NIDDM genes in mice: deleterious synergism by both parental genomes contributes to diabetogenic thresholds.

We used mouse genetics to model how polygenic thresholds for the transition from impaired glucose tolerance (IGT) to NIDDM are reached. NON/Lt and NZO/Hl are inbred mouse strains selected for IGT and polygenic obesity, respectively. Their F1 male progeny consistently developed NIDDM. Genetic analysis of F2 males from both cross directions identified an NON-derived diabetogenic locus, Nidd 1, on chromosome (Chr) 4 near the leptin receptor. This locus was associated with reduced plasma insulin, increased non-fasted blood glucose, and lower body weight. Another NON-derived diabetogenic locus on Chr 18 (Nidd2) that controls blood glucose was identified. An NZO-derived diabetogenic region on Chr 11 (Nidd3), possibly comprising two separate loci, reduced ability to sustain elevated plasma insulin and significantly reduced weight gain over time. Thus, the diabetogenic synergism between genetic loci from strains separately exhibiting subthreshold defects perturbing glucose homeostasis underscores the likely complexity of the inheritance of obesity-associated forms of NIDDM in humans.

The regulation of luteinizing hormone and prolactin in C57BL/6J mice: effects of estradiol implant size, duration of ovariectomy, and aging.

The induction of a LH surge by estradiol (E2) implants was characterized in ovariectomized C57BL/6J mice. Various times after ovariectomy mice were given a priming E2 implant, followed by an LH surge-inducing E2 implant, and were sampled 30 h later at darkness. The magnitude of the E2-induced LH surge was influenced by the postovariectomy interval, sizes of the implants, and age. Mice ovariectomized for 30-60 days before E2 implantation displayed larger surges than those ovariectomized for 4 days. Priming implants yielding 10 pg E2/ml plasma permitted the subsequent induction of vigorous LH surges, whereas no LH surges were observed with slightly larger priming implants that yielded 15 pg E2/ml. The size of the surge-inducing implant was correlated with the size of the subsequent LH surge. However, regardless of implant size, aging mice (8 vs. 13 months old) had smaller LH surges. Sequential daily LH surges were not observed under any conditions, suggesting that mice differ from rats and hamsters in their regulation of LH by E2. Plasma PRL was slightly elevated in the afternoon just before the LH surge, but returned to basal levels during the LH surge, indicating an uncoupling of the LH and PRL surges. The two-stage E2 implantation protocol for inducing LH surges by physiological levels of E2 allows more detailed examination of the priming vs. surge-inducing effects of E2.

Preparation and characterization of antisera and monoclonal antibodies to serotonergic and dopaminergic ligands.

In an attempt to produce polyclonal antisera and monoclonal antibodies to serotonin, SKF 38393 (D-1 agonist), dopamine, and haloperidol (D-2 antagonist) several procedures for the preparation of immunogenic ligand-protein carrier conjugates were investigated. The Mannich reaction utilizing formaldehyde as the chemical linker was used to prepare serotonin-protein conjugates; antibodies raised to this conjugate reacted specifically to the conjugated serotonin moiety but did not react to native serotonin. Chemical conjugations involving dimethylpimelylimidate or N-carboxymethyl derivatives for the coupling of serotonin, dopamine and SKF 38393 to carrier proteins produced antibodies primarily directed against the 'chemical coupling arm' and very little antibody activity against the ligand itself could be detected. Synthesis of a haloperidol derivative suitable for chemical coupling to a protein carrier via oxobutyric acid produced an immunogen which was capable of eliciting both polyclonal and monoclonal antibodies specific for the hapten. The pitfalls of the various chemical conjugation procedures and the difficulties of producing antibodies to free ligands are discussed.

Maximum life spans in mice are extended by wild strain alleles.

The genes that control basic aging mechanisms in mammals are unknown. By using two four-way crosses, each including a strain derived from wild, undomesticated stocks, we identified two quantitative trait loci that extend murine life spans by approximately 10%. In one cross, the longest-lived 18% of carriers of the D8Mit171 marker allele from the MOLD/Rk strain, Mus m. molossinus, outlived the longest lived 18% of noncarriers by 129 days (P = 5.4 x 10(-5)); in a second cross, carriers of the D10Mit267 allele from the CAST/Ei strain, Mus m. castaneus, outlived noncarriers by 125 days ( P = 1.6 x 10(-6)). In both crosses, P < 1.0 x 10(-4 )is considered significant. Because these life span increases required that all essential biological systems function longer than normal, these alleles most likely retarded basic aging mechanisms in multiple biological systems simultaneously.

