Physiological effects of housing density on C57BL/6J mice over a 9-month period.

The NRC has consistently recommended floor space for animals used in science and agriculture. For mice, the recommended floor space is 77.4 cm(2) (12 in(2)) for a 15- to 25-g mouse. The NRC noted that its recommendations were based on "best professional judgment" and encouraged alternatives that were data driven. As part of a continual effort of The Jackson Laboratory to ensure the health and well-being of production and research mice, while promoting cost-effective, state-of-the-art research, several density-driven studies have been conducted by lab researchers. The objectives of this study were to determine the effect of housing density on variables related to mouse physiology and air quality in cages and assess the value of specific measured variables in such studies. In the present study, we monitored C57BL/6J mice in individually ventilated cages from weaning until 9 mo of age. Housing densities were equivalent to 66.4 or 36.8 cm(2) per mouse (10.3 or 5.7 in(2)). Clinical physiological variables representing general health and well-being were measured. Hematological traits, plasma lipids, and glucose, growth, bone mineral density, and percent body fat did not differ between housing densities. In the more densely housed mice, however, adrenal glands were significantly smaller, heart rates were significantly less, and food consumption was less. Cage air microenvironment was evaluated for ammonia, carbon dioxide, temperature, and humidity in cages changed weekly or every 2 wk. The cage microenvironment remained within acceptable limits at the higher density of mice at both cage-changing frequencies. The results suggest that mice housed for as long as 9 mo at up to twice the density currently recommended by NRC show no measurable adverse effects. Continued re-evaluation of the recommendation by measuring additional relevant variables of health and general well-being, and studying additional strains of mice is warranted.

Gender- and compartment-specific bone loss in C57BL/6J mice: correlation to season?

Seasonal variation in bone mineral density (BMD) has been documented in humans, and has been attributed to changes in 25-hydroxyvitamin D [25(OH)D] synthesis. To test the hypothesis that seasonal changes in bone mass occur in laboratory mice, we measured body composition, femoral bone phenotypes, and serum bone markers in 16-wk-old male and female C57BL/6 (B6) mice during the summer (June-August) and winter (December-February) months at The Jackson Laboratory in Bar Harbor, Maine. Both male and female B6 mice had higher volumetric BMD in the summer than winter. Females showed reduced trabecular bone, whereas males showed changes in bone volume. Males, but not females, had higher insulin-like growth factor 1 in summer than in winter, and only males showed an increase in body weight during the winter. No seasonal differences in serum TRAP5b, osteocalcin, or 25(OH)D were noted for either sex. We conclude that seasonal variation in skeletal and body composition parameters in B6 mice is significant and must be considered when performing longitudinal phenotyping of the skeleton. Further studies are needed to determine the environmental factors that cue seasonal changes in body composition and the mechanisms that produce these changes.

Quantitative trait loci mapping for cholesterol gallstones in AKR/J and C57L/J strains of mice.

Quantitative trait locus (QTL) mapping was used to locate genes that determine the difference in cholesterol gallstone disease between the gallstone-susceptible strain C57L/J and the gallstone-resistant strain AKR/J. Gallstone weight was determined in 231 male (AKR x C57L) F(1) x AKR backcross mice fed a lithogenic diet containing 1% cholesterol, 0.5% cholic acid, and 15% butterfat for 8 wk. Mice having no stones and mice having the largest stones were genotyped at approximately 20-cM intervals to find the loci determining cholesterol gallstone formation. The major locus, Lith1, mapped near D2Mit56 and was confirmed by constructing a congenic strain, AK. L-Lith1(s). Another locus, Lith2, mapped near D19Mit58 and was also confirmed by constructing a congenic strain AK.L-Lith2(s). Other suggestive, but not statistically significant, loci mapped to chromosomes 6, 7, 8, 10, and X. The identification of these Lith genes will elucidate the pathophysiology of cholesterol gallstone formation.

Serum levels of soluble Fas correlate with indices of organ damage in systemic lupus erythematosus.

