derived n-3 PUFAs may regulate the protein expressions of AQP9 in glycerol metabolism-related organs in KK mice and 3T3-L1 adipocytes.

1354-P: Alternate-Day Intermittent Fasting Improves Diabetes and Protects Beta-Cell Function in Polygenic Mouse Models of T2DM

Type 2 diabetes (T2DM) , caused by the interaction of multiple genes and environmental factors, is characterized by hyperglycemia, insulin secretion deficiency and insulin resistance. Chronic hyperglycemia induces β-cell dysfunction and loss of β-cell mass/identity, increased apoptosis and β-cell dedifferentiation. Intermittent fasting (IF) , either alternate-day fasting (ADF) or time-restricted feeding, commonly used regimens for weight-loss, also induces metabolic benefits including reduced blood glucose, improved insulin sensitivity, reduced adiposity, inflammation and oxidative-stress, and increased fatty-acid oxidation; however, the mechanisms underlying these effects remain elusive. KK and KKAy, mouse models of polygenic T2DM spontaneously develop hyperglycemia, glucose intolerance, glucosuria, impaired insulin secretion and insulin resistance. To determine the long-term effects of IF on T2DM, 6-weeks old KK and KKAy mice were subjected to ADF for 16-weeks. While KKAy mice fed ad-libitum demonstrated severe hyperglycemia (˜500mg/dL) , increased plasma insulin, impaired glucose tolerance and insulin resistance at 8 weeks of age, KK mice showed blood glucose levels of ˜200mg/dL, but progressively became as severely diabetic as KKAy mice by 22-weeks. Strikingly, both KK and KKAy mice subjected to ADF showed reduced blood glucose and plasma insulin levels, decreased body weight gain despite increased food intake, reduced plasma triglycerides and cholesterol, and improved insulin sensitivity. They also demonstrated enhanced expression of the β-cell transcription factors PDX1 and NKX6.1, suggesting protection from loss of β-cell identity/dedifferentiation by IF. In addition, respiratory exchange ratio (RER) and movement were significantly enhanced in the ADF group, indicating peripheral benefits of IF. These results have important implications as an optional intervention for preservation of β-cell mass and function in T2DM. Disclosure S.Patel: None. Z.Yan: None. M.S.Remedi: None. Funding National Institutes of Health (R01DK123163)

Phenotypic and genotypic changes in obesity and type 2 diabetes of male KK mice with aging.

Research into the prevention and treatment of age-related metabolic diseases are important in the present-day situation of the aging population. We propose that an elderly diabetic mouse model may be useful to such research as it exhibits deterioration of glucose and lipid metabolism. Although the KK mouse strain is commonly used as a model of moderate obesity and type 2 diabetes, the utility of this strain as an elderly obese and diabetic model mouse for research into aging remains unclear. The present study aimed to investigate age-related changes of glucose and lipid metabolism in male KK mice fed a standard chow diet. We demonstrate that 40 weeks KK mice exhibit age-related dysfunctions, such as development of insulin resistance associated with pancreatic islet hypertrophy and decreased lipolysis in white adipose tissue (WAT) compared with 15 weeks KK mice. However, aging does not appear to cause mitochondrial dysfunction of brown adipose tissue. Unexpectedly, hyperglycemia, potential glucose uptake in insulin-sensitive organs, hepatic lipid accumulation, hypertrophy of adipocytes, and inflammation in epididymal WAT did not worsen but rather compensated in 40 weeks KK mice. Our data indicate that the use of male KK mice as an elderly obese and diabetic mouse model has some limitations and in order to represent a useful elderly obese and diabetic animal model, it may be necessary to induce deterioration of glucose and lipid metabolism in KK mice through breeding with high-sucrose or high-fat diets.