Memory T lymphocyte hyporesponsiveness to non-cognate stimuli: a key factor in age-related immunodeficiency.

Previous studies from our laboratory have suggested that aging leads to an accumulation of cells expressing high levels of CD44, thought to be a marker for memory lymphocytes, and that positively selected CD44hi T cells, from mice of any age, respond poorly to concanavalin A (Con A) in limiting dilution estimates of interleukin (IL)-2-producing cells. We now report the results of a more comprehensive analysis of memory T cell function, in old and young mice, to non-cognate activators (Con A and the staphylococcal enterotoxin SEB). We report that memory T cells, isolated by removing cells bearing the CD45RB determinant, contain very few cells able to respond to either Con A or SEB under limiting dilution culture conditions, whether the responses are measured by IL-2 or by IL-3 accumulation. As a control, we show that memory T cells do respond strongly, at limiting dilution, to recently encountered priming antigens, i.e. Schistosoma mansoni egg antigen; the limiting dilution culture protocol thus does not preclude activation of memory T cells when cognate stimuli are presented to antigen-specific cells. These data suggest that virgin and memory T cells may differ fundamentally in their activation requirements, and suggest further that the accumulation, with age, of memory T cells accounts for the low responsiveness of old mice to non-cognate mitogens.

Cellular determinants of age-related decrements in the T-cell mitogen response of B6CBAF1 mice.

Age-related changes in the cellular composition of the immune system that are associated with an impaired proliferative response to T-cell mitogens were identified for B6CBAF1 mice. The frequencies of precursors of Con A-induced IL-2-secreting cells (pHTL) and of Con A-induced cytotoxic cells (pCTL), determined by limiting dilution analysis, were lower for splenocytes from old mice, as were the proliferative responses to Con A and PHA, determined in conventional high cell density cultures for the same mice. The pHTL frequency correlated with the proliferative response to Con A (r2 = .94) and to PHA (r2 = .83) among old mice, but not among young; there were no correlations of pCTL frequency with proliferative responses. The reduced pHTL frequency in old mice resulted from: (a) an age-related doubling of the number of splenic B cells that diluted T cells, and (b) a 67% decline in the absolute number of Con A-reactive pHTL cells in the spleen that appeared despite the maintenance of normal numbers of total splenic CD4+ and CD8+ cells. Thus, both a decline in absolute pHTL numbers and an increase in the number of non-T cells in the spleen result in a diminished pHTL frequency that is closely linked to the impaired mitogen response observed for old B6CBAF1 mice.

Aging and the regulation of luteinizing hormone in C57BL/6J mice: impaired elevations after ovariectomy and spontaneous elevations at advanced ages.

The effects of age on the elevations of plasma luteinizing hormone (LH) after ovariectomy were studied in C57BL/6J mice. In longitudinal studies, mice ovariectomized at 4-6 months and sampled serially reached maximum plasma LH (200-300 ng/ml) after 1 month; these elevations were maintained to at least 15 months of age, when most intact mice have ceased cycling and have persistent vaginal cornification (PVC). In contrast, mice ovariectomized at 15 or 21 months had smaller LH elevations (to 150 ng/ml) 1 or 2 months after ovariectomy, as compared to younger (5- or 11-month-old) cycling mice whose LH was ca. 200 ng/ml 1 month after ovariectomy. The maintenance of LH elevations in chronically ovariectomized mice suggests that the smaller LH elevations in acutely ovariectomized mice of the same age may be an ovary-dependent aging phenomenon. Pilot studies detected no age effects on plasma LH turnover. Intact 20- to 26-month-old mice with leukocytic vaginal smears, low plasma estradiol (E2) and low progesterone had initially elevated plasma LH (ca. 125 ng/ml), if gross pathologic lesions were not present at necropsy. In contrast, old mice with cornified vaginal smears had higher plasma E2 and low (normal) plasma LH. The low plasma progesterone and elevated LH in older mice with leukocytic smears argues against a pseudopregnant status. Old mice of all vaginal smear types with pathologic lesions had low LH. Ovariectomy did not yield further increases of LH in old mice. The spontaneous elevation of plasma LH to ovariectomized levels in old intact mice with leukocytic vaginal smears and reduced plasma E2 implies that C57BL/6J mice eventually undergo ovarian changes analogous to menopause in humans.

