The mouse adrenal gland

The adrenal gland is a complex endocrine organ and its secreted hormones affect a multitude of essential systems, including stress response, immune function, cardio vascular regulation and anabolic or catabolic processes. In order to define age-dependent changes within the adrenal gland in the mouse, a systematic analysis of functional and growth-related parameters was performed by investigating the adrenal glands of male and female mice from weaning to adulthood (weeks 3, 5, 7, 9 and 11) at the organ, compartment, and cellular level. Therefore, an adrenal weight analysis was performed and the absolute volume of every adrenal zone was determined using unbiased stereological methods. At the cellular level, zona fasciculata (ZF) mean cell volume and total cell number were analysed at five distinct points in time. Furthermore, the amount of stored lipids and the expression of two enzymes involved in steroidogenesis, side-chain-cleavage enzyme (SCC) and 11-beta-hydroxylase, were explored in the adrenal glands at selected points in time. Serum aldosterone and corticosterone concentrations were monitored throughout the investigation period as functional readouts of the zona glomerulosa and the ZF, respectively. Results: Increasing weights of the adrenal glands were observed until week 7. Of note is a significant adrenal weight reduction by 25 % exclusively in male mice, between weeks 7 and 9. Adrenal glands in female mice principally displayed higher weights. At an age of 9 weeks, this weight difference reached a maximum, with female mice characterized by two-fold higher adrenal gland weights than male mice. The weight dimorphism was in particular due to an enlarged ZF in female mice. Also the X-Zone and the medulla, showed increased volumes if compared to male littermates. No gender-dependent volume difference was found in the zona glomerulosa. In addition to the gender-dependent growth effects, time-dependent growth features were observed in each zone, and every zone, with the exception of the X-zone in males, gradually increased in volume between weeks 3 and 7 of age. Time- and gender-dependent effects were also studied at the cellular level in the ZF, where effects of sex and age on the mean cell volumes as well as on the total cell number were present. From week 5 onwards, the ZF of female mice comprised significantly more cells if compared to their male counterparts. In both genders, the number of ZF cells reached a maximum at the age of 7 weeks and decreased significantly until week 9. In male mice, the mean volume of ZF cells increased between 3 and 11 weeks of age. In 3- to 7-week-old female mice ZF mean cell volumes were similar, however, between 7 and 9 weeks of age, a volume increase by approximately 50 % was observed. A positive association of ZF total cell number with the estimated total amounts of corticosterone in the circulation (ETAC) could be established, in contrast, no association of ZF mean cell volume and the ETAC was found. Serum aldosterone concentrations were similar in both genders, in contrast serum corticosterone levels were higher, at least as a tendency, in female mice. Moreover, dynamic changes of both steroid hormone concentrations were observed in a time-dependent manner. The aldosterone concentrations increased over time, whereas the corticosterone levels decreased significantly between weeks 3 and 11. The amount of stored lipids in the adrenal glands appeared to be higher in female mice if compared to male mice and the adrenal glands of 11-week-old male and female mice seem to store more lipids than 7-week-old mice. Also the expression of the two analysed enzymes, SCC and 11-beta-hydroxylase, increased between 3 and 11 weeks of age in both sexes, indicating that 11-week-old mice might have a higher steroidbiosynthesis capacity than the younger mice. In conclusion, this study demonstrates that, between weaning and adulthood dynamic processes are present on multiple levels of adrenal gland growth. Specific adrenal growth regulation occurs through the influence of age, sex, cell type, cell number and cell size. A particular finding of the study is, that growth is not a unidirectional process by constant increases of cell size and cell number and that decreases of cell number may be compensated by increases of cell volumes. Certainly, these dynamic processes can be assumed to affect also functional parameters, however the final quantity of corticosterone secreted can only be explained in parts as a function of ZF cell size or ZF cell number, respectively. In general, this work supports the systemic approach integrating multiple information from various levels being more and more relevant for future research projects

