Dennis F. Lawler

Diet restriction and ageing in the dog: major observations over two decades

This report reviews decade two of the lifetime diet restriction study of the dog. Labrador retrievers (n 48) were paired at age 6 weeks by sex and weight within each of seven litters, and assigned randomly within the pair to control-feeding (CF) or 25 % diet restriction (DR). Feeding began at age 8 weeks. The same diet was fed to all dogs; only the quantity differed. Major lifetime observations included 1.8 years longer median lifespan among diet-restricted dogs, with delayed onset of late life diseases, especially osteoarthritis. Long-term DR did not negatively affect skeletal maturation, structure or metabolism. Among all dogs, high static fat mass and declining lean body mass predicted death, most strongly at 1 year prior. Fat mass above 25 % was associated with increasing insulin resistance, which independently predicted lifespan and chronic diseases. Metabolizable energy requirement/lean body mass most accurately explained energy metabolism due to diet restriction; diet-restricted dogs required 17 % less energy to maintain each lean kilogram. Metabonomics-based urine metabolite trajectories reflected DR-related differences, suggesting that signals from gut microbiota may be involved in the DR longevity and health responses. Independent of feeding group, increased hazard of earlier death was associated with lower lymphoproliferative responses to phytohaemagglutinin, concanavalin A, and pokeweed mitogen; lower total lymphocytes, T-cells, CD4 and CD8 cells; lower CD8 percentages and higher B-cell percentages. When diet group was taken into account, PWM responses and cell counts and percentages remained predictive of earlier death.

Bilaterally asymmetric effects of quantitative trait loci (QTLs): QTLs that affect laxity in the right versus left coxofemoral (hip) joints of the dog (Canis familiaris)

In dogs hip joint laxity that can lead to degenerative joint disease (DJD) is frequent and heritable, providing a genetic model for some aspects of the human disease. We have used Portuguese water dogs (PWDs) to identify Quantitative trait loci (QTLs) that regulate laxity in the hip joint. A population of 286 PWDs, each characterized by ca. 500 molecular genetic markers, was analyzed for subluxation of the hip joint as measured by the Norberg angle, a quantitative radiographic measure of laxity. A significant directed asymmetry was observed, such that greater laxity was observed in the left than the right hip. This asymmetry was not heritable. However, the average Norberg angle was highly heritable as were the Norberg angles of either the right or left hips. After correction for pedigree effects, two QTLs were identified using the metrics of the left and right hips as separate data sets. Both are on canine chromosome 1 (CFA1), separated by about 95 Mb. One QTL, associated with the SSR marker FH2524 was significant for the left, but not the right hip. The other, associated with FH2598, was significant for the right but not the left hip. For both QTLs, some extreme phenotypes were best explained by specific interactions between haplotypes.

The influence of age on the canine immune system

Immune function was assessed in a group of 47 Labrador Retrievers, ranging in age from 0.8 to 11.5 years, in order to establish baseline data on canine immunosenescence. Natural killer cell activity, lymphocyte subset distributions, antibody production, and mitogen-induced lymphoproliferative responses, all of which have been demonstrated to undergo age-related changes in humans and mice, were chosen as indicators of immune function. Dogs were categorized by age as young (mean 2.4 years), middle-aged (mean 5.8 years), and old (mean 9.1 years). Natural killer cell activity was not affected significantly by age. Lymphocyte subset analysis revealed a significant age-related increase in the percentage of cells staining with a pan T-cell reagent, accompanied by a corresponding increase in the percentage of CD8 cells from youth to middle age. An age-related decrease in the percentage of B-cells was observed concomitant with the increases in T-cell percentages. A gender-related difference in pan T-cell distribution was also observed, with females having a higher percentage than males. Lymphoproliferative responses of both young and middle-aged dogs to the mitogens concanavalin A, phytohemagglutinin, pokeweed mitogen, and staphylococcal enterotoxin B were significantly higher than those of old dogs. In general, the mitogen responses of male dogs were affected more dramatically by age than those of females. A significant age-related decline in in vivo antibody responses to the protein antigen, keyhole limpet hemocyanin, was not observed, although the mean titers of the young dogs were higher than those of the old.

The influence of age and gender on the immune system: a longitudinal study in Labrador Retriever dogs.