Transient shortening of estrous cycles in aging C57BL/6J mice: effects of spontaneous pseudopregnancy, progesterone, L-dihydroxyphenylalanine, and hydergine.

Regular estrous cycles can be reinitiated in old acyclic female rats by pharmacologic, hormonal, and environmental manipulations. The most responsive acyclic states are persistent vaginal cornification (PVC) and spontaneous pseudopregnancy (SP). However, it is not known if the irregular cyclicity that precedes acyclicity during aging can also be alleviated. We found that transient shortening of estrous cycles follows smear sequences indicative of pseudopregnancy in C57BL/6J mice, aged 9-15 mo, suggesting a role for progesterone. This phenomenon was investigated through a limited model of pseudopregnancy in which intact aging mice with lengthened cycles were given progesterone implants (yielding 70 ng progesterone/ml plasma) that suppressed estrous cycles; upon removal of the implants, cycles were transiently shortened in aging mice. Therefore, we hypothesize that withdrawal from the progesterone elevations associated with SP is the mechanism in shortening subsequent estrous cycles. Effects of central-acting drugs, similar to those used to reinitiate cyclicity in acyclic old rats, were also examined. Hydergine, an ergot mixture with partial dopaminergic and serotonergic agonist activities, suppressed SP when fed to 10- to 12-mo-old, middle-aged mice. Hydergine did not otherwise affect estrous cycle length, prevent PVC, or reinitiate cycling in acyclic PVC mice. Feeding L-dihydroxyphenylalanine to middle-aged mice did not suppress SP, affect estrous cycle lengths, or reinitiate cycles from PVC.

Supernumerary ovarian grafts in aging C57BL/6J mice reveal complexities in the neuroendocrine impairments of acyclic mice.

Determinants of the age-related acyclic state, persistent vaginal cornification (PVC), were studied in reproductively senescent mice using a 2-stage ovarian transplantation procedure, whereby ovaries from young mice were grafted to older mice without removing their autogenous ovaries until 8 wk later. In contrast to the usual ("1-stage") procedure, in which the autogenous ovaries are removed at the time of grafting, the 2-stage approach is postulated to circumvent potential effects of the reduction in steroids during the ovariprival phase prior to vascularization of the grafted ovary, which may reverse age-related hypothalamic-pituitary impairments. The 2-stage transplantation procedure was validated in young C57BL/6J mice. Estrous cycles were not disrupted by removal of the autogenous ovaries 8 wk after the grafting, indicating that grafted ovaries began functioning before or within days after ovariectomy. No difference in estrous cycle frequency or distribution was detected between the young mice with 2-stage and those with 1-stage transplants for at least 3 mo after removal of the autogenous ovaries. Most older (15- to 18-mo-old) mice with PVC (70%) remained acyclic after receiving young ovaries by either the 1-stage or the 2-stage procedure, indicating that extra-ovarian, presumably neuroendocrine, impairments are sufficient to maintain acyclicity in most older mice once it is initiated. However, 30% of the older mice from each transplantation group began cycling after receiving young ovaries by either the 1-stage or 2-stage procedure, as observed before 1-stage transplants. Therefore, cycle reactivation was not a result of the transient ovariprival phase incurred during 1-stage transplantation.(ABSTRACT TRUNCATED AT 250 WORDS)

Differential sensitivity of virgin and memory T lymphocytes to calcium ionophores suggests a buoyant density separation method and a model for memory cell hyporesponsiveness to Con A.