Control of (pre)-analytical aspects in immunoassay measurements of metabolic hormones in rodents

The measurement of circulating hormones by immunoassay remains a cornerstone in preclinical endocrine research. For scientists conducting and interpreting immunoassay measurements of rodent samples, the paramount aim usually is to obtain reliable and meaningful measurement data in order to draw conclusions on biological processes. However, the biological variability between samples is not the only variable affecting the readout of an immunoassay measurement and a considerable amount of unwanted or unintended variability can be quickly introduced during the pre-analytical and analytical phase. This review aims to increase the awareness for the factors 'pre-analytical' and 'analytical' variability particularly in the context of immunoassay measurement of circulating metabolic hormones in rodent samples. In addition, guidance is provided how to gain control over these variables and how to avoid common pitfalls associated with sample collection, processing, storage and measurement. Furthermore, recommendations are given on how to perform a basic validation of novel single and multiplex immunoassays for the measurement of metabolic hormones in rodents. Finally, practical examples from immunoassay measurements of plasma insulin in mice address the factors 'sampling site and inhalation anesthesia' as frequent sources of introducing an unwanted variability during the pre-analytical phase. The knowledge about the influence of both types of variability on the immunoassay measurement of circulating hormones as well as strategies to control these variables are crucial, on the one hand, for planning and realization of metabolic rodent studies and, on the other hand, for the generation and interpretation of meaningful immunoassay data from rodent samples

Isoenergetic Feeding of Low Carbohydrate-High Fat Diets Does Not Increase Brown Adipose Tissue Thermogenic Capacity in Rats

Low-carbohydrate, high-fat (LC-HF) diets are popular for inducing weight loss in overweighed adults. Adaptive thermogenesis increased by specific effects of macronutrients on energy expenditure has been postulated to induce this weight loss. We studied brown adipose tissue (BAT) morphology and function following exposure to different LC-HF diets

Dissociation of somatic growth, time of sexual maturity, and life expectancy by overexpression of an RGD-deficient IGFBP-2 variant in female transgenic mice

Impaired growth is often associated with an extension of lifespan. However, the negative correlation between somatic growth and life expectancy is only true within, but not between, species. This can be observed because smaller species have, as a rule, a shorter lifespan than larger species. In insects and worms, reduced reproductive development and increased fat storage are associated with prolonged lifespan. However, in mammals the relationship between the dynamics of reproductive development, fat metabolism, growth rate, and lifespan are less clear. To address this point, female transgenic mice that were overexpressing similar levels of either intact (D-mice) or mutant insulin-like growth factor-binding protein-2 (IGFBP-2) lacking the Arg-Gly-Asp (RGD) motif (E-mice) were investigated. Both lines of transgenic mice exhibited a similar degree of growth impairment (-9% and -10%) in comparison with wild-type controls (C-mice). While in D-mice, sexual maturation was found to be delayed and life expectancy was significantly increased in comparison with C-mice, these parameters were unaltered in E-mice in spite of their reduced growth rate. These observations indicate that the RGD-domain has a major influence on the pleiotropic effects of IGFBP-2 and suggest that somatic growth and time of sexual maturity or somatic growth and life expectancy are less closely related than thought previously

9-PAHSA displays a weak anti-inflammatory potential mediated by specific antagonism of chemokine G protein-coupled receptors

