Modulation of Extravascular Binding of Recombinant Factor IX Impacts the Duration of Efficacy in Mouse Models.

BACKGROUND: There is an emerging concept that in addition to circulating coagulation factor IX (FIX), extravascular FIX contributes to hemostasis. OBJECTIVE: Our objective was to evaluate the efficacy of extravascular FIX using animal models of tail clip bleeding and ferric chloride-induced thrombosis. METHODS: Mutant rFIX proteins with described enhanced (rFIXK5R) or reduced (rFIXK5A) binding to extracellular matrix were generated and characterized using in vitro aPTT, one-stage clotting, and modified FX assays. Using hemophilia B mice, pharmacokinetic (PK) parameters and in vivo efficacy of these proteins were compared against rFIX wild-type protein (rFIXWT) in a tail clip bleeding and FeCl3-induced thrombosis model. Respective tissue disposition of FIX was evaluated using immunofluorescence. RESULTS: In vitro characterization demonstrated comparable clotting activity of rFIX proteins. The PK profile showed that rFIXK5A displayed the highest plasma exposure compared to rFIXWT and rFIXK5R. Immunofluorescence evaluation of liver tissue showed that rFIXK5R was detectable up to 24 hours, whereas rFIXWT and rFIXK5A were detectable only up to 15 minutes. In the tail clip bleeding model, rFIXK5R displayed significant hemostatic protection against bleeding incidence for up to 72 hours postintravenous administration of 50 IU/kg, whereas the efficacy of rFIXK5A was already reduced at 24 hours. Similarly, in the mesenteric artery thrombus model, rFIXK5R and rFIXWT demonstrated prolonged efficacy compared to rFIXK5A. CONCLUSION: Using two different in vivo models of hemostasis and thrombosis, we demonstrate that mutated rFIX protein with enhanced binding (rFIXK5R) to extravascular space confers prolonged hemostatic efficacy in vivo despite lower plasma exposure, whereas rFIXK5A rapidly lost its efficacy despite higher plasma exposure.

Preclinical pharmacology of RA15127343: In vitro and in vivo activity of a novel ultralong-acting basal insulin.

AIM: To report the in vitro and in vivo preclinical pharmacokinetic (PK) and pharmacodynamic (PD) properties of RA15127343, a novel ultralong-acting insulin analogue targeting once-weekly administration, in female Göttingen minipigs. METHODS: In vitro binding and activation of human insulin receptor isoforms (IR-A/IR-B), glucose uptake in rat myocytes, as well as mitogenic activity of RA15127343 were evaluated. In vivo, the PK and PD activities of RA15127343 were assessed in female, normoglycaemic Göttingen minipigs. The half-life (t1/2 ) and time to maximum plasma concentration (Tmax ) of subcutaneously (SC) administered RA15127343 (10/30/45/60 nmol/kg) were estimated. In vivo blood glucose and endogenous plasma C-peptide concentrations after single SC administration (10/30/45/60 nmol/kg) or repeated dosing (15 nmol/kg) were analysed. RESULTS: In comparison to human insulin, RA15127343 showed lower in vitro binding affinity (19.9/6.31 µM vs. 1.10/1.14 nM) and activation (2.054 µM/669.6 nM vs. 26.04/18.24 nM) of IR-A/IR-B, lower potency to activate glucose uptake (855.2 vs. 3.37 nM) and lower mitogenic activity (17.92 µM vs. 10.78 nM; proliferation in MCF7 cells). In vivo, the mean t1/2 and Tmax of RA15127343 after SC administration ranged from 48 to 59 and 30 to 39 hours, respectively. Blood glucose and plasma C-peptide concentrations were significantly lower with RA15127343 (single/repeated doses) versus vehicle. CONCLUSIONS: RA15127343 showed an ultra-long t1/2 with a slow onset of action. The preclinical pharmacological outcomes suggest RA15127343 could be a potential ultralong-acting insulin analogue with low risk of hypoglycaemia in humans.

Liver-Specific Knockdown of Class IIa HDACs Has Limited Efficacy on Glucose Metabolism but Entails Severe Organ Side Effects in Mice.