While aging studies employing a cross-sectional design have been informative in documenting many age-related alterations in immune function between different age cohorts within a population, longitudinal studies are invaluable for verifying changes at the level of the individual and for defining the precise periods of life during which particular changes occur. In the present study, a battery of immunological parameters were evaluated in a group of Labrador Retrievers as part of a comprehensive longitudinal aging study. Twenty-three dogs (14 females, 9 males; from 4 to 11 years of age) were evaluated annually for total WBC counts, lymphocyte subset distributions, natural killer cell activity and neutrophil phagocytic activity, and biannually for lymphoproliferative activity. An age-related decline in absolute numbers of lymphocytes, T-cells, CD4-cells and CD8-cells was observed in both genders. The distribution of lymphocyte subsets shifted with age, most dramatically in the females; percentages of B-cells declined while those of T-cells increased. Changes in percentages of CD4- and CD8-cells over the 8-year period were not dramatic; in females, percentages of CD8-cells increased significantly in early- to mid-life and then stabilized. Lymphoproliferative responses to mitogens declined over time in both genders. Males demonstrated higher levels of NK cytolytic activity than females; a marginal decline in activity with age was observed. No significant age-related changes in the phagocytic capacity of PMN were observed. These longitudinal findings help to discriminate between those immune parameters which change most dramatically in early-life versus those which either change more dramatically later in life or change gradually over the entire span of life. In addition they identify significant gender differences in several parameters and corroborate our previously published cross-sectional aging data in the same species.

Genetic Regulation of Osteoarthritis: A QTL Regulating Cranial and Caudal Acetabular Osteophyte Formation in the Hip Joint of the Dog (Canis familiaris)

Dogs, Canis familiaris, share more than 200 genetic disease phenotypes with humans [Patterson et al., 1982; Patterson, 2000]. Genes for specific diseases are often concentrated in purebred dogs (genetic isolates) due to founder effects, inbreeding and/or frequent use of ‘‘popular sires.’’ As a result, recessive, interactive, or polygenic modes of inheritance are more readily investigated, leading to identification of Quantitative Trait Loci (QTLs) [Chase et al., 2002, 2004]. The recent completion of the sequence for the canine genome has facilitated the comparison of the dog genome with human and othermammalian genomes, allowing the further investigative analysis of canine QTLs in other mammalian systems, notably the human and mouse. Here we examine changes that involve pathological remodeling of bone, altered joint conformation, and osteophyte formation, visible radiographically as osteoarthritis (OA) [Olsson, 1971; Riser, 1973]. We have related such changes in the coxofemoral (hip) joint of Portuguese Water Dogs (PWDs) to genotypes defined by SSR markers and associated themwith a specific region (haplotype) of the canine genome (QTL). Radiographs and blood for DNA were collected from 431 PWDs through the Georgie Project [http://www.georgieproject. com, Karen Miller director; Chase et al., 2002]. The dogs, ranging in age from 1.7 to 17 years (median age, 6 years), included 171 males and 260 females and represented a crosssection of the entire PWD population in the USA. They trace their ancestry to 31 founders through ca. 25 generations and consanguinities range from 0 to 0.6 with a mean of 0.2 [Chase et al., 1999]. We have associated marker alleles with a few infrequently used founders using the consanguinity between that founder and all dogs known to carry the allele. Permutation tests are used to establish the significance of each association [Alroy et al., 2000]. DNA was isolated from blood and characterized by PCR amplification and electrophoretic identification of the alleles of simple sequence repeat geneticmarkers [Francisco et al., 1996; Mellersh et al., 2000; Chase et al., 2002, 2004]. Osteoarthritis (OA) was scored from ventrodorsal radiographs of the pelvis as illustrated in Figure 2 of Chase et al. [2002]. In all, 431 dogs were scored for subchondral sclerosis of the cranial acetabular margin, osteophytes of the caudal and cranial acetabular margins, and femoral head osteophytes (illustrated in Fig. 1). The severity of each of these phenotypes was scored on an ordinal scale from 0 (none) to 3 (most severe). Left and right hips were scored independently. Scores ranged from0 to 22 out of a possiblemaximumof 24 (12 leftþ 12 right). Fifty percent of the dogs had a score greater than 0. Methods for estimatingheritability (h), identifying theQTL and estimating its effect on the variation of specific phenotypes (R) have been described previously [Chase et al., 2004]. Estimation of QTL significance used permutations, as described in Chase et al. [2002]. In the PWD population OA is heritable (h1⁄4 30%). About half of the population show some degree of OA. OA is significantly correlated with the Norberg Angle (an indicator of joint laxity). However, whereas the Norberg Angle is significantly greater for the right than the left hip, there is no significant difference in the OA scores between the right and left joints. OA also is significantly correlated with the 4th Principal Component (PC) defined by variation in the skeletal metrics of the pelvis and limb bones. There is no significant correlation with other PCs. We had identified two QTLs on autosome 1 (CFA 1) associated with the Norberg Angle [Chase et al., 2004] and several QTLs associated with PC4 (unpublished data). In addition, we have identified two QTLs related to autoimmune Addison’s disease (unpublished data). We analyzed these QTLs (11 in all) for association with OA. One QTL identified by the SSR marker, FH2320 on CFA 3 and associated with PC4, also was significantly associated withOA (P 0.002, corrected for pedigree effects and number of QTLs tested [Chase et al., 2002]). Table I presents the relevant region of the canine genome and its syntenic counterparts on the human and mouse genomes. We have characterized the effects of this QTL in greater detail. It accounts for about 16% of the OA variation (R1⁄4 16.4%), and involves primarily cranial and caudal acetabular marginal osteophytes. This same QTL affects PC4 through its effect on the ischial tuberosity, the width of which is segregating in the PWD population (R1⁄4 7.5% for the trait residual after removing the effects of PCs 1, 2, and 3). This QTL also