Introduction: 9-PAHSA belongs to a class of endogenous mammalian bioactive lipids, fatty acid esters of hydroxy fatty acids (FAHFA), that are present in circulation at nanomolar concentrations in mice and humans. Published preclinical data suggest beneficial effects of 9-PAHSA treatment on glucose metabolism as well as modulation of immune function. However, receptor molecules with high affinity towards these lipids have not been identified so far.Methods: In a broad screen of a panel of G protein-coupled receptors (GPCRs) we discovered that 9-PAHSA displays antagonist activity with an IC50 in the micromolar range on selected chemokine receptors, namely, CCR6, CCR7, CXCR4, and CXCR5. The potential immunomodulatory activities in a human cellular model of innate immunity were then investigated.Results and discussion: In our in vitro experiments, a weak anti-inflammatory potential for high concentrations of 9-PAHSA (10–100 µM) could be detected, as treatment reduced the LPS-induced secretion of certain chemokines, such as CXCL10, MIP-1 beta and MCP. Regarding metabolic effects, we re-investigated 9-PAHSA on glucose metabolism and insulin sensitivity in vitro and in mice confirming conclusions from our earlier study that FAHFAs lack glucoregulatory activity following an acute treatment. In conclusion, the specific interactions with a subset of chemokine receptors may contribute to weak anti-inflammatory properties of 9-PAHSA, but further studies are needed to confirm its in anti-inflammatory potential in vivo

Phenotype Selection Reveals Coevolution of Muscle Glycogen and Protein and PTEN as a Gate Keeper for the Accretion of Muscle Mass in Adult Female Mice

We have investigated molecular mechanisms for muscle mass accretion in a non-inbred mouse model (DU6P mice) characterized by extreme muscle mass. This extreme muscle mass was developed during 138 generations of phenotype selection for high protein content. Due to the repeated trait selection a complex setting of different mechanisms was expected to be enriched during the selection experiment. In muscle from 29-week female DU6P mice we have identified robust increases of protein kinase B activation (AKT, Ser-473, up to 2-fold) if compared to 11- and 54-week DU6P mice or controls. While a number of accepted effectors of AKT activation, including IGF-I, IGF-II, insulin/IGF-receptor, myostatin or integrin-linked kinase (ILK), were not correlated with this increase, phosphatase and tensin homologue deleted on chromosome 10 (PTEN) was down-regulated in 29-week female DU6P mice. In addition, higher levels of PTEN phosphorylation were found identifying a second mechanism of PTEN inhibition. Inhibition of PTEN and activation of AKT correlated with specific activation of p70S6 kinase and ribosomal protein S6, reduced phosphorylation of eukaryotic initiation factor 2α (eIF2α) and higher rates of protein synthesis in 29-week female DU6P mice. On the other hand, AKT activation also translated into specific inactivation of glycogen synthase kinase 3ß (GSK3ß) and an increase of muscular glycogen. In muscles from 29-week female DU6P mice a significant increase of protein/DNA was identified, which was not due to a reduction of protein breakdown or to specific increases of translation initiation. Instead our data support the conclusion that a higher rate of protein translation is contributing to the higher muscle mass in mid-aged female DU6P mice. Our results further reveal coevolution of high protein and high glycogen content during the selection experiment and identify PTEN as gate keeper for muscle mass in mid-aged female DU6P mice

Obesity in MENX Rats Is Accompanied by High Circulating Levels of Ghrelin and Improved Insulin Sensitivity

Ghrelin, the natural ligand of the growth hormone secretagogue receptor type la (GHS-R1a), is mainly secreted from the stomach and regulates food intake and energy homeostasis. p27 regulates cell cycle progression in many cell types. Here, we report that rats affected by the multiple endocrine neoplasia syndrome MENX, caused by a p27 mutation, develop pancreatic islet hyperplasia containing elevated numbers of ghrelin-producing epsilon-cells. The metabolic phenotype of MENX-affected rats featured high endogenous acylated and unacylated plasma ghrelin levels. Supporting increased ghrelin action, MENX rats show increased food intake, enhanced body fat mass, and elevated plasma levels of triglycerides and cholesterol. Ghrelin effect on food intake was confirmed by treating MENX rats with a GHS-R1a antagonist. At 7.5 months, MENX-affected rats show decreased mRNA levels of hypothalamic GHS-R1a, neuropeptide Y (NPY), and agouti-related protein (AgRP), suggesting that prolonged hyperghrelinemia may lead to decreased ghrelin efficacy. In line with ghrelin's proposed role in glucose metabolism, we find decreased glucose-stimulated insulin secretion in MENX rats, while insulin sensitivity is improved. In summary, we provide a novel nontransgenic rat model with high endogenous ghrelin plasma levels and, interestingly, improved glucose tolerance. This model might aid in identifying new therapeutic approaches for obesity and obesity-related diseases, including type 2 diabetes

