Adenylate cyclase 3: a potential genetic link between obesity and major depressive disorder.

Obesity and major depressive disorder (MDD) are both significant health issues that have been increasing in prevalence and are associated with multiple comorbidities. Obesity and MDD have been shown to be bidirectionally associated, and they are both influenced by genetics and environmental factors. However, the molecular mechanisms that link these two diseases are not yet fully understood. It is possible that these diseases are connected through the actions of the cAMP/protein kinase A (PKA) pathway. Within this pathway, adenylate cyclase 3 (Adcy3) has emerged as a key player in both obesity and MDD. Numerous genetic variants in Adcy3 have been identified in humans in association with obesity. Rodent knockout studies have also validated the importance of this gene for energy homeostasis. Furthermore, Adcy3 has been identified as a top candidate gene and even a potential blood biomarker for MDD. Adcy3 and the cAMP/PKA pathway may therefore serve as an important genetic and functional link between these two diseases. In this mini-review, we discuss the role of both Adcy3 and the cAMP/PKA pathway, including specific genetic mutations, in both diseases. Understanding the role that Adcy3 mutations play in obesity and MDD could open the door for precision medicine approaches and treatments for both diseases that target this gene.

Long-access heroin self-administration induces region specific reduction of grey matter volume and microglia reactivity in the rat.

In opioid use disorder (OUD) patients, a decrease in brain grey matter volume (GMV) has been reported. It is unclear whether this is the consequence of prolonged exposure to opioids or is a predisposing causal factor in OUD development. To investigate this, we conducted a structural MRI longitudinal study in NIH Heterogeneous Stock rats exposed to heroin self-administration and age-matched naïve controls housed in the same controlled environment. Structural MRI scans were acquired before (MRI1) and after (MRI2) a prolonged period of long access heroin self-administration resulting in escalation of drug intake. Heroin intake resulted in reduced GMV in various cortical and sub-cortical brain regions. In drug-naïve controls no difference was found between MRI1 and MRI2. Notably, the degree of GMV reduction in the medial prefrontal cortex (mPFC) and the insula positively correlated with the amount of heroin consumed and the escalation of heroin use. In a preliminary gene expression analysis, we identified a number of transcripts linked to immune response and neuroinflammation. This prompted us to hypothesize a link between changes in microglia homeostasis and loss of GMV. For this reason, we analyzed the number and morphology of microglial cells in the mPFC and insula. The number of neurons and their morphology was also evaluated. The primary motor cortex, where no GMV change was observed, was used as negative control. We found no differences in the number of neurons and microglia cells following heroin. However, in the same regions where reduced GMV was detected, we observed a shift towards a rounder shape and size reduction in microglia, suggestive of their homeostatic change towards a reactive state. Altogether these findings suggest that escalation of heroin intake correlates with loss of GMV in specific brain regions and that this phenomenon is linked to changes in microglial morphology.

Changes in environmental stress over COVID-19 pandemic likely contributed to failure to replicate adiposity phenotype associated with Krtcap3.

We previously identified keratinocyte-associated protein 3, Krtcap3, as an obesity-related gene in female rats where a whole body Krtcap3 knockout (KO) led to increased adiposity compared to wild-type (WT) controls when fed a high-fat diet (HFD). We sought to replicate this work to better understand the function of Krtcap3 but were unable to reproduce the adiposity phenotype. In the current work, WT female rats ate more compared to WT in the prior study, with corresponding increases in body weight and fat mass, while there were no changes in these measures in KO females between the studies. The prior study was conducted before the COVID-19 pandemic, while the current study started after initial lockdown orders and was completed during the pandemic in a generally less stressful environment. We hypothesize that the environmental changes impacted stress levels and may explain the failure to replicate our results. Analysis of corticosterone (CORT) at euthanasia showed a significant study-by-genotype interaction where WT had significantly higher CORT relative to KO in study 1, with no differences in study 2. These data suggest that decreasing Krtcap3 expression may alter the environmental stress response to influence adiposity. We also found that KO rats in both studies, but not WT, experienced a dramatic increase in CORT after their cage mate was removed, suggesting a separate connection to social behavioral stress. Future work is necessary to confirm and elucidate the finer mechanisms of these relationships, but these data indicate the possibility of Krtcap3 as a novel stress gene.NEW & NOTEWORTHY Obesity is linked to both genetics and environmental factors such as stress. Krtcap3 has previously been identified as a gene associated with adiposity, and our work here demonstrates that environmental stress may influence the role of Krtcap3 on both food intake and adiposity. Obesity is strongly influenced by stress in humans, so the identification of novel genes that link stress and obesity will greatly advance our understanding of the disease.