Strain-based and sex-biased differences in adrenal and pancreatic gene expression between KK/HlJ and C57BL/6\u2009J mice

BackgroundThe ever-increasing prevalence of diabetes and associated comorbidities serves to highlight the necessity of biologically relevant small-animal models to investigate its etiology, pathology and treatment. Although the C57BL/6 J model is amongst the most widely used mouse model due to its susceptibility to diet-induced obesity (DIO), there are a number of limitations namely [1] that unambiguous fasting hyperglycemia can only be achieved via dietary manipulation and/or chemical ablation of the pancreatic beta cells. [2] Heterogeneity in the obesogenic effects of hypercaloric feeding has been noted, together with sex-dependent differences, with males being more responsive. The KK mouse strain has been used to study aspects of the metabolic syndrome and prediabetes. We recently conducted a study which characterized the differences in male and female glucocentric parameters between the KK/HlJ and C57BL/6 J strains as well as diabetes-related behavioral differences (Inglis et al. 2019). In the present study, we further characterize these models by examining strain- and sex-dependent differences in pancreatic and adrenal gene expression using Affymetrix microarray together with endocrine-associated serum analysis.ResultsIn addition to strain-associated differences in insulin tolerance, we found significant elevations in KK/HlJ mouse serum leptin, insulin and aldosterone. Additionally, glucagon and corticosterone were elevated in female mice of both strains. Using 2-factor ANOVA and a significance level set at 0.05, we identified 10,269 pancreatic and 10,338 adrenal genes with an intensity cut-off of ≥2.0 for all 4 experimental groups. In the pancreas, gene expression upregulated in the KK/HlJ strain related to increased insulin secretory granule biofunction and pancreatic hyperplasia, whereas ontology of upregulated adrenal differentially expressed genes (DEGs) related to cell signaling and neurotransmission. We established a network of functionally related DEGs commonly upregulated in both endocrine tissues of KK/HlJ mice which included the genes coding for endocrine secretory vesicle biogenesis and regulation: PCSK2, PCSK1N, SCG5, PTPRN, CHGB and APLP1. We also identified genes with sex-biased expression common to both strains and tissues including the paternally expressed imprint gene neuronatin.ConclusionOur novel results have further characterized the commonalities and diversities of pancreatic and adrenal gene expression between the KK/HlJ and C57BL/6 J strains as well as differences in serum markers of endocrine physiology.

The KCNJ11-E23K Gene Variant Hastens Diabetes Progression by Impairing Glucose-Induced Insulin Secretion.

The ATP-sensitive K+ (KATP) channel controls blood glucose levels by coupling glucose metabolism to insulin secretion in pancreatic β-cells. E23K, a common polymorphism in the pore-forming KATP channel subunit (KCNJ11) gene, has been linked to increased risk of type 2 diabetes. Understanding the risk-allele-specific pathogenesis has the potential to improve personalized diabetes treatment, but the underlying mechanism has remained elusive. Using a genetically engineered mouse model, we now show that the K23 variant impairs glucose-induced insulin secretion and increases diabetes risk when combined with a high-fat diet (HFD) and obesity. KATP-channels in β-cells with two K23 risk alleles (KK) showed decreased ATP inhibition, and the threshold for glucose-stimulated insulin secretion from KK islets was increased. Consequently, the insulin response to glucose and glycemic control was impaired in KK mice fed a standard diet. On an HFD, the effects of the KK genotype were exacerbated, accelerating diet-induced diabetes progression and causing β-cell failure. We conclude that the K23 variant increases diabetes risk by impairing insulin secretion at threshold glucose levels, thus accelerating loss of β-cell function in the early stages of diabetes progression.

