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    Samples were taken after food had been withdrawn for 18 h and again 3 h after feeding. Direct observation confirmed that food had been eaten by each cat.

    The effects of nutritional restriction for 14 d were studied in nine nonobese healthy adult cats [6 f, 2 female neuter fn and 1 mn, median weight, 4.

    Short-term dietary restriction was followed by free access to the diet. Serum samples were taken throughout the study, including the 5-d maintenance period at the start.

    Weight was measured regularly and voluntary food intake was recorded daily. The same diet [Whiskas Calorie Control Diet, WALTHAM, containing per kg kJ energy, g protein and 98 g fat] was fed throughout the study and formulated to exceed minimum adult maintenance nutrient requirements except for energy where indicated.

    These restriction levels were based on similar studies in dogs Maxwell et al. Serum concentrations of IGF-I and -II, and IGFBP-2 were measured using validated in-house heterologous RIA.

    Recombinant human rh IGF-I and -II a gift from Pharmacia and Upjohn, Stockholm, Sweden were radiolabeled with [ I] Amersham International, Bucks.

    Monoclonal antibodies to human IGF-I Blood Products, Hertfordshire, UK and rat IGF-II [Upstate Biotechnology UBI , Lake Placid, NY] were incubated overnight with radiolabeled peptide and then separated Sac-Cel, Immunodiagnostic Systems, Tyne and Wear, UK and compared with a standard curve of rhIGF standards.

    For the IGF-I RIA, the intra- and interassay CV were 6. For the IGF-II RIA, the CV were 8. For comparison of extraction methods, acid gel chromatography was used to separate serum IGF from IGFBP as previously described Horner et al.

    Fractions were then assayed for IGF without further extraction. Serum IGFBP were examined using western ligand blotting WLB methods as described by Hossenlopp et al.

    The resulting autoradiographs were analyzed by scanning densitometry image densitometer GS and Molecular Analyst software, Bio Rad, Herts.

    Individual IGFBP were identified by immunoblotting using Enhanced Chemiluminescence Amersham International. Bovine IGFBP-2 antiserum kindly provided by Dr.

    IGFBP-2 concentrations were further analyzed through RIA using a validated commercial kit Diagnostic Systems Laboratories, Webster, TX,.

    The intra-assay CV was 9. Serum albumin Roche Cobas Mira and BCG kit, Roche Diagnostics, Lewes, E. Sussex, UK and insulin Nelson et al.

    A normal human serum NHS pool, collected from volunteers 3 m, 7 f, Normality of data distribution was determined through the use of the Shapiro-Wilks and Kolmogorov-Smirnov tests Conover and examination of the symmetry of box-and-whisker plots.

    Data were not normally distributed, due in part to study population size; therefore they were analyzed by nonparametric methods. These data were expressed as the median plus range.

    Paired observations, e. The Bonferroni correction was applied in cases in which multiple analyses were made. SPSS 6. Serial dilutions of feline serum were parallel to standard curves in the IGF and IGFBP-2 RIA as shown in Figure 1.

    Examination of extracted serum by WLB revealed residual IGFBP, predominantly IGFBP-3 and -2 data not shown. The potential interference of these residual binding proteins in the RIA for IGF was investigated by assessing recovery of unlabeled IGF from serum samples pre- and post-FAE, and also by comparison with acid gel chromatography.

    Serial dilution curves of feline serum squares showing parallel displacement from the recombinant human insulin-like growth factor IGF standard curves circles in each RIA.

    Serum samples from two cats are shown for IGF-I. Acid-gel chromatography of NHS and feline serum resulted in similar elution profiles data not shown.

    Furthermore, serial dilution curves of serum from two cats with high concentrations of IGF-I and from one cat with a low IGF-I concentration were parallel to recombinant human standard curves data not shown.

    Results for NHS and feline serum were similar and the assays were considered appropriate. Serum IGFBP concentrations were analyzed as described.

    IGFBP-3, a doublet at and kDa molecular weight, was the predominant binding protein Fig. Serum IGFBP-3 concentrations analyzed by WLB reflected body weight and IGF-I concentrations Fig.

    Compared with NHS, feline serum IGFBP-2 band intensity was increased by WLB analysis; this was confirmed by immunoblotting Fig.