The aging feline kidney: a model mortality antagonist?

Traditional thinking views apparently non-programmed disruptions of aging, which medical science calls geriatric diseases, as separate from ‘less harmful’ morphological and physiological aging phenotypes that are more universally expected with passage of time (loss of skin elasticity, graying of hair coat, weight gain, increased sleep time, behavioral changes, etc). Late-life disease phenotypes, especially those involving chronic processes, frequently are complex and very energy-expensive. A non-programmed process of homeostatic disruption leading into a death trajectory seems inconsistent with energy intensive processes. That is, evolutionary mechanisms do not favor complex and prolonged energy investment in death. Taking a different view, the naturally occurring feline (Felis silvestris catus) renal model suggests that at least some diseases of late life represent only the point of failure in essentially survival-driven adaptive processes. In the feline renal model, individuals that succumbed to failure most frequently displayed progressive tubular deletion and peritubular interstitial fibrosis, but had longer mean life span than cats that died from other causes. Additionally, among cats that died from non-renal causes, those that had degrees of renal tubular deletion and peritubular interstitial fibrosis also had longer mean life span than those cats with no changes, even though causes of death differed minimally between these latter two groups. The data indicate that selective tubular deletion very frequently begins early in adult life, without a clear initiating phase or event. The observations support a hypothesis that this prolonged process may be intrinsic and protective prior to an ultimate point of failure. Moreover, given the genetic complexity and the interplay with associated risk factors, existing data also do not support the ideas that these changes are simple compensatory responses and that breed- or strain-based ‘default’ diseases are inevitable results of increasing individual longevity. Emerging molecular technology offers the future potential to further evaluate and refine these observations. At present, the existence of plastic and adaptive aging programming is suggested by these findings.

Automated SPE-RP-HPLC fractionation of biofluids combined to off-line NMR spectroscopy for biomarker identification in metabonomics

NMR-based metabonomics is a valuable and straightforward approach to measuring hundreds of metabolites in complex biofluids. However, metabolite identification is sometimes limited by overlapped signals in NMR spectra. We describe a new methodology using an automated hyphenation of solid phase extraction (SPE) with RP-HPLC combined to NMR spectroscopy, which allowed identification of 72 metabolites of various molecular classes in human urine. This methodology was also successfully applied to the fractionation of a cat urine sample to aid identification of aromatic compounds and felinine. The SPE-RP-HPLC method appears to be a reliable tool to support biomarker discovery in metabonomic studies.

A longitudinal study of the influence of lifetime food restriction on development of osteoarthritis in the canine elbow.