Histone deacetylases (HDACs) are important regulators of epigenetic gene modification that are involved in the transcriptional control of metabolism. In particular class IIa HDACs have been shown to affect hepatic gluconeogenesis and previous approaches revealed that their inhibition reduces blood glucose in type 2 diabetic mice. In the present study, we aimed to evaluate the potential of class IIa HDAC inhibition as a therapeutic opportunity for the treatment +of metabolic diseases. For that, siRNAs selectively targeting HDAC4, 5 and 7 were selected and used to achieve a combinatorial knockdown of these three class IIa HDAC isoforms. Subsequently, the hepatocellular effects as well as the impact on glucose and lipid metabolism were analyzed in vitro and in vivo. The triple knockdown resulted in a statistically significant decrease of gluconeogenic gene expression in murine and human hepatocyte cell models. A similar HDAC-induced downregulation of hepatic gluconeogenesis genes could be achieved in mice using a liver-specific lipid nanoparticle siRNA formulation. However, the efficacy on whole body glucose metabolism assessed by pyruvate-tolerance tests were only limited and did not outweigh the safety findings observed by histopathological analysis in spleen and kidney. Mechanistically, Affymetrix gene expression studies provide evidence that class IIa HDACs directly target other key factors beyond the described forkhead box (FOXP) transcription regulators, such as hepatocyte nuclear factor 4 alpha (HNF4a). Downstream of these factors several additional pathways were regulated not merely including glucose and lipid metabolism and transport. In conclusion, the liver-directed combinatorial knockdown of HDAC4, 5 and 7 by therapeutic siRNAs affected multiple pathways in vitro, leading in vivo to the downregulation of genes involved in gluconeogenesis. However, the effects on gene expression level were not paralleled by a significant reduction of gluconeogenesis in mice. Combined knockdown of HDAC isoforms was associated with severe adverse effects in vivo, challenging this approach as a treatment option for chronic metabolic disorders like type 2 diabetes.

Nearly a Century of Insulin at Sanofi: Looking Back Over the Decades of Production and Development.

Almost a century ago, the first insulin was produced by Banting, Best, MacLeod and Collip in Toronto, thereby enabling life-saving treatment for people with diabetes. Since then, there have been many advancements in insulin production and development of new insulin analogues. In this article, we reflect on the rich heritage of Sanofi and its predecessor, Hoechst, in insulin production and development, from being one of the first companies to produce insulin in Europe in 1923, to modern-day insulin analogues and integrated care solutions at present-day Sanofi.

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.

The role of C16:0 ceramide in the development of obesity and type 2 diabetes: CerS6 inhibition as a novel therapeutic approach.

OBJECTIVE: Ectopic fat deposition is associated with increased tissue production of ceramides. Recent genetic mouse studies suggest that specific sphingolipid C16:0 ceramide produced by ceramide synthase 6 (CerS6) plays an important role in the development of insulin resistance. However, the therapeutic potential of CerS6 inhibition not been demonstrated. Therefore, we pharmacologically investigated the selective ablation of CerS6 using antisense oligonucleotides (ASO) in obese insulin resistance animal models. METHODS: We utilized ASO as therapeutic modality, CerS6 ASO molecules designed and synthesized were initially screened for in-vitro knock-down (KD) potency and cytotoxicity. ASOs with >85% inhibition of CerS6 mRNA were selected for further investigations. Most promising ASOs verified for in-vivo KD efficacy in healthy mice. CerS6 ASO (AAGATGAGCCGCACC) was found most active with hepatic reduction of CerS6 mRNA expression. Prior to longitudinal metabolic studies, we performed a dose titration target engagement analysis with CerS6 ASO in healthy mice to select the optimal dose. Next, we utilized leptin deficiency ob/ob and high fat diet (HFD) induced obese mouse models for pharmacological efficacy study. RESULTS: CerS6 expression were significantly elevated in the liver and brown adipose, this was correlated with significantly elevated C16:0 ceramide concentrations in plasma and liver. Treatment with CerS6 ASO selectively reduced CerS6 expression by ∼90% predominantly in the liver and this CerS6 KD resulted in a significant reduction of C16:0 ceramide by about 50% in both liver and plasma. CerS6 KD resulted in lower body weight gain and accompanied by a significant reduction in whole body fat and fed/fasted blood glucose levels (1% reduction in HbA1c). Moreover, ASO-mediated CerS6 KD significantly improved oral glucose tolerance (during oGTT) and mice displayed improved insulin sensitivity. Thus, CerS6 appear to play an important role in the development of obesity and insulin resistance. CONCLUSIONS: Our investigations identified specific and selective therapeutic valid ASO for CerS6 ablation in in-vivo. CerS6 should specifically be targeted for the reduction of C16:0 ceramides, that results in amelioration of insulin resistance, hyperglycemia and obesity. CerS6 mediated C16:0 ceramide reduction could be a potentially attractive target for the treatment of insulin resistance, obesity and type 2 diabetes.