Nociceptin attenuates the escalation of oxycodone self-administration by normalizing CeA-GABA transmission in highly addicted rats.

Approximately 25% of patients who are prescribed opioids for chronic pain misuse them, and 5 to 10% develop an opioid use disorder. Although the neurobiological target of opioids is well known, the molecular mechanisms that are responsible for the development of addiction-like behaviors in some but not all individuals are poorly known. To address this issue, we used a unique outbred rat population (heterogeneous stock) that better models the behavioral and genetic diversity that is found in humans. We characterized individual differences in addiction-like behaviors using an addiction index that incorporates the key criteria of opioid use disorder: escalated intake, highly motivated responding, and hyperalgesia. Using in vitro electrophysiological recordings in the central nucleus of the amygdala (CeA), we found that rats with high addiction-like behaviors (HA) exhibited a significant increase in γ-aminobutyric acid (GABA) transmission compared with rats with low addiction-like behaviors (LA) and naive rats. The superfusion of CeA slices with nociceptin/orphanin FQ peptide (N/OFQ; 500 nM), an endogenous opioid-like peptide, normalized GABA transmission in HA rats. Intra-CeA levels of N/OFQ were lower in HA rats than in LA rats. Intra-CeA infusions of N/OFQ (1 µg per site) reversed the escalation of oxycodone self-administration in HA rats but not in LA rats. These results demonstrate that the downregulation of N/OFQ levels in the CeA may be responsible for hyper-GABAergic tone in the CeA that is observed in individuals who develop addiction-like behaviors. Based on these results, we hypothesize that small molecules that target the N/OFQ system might be useful for the treatment of opioid use disorder.

Transcriptome-wide analyses of adipose tissue in outbred rats reveal genetic regulatory mechanisms relevant for human obesity.

Transcriptomic analysis in metabolically active tissues allows a systems genetics approach to identify causal genes and networks involved in metabolic disease. Outbred heterogeneous stock (HS) rats are used for genetic mapping of complex traits, but to-date, a systems genetics analysis of metabolic tissues has not been done. We investigated whether adiposity-associated genes and gene coexpression networks in outbred heterogeneous stock (HS) rats overlap those found in humans. We analyzed RNAseq data from adipose tissue of 415 male HS rats, correlated these transcripts with body weight (BW) and compared transcriptome signatures to two human cohorts: the "African American Genetics of Metabolism and Expression" and "Metabolic Syndrome in Men." We used weighted gene coexpression network analysis to identify adiposity-associated gene networks and mediation analysis to identify genes under genetic control whose expression drives adiposity. We identified 554 orthologous "consensus genes" whose expression correlates with BW in the rat and with body mass index (BMI) in both human cohorts. Consensus genes fell within eight coexpressed networks and were enriched for genes involved in immune system function, cell growth, extracellular matrix organization, and lipid metabolic processes. We identified 19 consensus genes for which genetic variation may influence BW via their expression, including those involved in lipolysis (e.g., Hcar1), inflammation (e.g., Rgs1), adipogenesis (e.g., Tmem120b), or no previously known role in obesity (e.g., St14 and Ms4a6a). Strong concordance between HS rat and human BW/BMI associated transcripts demonstrates translational utility of the rat model, while identification of novel genes expands our knowledge of the genetics underlying obesity.

High-fat diet negatively impacts both metabolic and behavioral health in outbred heterogeneous stock rats.