OBJECTIVE: To evaluate whether the levels of soluble form of the Fas apoptosis antigen (sCD95/sFas) varied from those of healthy control subjects in a group of patients with systemic lupus erythematosus (SLE). This was done to determine whether sFas has a role in either the disease activity or the organ damage in SLE. METHODS: Serum levels of sFas were measured over a period of 4 y (277 determinations) in 39 Arab patients with SLE and 22 age-, gender-, and race-matched healthy controls using double antibody ELISA. SLEDAI scores for disease activity and SLICC/ACR scores for cumulative organ damage were determined. Serum levels of acute phase reactants, complement, inflammatory cell counts, levels of autoantibodies, and kidney and liver function test results were obtained retrospectively from clinical records. RESULTS: sFas levels were significantly higher in patients with SLE (n = 39, 277 determinations) (0.60 ng/ml +/- 0.38) than in healthy controls (n = 22) (0.26 ng/ml +/- 0.11) (P < 0.00001). The levels of sFas correlated with SLICC/ACR (r = 0.36; P < 0.02), but not with SLEDAI. sFas correlated with renal and liver function tests measured by s-creatinine (r = 0.38; P < 0.0001), creatinine clearance (r = -0.30, P < 0.001), s-albumin (r = -0.28, P < 0.0001), and ALT (r = 0.35; P < 0.00001), but did not correlate with the levels of acute phase reactants. CONCLUSION: sFas is elevated in sera of SLE patient. Since sFas correlates with indices of organ damage but not with disease activity, it may be a marker of organ damage in SLE and may act to protect certain organs from further damage by inhibiting Fas-mediated apoptosis.

Quantitative trait loci analysis for the differences in susceptibility to atherosclerosis and diabetes between inbred mouse strains C57BL/6J and C57BLKS/J.

Mice from the inbred strain C57BLKS/J (BKS) exhibit increased susceptibility to both diabetes and atherosclerosis compared to C57BL/6J (B6) mice. To determine whether the differences in diabetes and atherosclerosis are related, we carried out a cross between B6-db/db and BKS. We selected 99 female F2-db/db progeny, tested the progeny for plasma lipids, plasma glucose, and fatty-streak lesions, and used quantitative trait loci (QTL) analysis to identify the chromosomal regions associated with these phenotypes. No major QTL were found for total cholesterol, VLDL-cholesterol, or triglycerides. Two suggestive QTL were found for HDL-cholesterol (LOD scores of 2. 7 and 2.8), and two suggestive loci were found for plasma glucose (LOD scores of 2.3 and 2.0). Lesion size was not correlated with plasma lipid levels or glucose. Lesion size was determined by a locus at D12Mit49 with a LOD score of 2.5 and a significant likelihood ratio statistic. The gene for apolipoprotein apoB lies within the region, but apoB levels were similar in strains B6 and BKS. The QTL on Chr 12 was confirmed by constructing a congenic strain with BKS alleles in the QTL region on a B6 genetic background. We conclude that susceptibilities to diabetes and atherosclerosis are not conferred by the same genes in these strains and that a major gene on Chr 12, which we name Ath6, determines the difference in atherosclerosis susceptibility.

Strain distribution pattern for SSLP markers in the SWXJ recombinant inbred strain set: chromosomes 7 to X.

The SWXJ recombinant inbred (RI) set was developed for genetic analysis of heritable ovarian tumors. In this report we present data for 223 simple sequence length polymorphisms spanning Chromosomes (Chrs) 7-X to complete the genetic marking of this RI set. The strain distribution patterns (SDP) for these loci were combined with data from 19 other polymorphic genes, resulting in densely marked maps for Chrs 7-X. Combined with the 165 loci for Chr 1-6 reported previously (Svenson et al., Mamm. Genome 6, 867, 1995), the SWXJ RI set represents a powerful tool for mapping genes in neoplastic as well as other heritable disorders.

Strain distribution pattern for SSLP markers in the SWXJ recombinant inbred strain set: chromosomes 1 to 6.

We typed 147 simple sequence length polymorphisms in the SWXJ recombinant inbred (RI) strain set spanning Chromosomes (Chrs) 1-6. The strain distribution pattern for these loci was combined with data from 18 previously typed loci for SWXJ, resulting in new chromosome maps for this RI set, with an average density of 3.5 cM between loci. This is the first systematic effort to develop a more highly resolved genetic map for the SWXJ RI set and thereby improves the usefulness of this genetic tool for mapping genes underlying both simple and complex genetic disorders.

Identification of four chromosomal loci determining obesity in a multifactorial mouse model.

