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

BACKGROUND: The 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. RESULTS: In 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. CONCLUSION: Our 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.

Strain and sex-based glucocentric & behavioral differences between KK/HlJ and C57BL/6J mice.

INTRODUCTION: Small-animal models are the most widely used preclinical model for studying the etiology, pathology and treatment of diabetes, prediabetes and diabetic comorbidities. Diabetic patients are burdened with higher rates of depression, anxiety and cognitive decline due to inadequate control of blood glucose levels, vascular damage and aberrant CNS insulin signaling. The C57BL/6J model is amongst the most widely used mouse model due to its susceptibility to diet-induced obesity (DIO). This strain has also been well-characterized in behavioral research studies. However the C57BL/6J model has a number of limitations: [1] overt fasting hyperglycemia can only be induced by dietary manipulation and/or chemical ablation of the pancreatic beta cells. [2] There is heterogeneity in the obesogenic response to hypercaloric feeding as well as sex-dependent differences, with males being more responsive. The KK inbred strain has been used to study aspects of the metabolic syndrome and prediabetes due to inherent glucose intolerance, hyperinsulinemia and insulin resistance. However KK/HlJ mice are less well-characterized and there have been fewer behavioral studies reported. The aim of this study was to examine differences in male and female glucocentric parameters between KK/HlJ and C57BL/6J mice, and to compare their performance in a variety of standard behavioral tests relating to general, anxiogenic and cognitive paradigms. METHODS: Strain differences in male and female KK/HlJ and C57BL/6J mouse adiposity, glucose and insulin parameters were studied together with group differences in standard Open Field, Object Recognition, Elevated Plus Maze, Light-Dark Transition, Porsolt test, Marble Burying, Social Recognition and Morris Water Maze tests. Correlations between behavioral variables were analyzed. RESULTS AND CONCLUSION: In addition to being uniformly larger, hyperinsulinemic and more insulin intolerant than C57BL/6J mice, we observed marked strain and sex-differences in KK/HlJ behavior. KK/HlJ mice exhibited less locomotor and vertical exploratory behavior in comparison to C57BL/6J, whereas object exploration and novel object discrimination were superior in KK/HlJ mice. Female KK/HlJ mice were faster swimmers, whereas the males exhibited greater spatial cognition and place-learning during the MWM test.

Effect of developmental NMDAR antagonism with CGP 39551 on aspartame-induced hypothalamic and adrenal gene expression.

RATIONALE: Aspartame (L-aspartyl phenylalanine methyl ester) is a non-nutritive sweetener (NNS) approved for use in more than 6000 dietary products and pharmaceuticals consumed by the general public including adults and children, pregnant and nursing mothers. However a recent prospective study reported a doubling of the risk of being overweight amongst 1-year old children whose mothers consumed NNS-sweetened beverages daily during pregnancy. We have previously shown that chronic aspartame (ASP) exposure commencing in utero may detrimentally affect adulthood adiposity status, glucose metabolism and aspects of behavior and spatial cognition, and that this can be modulated by developmental N-methyl-D-aspartate receptor (NMDAR) blockade with the competitive antagonist CGP 39551 (CGP). Since glucose homeostasis and certain aspects of behavior and locomotion are regulated in part by the NMDAR-rich hypothalamus, which is part of the hypothalamic-pituitary-adrenal- (HPA) axis, we have elected to examine changes in hypothalamic and adrenal gene expression in response to ASP exposure in the presence or absence of developmental NMDAR antagonism with CGP, using Affymetrix microarray analysis. RESULTS: Using 2-factor ANOVA we identified 189 ASP-responsive differentially expressed genes (DEGs) in the adult male hypothalamus and 2188 in the adrenals, and a further 23 hypothalamic and 232 adrenal genes significantly regulated by developmental treatment with CGP alone. ASP exposure robustly elevated the expression of a network of genes involved in hypothalamic neurosteroidogenesis, together with cell stress and inflammatory genes, consistent with previous reports of aspartame-induced CNS stress and oxidative damage. These genes were not differentially expressed in ASP mice with CGP antagonism. In the adrenal glands of ASP-exposed mice, GABA and Glutamate receptor subunit genes were amongst those most highly upregulated. Developmental NMDAR antagonism alone had less effect on adulthood gene expression and affected mainly hypothalamic neurogenesis and adrenal steroid metabolism. Combined ASP + CGP treatment mainly upregulated genes involved in adrenal drug and cholesterol metabolism. CONCLUSION: ASP exposure increased the expression of functional networks of genes involved in hypothalamic neurosteroidogenesis and adrenal catecholamine synthesis, patterns of expression which were not present in ASP-exposed mice with developmental NMDAR antagonism.

Differential effects of early-life NMDA receptor antagonism on aspartame-impaired insulin tolerance and behavior.

We have previously showed that lifetime exposure to aspartame, commencing in utero via the mother's diet, may impair insulin tolerance and cause behavioral deficits in adulthood via mechanisms which are incompletely understood. The role of the CNS in regulating glucose homeostasis has been highlighted by recent delineation of the gut-brain axis, in which N-methyl-d-aspartic acid receptors (NMDARs) are important in maintaining glucose homeostasis, in addition to regulating certain aspects of behavior. Since the gut-brain axis can be modulated by fetal programming, we hypothesized that early-life NMDAR antagonism may affect aspartame-induced glucose deregulation in adulthood, and may alter the aspartame behavioral phenotype. Accordingly, C57Bl/6J mice were chronically exposed to aspartame commencing in utero, in the presence and absence of maternal administration of the competitive NMDAR antagonist CGP 39551, from conception until weaning. Drug/diet interactions in adulthood glucocentric and behavioral parameters were assessed. Aspartame exposure elevated blood glucose and impaired insulin-induced glucose disposal during an insulin tolerance test, which could be normalized by NMDAR antagonism. The same effects were not observed in control diet mice, suggesting an early-life drug/diet interaction. Behavioral analysis of adult offspring indicated that NMDAR antagonism of control diet mice caused hyperlocomotion and impaired spatial navigation. Conversely hypolocomotion, reduced exploratory activity and increased anxiety-related behavior were apparent in aspartame diet mice with early-life NMDAR antagonism. CONCLUSION: significant drug/diet interactions in glucocentric and behavioral parameters were identified in aspartame-exposed mice with early-life NMDAR antagonism. This suggests a possible involvement of early NMDAR interactions in aspartame-impaired glucose homeostasis and behavioral deficits.