Obesity is influenced by genetics and diet and has wide ranging comorbidities, including anxiety and depressive disorders. Outbred heterogeneous stock (HS) rats are used for fine-genetic mapping of complex traits and may be useful for understanding gene by diet interactions. In this study, HS rats were fed diets containing 60% kcal from fat (high-fat diet, HFD) or 10% kcal from fat (low-fat diet, LFD) and tested for metabolic (study 1) and behavioral (study 2) outcomes. In study 1, we measured glucose tolerance, fasting glucose and insulin, fat pad weights and despair-like behavior in the forced swim test (FST). In study 2, we assessed anxiety-like (elevated plus maze, EPM; open field test, OFT) and despair-like/coping (splash test, SpT; and FST) behaviors. Body weight and food intake were measured weekly in both studies. We found negative effects of HFD on metabolic outcomes, including increased body weight and fat pad weights, decreased glucose tolerance, and increased fasting insulin. We also found negative effects of HFD on despair-like/coping and anxiety-like behaviors. These include increased immobility in the FST, decreased open arm time in the EPM, and increased movement and rest episodes and decreased rearing in the OFT. The diet-induced changes in EPM and OFT were independent of overall locomotion. Additionally, diet-induced changes in OFT behaviors were independent of adiposity, while adiposity was a confounding factor for EPM and FST behavior. This work establishes the HS as a model to study gene by diet interactions affecting metabolic and behavioral health.

Region-Based Convolutional Neural Nets for Localization of Glomeruli in Trichrome-Stained Whole Kidney Sections.

Background Histologic examination of fixed renal tissue is widely used to assess morphology and the progression of disease. Commonly reported metrics include glomerular number and injury. However, characterization of renal histology is a time-consuming and user-dependent process. To accelerate and improve the process, we have developed a glomerular localization pipeline for trichrome-stained kidney sections using a machine learning image classification algorithm.Methods We prepared 4-µm slices of kidneys from rats of various genetic backgrounds that were subjected to different experimental protocols and mounted the slices on glass slides. All sections used in this analysis were trichrome stained and imaged in bright field at a minimum resolution of 0.92 µm per pixel. The training and test datasets for the algorithm comprised 74 and 13 whole renal sections, respectively, totaling over 28,000 glomeruli manually localized. Additionally, because this localizer will be ultimately used for automated assessment of glomerular injury, we assessed bias of the localizer for preferentially identifying healthy or damaged glomeruli.Results Localizer performance achieved an average precision and recall of 96.94% and 96.79%, respectively, on whole kidney sections without evidence of bias for or against glomerular injury or the need for manual preprocessing.Conclusions This study presents a novel and robust application of convolutional neural nets for the localization of glomeruli in healthy and damaged trichrome-stained whole-renal section mounts and lays the groundwork for automated glomerular injury scoring.

Tpcn2 knockout mice have improved insulin sensitivity and are protected against high-fat diet-induced weight gain.

Type 2 diabetes is a complex disorder affected by multiple genes and the environment. Our laboratory has shown that in response to a glucose challenge, two-pore channel 2 ( Tpcn2) knockout mice exhibit a decreased insulin response but normal glucose clearance, suggesting they have improved insulin sensitivity compared with wild-type mice. We tested the hypothesis that improved insulin sensitivity in Tpcn2 knockout mice would protect against the negative effects of a high fat diet. Male and female Tpcn2 knockout (KO), heterozygous (Het), and wild-type (WT) mice were fed a low-fat (LF) or high-fat (HF) diet for 24 wk. HF diet significantly increases body weight in WT mice relative to those on the LF diet; this HF diet-induced increase in body weight is blunted in the Het and KO mice. Despite the protection against diet-induced weight gain, however, Tpcn2 KO mice are not protected against HF-diet-induced changes in glucose or insulin area under the curve during glucose tolerance tests in female mice, while HF diet has no significant effect on glucose tolerance in the male mice, regardless of genotype. Glucose disappearance during an insulin tolerance test is augmented in male KO mice, consistent with our previous findings suggesting enhanced insulin sensitivity in these mice. Male KO mice exhibit increased fasting plasma total cholesterol and triglyceride concentrations relative to WT mice on the LF diet, but this difference disappears in HF diet-fed mice where there is increased cholesterol and triglycerides across all genotypes. These data demonstrate that knockout of Tpcn2 may increase insulin action in male, but not female, mice. In addition, both male and female KO mice are protected against diet-induced weight gain, but this protection is likely independent from glucose tolerance, insulin sensitivity, and plasma lipid levels.