Pharmacologic inhibition of serotonin htr2b ameliorates hyperglycemia and the altered expression of hepatic FGF21, Sdf2l1, and htr2a in db/db mice and KKAy mice

Plasma fibroblast growth factor 21 (FGF21) levels and hepatic FGF21, serotonin 2a receptor (htr2a), and stromal cell-derived factor 2 like 1 (Sdf2l1) expression are increased in insulin-resistant C57BL6J mice fed a high-fat diet. Here we show that plasma FGF21 levels and hepatic FGF21, Sdf2l1, and htr2a expression were decreased in 6-week-old db/db mice compared with C57BL6J mice, whereas they were increased in 6-week-old KKAy mice compared with KK mice. Expression of hepatic htr2b was increased in db/db mice and KKAy mice compared with controls. Treatment with the selective htr2b antagonist SB204741 suppressed the hyperglycemia in either db/db mice or KKAy mice. Treatment with SB20471 reversed the decreases in plasma FGF21 levels and hepatic FGF21, Sdf2l1, and htr2a expression in db/db mice, whereas it suppressed the increases in plasma FGF21 levels and hepatic FGF21, Sdf2l1, and htr2a expression in KKAy mice. Moreover, treatment with SB204741 increased plasma FGF21 levels and expression of hepatic FGF21, htr2a, and Sdf2l1 in C57BL6J mice, whereas it decreased plasma FGF21 levels and hepatic FGF21 expression in KK mice. These findings suggest that pharmacologic inhibition of htr2b ameliorates the hyperglycemia and altered expression of hepatic FGF21, Sdf2l1 and htr2a in obese and diabetic db/db and KKAy mice.

The sodium-glucose cotransporter 2 inhibitor tofogliflozin prevents diabetic kidney disease progression in type 2 diabetic mice.

Trials on cardiovascular and renal outcomes in patients with type 2 diabetes have consistently demonstrated that sodium-glucose cotransporter 2 (SGLT2) inhibitors reduce the risk of diabetic kidney disease (DKD) progression. However, their renal protective mechanisms have yet to be completely understood and the effect on albuminuria reduction in animal models is controversial. We investigated these issues using KK and KK-Ay mice as a control (CTRL) and as a model for type 2 diabetes (DKD), respectively. KK-Ay mice were treated with 0.015% tofogliflozin, which is an SGLT2 inhibitor, starting at seven weeks of age for eight weeks. Compared with the CTRL mice, the DKD mice had higher HbA1c levels and albuminuria. Although tofogliflozin treatment significantly lowered HbA1c levels, it did not reverse albuminuria. Tofogliflozin treatment enhanced damage in both the glomerular (i.e., enlarged mesangial area, increased foot process effacement rate, and decreased number of WT-1-positive cells) and tubulointerstitial (increased protein levels of KIM-1 and MCP-1, increased number of macrophages, and abnormal mitochondrial morphology) areas. Our results suggest that tofogliflozin may prevent glomerular and tubulointerstitial damage, partly by ameliorating hyperglycemia, renal inflammation, and abnormalmitochondrial morphology.

Repetitive Ozone Exposures and Evaluation of Pulmonary Inflammation and Remodeling in Diabetic Mouse Strains.

Background:Epidemiological studies support the hypothesis that diabetes alters pulmonary responses to air pollutants like ozone (). The mechanism(s) underlying these associations and potential links among diabetes, , and lung inflammation and remodeling are currently unknown.Objectives:The goal was to determine whether pulmonary responses to repetitive ozone exposures are exacerbated in murine strains that are hyperglycemic and insulin resistant.Methods:Normoglycemic and insulin-sensitive C57BL/6J mice; hyperglycemic, but mildly insulin-resistant, KK mice; and hyperglycemic and markedly insulin-resistant KKAy mice were used for ozone exposure studies. All animals were exposed to filtered air (FA) or repetitive ozone ( , 4 h/d, for 13 consecutive weekdays). Tissue analysis was performed 24 h following the final exposure. This analysis included bronchoalveolar lavage (BAL) for cell and fluid analysis, and tissue for pathology, immunohistology, mRNA, and hydroxyproline.Results:Following repetitive exposure, higher bronchoalveolar lavage fluid inflammatory cells were observed in all mice (), with a notable influx of neutrophils and eosinophils in KK and KKAy mice. Although the lungs of -exposed C57BL/6J and KK mice had minimal centriacinar histological changes without fibrosis, the lungs of -exposed KKAy mice contained marked epithelial hyperplasia in proximal alveolar ducts and adjacent alveoli with associated centriacinar fibrosis. Fibrosis in -exposed KKAy lungs was confirmed with immunohistochemistry, tissue hydroxyproline content, and tissue mRNA expression of fibrosis-associated genes (Ccl11, Il13, and Mmp12). Immunofluorescence staining and confocal microscopy revealed alterations in the structure and composition of the airway and alveolar epithelium in regions of fibrosis.Discussion:Our results demonstrate that in diabetic animal strains repetitive ambient ozone exposure led to early and exaggerated pulmonary inflammation and remodeling. Changes in distal and interstitial airspaces and the activation of Th2 inflammatory and profibrotic pathways in experimental animals provide a preliminary, mechanistic framework to support the emerging epidemiological associations among air pollution, diabetes, and lung disease. https://doi.org/10.1289/EHP7255