Previous work from this laboratory has indicated that murine memory T cells differ from virgin T cells in that the former are more resistant to agents that alter intracellular [Ca]i. We have used this difference to devise a method for separating virgin from memory T cells by centrifugation over an ionomycin-containing Percoll step gradient after brief exposure to 2 microM ionomycin. Under these conditions, those T cells that are most sensitive to ionomycin-induced changes in [Ca]i become more dense and therefore travel further into the Percoll/ionomycin gradient than cells that are more resistant to ionomycin. We show that the ionomycin-resistant cell population is enriched for cells that express high levels of Pgp-1 (CD44), and low levels of CD45RB, and thus appears to consist largely of memory T cells. Both CD4 and CD8 cells can be divided into Pgp-1hi and Pgp-1lo subsets in this way. Cells recovered from such a gradient and washed to remove the ionomycin appear normally functional, i.e., neither more nor less responsive to mitogens and costimuli than untreated cells. Limiting dilution methods show that the ionomycin-sensitive (virgin) subset contains most of the Con A-responsive precursors for cytotoxicity, and most of the cells able to produce IL-2 in responses to Con A or staphylococcal enterotoxin B. Ag-specific helper memory cells are, however, found predominantly in the ionomycin-resistant fraction of the spleen and draining lymph nodes of mice infected with Schistosoma mansoni. Changes in resistance to calcium signal development may represent a fundamental distinction between virgin and memory T cells, and could contribute to differences in activation requirements between these two cell subsets.

Physiologic and endocrinologic characterization of male sex-biased diabetes in C57BLKS/J mice congenic for the fat mutation at the carboxypeptidease E locus.

The fat gene in mice represents a recessive mutation at the carboxypeptidase E (Cpe) locus. The mutant allele (Cpe(fat)) encodes a highly unstable enzyme and produces an obesity phenotype characterized by attenuated processing of prohormones such as proinsulin that require this exopeptidase for full maturation. This article presents a preliminary physiologic and endocrinologic characterization of the stock of C57BLKS/LtJ-Cpe(fat)/Cpe(fat) mice at the backcross generation (N10) currently distributed by The Jackson Laboratory. Although previously reported not to be diabetogenic at N5, an additional five backcrosses to the C57BLKS/J background resulted in a male-biased development of both obesity and diabetes. Major differences distinguishing this mutant stock from the phenotypes produced by either the diabetes (Lepr(db)) or obese (Lep(ob)) mutations on the same inbred strain background are lack of hyperphagia and hypercorticism, sensitivity of diabetic males to exogenous insulin, and a milder and male-biased diabetes syndrome that is not associated with widespread beta-cell necrosis and islet atrophy, and that often remits with age.

Lifespan extension and delayed immune and collagen aging in mutant mice with defects in growth hormone production.

Single-gene mutations that extend lifespan provide valuable tools for the exploration of the molecular basis for age-related changes in cell and tissue function and for the pathophysiology of age-dependent diseases. We show here that mice homozygous for loss-of-function mutations at the Pit1 (Snell dwarf) locus show a >40% increase in mean and maximal longevity on the relatively long-lived (C3H/HeJ x DW/J)F(1) background. Mutant dw(J)/dw animals show delays in age-dependent collagen cross-linking and in six age-sensitive indices of immune system status. These findings thus demonstrate that a single gene can control maximum lifespan and the timing of both cellular and extracellular senescence in a mammal. Pituitary transplantation into dwarf mice does not reverse the lifespan effect, suggesting that the effect is not due to lowered prolactin levels. In contrast, homozygosity for the Ghrhr(lit) mutation, which like the Pit1(dw) mutation lowers plasma growth hormone levels, does lead to a significant increase in longevity. Male Snell dwarf mice, unlike calorically restricted mice, become obese and exhibit proportionately high leptin levels in old age, showing that their exceptional longevity is not simply due to alterations in adiposity per se. Further studies of the Pit1(dw) mutant, and the closely related, long-lived Prop-1(df) (Ames dwarf) mutant, should provide new insights into the hormonal regulation of senescence, longevity, and late life disease.

Graafian follicle growth and replacement in anovulatory ovaries of ageing C57BL/6J mice.

The growth of preantral follicles and replacement of large Graafian follicles was studied in reproductively senescent C57BL/6J mice immediately after the loss of oestrous cyclicity when the ovary is approaching exhaustion of its follicular reserve. Granulosa cells were labelled in vivo by injection of tritiated thymidine and were evaluated by autoradiography. At 1 h after injection, labelling was confined almost exclusively to the cumulus and neighbouring cells whereas the distribution of labelled granulosa cells in preantral follicles was approximately uniform. The proportion of labelled mural cells in Graafian follicles rose from initially low values to maximal levels 3 days later; this suggested that those follicles that were Graafian at the time of injection had been replaced by recruitment of preantral stages. The rate of growth of preantral follicles was similar in senescent anovulatory mice and in young adult animals. A simple model was constructed to illustrate how persistent vaginal cornification in ageing mice is sustained by a stream of Graafian follicles and why some of the latter did not respond fully to an ovulatory dose of hCG.