Spontaneous one-kidney rats are more susceptible to develop hypertension by DOCA-NaCl and subsequent kidney injury compared with uninephrectomized rats.

There is little clinical data of how hypertension may influence individuals with nephron deficiency in the context of being born with a single kidney. We recently developed a new rat model (the heterogeneous stock-derived model of unilateral renal agenesis rat) that is born with a single kidney and exhibits progressive kidney injury and decline in kidney function with age. We hypothesized that DOCA-salt would induce a greater increase in blood pressure and therefore accelerate the progression of kidney injury in rats born with a solitary kidney compared with rats that have undergone unilateral nephrectomy. Time course evaluation of blood pressure, kidney injury, and renal hemodynamics was performed in the following six groups of animals from weeks 13 to 18: 1) DOCA-treated rats with a solitary kidney (DOCA+S group), 2) placebo-treated rats with a solitary kidney, 3) DOCA-treated control rats with two kidneys (DOCA+C group), 4) placebo-treated control rats with two kidneys, 5) DOCA-treated rats with two kidneys that underwent uninephrectomy (DOCA+UNX8 group), and 6) placebo-treated rats with two kidneys that underwent uninephrectomy. DOCA+S rats demonstrated a significant rise (P < 0.05) in blood pressure (192 ± 4 mmHg), proteinuria (205 ± 31 mg/24 h), and a decline in glomerular filtration rate (600 ± 42 µl·min(-1)·g kidney weight(-1)) relative to the DOCA+UNX8 (173 ± 3 mmHg, 76 ± 26 mg/24 h, and 963 ± 36 µl·min(-1)·g kidney weight(-1)) and DOCA+C (154 ± 2 mmHg, 7 ± 1 mg/24 h, and 1,484 ± 121 µl·min(-1)·g kidney weight(-1)) groups. Placebo-treated groups showed no significant change among the three groups. An assessment of renal injury markers via real-time PCR/Western blot analysis and histological analysis was concordant with the measured physiological parameters. In summary, congenital solitary kidney rats are highly susceptible to the induction of hypertension compared with uninephrectomized rats, suggesting that low nephron endowment is an important driver of elevated blood pressure, hastening nephron injury through the transmission of elevated systemic blood pressure and thereby accelerating decline in kidney function.

QTL mapping in outbred populations: successes and challenges.

Quantitative trait locus (QTL) mapping in animal populations has been a successful strategy for identifying genomic regions that play a role in complex diseases and traits. When conducted in an F2 intercross or backcross population, the resulting QTL is frequently large, often encompassing 30 Mb or more and containing hundreds of genes. To narrow the locus and identify candidate genes, additional strategies are needed. Congenic strains have proven useful but work less well when there are multiple tightly linked loci, frequently resulting in loss of phenotype. As an alternative, we discuss the use of highly recombinant outbred models for directly fine-mapping QTL to only a few megabases. We discuss the use of several currently available models such as the advanced intercross (AI), heterogeneous stocks (HS), the diversity outbred (DO), and commercially available outbred stocks (CO). Once a QTL has been fine-mapped, founder sequence and expression QTL mapping can be used to identify candidate genes. In this regard, the large number of alleles found in outbred stocks can be leveraged to identify causative genes and variants. We end this review by discussing some important statistical considerations when analyzing outbred populations. Fine-resolution mapping in outbred models, coupled with full genome sequence, has already led to the identification of several underlying causative genes for many complex traits and diseases. These resources will likely lead to additional successes in the coming years.

Long-access heroin self-administration induces region specific reduction of grey matter volume and microglia reactivity in the rat.