Sodium 4‑phenylbutyrate inhibits protein glycation

The production and accumulation of advanced glycation end-products (AGEs) are hypothesized to have a causal role in the development of the complications associated with aging and lifestyle-related diseases, such as diabetes, atherosclerosis and hyperlipidemia. Therefore, it is important to reduce the production and accumulation of AGEs. In the present study, the ability of sodium 4-phenylbutyrate (PBA) on inhibition of glycation was assessed. In vitro, PBA inhibited the glycation of albumin and collagen by up to 42.1 and 36.9%, respectively. Furthermore, when spontaneously diabetic KK mice were administered PBA (20 mg/day) or vehicle orally, glycosuria developed rapidly in the control mice, but after 6 weeks, only one treated mouse was glycosuric. In addition, the weight gain and HbA1c levels were significantly lower in the treated mice compared with the untreated mice (weight gain, 36.0 g vs. 39.4 g, P<0.01; HbA1C level, 3.96 vs. 4.78%, P<0.01; respectively). These results suggested that PBA also inhibited glycation in vivo. Further studies are required to determine whether PBA may be effective for the therapy or prevention of aging or lifestyle-related diseases caused by the accumulation of AGEs. The method of administration and the side-effects of PBA have already been established as PBA is already used clinically. Therefore, the repurposing of PBA for reducing AGE levels may be a potential option to reduce complications associated with aging.

2089-P: Islet GLUT2 Plasticity Improves Glycemic Control in Insulin-Resistant KK Mice but Not T2DM KKAy Mice

Beta cells secrete insulin in response to glucose entry by GLUT transporters. GLUT are reduced in beta cells from T2DM rodents. We hypothesized that glucose entry into islets would be reduced in T2DM mice. Islets were isolated from euglycemic mice (C57/Bl6, ICR), insulin-resistant but euglycemic mice (4 and 8 wk KK), prediabetic mice (4 wk KKAy), and T2DM mice (8 wk KKAy). Islets were exposed to the fluorescent glucose analogue 2-NBDG in presence or absence of high glucose or the GLUT inhibitor phloretin. Islet fluorescence was visualized using confocal microscopy and quantified from 3-D images using FIJI Image J. GLUT2 protein was determined in isolated islets by ELISA. Islet GLUT2 location was determined using IF sections with blinded observers assigning a score from 0 (cytosol) to 10 (cell membrane). *p&amp;lt;0.05. Using islets from euglycemic mice, 2-NBDG entry was competitively reduced 59% by high glucose and 30% by phloretin. Despite a 44% decrease in islet GLUT2 protein in prediabetic mice (KK, 4.0±0.5; KKAy, 2.3±0.3*), 2-NBDG entry into islets was similar (KK: 7.32±0.86; KKAy: 7.25±0.86). More GLUT2 transporters were located in the membrane of islets from KKAy (5.88±2.14*) as compared to KK (4.44±3.06) mice. Phloretin significantly decreased 2-NBDG entry in both KK (6.31±0.61*) and KKAy (6.52±0.69*) mice. By 8 wks of age, islet GLUT2 protein had decreased by 51% in KK mice (1.97±0.50*) with no change in KKAy mice (2.58±0.22), yet 2-NBDG entry was not altered (KK: 7.32±0.63; KKAy: 7.35±0.62). More GLUT2 was located in the membrane of 8 wk KK (7.08±1.68*) as compared to 4 wk KK mice. Membrane GLUT2 was unchanged in KKAy mice. Phloretin increased 2-NBDG entry into islets from both KK (8.05±0.48*) and KKAy (7.95±0.79*) mice. With age, HbA1c did not change in KK mice (4.53-4.97%) but increased in KKAy mice (5.27-6.57%). Plasticity in islet GLUT2 content, location and transport was evident in islets from KK mice but not KKAy mice. Plasticity may contribute to better glycemic control in aging KK mice. Disclosure K.J. LePard: None. K.L. Sondgeroth: None. Funding Midwestern University (to K.J.L.)