Acute and Repeated Treatment with 5-PAHSA or 9-PAHSA Isomers Does Not Improve Glucose Control in Mice.

Fatty acid esters of hydroxylated fatty acids (FAHFAs) were discovered as a novel class of endogenous mammalian lipids whose profound effects on metabolism have been shown. In the current study, in vitro and in vivo the metabolic effects of two of these FAHFAs, namely palmitic acid-5- (or -9) -hydroxy-stearic acid (5- or 9-PAHSA, respectively) were profiled. In DIO mice fed with differentially composed low- or high-fat diets, acute and subchronic treatment with 5-PAHSA and 9-PAHSA alone, or in combination, did not significantly improve the deranged metabolic status. Neither racemic 5- or 9-PAHSA, nor the enantiomers were able to: (1) increase basal or insulin-stimulated glucose uptake in vitro, (2) stimulate GLP-1 release from GLUTag cells, or (3) induce GSIS in rat, mouse, or human islets or in a human pancreatic ß cell line. Therefore, our data do not support the further development of PAHSAs or their derivatives for the control of insulin resistance and hyperglycemia.

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.

Dietary sugars, not lipids, drive hypothalamic inflammation.

OBJECTIVE: The hypothalamus of hypercaloric diet-induced obese animals is featured by a significant increase of microglial reactivity and its associated cytokine production. However, the role of dietary components, in particular fat and carbohydrate, with respect to the hypothalamic inflammatory response and the consequent impact on hypothalamic control of energy homeostasis is yet not clear. METHODS: We dissected the different effects of high-carbohydrate high-fat (HCHF) diets and low-carbohydrate high-fat (LCHF) diets on hypothalamic inflammatory responses in neurons and non-neuronal cells and tested the hypothesis that HCHF diets induce hypothalamic inflammation via advanced glycation end-products (AGEs) using mice lacking advanced glycation end-products (AGEs) receptor (RAGE) and/or the activated leukocyte cell-adhesion molecule (ALCAM). RESULTS: We found that consumption of HCHF diets, but not of LCHF diets, increases microgliosis as well as the presence of N(ε)-(Carboxymethyl)-Lysine (CML), a major AGE, in POMC and NPY neurons of the arcuate nucleus. Neuron-secreted CML binds to both RAGE and ALCAM, which are expressed on endothelial cells, microglia, and pericytes. On a HCHF diet, mice lacking the RAGE and ALCAM genes displayed less microglial reactivity and less neovasculature formation in the hypothalamic ARC, and this was associated with significant improvements of metabolic disorders induced by the HCHF diet. CONCLUSIONS: Combined overconsumption of fat and sugar, but not the overconsumption of fat per se, leads to excessive CML production in hypothalamic neurons, which, in turn, stimulates hypothalamic inflammatory responses such as microgliosis and eventually leads to neuronal dysfunction in the control of energy metabolism.

TNFα drives mitochondrial stress in POMC neurons in obesity.

Consuming a calorically dense diet stimulates microglial reactivity in the mediobasal hypothalamus (MBH) in association with decreased number of appetite-curbing pro-opiomelanocortin (POMC) neurons; whether the reduction in POMC neuronal function is secondary to the microglial activation is unclear. Here we show that in hypercaloric diet-induced obese mice, persistently activated microglia in the MBH hypersecrete TNFα that in turn stimulate mitochondrial ATP production in POMC neurons, promoting mitochondrial fusion in their neurites, and increasing POMC neuronal firing rates and excitability. Specific disruption of the gene expressions of TNFα downstream signals TNFSF11A or NDUFAB1 in the MBH of diet-induced obese mice reverses mitochondrial elongation and reduces obesity. These data imply that in a hypercaloric environment, persistent elevation of microglial reactivity and consequent TNFα secretion induces mitochondrial stress in POMC neurons that contributes to the development of obesity.

Impairment of Host Liver Repopulation by Transplanted Hepatocytes in Aged Rats and the Release by Short-Term Growth Hormone Treatment.