OBJECTIVE To report the effects of age and lifetime calorie restriction on development and progression of osteoarthritis (OA) in elbow joints of Labrador retrievers. STUDY DESIGN Longitudinal cohort study. ANIMALS Labrador retriever dogs (n=48). METHODS Puppies from 7 litters were allotted to 2 groups of 24 dogs each. Diet-restricted (DR) dogs received 25% fewer calories than control-fed (CF) pair mates. Elbow radiographs were taken at 6 and 8 years of age and end of life (EOL). Gross and histopathologic evaluations for OA occurred at EOL. RESULTS There was no statistical difference in radiographic OA frequency between groups at any of the time points. Radiographic OA severity was greater for CF dogs at 6 years only (P<.05). There was no significant difference between feeding groups for histopathologic prevalence or severity of OA. Similarly, there were no differences in gross OA lesions between the groups (P>.05). Fragmented medial coronoid process, un-united anconeal process, and osteochondrosis were not present in any elbow. CONCLUSION No differences in prevalence or severity of radiographic and histopathologic elbow OA were found between feeding groups. Diet restriction resulted in a 1.8-year extension in median lifespan but no additional incremental worsening of elbow disease. Evaluation at time points <6 years may have revealed larger differences in OA prevalence and severity between the dietary groups. CLINICAL RELEVANCE These findings support calorie restriction as a clinical tool to slow progression of elbow OA.

The Effects of Lifetime Food Restriction on the Development of Osteoarthritis in the Canine Shoulder

OBJECTIVE To report effects of age and lifetime food restriction on development and progression of shoulder joint osteoarthritis (OA) in Labrador retriever dogs. STUDY DESIGN Longitudinal life-span, cohort study. ANIMALS Labrador retriever dogs (n=48). METHODS Littermates were paired (gender, weight) to make 24 pairs of genetically similar dogs. Each diet-restricted (DR) pair-mate was fed daily 75% of the same diet consumed by its control-fed (CF) pair-mate for life. Shoulders were evaluated radiographically at years 6, 8, and end of life (EOL). At EOL shoulders were evaluated grossly and by histopathology for OA. RESULTS Radiographic evidence of shoulder OA was identified in 78% of dogs. Severity of radiographic shoulder OA at 6 (P<.03) and 8 years (P<.02) was significantly lower among DR dogs compared with CF dogs. Pooled gross evaluation results revealed 40 of 46 dogs had cartilage erosion on the caudal aspect of the humeral head. By EOL, 91% of dogs had histopathologic changes consistent with OA. CONCLUSION There was a high overall prevalence of radiographic, gross, and histologic OA among dogs. Substantial disparity was found between radiographic evidence of OA (at EOL) and characteristic changes visible by gross and histologic examination. CLINICAL RELEVANCE Radiographic evaluation correlates poorly with severity of shoulder joint pathology. The benefits of DR on shoulder OA are consistent with the demonstrated effect of DR in delaying species- and strain-specific diseases of aging.

Metabolic phenotype modulation by caloric restriction in a lifelong dog study.

Modeling aging and age-related pathologies presents a substantial analytical challenge given the complexity of gene-environment influences and interactions operating on an individual. A top-down systems approach is used to model the effects of lifelong caloric restriction, which is known to extend life span in several animal models. The metabolic phenotypes of caloric-restricted (CR; n = 24) and pair-housed control-fed (CF; n = 24) Labrador Retriever dogs were investigated by use of orthogonal projection to latent structures discriminant analysis (OPLS-DA) to model both generic and age-specific responses to caloric restriction from the ¹H NMR blood serum profiles of young and older dogs. Three aging metabolic phenotypes were resolved: (i) an aging metabolic phenotype independent of diet, characterized by high levels of glutamine, creatinine, methylamine, dimethylamine, trimethylamine N-oxide, and glycerophosphocholine and decreasing levels of glycine, aspartate, creatine and citrate indicative of metabolic changes associated largely with muscle mass; (ii) an aging metabolic phenotype specific to CR dogs that consisted of relatively lower levels of glucose, acetate, choline, and tyrosine and relatively higher serum levels of phosphocholine with increased age in the CR population; (iii) an aging metabolic phenotype specific to CF dogs including lower levels of liproprotein fatty acyl groups and allantoin and relatively higher levels of formate with increased age in the CF population. There was no diet metabotype that consistently differentiated the CF and CR dogs irrespective of age. Glucose consistently discriminated between feeding regimes in dogs (≥312 weeks), being relatively lower in the CR group. However, it was observed that creatine and amino acids (valine, leucine, isoleucine, lysine, and phenylalanine) were lower in the CR dogs (<312 weeks), suggestive of differences in energy source utilization. ¹H NMR spectroscopic analysis of longitudinal serum profiles enabled an unbiased evaluation of the metabolic markers modulated by a lifetime of caloric restriction and showed differences in the metabolic phenotype of aging due to caloric restriction, which contributes to longevity studies in caloric-restricted animals. Furthermore, OPLS-DA provided a framework such that significant metabolites relating to life extension could be differentiated and integrated with aging processes.