Genetic Disruption of System xc-Mediated Glutamate Release from Astrocytes Increases Negative-Outcome Behaviors While Preserving Basic Brain Function in Rat.

The importance of neuronal glutamate to synaptic transmission throughout the brain illustrates the immense therapeutic potential and safety risks of targeting this system. Astrocytes also release glutamate, the clinical relevance of which is unknown as the range of brain functions reliant on signaling from these cells hasn't been fully established. Here, we investigated system xc- (Sxc), which is a glutamate release mechanism with an in vivo rodent expression pattern that is restricted to astrocytes. As most animals do not express Sxc, we first compared the expression and sequence of the obligatory Sxc subunit xCT among major classes of vertebrate species. We found xCT to be ubiquitously expressed and under significant negative selective pressure. Hence, Sxc likely confers important advantages to vertebrate brain function that may promote biological fitness. Next, we assessed brain function in male genetically modified rats (MSxc) created to eliminate Sxc activity. Unlike other glutamatergic mechanisms, eliminating Sxc activity was not lethal and didn't alter growth patterns, telemetry measures of basic health, locomotor activity, or behaviors reliant on simple learning. However, MSxc rats exhibited deficits in tasks used to assess cognitive behavioral control. In a pavlovian conditioned approach, MSxc rats approached a food-predicted cue more frequently than WT rats, even when this response was punished. In attentional set shifting, MSxc rats displayed cognitive inflexibility because of an increased frequency of perseverative errors. MSxc rats also displayed heightened cocaine-primed drug seeking. Hence, a loss of Sxc-activity appears to weaken control over nonreinforced or negative-outcome behaviors without altering basic brain function.SIGNIFICANCE STATEMENT Glutamate is essential to synaptic activity throughout the brain, which illustrates immense therapeutic potential and risk. Notably, glutamatergic mechanisms are expressed by most types of brain cells. Hence, glutamate likely encodes multiple forms of intercellular signaling. Here, we hypothesized that the selective manipulation of astrocyte to neuron signaling would alter cognition without producing widespread brain impairments. First, we eliminated activity of the astrocytic glutamate release mechanism, Sxc, in rat. This impaired cognitive flexibility and increased expression of perseverative, maladaptive behaviors. Notably, eliminating Sxc activity did not alter metrics of health or noncognitive brain function. These data add to recent evidence that the brain expresses cognition-specific molecular mechanisms that could lead to highly precise, safe medications for impaired cognition.

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.

Protein-coding mutation in Adcy3 increases adiposity and alters emotional behaviors sex-dependently in rats.

Objective: Adenylate cyclase 3 (Adcy3) has been linked to both obesity and major depressive disorder (MDD). Our lab identified a protein-coding variant in the 2nd transmembrane (TM) helix of Adcy3 in rats, and similar obesity variants have been identified in humans. The current study investigates the role of a TM variant in adiposity and behavior. Methods: We used CRISPR-SpCas9 to mutate the TM domain of Adcy3 in WKY rats (Adcy3mut/mut). We also created a heterozygous knockout rat in the same strain (Adcy3+/-). Wild-type (WT), Adcy3+/-, and Adcy3mut/mut rats were fed a high-fat diet for 12 weeks. We measured body weight, fat mass, glucose tolerance, food intake, metabolism, emotion-like behaviors, and memory. Results: Adcy3+/- and Adcy3mut/mut rats weighed more than WT rats due to increased fat mass. There were key sex differences: adiposity was driven by increased food intake in males but by decreased energy expenditure in females. Adcy3mut/mut males displayed increased passive coping and decreased memory while Adcy3mut/mut females displayed increased anxiety-like behavior. Conclusions: These studies show that the ADCY3 TM domain plays a role in protein function, that Adcy3 may contribute to the relationship between obesity and MDD, and that sex influences the relationships between Adcy3, metabolism, and behavior.

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.

Keratinocyte-associated protein 3 plays a role in body weight and adiposity with differential effects in males and females.

Despite the obesity crisis in the United States, the underlying genetics are poorly understood. Our lab previously identified Keratinocyte-associated protein 3, Krtcap3, as a candidate gene for adiposity through a genome-wide association study in outbred rats, where increased liver expression of Krtcap3 correlated with decreased fat mass. Here we seek to confirm that Krtcap3 expression affects adiposity traits. To do so, we developed an in vivo whole-body Krtcap3 knock-out (KO) rat model. Wild-type (WT) and KO rats were placed onto a high-fat (HFD) or low-fat diet (LFD) at 6 weeks of age and were maintained on diet for 13 weeks, followed by assessments of metabolic health. We hypothesized that Krtcap3-KO rats will have increased adiposity and a worsened metabolic phenotype relative to WT. We found that KO male and female rats have significantly increased body weight versus WT, with the largest effect in females on a HFD. KO females also ate more and had greater adiposity, but were more insulin sensitive than WT regardless of diet condition. Although KO males weighed more than WT under both diet conditions, there were no differences in eating behavior or fat mass. Interestingly, KO males on a HFD were more insulin resistant than WT. This study confirms that Krtcap3 plays a role in body weight regulation and demonstrates genotype- and sex-specific effects on food intake, adiposity, and insulin sensitivity. Future studies will seek to better understand these sex differences, the role of diet, and establish a mechanism for Krtcap3 in obesity.

Maternal diet during gestation and lactation modifies the severity of salt-induced hypertension and renal injury in Dahl salt-sensitive rats.

Environmental exposure of parents or early in life may affect disease development in adults. We found that hypertension and renal injury induced by a high-salt diet were substantially attenuated in Dahl SS/JrHsdMcwiCrl (SS/Crl) rats that had been maintained for many generations on the grain-based 5L2F diet compared with SS/JrHsdMcwi rats (SS/Mcw) maintained on the casein-based AIN-76A diet (mean arterial pressure, 116±9 versus 154±25 mm Hg; urinary albumin excretion, 23±12 versus 170±80 mg/d). RNAseq analysis of the renal outer medulla identified 129 and 82 genes responding to a high-salt diet uniquely in SS/Mcw and SS/Crl rats, respectively, along with minor genetic differences between the SS substrains. The 129 genes responding to salt in the SS/Mcw strain included numerous genes with homologs associated with hypertension, cardiovascular disease, or renal disease in human. To narrow the critical window of exposure, we performed embryo-transfer experiments in which single-cell embryos from 1 colony (SS/Mcw or SS/Crl) were transferred to surrogate mothers from the other colony, with parents and surrogate mothers maintained on their respective original diet. All offspring were fed the AIN-76A diet after weaning. Salt-induced hypertension and renal injury were substantially exacerbated in rats developed from SS/Crl embryos transferred to SS/Mcw surrogate mothers. Conversely, salt-induced hypertension and renal injury were significantly attenuated in rats developed from SS/Mcw embryos transferred to SS/Crl surrogate mothers. Together, the data suggest that maternal diet during the gestational-lactational period has substantial effects on the development of salt-induced hypertension and renal injury in adult SS rats.