Hepatocyte transplantation is an alternative to whole liver transplantation. Yet, efficient liver repopulation by transplanted hepatocytes is low in livers of old animals. This restraint might be because of the poor proliferative capacity of aged donor hepatocytes or the regenerative impairment of the recipient livers. The age-dependent liver repopulation by transplanted wild-type hepatocytes was investigated in juvenile and senescent rats deficient in dipeptidyl-peptidase IV. Repopulation was quantified by flow cytometry and histochemical estimation of dipeptidyl-peptidase IV enzyme activity of donor cells in the negative host liver. As a potential pathway involved, expression of cell cycle proteins was assessed. Irrespective of the age of the donor hepatocytes, large cell clusters appeared in juvenile, but only small clusters in senescent host livers. Because juvenile and senescent donor hepatocytes were likewise functional, host-derived factor(s) impaired senescent host liver repopulation. Growth hormone levels were significantly higher in juvenile than in senescent rats, suggesting that growth hormone might promote host liver repopulation. Indeed, short-term treatment with growth hormone augmented senescent host liver repopulation involving the growth hormone-mediated release of the transcriptional blockade of genes associated with cell cycle progression. Short-term growth hormone substitution might improve liver repopulation by transplanted hepatocytes, thus augmenting the therapeutic benefit of clinical hepatocyte transplantation in older patients.

Role of selected amino acids on plasma IGF-I concentration in infants.

PURPOSE: Insulin-like growth factor-I (IGF-I) is related to growth and its secretion is modified by protein intake in early infancy. We examined the relationship of dietary protein and circulating amino acids on plasma IGF-I levels and early growth. METHODS: Healthy formula-fed infants (n = 213) were randomly assigned to receive either a protein-reduced infant formula with alpha-lactalbumin-enriched whey and free tryptophan and phenylalanine (IF) or an isocaloric standard formula without free amino acids (CF) for the first 120 days of life. A group of breastfed (BF) infants was studied as a non-randomized reference cohort. Biochemical variables were measured shortly after birth (subpopulation) and at an age of 120 days. A path analysis was used to explore the relationship between IGF-I, insulin and amino acids. Results are derived from secondary analyses of a randomized controlled trial. RESULTS: Plasma concentrations of IGF-I at 120 days were significantly higher in IF than in CF infants [58.5 (15.0) vs. 53.7 (9.95) ng/mL; p = 0.020]. BF infants showed lower IGF-I concentrations of 41.6 (10.7) ng/mL. All amino acids but Thr and Cit had a more marked effect on insulin than on IGF-I level. Considering weight, sex and feeding group, Trp explained an equal percentage of variance of IGF-I and insulin (total R 2 12.5 % of IGF-I and 12.3 % of insulin), while branched-chain AA explained an up to twofold higher variance of insulin than IGF-I. Compared to CF, IF explained 18.9 % of the IGF-I level (p = 0.03), while for insulin no direct effect was detectable. CONCLUSION: Higher IGF-I concentrations and growth velocities in infants receiving protein-reduced IF indicate that the protein concentration of an infant formula alone does not control IGF-I levels and growth. Other components (e.g., selected amino acids) of infant formulae might control directly or indirectly via insulin influence IGF-I.

Early effects of a high-caloric diet and physical exercise on brain volumetry and behavior: a combined MRI and histology study in mice.

Excessive intake of high-caloric diets as well as subsequent development of obesity and diabetes mellitus may exert a wide range of unfavorable effects on the central nervous system (CNS) in the long-term. The potentially harmful effects of such diets were suggested to be mitigated by physical exercise. Here, we conducted a study investigating early effects of a cafeteria-diet on gray and white brain matter volume by means of voxel-based morphometry (VBM) and region-of-interest (ROI) analysis. Half of the mice performed voluntary wheel running to study if regular physical exercise prevents unfavorable effects of a cafeteria-diet. In addition, histological analyses for myelination and neurogenesis were performed. As expected, wheel running resulted in a significant increase of gray matter volume in the CA1-3 areas, the dentate gyrus and stratum granulosum of the hippocampus in the VBM analysis, while a positive effect of the cafeteria-diet was shown for the whole hippocampal CA1-3 area only in the ROI analysis, indicating a regional volume effect. It was earlier found that hippocampal neurogenesis may be related to volume increases after exercise. Interestingly, while running resulted in a significant increase in neurogenesis assessed by doublecortin (DCX)-labeling, this was not true for cafeteria diet. This indicates different underlying mechanisms for gray matter increase. Moreover, animals receiving cafeteria diet only showed mild deficits in long-term memory assessed by the puzzle-box paradigm, while executive functioning and short term memory were not affected. Our data therefore highlight that high caloric diet impacts on the brain and behavior. Physical exercise seems not to interact with these mechanisms.

