Bone development and age-related bone loss in male C57BL/6J mice

The objective of this study was to examine changes in the long bones of male C57BL/6J mice with growth and aging, and to consider the applicability of this animal for use in studying Type II osteoporosis. Male C57BL/6J mice were aged in our colony between 4 and 104 weeks (n=9-15/group). The right femur and humeri were measured for length and subjected to mechanical testing (3-point flexure) and compositional analysis. The left femurs were embedded and thick slices at the mid-diaphysis were assessed for morphology, formation indices, and bone structure. In young mice, rapid growth was marked by substantial increases in bone size, mineral mass, and mechanical properties. Maturity occurred between 12 and 42 weeks of age with the maintenance of bone mass and mechanical properties. From peak levels, mice aged for 104 weeks experienced decreased whole femur mass (12.1 and 18.6% for dry and ash mass, respectively), percentage mineralization (7.4%), diminished whole bone stiffness (29.2%), energy to fracture (51.8%), and decreased cortical thickness (20.1%). Indices of surface-based formation decreased rapidly from the onset of the study. However, the periosteal perimeter and, consequently, the cross-sectional moments of inertia continued to increase through 104 weeks, thus maintaining structural properties. This compensated for cortical thinning and increased brittleness due to decreased mineralization and stiffness. The shape of the mid-diaphysis became increasingly less elliptical in aged mice, and endocortical resorption and evidence of subsequent formation were present in 20-50% of femurs aged > or =78 weeks. This, combined with the appearance of excessive endocortical resorption after 52 weeks, indicated a shift in normal mechanisms regulating bone shape and location, and was suggestive of remodeling. The pattern of bone loss at the femoral mid-diaphysis in this study is markedly similar to that seen in cortical bone in the human femoral neck in Type II osteoporosis. This study has thus demonstrated that the male C57BL/6J mouse is a novel and appropriate model for use in studying endogenous, aging-related osteopenia and may be a useful model for the study of Type II osteoporosis.     

Age-related changes in trabecular architecture differ in female and male C57BL/6J mice.

We used microCT and histomorphometry to assess age-related changes in bone architecture in male and female C57BL/6J mice. Deterioration in vertebral and femoral trabecular microarchitecture begins early, continues throughout life, is more pronounced at the femoral metaphysis than in the vertebrae, and is greater in females than males. INTRODUCTION: Despite widespread use of mice in the study of musculoskeletal disease, the age-related changes in murine bone structure and the relationship to whole body BMD changes are not well characterized. Thus, we assessed age-related changes in body composition, whole body BMD, and trabecular and cortical microarchitecture at axial and appendicular sites in mice. MATERIALS AND METHODS: Peripheral DXA was used to assess body composition and whole body BMD in vivo, and microCT and histomorphometry were used to measure trabecular and cortical architecture in excised femora, tibia, and vertebrae in male and female C57BL/6J mice at eight time-points between 1 and 20 mo of age (n = 6-9/group). RESULTS: Body weight and total body BMD increased with age in male and female, with a marked increase in body fat between 6 and 12 mo of age. In contrast, trabecular bone volume (BV/TV) was greatest at 6-8 wk of age and declined steadily thereafter, particularly in the metaphyseal region of long bones. Age-related declines in BV/TV were greater in female than male. Trabecular bone loss was characterized by a rapid decrease in trabecular number between 2 and 6 mo of age, and a more gradual decline thereafter, whereas trabecular thickness increased slowly over life. Cortical thickness increased markedly from 1 to 3 mo of age and was maintained or slightly decreased thereafter. CONCLUSIONS: In C57BL/6J mice, despite increasing body weight and total body BMD, age-related declines in vertebral and distal femoral trabecular bone volume occur early and continue throughout life and are more pronounced in females than males. Awareness of these age-related changed in bone morphology are critical for interpreting the skeletal response to pharmacologic interventions or genetic manipulation in mice.     

Expression of RANKL and OPG correlates with age-related bone loss in male C57BL/6 mice.