In opioid use disorder (OUD) patients, a decrease in brain grey matter volume (GMV) has been reported. It is unclear whether this is the consequence of prolonged exposure to opioids or is a predisposing causal factor in OUD development. To investigate this, we conducted a structural MRI longitudinal study in NIH Heterogeneous Stock rats exposed to heroin self-administration and age-matched naïve controls housed in the same controlled environment. Structural MRI scans were acquired before (MRI 1 ) and after (MRI 2 ) a prolonged period of long access heroin self-administration resulting in escalation of drug intake. Heroin intake resulted in reduced GMV in various cortical and sub-cortical brain regions. In drug-naïve controls no difference was found between MRI 1 and MRI 2 . Notably, the degree of GMV reduction in the medial prefrontal cortex (mPFC) and the insula positively correlated with the amount of heroin consumed and the escalation of heroin use. In a preliminary gene expression analysis, we identified a number of transcripts linked to immune response and neuroinflammation. This prompted us to hypothesize a link between changes in microglia homeostasis and loss of GMV. For this reason, we analyzed the number and morphology of microglial cells in the mPFC and insula. The number of neurons and their morphology was also evaluated. The primary motor cortex, where no GMV change was observed, was used as negative control. We found no differences in the number of neurons and microglia cells following heroin. However, in the same regions where reduced GMV was detected, we observed a shift towards a rounder shape and size reduction in microglia, suggestive of their homeostatic change towards a reactive state. Altogether these findings suggest that escalation of heroin intake correlates with loss of GMV in specific brain regions and that this phenomenon is linked to changes in microglial morphology.

Environmental enrichment promotes adaptive responding during tests of behavioral regulation in male heterogeneous stock rats.

Organisms must regulate their behavior flexibly in the face of environmental challenges. Failure can lead to a host of maladaptive behavioral traits associated with a range of neuropsychiatric disorders, including attention deficit hyperactivity disorder, autism, and substance use disorders. This maladaptive dysregulation of behavior is influenced by genetic and environmental factors. For example, environmental enrichment produces beneficial neurobehavioral effects in animal models of such disorders. The present study determined the effects of environmental enrichment on a range of measures related to behavioral regulation using a large cohort of male, outbred heterogeneous stock (HS) rats as subjects. Subjects were reared from late adolescence onwards either in pairs in standard housing with minimal enrichment (n = 200) or in groups of 16 in a highly enriched environment consisting of a large multi-level cage filled with toys, running wheels, and shelters (n = 64). Rats were subjected to a battery of tests, including: (i) locomotor response to novelty, (ii) light reinforcement, (iii) social reinforcement, (iv) reaction time, (v) a patch-depletion foraging test, (vi) Pavlovian conditioned approach, (vii) conditioned reinforcement, and (viii) cocaine conditioned cue preference. Results indicated that rats housed in the enriched environment were able to filter out irrelevant stimuli more effectively and thereby regulate their behavior more efficiently than standard-housing rats. The dramatic impact of environmental enrichment suggests that behavioral studies using standard housing conditions may not generalize to more complex environments that may be more ethologically relevant.

Reversal of biological age in multiple rat organs by young porcine plasma fraction.

Young blood plasma is known to confer beneficial effects on various organs in mice and rats. However, it was not known whether plasma from young adult pigs rejuvenates old rat tissues at the epigenetic level; whether it alters the epigenetic clock, which is a highly accurate molecular biomarker of aging. To address this question, we developed and validated six different epigenetic clocks for rat tissues that are based on DNA methylation values derived from n = 613 tissue samples. As indicated by their respective names, the rat pan-tissue clock can be applied to DNA methylation profiles from all rat tissues, while the rat brain, liver, and blood clocks apply to the corresponding tissue types. We also developed two epigenetic clocks that apply to both human and rat tissues by adding n = 1366 human tissue samples to the training data. We employed these six rat clocks to investigate the rejuvenation effects of a porcine plasma fraction treatment in different rat tissues. The treatment more than halved the epigenetic ages of blood, heart, and liver tissue. A less pronounced, but statistically significant, rejuvenation effect could be observed in the hypothalamus. The treatment was accompanied by progressive improvement in the function of these organs as ascertained through numerous biochemical/physiological biomarkers, behavioral responses encompassing cognitive functions. An immunoglobulin G (IgG) N-glycosylation pattern shift from pro- to anti-inflammatory also indicated reversal of glycan aging. Overall, this study demonstrates that a young porcine plasma-derived treatment markedly reverses aging in rats according to epigenetic clocks, IgG glycans, and other biomarkers of aging.

Genome-wide association study reveals multiple loci for nociception and opioid consumption behaviors associated with heroin vulnerability in outbred rats.