We previously described a new mouse model for multigenic obesity, designated BSB. We now report the use of a complete linkage map approach to identify loci contributing to body fat and other traits associated with obesity in this model. Four loci exhibiting linkage with body fat, or with the weights of four different fat depots, residing on mouse chromosomes 6, 7, 12, and 15, were identified and confirmed by analysis of additional BSB mice. Each of the four loci differed with respect to their effects on the percent of body fat, specific fat depots and plasma lipoproteins. The loci exhibited allele-specific, non-additive interactions. A locus for hepatic lipase activity was co-incident with the body fat and total cholesterol loci on chromosome 7, providing a possible mechanism linking plasma lipoproteins and obesity. The chromosome 7 locus affecting body fat, total cholesterol and hepatic lipase activity was isolated in congenic strains whose donor strain regions overlap with the chromosome 7 BSB locus. These results provide candidate genes and candidate loci for the analysis of human obesity.

Linkage mapping of 40 randomly isolated liver cDNA clones in the mouse.

We report the chromosomal mapping of 43 loci for 40 randomly isolated mouse liver cDNA clones by linkage analysis in an interspecific backcross of ((C57BL/6J x Mus spretus) x C57BL/6J). The clones were sequenced from both sides and a subset was examined for expression in various mouse tissues. Fifteen of the 40 mapped cDNA clones are either identical or strongly related to known sequences in GenBank, while 25 represent new genes. Additional loci mapped in this cross include 53 simple sequence repeat polymorphisms and 40 restriction fragment length variants from previously characterized cDNA markers. Nine homologous human genes were identified for 7 mouse liver cDNA clones. One clone that maps to mouse chromosome 3 (D3Ucla1) identified a novel homologous segment (synteny) on human chromosome 18q23 (D18S372E). These studies provide linkage mapping and initial characterization of random cDNA clones that may provide a resource for the positional candidate cloning of disease genes.

Coincidence of genetic loci for plasma cholesterol levels and obesity in a multifactorial mouse model.

We have examined backcross progeny derived from a cross of Mus spretus with C57BL/6J, that range from 1 to 50% carcass lipid (n = 215), and from 22 to 130 mg/dl plasma total cholesterol (n = 238). Statistical analysis revealed that distal mouse chromosome 7 exhibits significant linkage both to plasma total cholesterol (likelihood of the odds [LOD] 5.8) and to carcass lipid (LOD 3.8). A locus on chromosome 6 also shows significant linkage to plasma total cholesterol (LOD 5.6), but no linkage to carcass lipid. Neither chromosomal region contains any previously mapped genes likely to influence lipoprotein metabolism, indicating that novel genetic factors contributing to plasma lipoprotein levels have been identified.

Mechanisms controlling competence gene expression in murine fibroblasts stimulated with minimally modified LDL.

Mildly oxidized low density lipoprotein (minimally modified low density lipoprotein [MM-LDL] is capable of inducing gene expression in cells of the artery wall. In this study, we investigated the mechanisms that control the mRNA expression of JE, KC, c-myc, and c-fos in quiescent mouse L-cell fibroblasts stimulated with MM-LDL. The data demonstrate that MM-LDL induces increases greater than or equal to 20-fold in the levels of transcripts of these genes within 15-60 minutes. Of the four genes examined, JE and KC mRNA showed the greatest response to MM-LDL. The pattern of induction by MM-LDL is distinct from that observed in fibroblasts stimulated with serum, a known inducer of these genes. Treatment with cycloheximide (10 micrograms/ml) did not block the MM-LDL-induced increase in the mRNA levels of these genes. The increase of JE and KC mRNA levels in response to MM-LDL could be blocked by treatment with actinomycin D (5 micrograms/ml). In nuclear runoff studies, MM-LDL increased the transcription rate of JE and KC at 4 hours by 13-fold and fivefold, respectively. Small but reproducible stimulations of c-fos and c-myc transcription by MM-LDL were also observed. In addition, the half-life of JE mRNA was increased after addition of MM-LDL to fibroblasts, suggesting that the MM-LDL-induced accumulation of these mRNAs might be accomplished by both transcriptional and posttranscriptional mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Identification of a zinc finger protein that binds to the sterol regulatory element.

