PD Mice Research Articles

Role of PTH1R internalization in osteoblasts and bone mass using a phosphorylation-deficient knock-in mouse model

Phosphorylation, internalization, and desensitization of G protein-coupled receptors, such as the parathyroid hormone (PTH) and PTH-related peptide (PTHrP) receptor (PTH1R), are well characterized and known to regulate the cellular responsiveness in vitro. However, the role of PTH1R receptor phosphorylation in bone formation and osteoblast functions has not yet been elucidated. In previous studies, we demonstrated impaired internalization and sustained cAMP stimulation of a phosphorylation-deficient (pd) PTH1R in vitro, and exaggerated cAMP and calcemic responses to s.c. PTH infusion in pdPTH1R knock-in mouse model. In this study, we examined the impact of impaired PTH1R phosphorylation on the skeletal phenotype of mice maintained on normal, low, and high calcium diets. The low calcium diet moderately reduced (P<0.05) bone volume and trabecular number, and increased trabecular spacing in both wild-type (WT) and pd mice. The effects, however, seem to be less pronounced in the female pd compared to WT mice. In primary calvarial osteoblasts isolated from 2-week-old pd or WT mice, PTH and PTHrP decreased phosphorylated extracellular signal-regulated kinases 1/2 (pERK1/2), a member of mitogen-activated protein kinase, and cyclin D1, a G₁/S phase cyclin, in vitro. In contrast to WT osteoblasts, down-regulation of cyclin D1 was sustained for longer periods of time in osteoblasts isolated from the pd mice. Our results suggest that adaptive responses of intracellular signaling pathways in the pd mice may be important for maintaining bone homeostasis.

The effects of pyridoxine deficiency and supplementation on hematological profiles, lymphocyte function, and hepatic cytochrome P450 in B6C3F1 mice

Pyridoxine, a B6 vitamin, is a co-factor in a variety of enzymatic reactions involved in intermediary metabolism. The effects of pyridoxine deficiency and supplementation on hematological profiles, lymphocyte function, and hepatic CYP1A1 and CYP2E1 were investigated in B6C3F1 mice fed a diet containing either 0 (i.e., pyridoxine-deficient diet, [PD]); or 7 mg pyridoxine-HCl/kg (i.e., control diet, [CD]) for 8 or 13 weeks followed by administration of 500 μg pyridoxine-HCl (IP) daily for either 2 (PD-S2 and CD-S2) or 3 (PD-S3 and CD-S3) consecutive days. Results demonstrated that erythrocyte aspartate aminotransferase activity coefficient (EAST-AC) values, which reflect host pyridoxine status, were significantly higher in PD mice than in CD mice, and dropped to control levels after supplementation. PD mice had significantly reduced weight gains, mean corpuscular volume (MCV), hemoglobin (HGB), and hematocrit (HCT) levels compared to CD mice after 8 and 13 weeks on the prescribed diet. In addition, PD mice had significantly lower circulating levels of total white blood cells, but higher red blood cell numbers after 8 weeks (compared to CD mice). Pyridoxine supplementation for 3 days restored HGB levels in PD mice to that of the unsupplemented CD controls; HCT, MCV and MCH levels were also increased in PD-S3 mice compared to their unsupplemented PD counterparts, but failed to reach comparable levels to those seen in mice fed a control diet. The pyridoxine-deficient diet also resulted in decreased mitogen stimulated T-lymphocyte proliferation after a 13-week feeding regimen and increased hepatic CYP1A1 activity that was reversed by pyridoxine supplementation. These studies demonstrate in a murine model that pyridoxine deficiency can cause multiple alterations that, in many cases, can be reversed by supplementation.

Tissue Mineral Distributions are Differentially Modified by Dietary Protein Deficiency and a Murine Nematode Infection

This study was designed to investigate whether mineral concentrations in the spleen, serum, and liver were modified by challenge infection with a gastrointestinal nematode, by infection dose, or by protein deficiency despite adequate dietary intakes of minerals. BALB/c mice fed protein-sufficient (PS, 24%) or protein-deficient (PD, 3%) diets were infected with 100 L3 of Heligmosomoides bakeri, drug-treated, and then re-infected with either 0, 100, or 200 L3. Protein deficiency and infection, but not dose, independently modified tissue mineral distributions. H. bakeri infection lowered serum iron concentrations in both diet groups. Despite this, PD mice had elevated iron and calcium concentrations and Ca/Zn ratio in the spleen as well as Fe/Zn ratio in liver, but they had reduced calcium, zinc, copper, and sulfur concentrations, and Cu/Zn ratio in the liver. Infection reduced calcium and iron concentrations and the Ca/Zn ratio in the spleen. We suggest that tissue mineral distribution is a consequence of Th2 immune and inflammatory responses induced by infection in PS mice and the switch to predominant Th1 inflammation in PD, nematode-infected mice.