Osteoblasts regulate the recruitment and activity of osteoclasts through expression of RANKL and osteoprotegerin (OPG). To determine whether expression of RANKL and OPG change with age and how these changes relate to the bone loss of aging, we measured bone mass and cancellous volume, and expression of RANKL, OPG, alkaline phosphatase (AP), osteocalcin (OC), and alpha I collagen (COLL) in whole bone and osteoblast-like cells in culture using 6-week- (young), 6-month- (adult), and 24-month-old (old) mice. Cancellous volume decreased by 20% from young to adult and by 52% from adult to old. RANKL mRNA levels in whole bone were 2.1-fold and 4.4-fold higher in adult and old mice, respectively, compared with young mice, whereas OPG mRNA levels decreased with age slightly. RANKL expression was negatively (r = -0.99) and OPG was positively (r = 0.92) correlated with cancellous bone volume. Expression of RANKL was higher and OPG lower in cells from older animals early in culture (day 7). With cell maturation, RANKL mRNA levels in cells from young and adult mice increased, whereas levels in cells from old animals decreased. By 21 and 28 days of culture, no differences were found in RANKL mRNA in osteoblast-like cells among different age groups. We conclude that expression of RANKL and OPG change with age in whole bone and in cultured osteoblast-like cells. These changes favor increased osteoclast over osteoblast activity, and may explain, in part, the imbalance in bone formation and resorption associated with aging.     

Pharmacological inhibition of PPARγ increases osteoblastogenesis and bone mass in male C57BL/6 mice

Infiltration of bone marrow with fat is a prevalent feature in people with age‐related bone loss and osteoporosis, which correlates inversely with bone formation and positively with high expression levels of peroxisomal proliferator‐activated receptor gamma (PPARγ). Inhibition of PPARγ thus represents a potential therapeutic approach for age‐related bone loss. In this study, we examined the effect of PPARγ inhibition on bone in skeletally mature C57BL/6 male mice. Nine‐month‐old mice were treated with a PPARγ antagonist, bisphenol‐A‐diglycidyl ether (BADGE), alone or in combination with active vitamin D (1,25[OH]₂D₃) for 6 weeks. Micro‐computed tomography and bone histomorphometry indicated that mice treated with either BADGE or BADGE + 1,25(OH)₂D₃ had significantly increased bone volume and improved bone quality compared with vehicle‐treated mice. This phenotype occurred in the absence of alterations in osteoclast number. Furthermore, the BADGE + 1,25(OH)₂D₃‐treated mice exhibited higher levels of unmineralized osteoid. All of the treated groups showed a significant increase in circulating levels of bone formation markers without changes in bone resorption markers, while blood glucose, parathyroid hormone, and Ca⁺ remained normal. Furthermore, treatment with BADGE induced higher levels of expression of vitamin D receptor within the bone marrow. Overall, treated mice showed higher levels of osteoblastogenesis and bone formation concomitant with decreased marrow adiposity and ex vivo adipogenesis. Taken together, these observations demonstrate that pharmacological inhibition of PPARγ may represent an effective anabolic therapy for osteoporosis in the near future. © 2013 American Society for Bone and Mineral Research.     

SEM and TEM study of the hierarchical structure of C57BL/6J and C3H/HeJ mice trabecular bone

Transmission electron microscopy (TEM) and scanning electron microscopy (SEM) were used to study the hierarchical structure of trabecular bone from C57BL/6J (low bone mass) and C3H/HeJ mice (high bone mass). Bone was harvested from two different anatomical locations: femoral metaphysis and L5 vertebra. This investigation focused on three structural scales: the mesostructural (porous network of trabecular struts), the microstructural (collagen fibril arrangements in trabecular packets), and the nanostructural (collagen fibril and apatite crystals) levels. At the mesostructural level, no distinct differences were found in the trabecular structure of femoral metaphysis but thinner trabecular struts were observed in L5 vertebra for C57BL/6J mice strain. At the microstructural level, the collagen fibrils forming the rotated, twisted, and orthogonal plywood arrangements were distinguished as well as atypical arrangements. At the nanostructural level, the shape and size of apatite crystals, and their arrangement with respect to collagen fibrils were studied. In spite of very different bone mass densities, both mice strains had similar structures at the nanostructural and microstructural levels.     