The increased prevalence of opioid use disorder (OUD) makes it imperative to disentangle the biological mechanisms contributing to individual differences in OUD vulnerability. OUD shows strong heritability, however genetic variants contributing toward vulnerability remain poorly defined. We performed a genome-wide association study using over 850 male and female heterogeneous stock (HS) rats to identify genes underlying behaviors associated with OUD such as nociception, as well as heroin-taking, extinction and seeking behaviors. By using an animal model of OUD, we were able to identify genetic variants associated with distinct OUD behaviors while maintaining a uniform environment, an experimental design not easily achieved in humans. Furthermore, we used a novel non-linear network-based clustering approach to characterize rats based on OUD vulnerability to assess genetic variants associated with OUD susceptibility. Our findings confirm the heritability of several OUD-like behaviors, including OUD susceptibility. Additionally, several genetic variants associated with nociceptive threshold prior to heroin experience, heroin consumption, escalation of intake, and motivation to obtain heroin were identified. Tom1 , a microglial component, was implicated for nociception. Several genes involved in dopaminergic signaling, neuroplasticity and substance use disorders, including Brwd1 , Pcp4, Phb1l2 and Mmp15 were implicated for the heroin traits. Additionally, an OUD vulnerable phenotype was associated with genetic variants for consumption and break point, suggesting a specific genetic contribution for OUD-like traits contributing to vulnerability. Together, these findings identify novel genetic markers related to the susceptibility to OUD-relevant behaviors in HS rats.

A revamped rat reference genome improves the discovery of genetic diversity in laboratory rats.

The seventh iteration of the reference genome assembly for Rattus norvegicus-mRatBN7.2-corrects numerous misplaced segments and reduces base-level errors by approximately 9-fold and increases contiguity by 290-fold compared with its predecessor. Gene annotations are now more complete, improving the mapping precision of genomic, transcriptomic, and proteomics datasets. We jointly analyzed 163 short-read whole-genome sequencing datasets representing 120 laboratory rat strains and substrains using mRatBN7.2. We defined ∼20.0 million sequence variations, of which 18,700 are predicted to potentially impact the function of 6,677 genes. We also generated a new rat genetic map from 1,893 heterogeneous stock rats and annotated transcription start sites and alternative polyadenylation sites. The mRatBN7.2 assembly, along with the extensive analysis of genomic variations among rat strains, enhances our understanding of the rat genome, providing researchers with an expanded resource for studies involving rats.

The Wistar Kyoto Rat: A Model of Depression Traits.

There is an ongoing debate about the value of animal research in psychiatry with valid lines of reasoning stating the limits of individual animal models compared to human psychiatric illnesses. Human depression is not a homogenous disorder; therefore, one cannot expect a single animal model to reflect depression heterogeneity. This limited review presents arguments that the Wistar Kyoto (WKY) rats show intrinsic depression traits. The phenotypes of WKY do not completely mirror those of human depression but clearly indicate characteristics that are common with it. WKYs present despair- like behavior, passive coping with stress, comorbid anxiety, and enhanced drug use compared to other routinely used inbred or outbred strains of rats. The commonly used tests identifying these phenotypes reflect exploratory, escape-oriented, and withdrawal-like behaviors. The WKYs consistently choose withdrawal or avoidance in novel environments and freezing behaviors in response to a challenge in these tests. The physiological response to a stressful environment is exaggerated in WKYs. Selective breeding generated two WKY substrains that are nearly isogenic but show clear behavioral differences, including that of depression-like behavior. WKY and its substrains may share characteristics of subgroups of depressed individuals with social withdrawal, low energy, weight loss, sleep disturbances, and specific cognitive dysfunction. The genomes of the WKY and WKY substrains contain variations that impact the function of many genes identified in recent human genetic studies of depression. Thus, these strains of rats share characteristics of human depression at both phenotypic and genetic levels, making them a model of depression traits.

Changes in Environmental Stress over COVID-19 Pandemic Likely Contributed to Failure to Replicate Adiposity Phenotype Associated with Krtcap3.