Cholesterol balance in mammalian cells is maintained in part by sterol-mediated repression of gene transcription for the low density lipoprotein receptor and enzymes in the cholesterol biosynthetic pathway. A promoter sequence termed the sterol regulatory element (SRE) is essential for this repression. With the use of an oligonucleotide containing the SRE to screen a human hepatoma complementary DNA expression library, a clone for a DNA binding protein was isolated that binds to the conserved SRE octanucleotide in both a sequence-specific and a single-strand--specific manner. This protein contains seven highly conserved zinc finger repeats that exhibit striking sequence similarity to retroviral nucleic acid binding proteins (NBPs). We have designated the protein "cellular NBP" (CNBP). CNBP is expressed in a wide variety of tissues, is up regulated by sterols, and exhibits binding specificity that correlates with in vivo function. These properties are consistent with a role in sterol-mediated control of transcription.

Biosynthetic relationships between three rat apolipoprotein B peptides.

Rat liver is unique in secreting very low density lipoproteins (VLDL) with three size-isoforms of apolipoprotein B: PI and PIII correspond to B-100 and B-48, respectively, while PII is slightly smaller than PI and has no counterpart in other species. Antibodies against a fusion protein corresponding to the extreme C-terminal region of PI fail to react with PII, suggesting that the latter lacks this moiety. [35S]Methionine-labeled perfused rat liver and isolated hepatocytes secrete labeled PII, but intracellular apoB contains only PI and PIII. The absence of labeled PII from Golgi VLDL, and the absence of continued PII production within the plasma compartment, strongly suggest that PIII-containing VLDL are formed by a one-time proteolytic processing of a certain proportion of PI-containing VLDL at the time of secretion. In contrast, polysome run-off translation experiments and analysis of polysome-bound nascent apoB chains show that both rat liver and intestinal polysomes release PIII-sized peptides directly at the appropriate point of elongation, in a manner incompatible with their formation by posttranslational processing. These results strongly suggest that the large (PI, B-100) and small (PIII, B-48) apoB peptides are translated from separate mRNAs. Thus, although both PII and PIII are C-terminally truncated products of PI, the mechanisms involved are entirely different.

Hormone-sensitive lipase: sequence, expression, and chromosomal localization to 19 cent-q13.3.

Hormone-sensitive lipase, a key enzyme in fatty acid mobilization, overall energy homeostasis, and possibly steroidogenesis, is acutely controlled through reversible phosphorylation by catecholamines and insulin. The 757-amino acid sequence predicted from a cloned rat adipocyte complementary DNA showed no homology with any other known lipase or protein. The activity-controlling phosphorylation site was localized to Ser563 in a markedly hydrophilic domain, and a lipid-binding consensus site was tentatively identified. One or several messenger RNA species (3.3, 3.5, or 3.9 kilobases) were expressed in adipose and steroidogenic tissues and heart and skeletal muscle. The human hormone-sensitive lipase gene mapped to chromosome 19 cent-q13.3.

Impaired glucose handling in active rheumatoid arthritis: relationship to peripheral insulin resistance.

Glucose metabolism was studied after an intravenous glucose loading in normal-weighted, previously untreated patients (n = 14) with active rheumatoid arthritis (RA). The patients displayed an enhanced insulin response and impaired glucose handling compared with healthy controls (P less than .001). The insulin sensitivity, measured as the glucose utilization rate during steady state of euglycemia (M) was significantly decreased (P less than .01) among the patients compared to the controls (5.5 +/- 1.9 mg/kg BW/min [mean +/- SD] and 7.2 +/- 1.2, respectively). The corresponding values for the metabolic clearance rate (MCR) were 5.8 +/- 0.6 mL/kg BW/min and 8.2 +/- 0.4, respectively (P less than .01). In the patient group the k value correlated with the peripheral insulin sensitivity (P less than .01), which, in turn, was inversely related to the acute phase reaction (P less than .05). During 1 week of potent anti-inflammatory treatment with corticosteroids (prednisolone 20 mg daily) the k value improved P less than .001), the insulin sensitivity tended to improve and the insulin response increased (P less than .001) after an intravenous glucose loading. Five patients who had a remission of their disease on sulphasalazine as antirheumatic therapy were reexamined. A normalization of the inflammatory activity as well as the glucose handling and insulin sensitivity was achieved. The data obtained indicate that impaired glucose handling in active RA is related to insulin resistance. The linkage between inflammatory indices and glucose metabolism might reflect a special consequence of inflammation, but the influence of nonspecific disease manifestations, ie, malnutrition, inactivity, and myopenia, has to be considered.