Lower bone cellular activities in male and female mature C3H/HeJ mice are associated with higher bone mass and different pyridinium crosslink profiles compared to C57BL/6J mice

The female inbred strains of C3H/HeJ (C3H) and C57BL/6J mice (B6), having high and low femoral peak bone mass, respectively, were proposed as models for studying the genetic regulation of bone mass. Here, we compared the known bone phenotype, in 4.5-month-old C3H versus B6 mice, in both genders. Femoral bone mineral content, trabecular bone mass, and thickness at the distal metaphysis were higher in C3H mice. In the long bones, deoxypyridinoline content was lower and pyridinoline/deoxypyridinoline ratios were greater in C3H. Intrafibrillar collagen packing is different not only within strains but also within sexes. Bone resorption activity, evaluated by urinary pyridinium crosslinks and active resorption surfaces in the femoral metaphysis, was lower in C3H. Bone formation activity, evaluated by serum osteocalcin and alkaline phosphatase (ALP) levels, as well as histomorphometric indices of bone formation in the femoral metaphysis and the cortical tibia, was lower in C3H. Conversely, the ALP- and Von Kossa-positive colony-forming units were more numerous in bone marrow cell cultures originating from male C3H. In both strains, resorption and formation activities were lower in males than in females. In C3H, males had lower bone mass than females whereas the opposite was seen in B6. In conclusion, we found that the lower cellular activities in C3H were associated with high cancellous bone mass and pyridinium crosslink levels, which might account for the more mineralized bone in C3H mice compared to that in B6 mice.     

Genetic effects for femoral biomechanics, structure, and density in C57BL/6J and C3H/HeJ inbred mouse strains.

Genome-wide QTL analysis for bone density, structure, and biomechanical phenotypes was performed in 999 (B6xC3H)F2 mice. Multivariate phenotypes were also derived to test for pleiotropic QTL effects. Highly significant QTLs were detected with pleiotropic effects on many of these phenotypes, and QTLs with unique effects on specific phenotypes were found as well. INTRODUCTION: The inbred C57BL/6J (B6) and C3H/HeJ (C3H) mouse strains were previously shown to segregate quantitative trait loci (QTLs) for femoral bone density. MATERIALS AND METHODS: The 999 s filial (F2) mouse progeny were further phenotyped for measures of femoral biomechanics (load to failure, Fu; work to failure, U; stiffness, S), structure (polar moment of inertia, Ip; moment of inertia ratio, Ir), and more specific femoral midshaft bone density measures (cortical and total vBMD). Two novel multivariate phenotypes were computed using principal component analysis, thus aiding in the exploration of pleiotropic effects of the QTLs detected. RESULTS AND CONCLUSIONS: Results of a genome-wide analysis provided strong evidence of pleiotropic QTL effects on chromosome 4, with six of the seven primary phenotypic measures, representing femoral biomechanics, density, and structure, producing LOD scores greater than 8. Chromosomes 1, 8, 13, and 14 were also identified as harboring QTLs that affect phenotypes in two of the three aspects of bone properties. QTLs uniquely contributing to variability in biomechanical measures were identified on chromosomes 10 and 12, whereas a QTL solely affecting structure was found on chromosome 17. Analysis of the evidence for pleiotropic effects using principal component analysis revealed pleiotropic QTLs on chromosomes 4 and 14, influencing nearly all the bone phenotypes measured and revealed QTLs on chromosomes 1, 8, 13, and 17 with pleiotropic effects restricted to either density or the structure and stiffness phenotypes. The use of multivariate phenotypes has allowed us to identify pleiotropic effects of several QTLs previously linked in studies of other mouse strains and in human studies of bone mineral density and femoral structure, which will provide important insight regarding the importance of allelic variation on the entire skeleton.     

Bone brittleness varies with genetic background in A/J and C57BL/6J inbred mice.

The contribution of genetic and environmental factors to variations in bone quality are understood poorly. We tested whether bone brittleness varies with genetic background using the A/J and C57BL/6J inbred mouse strains. Whole bone four-point bending tests revealed a 70% decrease in postyield deflection of A/J femurs compared with C57BL/6J, indicating that A/J femurs failed in a significantly more brittle manner. Cyclic loading studies indicated that A/J femurs accumulated damage differently than C57BL/6J femurs, consistent with their increased brittleness. Differences in matrix composition also were observed between the two mouse strains. A/J femurs had a 4.5% increase in ash content and an 11.8% decrease in collagen content. Interestingly, a reciprocal relationship was observed between femoral geometry and material stiffness; this relationship may have contributed to the brittle phenotype of A/J femurs. A/J femurs are more slender than those of C57BL/6J femurs; however, their 47% smaller moment of inertia appeared to be compensated by an increased tissue stiffness at the expense of altered tissue damageability. Importantly, these differences in whole bone mechanical properties between A/J and C57BL/6J femurs could not have been predicted from bone mass or density measures alone. The results indicated that bone brittleness is a genetically influenced trait and that it is associated with genetically determined differences in whole bone architecture, bone matrix composition, and mechanisms of cyclical damage accumulation.     