Abstract PR03: Modeling the genetic heterogeneity of KRAS mutant lung adenocarcinomas for therapeutic discovery

Abstract Lung adenocarcinoma (ADC) is the most common histologic subtype of lung cancer. Oncogenic driver mutations in KRAS are found in 36% of lung ADC cases and with a lack of specific KRAS inhibitors, this subset presents a significant clinical challenge. There is therefore an urgency to identify actionable co-occurring mutations as an alternative therapeutic approach. One of the most critical stress-response pathways in mammals is mediated by the transcription factor Nuclear Factor Erythroid-2-Related Factor 2 (NFE2L2/NRF2), which is negatively regulated by Kelch-like ECH-Associated Protein 1 (KEAP1). Loss-of-function mutations in KEAP1 have been identified in 19% of ADC, and often co-occur with KRAS mutations, but appear to be largely mutually exclusive with TP53 mutations. To best model these genetic subgroups of KRAS mutant lung ADC, we generated genetically engineered mouse models (GEMMs) whereby oncogenic KrasG12D was expressed either in combination with loss of p53 (KP mice) or Keap1 (KK mice). Critically, loss of Keap1 promoted KrasG12D-induced malignant transformation, which was associated with a unique metabolic program that could be exploited using pharmacologic inhibitors. Furthermore, bronchiolar epithelial cells appeared to be more sensitive than alveolar epithelial cells to transformation following Keap1 loss. Interestingly, this cell-type bias appeared to directly modulate the immune phenotype of lung tumors. Taken together, we have generated powerful in vivo models that enable us to address the relationship between tumor cells and resident immune cells to evaluate novel treatment strategies aimed at improving the long-term outcomes for patients carrying KRAS alterations. This abstract is also being presented as Poster B10. Citation Format: Sarah A. Best, Sheryl Ding, Boris Reljic, Ji-Ying Song, Kate D. Sutherland. Modeling the genetic heterogeneity of KRAS mutant lung adenocarcinomas for therapeutic discovery [abstract]. In: Proceedings of the AACR Special Conference on Targeting RAS-Driven Cancers; 2018 Dec 9-12; San Diego, CA. Philadelphia (PA): AACR; Mol Cancer Res 2020;18(5_Suppl):Abstract nr PR03.

Persistent proliferation of keratinocytes and prolonged expression of pronociceptive inflammatory mediators might be associated with the postoperative pain in KK mice.

Epidermal keratinocytes play a vital role in restoration of the intact skin barrier during wound healing. The negative effect of hyperglycemia may prolong the wound healing process. Epidermal keratinocytes have been demonstrated to modulate and directly initiate nociceptive responses in rat models of fractures and chemotherapy-induced neuropathic pain. However, it is unclear whether epidermal keratinocytes are involved in the development and maintenance of incisional pain in nondiabetic or diabetic animals. In the current study, using behavioral tests and immunohistochemistry, we investigated the differential keratinocytes proliferation and expression of pronociceptive inflammatory mediators in keratinocytes in C57BL/6J mice and diabetic KK mice. Our data showed that plantar incision induced postoperative pain hypersensitivity in both C57BL/6J mice and KK mice, while the duration of postoperative pain hypersensitivity in KK mice was longer than that in C57BL/6J mice. Moreover, plantar incision induced the keratinocytes proliferation and expression of IL-1β and TNF-α in keratinocytes in both C57BL/6J mice and KK mice. Interestingly, compared to C57BL/6J mice, the slower and more persistent proliferation of keratinocytes and expression of IL-1β and TNF-α in keratinocytes were observed in KK mice. Together, our study suggested that plantar incision may induce the differential keratinocytes proliferation and expression of IL-1β and TNF-α in kertinocytes in diabetic and nondiabetic animals, which might be associated with the development and maintenance differences in diabetic and nondiabetic postoperative pain.