Serum lipoprotein in active rheumatoid arthritis and other chronic inflammatory arthritides. I. Relativity to inflammatory activity.

Lipoprotein metabolism was investigated in 69 patients with untreated active rheumatoid arthritis (n = 48) and in seronegative spondylarthropathies (n = 21). The patients had high inflammatory activity as measured by erythrocyte sedimentation rate and C-reactive protein (CRP). Serum cholesterol and cholesterol levels in the very-low-density lipoprotein (VLDL), low-density lipoprotein, and high-density lipoprotein fractions were reduced by 20% to 30% compared with healthy controls; and triglyceride levels in VLDL and high-density lipoprotein were reduced by 10% to 30%. There were significant correlations between the inflammatory activity and certain lipoprotein lipids, ie, between CRP and VLDL triglycerides, VLDL cholesterol, and serum triglycerides. The fractional elimination rate (K2) measured by an intravenous fat tolerance test was 30% higher in the patients than in the controls despite reduced tissue lipoprotein lipase activities. There was correlation between CRP and the K2 value. These findings suggest that it is the degree of inflammatory activity that governs the altered lipoprotein metabolism in untreated active chronic inflammatory arthritides. The relationships between CRP and VLDL and between CRP and K2 suggest that the VLDL particles may be altered by inflammatory process, and that the increased elimination may take place through the "scavenger pathway."

Serum lipoprotein in active rheumatoid arthritis and other chronic inflammatory arthritides. II. Effects of anti-inflammatory and disease-modifying drug treatment.

Serum lipids and lipoprotein patterns were prospectively analyzed in 33 previously untreated patients with active chronic inflammatory arthritides during different anti-inflammatory and disease-modifying drug regimens. Before treatment the lipoprotein pattern was characterized by low cholesterol concentrations in all lipoprotein fractions and low triglyceride concentrations in the very-low-density lipoprotein fraction as well as in the high-density lipoprotein fraction. During treatment with prednisolone combined with azathioprine or cyclophosphamide (n = 10), a reduction of the disease activity was achieved and the lipoprotein pattern was normalized; similar results were noted in a small group of patients (n = 4) treated with prednisolone alone while nonsteroidal anti-inflammatory drug therapy (n = 9) neither significantly affected the lipoprotein pattern nor the inflammatory activity measured by the acute-phase reactants. The long-term treatment with penicillamine (n = 4) and chloroquine (n = 6) induced both a clinical remission of the disease and a reduction of the inflammatory activity. The lipoprotein concentrations started to reverse to the normal values during penicillamine treatment. In contrast, in the chloroquine-treated group the alterations in lipoprotein lipid concentrations were further pronounced, ie, the cholesterol and triglyceride concentrations in serum and the very-low-density lipoprotein fraction decreased.

Impaired glucose handling in active rheumatoid arthritis: effects of corticosteroids and antirheumatic treatment.

Forty-two patients with active rheumatoid arthritis were studied serially with respect to glucose metabolism after the institution of different anti-inflammatory and antirheumatic therapies. Sixteen patients received 20 mg of prednisolone daily. After 1 week of treatment the mean k value in glucose tolerance tests increased from 1.0 +/- 0.1 (SEM) to 1.6 +/- 0.1 (P less than .001). The corticosteroid therapy thus restored the glucose tolerance to normal and significantly enhanced the insulin response (P less than .01). Corticosteroids also normalized the growth hormone response to glucose infusion but had no effect on plasma glucagon. Treatment with nonsteroidal anti-inflammatory drugs did not affect the k values nor the hormonal pattern either after short-term treatment or after three months of therapy, except for causing a minor increase in the plasma glucagon levels both before and after glucose infusion. The long-term effects of treatment with penicillamine (n = 4), chloroquine (n = 7), and immunosuppressive agents [corticosteroids combined with azathioprine or cyclophosphamide (n = 7)], were an improvement of the clinical state, a reduction of the inflammatory activity, and a reversal of the glucose handling to normal.