Strain differences in bone density and calcium metabolism between C3H/HeJ and C57BL/6J mice.

Previous reports indicate that peak bone density is significantly higher in C3H/HeJ (C3H) than in C57BL/6J (C57BL) mice, making these two inbred strains useful models for studying the genetic basis for peak bone density. The following study was undertaken to examine whether strain differences in the bone density of C3H and C57BL mice are associated with differences in intestinal calcium (Ca) absorption. Calcium absorption was measured by the balance technique and animals received two injections of fluorochromes 5 days apart before killing. Subsequently, the femurs were removed and, following measurement of volumetric density, the left femur was divided into three equal parts and the middle third served as the femoral cortical diaphysis. Femur diaphyseal volumetric bone density, ash, and Ca content were 10%, 29%, and 29% higher in C3H than in C57BL mice (p < 0.001), respectively. Bone length, periosteal mineral apposition rate, and periosteal bone formation rate of femoral diaphyseal cortical bone were not significantly different between the two strains of mice, but the marrow area of C57BL mice was almost twofold that of C3H mice (p < 0.0001). Intestinal Ca absorption and 1,25-dihydroxyvitamin D [1,25(OH)2D]-stimulated Ca2+ uptake by intestinal mucosal cells were 38% and 51% higher in C3H than in C57BL mice p < 0.001), respectively. Serum Ca and 1,25(OH)2D levels were 6% and 32% higher in C3H than in C57BL mice (p < 0.001), respectively, and the number of intestinal-occupied vitamin D receptors was 51% higher in C3H than in C57BL mice (p < 0.01). In a second experiment, three groups of C3H mice and three groups of C57BL mice were fed diets that contained 0.4%, 0.1%, or 0.02% Ca, and serum Ca, 1,25(OH)2D, parathyroid hormone (PTH), and intestinal Ca absorption measured. At all dietary Ca levels, C3H mice maintained positive Ca absorption and absorbed significantly more Ca than C57BL mice. In contrast, at low dietary Ca levels (0.1% and 0.02% Ca), C57BL mice maintained negative Ca absorption. Low dietary Ca increased serum PTH significantly in C57BL but not in C3H mice, and decreased serum 1,25(OH)2D and Ca levels in both strains of mice. Our findings indicate that the C57BL mice relied more on the mobilization of Ca from bone to maintain extracellular Ca homeostasis than the C3H mice. We conclude that strain differences in bone mass and density between C3H and C57BL mice is expressed, in part, through the vitamin D and PTH endocrine systems and their effects on the maintenance of extracellular Ca homeostasis.     

12 C57BL/6J小鼠肝癌动物模型的建立

目的:建立C57BL/6J小鼠肝癌动物模型，为深入开展肝细胞癌的实验研究打下基础。方法:取90只C57BL/6J小鼠，联合采用二甲基亚硝胺（DEN）/四氯化碳（CCl4）/乙醇诱导20周，观察其肝癌的发生情况。另取10只同种小鼠作为正常对照组。RT-PCR法检测病变肝组织中AFP基因表达。结果:实验组90只小鼠共死亡8只；病理学检查发现，存活的82只小鼠中有71只发生肝癌，诱癌成功率为78.9%（71/90），肝癌小鼠的肝癌组织学类型以中、高分化为主；另11只有不同程度的药物中毒性肝炎或肝硬化（12.2%）；对照组小鼠肝脏均未发现明显异常。成癌组中的肝癌组织RT-PCR检测可见AFP基因表达，非肝癌组织及对照组于组织中无AFP表达。结论:采用DEN/CCl4/乙醇联合能诱导出C57BL/6J甲胎蛋白分泌型小鼠肝癌，诱导时间相对较短，癌变率高，部分病变肝组织中伴有肝炎、肝硬化病理学改变，是较理想的研究肝癌的实验动物模型。     