Heritability of life span in mice and its implication for direct and indirect selection for longevity.

We found high narrow-sense heritability of life span based on the regression of offspring on average parental (midparent) life spans. In two mouse populations prepared using the 4-way-cross design, mean +/- SE heritabilities were 62 +/- 11% (P < 0.001) and 44 +/- 15% (P < 0.01). To reflect inherited rates of aging, rather than resistance to early disease, data from the first 25% to die were deleted, so that only about 40% of families were used for offspring-midparent regressions. Heritabilities still remained high, 38% and 55%, for the same two populations, respectively. Populations studied in two other experiments did not show nearly as high heritabilities; in one case probably due to environmental stress, and in the other probably because the strains used did not have sufficient additive variance in genes regulating longevity. Significant heritabilities occurred only when a wild derived inbred strain was included in the 4-way cross. The age when a female ceased to reproduce appeared to be related to the life spans of her offspring, but only weakly, not approaching significance for any individual experiment. The age when a female became infertile was related to her life span, but the relationship disappeared when short-lived mice were excluded from the analysis. Our findings indicate that, in sufficiently diverse mouse populations, selection for increased longevity should be possible and that the direct selection for parental life span will be a more efficient strategy than selection for female reproductive life span.

Preparation and characterization of antisera and monoclonal antibodies to haloperidol.

For the first time a library, of monoclonal antibodies (MoAbs) to the butyrophenone haloperidol (D-2 antagonist) has been prepared. Synthesis of a haloperidol derivative suitable for chemical coupling to a protein carrier via oxobutyric acid produced an immunogen which was used to develop two polyclonal antisera and twelve MoAbs specific for the hapten. Our library of MoAbs can be grouped into three classes; 1) high affinity and specificity for free 3H-haloperidol, 2) moderate affinity with significant cross-reactivity to other butyrophenone ligands, and 3) a group which binds poorly to free 3H-haloperidol but instead recognizes the ligand only when it is coupled to carrier protein. Clone (189(2)-6) was found to have the highest equilibrium binding affinity (Kd = 4 nM) and is far more specific than the currently available antisera to haloperidol. This MoAb has significantly lower affinity for all of the common metabolites of haloperidol. This capability makes 189(2)-6 a candidate for further development with regard to use in clinical radioimmuno-assays of therapeutic drug levels. In addition, one of the anti-haloperidol Moabs (190(2)-6) binds more tightly to spiperone than to haloperidol and displays a qualitative correlation in the rank order of neuroleptic binding affinity for a limited series of analogs when compared to membrane bound D-2 receptor binding.

Estradiol-induced adult anovulatory syndrome in female C57BL/6J mice: age-like neuroendocrine, but not ovarian, impairments.

Long-term effects of elevated plasma estradiol (E2) on ovarian and neuroendocrine functions were examined in 4-month-old cycling female C57BL/6J mice injected s.c. with 0.2 or 0.05 mg estradiol valerate (EV), or oil. Within 7 days, EV-injected mice became permanently acyclic, exhibiting the persistent vaginal cornification (PVC) characteristic of reproductive senescence in rodents. Four months after injection, ovaries from EV-injected mice exhibited no corpora lutea, but ovulated in response to an injection of human chorionic gonadotropin (hCG) (as do older, spontaneously PVC mice). When grafted into young mice, ovaries from EV-injected mice supported as many estrous cycles as ovaries from oil-injected controls. EV did not alter the suppression of luteinizing hormone (LH) by E2, LH response to injected LH releasing hormone (LHRH), or plasma prolactin (Prl). However, EV-injected mice exhibited impairments in LH regulation similar to those seen in old, acyclic mice. Plasma LH 30 days after ovariectomy was 40% lower, and E2-induced LH surges were 60% lower, in EV-injected mice versus controls. Furthermore, EV-injected mice were unable to support estrous cycles given young ovarian grafts, in contrast to controls. Effects of sustained but physiological levels (15-20 pg/ml) of plasma E2, were examined in intact cycling mice given sham or E2 implants. Six weeks after implantation, the implants were removed; only 50% of the E2-implanted mice subsequently exhibited estrous cycles, compared with 100% of sham-implanted controls. Furthermore, those E2-implanted mice which did cycle had fewer cycles than controls.(ABSTRACT TRUNCATED AT 250 WORDS)