Lifelong Obesity in a Polygenic Mouse Model Prevents Age- and Diet-Induced Glucose Intolerance– Obesity Is No Road to Late-Onset Diabetes in Mice

AIMS/HYPOTHESIS: Visceral obesity holds a central position in the concept of the metabolic syndrome characterized by glucose intolerance in humans. However, until now it is unclear if obesity by itself is responsible for the development of glucose intolerance. METHODS: We have used a novel polygenic mouse model characterized by genetically fixed obesity (DU6) and addressed age- and high fat diet-dependent glucose tolerance. RESULTS: Phenotype selection over 146 generations increased body weight by about 2.7-fold in male 12-week DU6 mice (P<0.0001) if compared to unselected controls (Fzt:DU). Absolute epididymal fat mass was particularly responsive to weight selection and increased by more than 5-fold (P<0.0001) in male DU6 mice. At an age of 6 weeks DU6 mice consumed about twice as much food if compared to unselected controls (P<0.001). Absolute food consumption was higher at all time points measured in DU6 mice than in Fzt:DU mice. Between 6 and 12 weeks of age, absolute food intake was reduced by 15% in DU6 mice (P<0.001) but not in Fzt:DU mice. In both mouse lines feeding of the high fat diet elevated body mass if compared to the control diet (P<0.05). In contrast to controls, DU6 mice did not display high fat diet-induced increases of epididymal and renal fat. Control mice progressively developed glucose intolerance with advancing age and even more in response to the high fat diet. In contrast, obese DU6 mice did neither develop a glucose intolerant phenotype with progressive age nor when challenged with a high fat diet. CONCLUSIONS/INTERPRETATION: Our results from a polygenic mouse model demonstrate that genetically pre-determined and life-long obesity is no precondition of glucose intolerance later in life

A siRNA mediated hepatic dpp4 knockdown affects lipid, but not glucose metabolism in diabetic mice.

Systemic inhibition of dipeptidyl peptidase 4 (dpp4) represents an effective and established treatment option for type 2 diabetes (T2D). The current study investigated in mice if a liver selective knock-down of dpp4 by therapeutic siRNAs could be a novel, similarly effective treatment option for T2D. Furthermore, the potential effects on hepatic steatosis, inflammation and lipid metabolism were investigated after hepato-selective knock-down of dpp4. The knock-down efficiency and IC50 values of siRNAs targeting dpp4 were analyzed in PC3 cells. In two independent studies, either db/db mice or C57BL/6J mice were injected intravenously with a liposomal formulation of siRNAs targeting either dpp4 or a non-targeting control, followed by metabolically characterization. In comparator groups, additional cohorts of mice were treated with an oral dpp4 inhibitor. In both animal studies, we observed a robust knock-down (~75%) of hepatic dpp4 with a potent siRNA. Hepatic dpp4 knockdown did not significantly affect glucose metabolism or circulating incretin concentrations in both animal studies. However, in obese and diabetic db/db mice hepatic steatosis was reduced and hepatic mRNA expression of acaca, scd1, fasn and pparg was significantly lower after siRNA treatment. Systemic inhibition of the enzymatic dpp4 activity by an oral dpp4 inhibitor significantly improved glucose handling in db/db mice but did not affect hepatic endpoints. These data demonstrate that a targeted reduction of dpp4 expression in the liver may not be sufficient to improve whole-body glucose metabolism in obese and diabetic mice but may improve hepatic lipid metabolism