Diet-induced type II diabetes in C57BL/6J mice

We investigated the effects of diet-induced obesity on glucose metabolism in two strains of mice, C57BL/6J and A/J. Twenty animals from each strain received ad libitum exposure to a high-fat high-simple-carbohydrate diet or standard Purina Rodent Chow for 6 mo. Exposure to the high-fat, high-simple-carbohydrate, low-fiber diet produced obesity in both A/J and C57BL/6J mice. Whereas obesity was associated with only moderate glucose intolerance and insulin resistance in A/J mice, obese C57BL/6J mice showed clear-cut diabetes with fasting blood glucose levels of greater than 240 mg/dl and blood insulin levels of greater than 150 microU/ml. C57BL/6J mice showed larger glycemic responses to stress and epinephrine in the lean state than AJ mice, and these responses were exaggerated by obesity. These data suggest that the C57BL/6J mouse carries a genetic predisposition to develop non-insulin-dependent (type II) diabetes. Furthermore, altered glycemic response to adrenergic stimulation may be a biologic marker for this genetic predisposition to develop type II diabetes.     

A time course of bone response to jump exercise in C57BL/6J mice

 Exercise, by way of mechanical loading, provides a physiological stimulus to which bone tissue adapts by increased bone formation. The mechanical stimulus due to physical activity depends on both the magnitude and the duration of the exercise. Earlier studies have demonstrated that jump training for 4 weeks produces a significant bone formation response in C57BL/6J mice. An early time point with significant increase in bone formation response would be helpful in: (1) designing genetic quantitative trait loci (QTL) studies to investigate genes regulating the bone adaptive response to mechanical stimulus; and (2) mechanistic studies to investigate early stimulus to bone tissue. Consequently, we investigated the bone structural response after 2, 3, and 4 weeks of exercise with a loading cycle of ten jumps a day. We used biochemical markers and peripheral quantitative computed tomography (pQCT) of excised femur to measure bone density, bone mineral content (BMC), and area. Four-week-old mice were separated into control (n= 6) and jump groups (n= 6), and the latter groups of mice were subjected to jump exercise of 2-week, 3-week, and 4-week duration. Data (pQCT) from a mid-diaphyseal slice were used to compare bone formation parameters between exercise and control groups, and between different time points. There was no statistically significant change in bone response after 2 weeks of jump exercise as compared with the age-matched controls. After 3 weeks of jump exercise, the periosteal circumference, which is the most efficient means of measuring adaptation to exercise, was increased by 3% (P < 0.05), and total and cortical area were increased by 6% (P < 0.05) and 11% (P < 0.01), respectively. Total bone mineral density (BMD) increased by 11% (P < 0.01). The biggest changes were observed in cortical and total BMC, with the increase in total BMC being 12% (P < 0.01). Interestingly, the increase in BMC was observed throughout the length of the femur and was not confined to the mid-diaphysis. Consistent with earlier studies, mid-femur bone mass and area remained significantly elevated in the 4-week exercise group when compared with the control group of mice. The levels of the biochemical markers osteocalcin, skeletal alkaline phosphatase, and C-telopeptide were not significantly different between the exercise and control groups, indicating the absence of any systemic response due to the exercise. We conclude that a shorter exercise regimen, of 3 weeks, induced a bone response that was greater than or equal to that of 4 weeks of jump exercise reported earlier. Received: October 1, 2001 / Accepted: January 18, 2002     

Mapping of putative ether-anesthesia resistance gene using C57BL/6J and MSM/Ms mouse strains

Purpose We attempted to identify the locations of major mouse genes responsible for sensitivity to diethylether (ether) anesthesia, using microsatellite linkage analyses including Quantitative Trait Locus (QTL) analysis.Methods To determine the locations of ether anesthesia resistance genes on chromosomes, an ether anesthesia-resistant mouse strain, C57BL/6J (C57BL), and an ether anesthesia-sensitive mouse strain, MSM/Ms (MSM), were used. The sensitivity of mice to ether anesthesia was determined from the latency time required to lose the righting reflex during exposure to 4% ether vapor in air. The (C57BL × MSM) F₁ mice were found to be resistant to ether, showing that the resistant phenotype is genetically dominant. Twelve resistant and 12 sensitive mice were then selected from the 196 backcrossed F₂ mice (F₁ × MSM) at 11–16 weeks of age. Genomic DNA samples were extracted from the tails for mapping ether anesthesia-related genes using microsatellite linkage analyses.Results One major putative gene related to resistance to ether anesthesia was restricted in the region 23 to 37cM from the centromere in chromosome 7 by primary and secondary linkage analyses. The QTL analysis narrowed the position of the gene to 29.0cM, with a maximum logarithm of odds (LOD) score of 3.03, and it was termed Etan1 (ether-anesthesia 1).Conclusion Microsatellite linkage analyses, including QTL analysis, determined the location of the ether-resistance gene, Etan1, within a narrow range. Our findings should be helpful for further experiments, such as cloning of the gene governing the sensitivity to ether anesthesia in mice.     