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.

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 1a (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 ε-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.

Low-carbohydrate, high-fat diets have sex-specific effects on bone health in rats.

PURPOSE: Studies in humans suggest that consumption of low-carbohydrate, high-fat diets (LC-HF) could be detrimental for growth and bone health. In young male rats, LC-HF diets negatively affect bone health by impairing the growth hormone/insulin-like growth factor axis (GH/IGF axis), while the effects in female rats remain unknown. Therefore, we investigated whether sex-specific effects of LC-HF diets on bone health exist. METHODS: Twelve-week-old male and female Wistar rats were isoenergetically pair-fed either a control diet (CD), "Atkins-style" protein-matched diet (LC-HF-1), or ketogenic low-protein diet (LC-HF-2) for 4 weeks. In females, microcomputed tomography and histomorphometry analyses were performed on the distal femur. Sex hormones were analysed with liquid chromatography-tandem mass spectrometry, and endocrine parameters including GH and IGF-I were measured by immunoassay. RESULTS: Trabecular bone volume, serum IGF-I and the bone formation marker P1NP were lower in male rats fed both LC-HF diets versus CD. LC-HF diets did not impair bone health in female rats, with no change in trabecular or cortical bone volume nor in serum markers of bone turnover between CD versus both LC-HF diet groups. Pituitary GH secretion was lower in female rats fed LC-HF diet, with no difference in circulating IGF-I. Circulating sex hormone concentrations remained unchanged in male and female rats fed LC-HF diets. CONCLUSION: A 4-week consumption of LC-HF diets has sex-specific effects on bone health-with no effects in adult female rats yet negative effects in adult male rats. This response seems to be driven by a sex-specific effect of LC-HF diets on the GH/IGF system.

Effects of a high-caloric diet and physical exercise on brain metabolite levels: a combined proton MRS and histologic study.

Excessive intake of high-caloric diets as well as subsequent development of obesity and diabetes mellitus may exert a wide range of unfavorable effects on the central nervous system (CNS). It has been suggested that one mechanism in this context is the promotion of neuroinflammation. The potentially harmful effects of such diets were suggested to be mitigated by physical exercise. Here, we conducted a study investigating the effects of physical exercise in a cafeteria-diet mouse model on CNS metabolites by means of in vivo proton magnetic resonance spectroscopy ((1)HMRS). In addition postmortem histologic and real-time (RT)-PCR analyses for inflammatory markers were performed. Cafeteria diet induced obesity and hyperglycemia, which was only partially moderated by exercise. It also induced several changes in CNS metabolites such as reduced hippocampal glutamate (Glu), choline-containing compounds (tCho) and N-acetylaspartate (NAA)+N-acetyl-aspartyl-glutamic acid (NAAG) (tNAA) levels, whereas opposite effects were seen for running. No association of these effects with markers of central inflammation could be observed. These findings suggest that while voluntary wheel running alone is insufficient to prevent the unfavorable peripheral sequelae of the diet, it counteracted many changes in brain metabolites. The observed effects seem to be independent of neuroinflammation.

Validation of serum IGF-I as a biomarker to monitor the bioactivity of exogenous growth hormone agonists and antagonists in rabbits.