Impact of discontinuation of fish oil after pioglitazone–fish oil combination therapy in diabetic KK mice

Pioglitazone is one of the thiazolidinediones (TZDs) and an insulin-sensitive drug for type 2 diabetes. In our previous study, a combination of pioglitazone and fish oil rich in n-3 polyunsaturated fatty acids (PUFAs) was shown to inhibit pioglitazone-induced side effects, such as accumulation of subcutaneous fat and body weight gain. However, the effects of the discontinuation of fish oil after combination treatment with TZD and fish oil are not clear. In this study, discontinuation of fish oil for 4 weeks showed several unfavorable effects: (1) return of plasma adiponectin level, (2) reversal of the inhibition of lipogenesis and activation of fatty acid β-oxidation in liver, (3) increase in hypertrophic adipocytes in epidydimal white adipose tissue (WAT) and (4) accumulation of lipids in brown adipose tissue (BAT). However, insulin resistance was ameliorated by pioglitazone with or without fish oil treatment and the discontinuation of fish oil. These findings indicate that discontinuation of n-3 PUFA after combination therapy with TZDs adversely affects lipid metabolism and energy homeostasis in liver, epididymal WAT and BAT.

Monosodium Glutamate (MSG)-Induced Animal Model of Type 2 Diabetes.

In 1976, an animal model of type 2 diabetes (T2DM) was described by Cameron et al. using injection of monosodium glutamate (MSG) in KK mice during the neonatal period. Some years later, similar models have been developed by various doses and durations and the main of these models exhibited obesity and features of diabetes mellitus, including glycosuria, hyperglycemia, hyperinsulinemia, decreased glucose tolerance, and insulin sensitivity. Studies indicated that MSG treatment of newborn animals generates necrosis of neuronal cells of the hypothalamic ventromedial nucleus and arcuate nucleus. Neonatal MSG-treatment was related to normoglycemic-normoinsulinemic state at young ages and development of obesity and hyperinsulinemia at adult ages. Following observation of a severe hypertrophy of pancreatic islets due to the proliferation of β-cells in MSG-treated mice, this model has been proposed as a useful animal model of human T2DM. A higher dose of MSG (≥4mg/g body weight) accompanied by a longer follow-up duration (>6months) are needed to establish a typical animal model of T2DM.

Benefits of combination low-dose pioglitazone plus fish oil on aged type 2 diabetes mice

The elderly patients with type 2 diabetes suffer more adverse drug events than young adults due to pharmacokinetic and pharmacodynamic changes associated with aging. Reducing the risks of these medication-related problems are equally important for the clinical care of older type 2 diabetes patients. Pioglitazone is used for treating type 2 diabetes as an oral antidiabetic drug. Despite pioglitazone is used helpful insulin sensitizers, the accumulation of subcutaneous fat is considered a major adverse effect of pioglitazone therapy. We investigated to reduce the adverse effect of pioglitazone by combination with fish oil rich in eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) in aged diabetic KK mice. The accumulation of subcutaneous fat associated with high-dose pioglitazone is reduced by fish oil, suppressing lipogenesis and stimulating fatty acid β-oxidation in the liver. Our data suggest that adding fish oil to low-dose pioglitazone results in antidiabetic efficacy similar to that of the high-dose without concomitant body weight gain.