Analysis of differential survival of syngeneic islets transplanted into hyperglycemic C57BL/6J versus C57BL/KsJ mice

C57BL/KsJ (BKs) male mice were more sensitive to diabetes induction by administration of multiple low-doses of streptozotocin (Sz) than were C57BL/6J (B6) male mice. Analysis of islet size and insulin content of the two parental strains did not indicate that differences in drug sensitivity could be attributed to an effect of genetic background on islet size or insulin content. 50 BKs islets implanted into the spleens of BKs male mice made diabetic by Sz were eliminated within 12 days posttransplantation, whereas an equal number of B6 islets implanted into the spleens of diabetic B6 recipients were retained, even though the numbers of islets implanted were insufficient to effect remission from hyperglycemia. In contrast to the rapid loss of islets implanted into spleens of hyperglycemic BKs recipients, BKs islets implanted into spleens of normoglycemic recipients were not eliminated, thus suggesting that the basis for the differential survival between the B6 and BKs strains reflected their ability to survive hyperglycemic stress rather than a differential ability to replicate. Since BKs beta cells have been shown to respond to hyperglycemia by expression of an endogenous retroviral gene that cannot be expressed by B6 beta cells, the possibility that this differential survival represents a strain difference in autoreactivity against islet cells is raised.     

Ultrafine mapping of SNPs from mouse strains C57BL/6J, DBA/2J, and C57BLKS/J for loci contributing to diabetes and atherosclerosis susceptibility

The inbred mouse strain C57BLKS/J (BKS) carrying a mutation of the leptin receptor lepr(-/-) (BKS-db) is a classic mouse model of type 2 diabetes. While BKS was originally presumed to be a substrain of C57BL/6J (B6), it has become apparent that its genome contains introgressed regions from a DBA/2 (DBA)-like strain and perhaps other unidentified sources. It has been hypothesized that the strikingly enhanced diabetes susceptibility of BKS-db compared with B6-db is conferred by this introgressed DNA. Using high-density single nucleotide polymorphisms, we have mapped the DBA and other contaminating DNA regions present in BKS. Thus, approximately 70% of its genome appears to derive from B6, with approximately 20% from DBA and another 9% from an unidentified donor. Comparison with 56 diverse inbred strains suggests that this donor may be a less common inbred strain or an outbred or wild strain. Using expression data from a B6 x DBA cross, we identified differentially regulated genes between these two strains. Those cis-regulated genes located on DBA-like blocks in BKS constitute primary candidates for genes contributing to diabetes susceptibility in the BKS-db strain. To further prioritize these candidates, we identified those cis-acting expression quantitative trait loci whose expression significantly correlates with diabetes-related phenotypes.     

Increased glycogen synthase kinase-3 activity in diabetes- and obesity-prone C57BL/6J mice.

Although the precise mechanisms contributing to insulin resistance and type 2 diabetes are unknown, it is believed that defects in downstream components of the insulin signaling pathway may be involved. In this work, we hypothesize that a serine/threonine kinase, glycogen synthase kinase-3 (GSK-3), may be pertinent in this regard. To test this hypothesis, we examined GSK-3 activity in two inbred mouse strains known to be susceptible (C57BL/6J) or resistant (A/J) to diet-induced obesity and diabetes. Examination of GSK-3 in fat, liver, and muscle tissues of C57BL/6J mice revealed that GSK-3 activity increased twofold in the epididymal fat tissue and remained unchanged in muscle and liver of mice fed a high-fat diet, compared with their low-fat diet-fed counterparts. In contrast, GSK-3 activity did not change in the epididymal fat tissue of A/J mice, regardless of the type of diet they were fed. In addition, both basal and diet-induced GSK-3 activity was higher (2.3- and 3.2-fold, respectively) in the adipose tissue of C57BL/6J mice compared with that in A/J mice. Taken together, our studies suggest an unsuspected link between increased GSK-3 activity and development of insulin resistance and type 2 diabetes in fat tissue of C57BL/6J mice, and implicate GSK-3 as a potential factor contributing to susceptibility of C57BL/6J mice to diet-induced diabetes.     