Mechanisms underlying reduced expulsion of a murine nematode infection during protein deficiency

Balb/c mice infected with the gastrointestinal nematode Heligmosomoides bakeri were fed protein sufficient (PS, 24%) or deficient (PD, 3%) diets to investigate whether diet, infection or dose of larval challenge (0, 100 or 200 larvae) influenced gut pathophysiology and inflammation. Among the PS mice, worms were more posteriorad in the intestine of mice infected with 200 compared with 100 larvae, suggesting active expulsion in the more heavily infected mice. This was consistent with the positive correlation between worm numbers and fluid leakage in PS mice; similar patterns were not detected in the PD mice. Infection also induced villus atrophy, which was more pronounced in PS than in PD mice. Our cytokine screening array indicated that infection in PD mice elevated a wide range of pro-inflammatory cytokines and chemokines. Whereas serum leptin concentrations were higher in PD mice, monocyte chemotactic protein-5 (MCP-5) in serum increased with increasing larval dose and concentrations were lower in PD than PS mice. We suggest that elevated MCP-5 together with villus atrophy may contribute to the apparent dose-dependent expulsion of H. bakeri from PS mice but that delayed expulsion in PD mice appeared related to a predominant Th1 cytokine profile that may be driven by leptin.

TREADMILL EXERCISE TRAINING INDUCES ANGIOGENESIS and IMPROVES ENDURANCE and NEURONAL INDICATORS IN CHRONIC MOUSE MODEL OF PARKINSON'S DISEASE.

Purpose/Hypothesis: The goal of this study was to determine whether a 4-week treadmill running protocol could induce angiogenesis and improve endurance and neuronal indicators in a chronic MPTP/probenecid mouse model of Parkinson's disease. Number of Subjects: Twenty-three C57BL/6 mice were randomly assigned in 4 groups: sedentary control, exercise-trained control, sedentary PD, and exercise-trained PD. Materials/ Methods: The PD was induced by 10 chronic injections of 25 mg/kg MPTP and 250 mg/kg probenecid, which exhibited many symptoms of human PD. Exercised groups of mice were trained to run 40 min, 5 days/week for 4 weeks at an average speed of 18 m/min on a treadmill. Citrate synthase activity, cardiovascular parameters, dopamine level in sub-stantia nigra, tyrosine hydroxylase contents and blood vessels density in the striatum were analyzed and compared between the animal groups. Results: Compared to the respective sedentary groups, the soleus muscle citrate synthase activity increased by 21% in the exercise-trained PD group and 18% in the exercise-trained control group (p < 0.05). Citrate synthase activity in the gastrocnemius produced similar results. Resting heart rates declined significantly in both exercise-trained groups. PD showed a decrease in the resting heart rate by about 10%. Substantia nigra tyrosine hydroxylase content and striatal dopamine levels were increased nearly 30% in the exercise-trained PD over the sedentary PD mice, whereas these levels were indifferent between sedentary and exercise-trained controls. Exercise also resulted in a significant increase in blood vessel density in the striatum of trained PD animals compared to the sedentary group (p < 0.003). Conclusions: In summary, our study demonstrated that improvement in endurance and neuronal parameters as well as angiogenesis could be achieved in PD mice with a 4-week exercise training protocol. Clinical Relevance: According to our results, aerobic exercise could be beneficial for limited functional recovery in subjects with debilitating neurodegener-ative disorders, including Parkinson's disease

Effects of protein depletion on NK cell cytotoxicity and bone marrow cellularity.

Tumor bearing patients have repeatedly been shown to develop states of protein (PD). PD has frequently been associated with depression of acquired immunity. In contrast, little is known about the effects of PD on natural killer (NK) cell cytotoxicity and on bone marrow cellularity and how modulation of these parameters by maleic anhydride divinyl ether copolymer (MVE-2) would be affected. BALB/c mice 6-10 weeks old were fed normal diets (18% protein) (ND mice) or protein deficient isocaloric diets (PD mice) for 35 days. On days 5, 9, 15, 23, and 35 assays were performed. Three days prior to each assay day, some groups received MVE-2 i.p. In PD mice spontaneous NK activity was reduced by some 80%. After MVE-2 the levels only rose to half the amount of activity of ND mice, although in both groups an increase of NK activity took place. BMC levels of PD mice were also strongly decreased. After MVE-2, increases of BMC in PD mice were quite weak and did not reach the level of normal unstimulated BMC. In PD mice we found body weight loss of 46%, reduction in albumin and total protein of 22% and complete disappearance of prealbumin after 5 days of diets. Preliminary results indicate that repletion with amino acids (NeoAminomel L12.5 o.K.H. Salvia, Boehringer Mannheim Co.) is able restore NK activities and BMC levels.

Impairment of macrophage migration inhibitory factor synthesis and macrophage migration in protein-malnourished mice

Weanling CD2F 1 mice were fed isocaloric diets that were protein sufficient (PS; containing 27% casein) or protein deficient (PD; containing 8% casein). Weight measurements demonstrated that the growth of PD mice was significantly impaired, thus indicating that the PD diet induced protein malnutrition. The cellular immune responsiveness of these mice was assessed from Day 21 to Day 49 of the diet using, as indicators, in vitro production of migration inhibitory factor (MIF) by splenic lymphocytes and MIF responsiveness of peritoneal macrophages. PD lymphocytes, when stimulated with the polyclonal activator concanavalin A, produced significantly less MIF than did PS lymphocytes. The amount of MIF produced by PD lymphocytes, however, increased throughout the study, possibly indicating delayed maturation of MIF synthetic capacity in PD mice. Normal CD2F 1 mouse macrophages were used for these assays. MIF responsiveness of PD and PS macrophages was not significantly different when assayed using MIF produced by normal CD2F 1 mouse lymphocytes. As compared to that of PS macrophages, the migratory ability of PD macrophages decreased progressively throughout the study. This impaired migratory ability did not interfere with MIF responsiveness of PD macrophages.