The development of new growth hormone (GH) agonists and growth hormone antagonists (GHAs) requires animal models for pre-clinical testing. Ideally, the effects of treatment are monitored using the same pharmacodynamic marker that is later used in clinical practice. However, intact rodents are of limited value for this purpose because serum IGF-I, the most sensitive pharmacodynamic marker for the action of GH in humans, shows no response to treatment with recombinant human GH and there is little evidence for the effects of GHAs, except when administered at very high doses or when overexpressed. As an alternative, more suitable model, we explored pharmacodynamic markers of GH action in intact rabbits. We performed the first validation of an IGF-I assay for the analysis of rabbit serum and tested precision, sensitivity, linearity and recovery using an automated human IGF-I assay (IDS-iSYS). Furthermore, IGF-I was measured in rabbits of different strains, age groups and sexes, and we monitored IGF-I response to treatment with recombinant human GH or the GHA Pegvisomant. For a subset of samples, we used LC-MS/MS to measure IGF-I, and quantitative western ligand blot to analyze IGF-binding proteins (IGFBPs). Although recovery of recombinant rabbit IGF-I was only 50% in the human IGF-I assay, our results show that the sensitivity, precision (1.7-3.3% coefficient of variation) and linearity (90.4-105.6%) were excellent in rabbit samples. As expected, sex, age and genetic background were major determinants of IGF-I concentration in rabbits. IGF-I and IGFBP-2 levels increased after single and multiple injections of recombinant human GH (IGF-I: 286±22 versus 434±26 ng/ml; P<0.01) and were highly correlated (P<0.0001). Treatment with the GHA lowered IGF-I levels from the fourth injection onwards (P<0.01). In summary, we demonstrated that the IDS-iSYS IGF-I immunoassay can be used in rabbits. Similar to rodents, rabbits display variations in IGF-I depending on sex, age and genetic background. Unlike in rodents, the IGF-I response to treatment with recombinant human GH or a GHA closely mimics the pharmacodynamics seen in humans, suggesting that rabbits are a suitable new model to test human GH agonists and antagonists.

Mice selected for extremes in stress reactivity reveal key endophenotypes of major depression: a translational approach.

Clear evidence has linked dysregulated hypothalamus-pituitary-adrenocortical (HPA) axis function to the aetiology and pathophysiology of major depression (MD), as observed in the majority of patients. Increased stress reactivity and hyperactivity of the HPA axis seem characteristic for psychotic/melancholic depression, while the atypical subtype of depression has been connected with the opposing phenotypes. However, the underlying molecular-genetic mechanisms are poorly understood. In the present study, mouse lines selectively bred for extremes in stress reactivity (SR), i.e. presenting high (HR) or low (LR) corticosterone secretion in response to stressors, were used to characterise the molecular alterations on all levels of the HPA axis. Results were contrasted with clinical phenotypes of MD patients from the Munich Antidepressant Response Signature project, stratified according to their cortisol response in the Dex/CRH test. Distinct differences between HR and LR mice were found in the expression of HPA axis-related genes in the adrenals, pituitary and selected brain areas. Moreover, HR animals presented an enhanced adrenal sensitivity, increased stress-induced neuronal activation in the PVN and an overshooting Dex/CRH test response, whereas LR animals showed a blunted response in these paradigms. Interestingly, analogous neuroendocrine, morphometric, psychopathological and behavioural differences were observed between the respective high and low HPA axis responder groups of MD patients. Our findings suggests that (i) the SR mouse model can serve as a valuable tool to elucidate HPA axis-related mechanisms underlying affective disorders and (ii) a stratification of MD patients according to their HPA axis-related neuroendocrine function should be considered for clinical research and treatment.

Effects of low-carbohydrate, high-fat diets on apparent digestibility of minerals and trace elements in rats.

OBJECTIVE: Ketogenic low-carbohydrate, high-fat (LCHF) diets reduce growth and bone mineral density in children with epilepsy and in rats. Part of this effect might be due to a reduced availability of calcium in high-fat diets. The aim of this study was to determine mineral digestibility by total collection method in LCHF diets compared with a chow diet and a standard high-fat diet (HFD, high in fat and carbohydrates). METHODS: Twelve-wk-old male Wistar rats were pair-fed isoenergetic amounts of either six different LCHF diets based on tallow and casein (crude fat 75%-50%, crude protein 10%-35%), with chow or with a HFD diet. Mineral-to-energy ratio was matched in all diets. Circulating parathyroid hormone was measured by immunoassay. RESULTS: The apparent digestibility of calcium was reduced in all HFDs (high-fat diets, LCHF diets and the HFD diet) by at least 30% compared with the chow diet (P < 0.001). Fecal calcium excretion correlated positively with fecal fat excretion, presumably because of formation of calcium soaps. Apparent digestibility of phosphorous was higher in all HFDs. This resulted in a decrease of the ratio of apparently digested calcium to apparently digested phosphorous in all HFDs below a ratio of 1:1. Plasma parathyroid hormone was not affected by any diet. CONCLUSION: The alteration of apparent calcium and phosphorus digestibility may affect the impact of HFDs on bone metabolism.