A pentadecapeptide fragment of islet neogenesis-associated protein increases beta-cell mass and reverses diabetes in C57BL/6J mice

OBJECTIVE: The objective of this study was to demonstrate that islet neogenesis-associated protein (INGAP) peptide, a pentadecapeptide containing the biologically active portion of native INGAP, increases functional beta-cell mass in normal animals and can be used therapeutically to reverse hyperglycemia in streptozotocin-induced diabetes. SUMMARY BACKGROUND DATA: INGAP, a 175 amino acid pancreatic acinar cell protein, has been suggested to be implicated in beta-cell mass expansion. METHODS: In the first part of this study, normoglycemic hamsters were administered either 500 microg INGAP peptide (n = 30) or saline (n = 20) intraperitoneally daily and sacrificed after 10 or 30 days of treatment. Blood glucose and insulin levels were measured, and a histologic and morphometric analysis of the pancreas was performed to determine the effect of INGAP peptide on the endocrine pancreas. In the second part of the study, 6- to 8-week-old C57BL/6J mice (n = 8) were administered multiple low doses of the beta-cell toxin streptozotocin (STZ) inducing insulitis and hyperglycemia. The mice were then injected with INGAP peptide (n = 4) or saline (n = 4) for 39 days and sacrificed at 48 days. Two additional groups of diabetic mice were administered either a peptide composed of a scrambled sequence of amino acids from INGAP peptide (n = 5) or exendin-4 (n = 5), an incretin that has been associated with amelioration of hyperglycemia. RESULTS: Islet cell neogenesis was stimulated in INGAP-treated hamsters by 10 days. At 30 days, the foci of new endocrine cells had the appearance of mature islets. There was a 75% increase in islet number, with normal circulating levels of blood glucose and insulin. Administration of INGAP peptide to diabetic mice reversed the diabetic state in all animals, and this was associated with increased expression of PDX-1 in duct cells and islet cell neogenesis with a reduction of insulitis in the new islets. Diabetic mice treated with exendin-4 or a scrambled INGAP peptide did not revert from hyperglycemia. CONCLUSION: Because there is a deficiency of beta-cell mass in both type-1 and type-2 diabetes, INGAP peptide stimulation of fully functional neoislet differentiation may provide a novel approach for diabetes therapy.     

Tail suspension induces bone loss in skeletally mature mice in the C57BL/6J strain but not in the C3H/HeJ strain.

We assessed the effects of tail-suspension in two skeletal genetic backgrounds, the high C3H/HeJ (C3H) and low C57BL/6J (B6) bone masses inbred mice (male, 4-months old). Cancellous bone mass and structural parameters were evaluated in distal femoral metaphysis by three dimensional microcomputed tomography. Bone cellular activities were evaluated by histomorphometry and measurements of alkaline phosphatase activity (ALP) and osteocalcin in blood and deoxypyridinoline (D-pyr) in urine. In C3H mice, 2- and 3-week unloading experiments were performed. After an early and transient decrease in body weight, a 2-week suspension period resulted in stimulation of both bone formation rate by 45% and active osteoclastic surfaces by 19%. D-pyr did not change, but ALP and osteocalcin levels increased by 18% and 72%, respectively, in 2-week suspended mice, and osteocalcin remained elevated by 30% in the 3-week suspended mice. Such cellular modifications allowed the C3H mice to maintain their initial bone mass and trabecular structural parameters even after a 3-week suspension period. In B6 mice, 1- and 2-week unloading experiments were performed. Tail suspension resulted in decreased body weight during the first days followed by an incomplete recovery during the second week of unloading. The resorption activity was unaffected by any suspension time period, whereas a decrease of 42.5% in bone formation rate and of 21.5% in ALP were seen by the end of the first week of suspension, both values being restored after a 2-week suspension period. At this latter time, trabeculae were thinner, leading to a 24.5% cancellous bone loss. Trabecular number and connectivity, rod-plate index, and degree of anisotropy were not modified. We concluded that C3H mice constituted a unique model in which genetic background overwhelmed the usual effects of reduced biomechanical usage in bone, whereas B6 mice, compared with the standardized rat model, offered an alternative model of bone loss in a mature skeleton.