We previously identified Keratinocyte-associated protein 3, Krtcap3, as an obesity-related gene in female rats where a whole-body Krtcap3 knock-out (KO) led to increased adiposity compared to wild-type (WT) controls when fed a high-fat diet (HFD). We sought to replicate this work to better understand the function of Krtcap3 but were unable to reproduce the adiposity phenotype. In the current work, WT female rats ate more compared to WT in the prior study, with corresponding increases in body weight and fat mass, while there were no changes in these measures in KO females between the studies. The prior study was conducted before the COVID-19 pandemic, while the current study started after initial lock-down orders and was completed during the pandemic with a generally less stressful environment. We hypothesize that the environmental changes impacted stress levels and may explain the failure to replicate our results. Analysis of corticosterone (CORT) at euthanasia showed a significant study by genotype interaction where WT had significantly higher CORT relative to KO in Study 1, with no differences in Study 2. These data suggest that decreasing Krtcap3 expression may alter the environmental stress response to influence adiposity. We also found that KO rats in both studies, but not WT, experienced a dramatic increase in CORT after their cage mate was removed, suggesting a separate connection to social behavioral stress. Future work is necessary to confirm and elucidate the finer mechanisms of these relationships, but these data indicate the possibility of Krtcap3 as a novel stress gene.

Chronic stress from adolescence to adulthood increases adiposity and anxiety in rats with decreased expression of Krtcap3.

We previously identified Keratinocyte-associated protein 3, Krtcap3, as a novel adiposity gene, but subsequently found that its impact on adiposity may depend on environmental stress. To more thoroughly understand the connection between Krtcap3, adiposity, and stress, we exposed wild-type (WT) and Krtcap3 knock-out (KO) rats to chronic stress then measured adiposity and behavioral outcomes. We found that KO rats displayed lower basal stress than WT rats under control conditions and exhibited metabolic and behavioral responses to chronic stress exposure. Specifically, stress-exposed KO rats gained more weight, consumed more food when socially isolated, and displayed more anxiety-like behaviors relative to control KO rats. Meanwhile, there were minimal differences between control and stressed WT rats. At study conclusion stress-exposed KO rats had increased corticosterone (CORT) relative to control KO rats with no differences between WT rats. In addition, KO rats, independent of prior stress exposure, had an increased CORT response to removal of their cage-mate (psychosocial stress), which was only seen in WT rats when exposed to chronic stress. Finally, we found differences in expression of the glucocorticoid receptor, Nr3c1, in the pituitary and colon between control and stress-exposed KO rats that were not present in WT rats. These data support that Krtcap3 expression affects stress response, potentially via interactions with Nr3c1, with downstream effects on adiposity and behavior. Future work is necessary to more thoroughly understand the role of Krtcap3 in the stress response.

Genetic Mapping of Multiple Traits Identifies Novel Genes for Adiposity, Lipids, and Insulin Secretory Capacity in Outbred Rats.

Despite the successes of human genome-wide association studies, the causal genes underlying most metabolic traits remain unclear. We used outbred heterogeneous stock (HS) rats, coupled with expression data and mediation analysis, to identify quantitative trait loci (QTLs) and candidate gene mediators for adiposity, glucose tolerance, serum lipids, and other metabolic traits. Physiological traits were measured in 1,519 male HS rats, with liver and adipose transcriptomes measured in >410 rats. Genotypes were imputed from low-coverage whole-genome sequencing. Linear mixed models were used to detect physiological and expression QTLs (pQTLs and eQTLs, respectively), using both single nucleotide polymorphism (SNP)- and haplotype-based models for pQTL mapping. Genes with cis-eQTLs that overlapped pQTLs were assessed as causal candidates through mediation analysis. We identified 14 SNP-based pQTLs and 19 haplotype-based pQTLs, of which 10 were in common. Using mediation, we identified the following genes as candidate mediators of pQTLs: Grk5 for fat pad weight and serum triglyceride pQTLs on Chr1, Krtcap3 for fat pad weight and serum triglyceride pQTLs on Chr6, Ilrun for a fat pad weight pQTL on Chr20, and Rfx6 for a whole pancreatic insulin content pQTL on Chr20. Furthermore, we verified Grk5 and Ktrcap3 using gene knockdown/out models, thereby shedding light on novel regulators of obesity.