    This was a consistent finding throughout the study. A representative autoradiograph of serum insulin-like growth factor binding protein IGFBP profiles of eight cats with individual weight, sex, IGF-I and -II concentrations given in the table below.

    The lane marked N contains normal human serum pool. Molecular weight markers closed arrowheads and the band representing IGFBP-3 open arrowhead are indicated.

    Serum IGF concentrations and IGFBP-3 band intensities were greater in the male neutered mn cats than in the females f represented in this figure.

    A representative autoradiograph A and corresponding insulin-like growth factor binding protein IGFBP -2 immunoblot B of samples from one neutered female cat during restriction to Samples were collected on the day of study indicated beneath each lane.

    Samples collected during Lane N contains normal human serum pool. Molecular weight markers closed arrow heads and IGFBP-2 open arrowhead are indicated.

    Serum IGFBP band intensities did not change during the study. With validation, normal ranges for IGF-I and IGF-II were established for the study population of 46 cats.

    There were no significant correlations between age and IGF concentration. There were no changes in serum IGF or IGFBP concentrations, including IGFBP-1, with overnight food withdrawal or 3 h after refeeding data not shown.

    Feeding records indicated incomplete ration uptake during the study Table 1. Daily energy calculated vs. Data are shown as median actual intakes with the range in brackets during the 5-d maintenance period M , energy restriction Restricted and free access Refed.

    Serum samples were taken and weight recorded on d 1 and 5 during maintenance feeding at the start of the study to establish a baseline. Serum IGF concentrations and body weights did not differ significantly at the start of the study during the 5-d maintenance feeding period.

    Body weights at the start of each restriction period did not differ significantly as a result of weight gain during the intervening d refeeding period.

    However, concentrations normalized with refeeding; thus there was no difference at the start of each restriction period. Serum IGF-I concentrations fell significantly only after 14 d of Serum insulin-like growth factor IGF -I and albumin concentrations in nine cats during short-term dietary restriction and refeeding.

    Data are shown as the median line with interquartile values shaded as the range. Closed brackets indicate significant change in serum IGF-I concentrations with Serum IGF-II concentrations were not altered during each period of dietary restriction or refeeding.

    Densitometric analysis of WLB showed no significant change in serum IGFBP band intensities with dietary restriction or refeeding Fig.

    There were no changes in serum albumin Fig. Serum insulin concentrations fell from The absence of raised IGFBP-1 concentrations after overnight food withdrawal in adult cats is also seen in adult dogs Maxwell et al.

    Similarly, feline serum IGFBP-1 concentrations did not change during prolonged dietary restriction or ad libitum consumption of the diet, but we have not assessed the influence of prolonged food withdrawal.

    Although we have not positively identified the kDa band on WLB autoradiographs as IGFBP-1, specific immunoblotting has shown that it is not IGFBP It is also unlikely to be a fragment of IGFBP-3 because under the denaturing WLB conditions used, the lower-molecular-weight fragments of IGFBP-3 do not bind radiolabeled IGF-I Suikkari and Baxter In humans Busby et al.

    During overnight or longer-term food withdrawal, concentrations of insulin fall and IGFBP-1 increases, whereas rising postprandial insulin concentrations down-regulate hepatic IGFBP-1 mRNA transcription.

    Feline serum IGFBP-1 concentrations may not rise with overnight food withdrawal because of an insufficient decrease in serum insulin concentrations, which could be compounded by increased sensitivity to the suppressive effects of insulin.

    It also may not be regulated by insulin. A significant decrease in serum IGF-I concentrations was seen only after 14 d of The apparent discrepancy is partially resolved by analyzing the feeding records and expressing restriction concentrations as a percentage of intake during each preceding maintenance period Table 1.

    Cats did not eat their full diet allowances during each maintenance feeding period. Comparison with studies in other species Oster et al.

    Hirsch et al. It is possible that dietary energy requirements for our study population were overestimated, perhaps because of the effect of kenneling on activity.

    Alternatively, the significant decrease in serum IGF-I concentrations during the second period Although this explanation is unlikely due to normalization of serum IGF-I concentrations with the intervening refeeding period, a crossover study would address this question.

    During It is possible therefore that feline serum IGF-I concentrations may be more sensitive to dietary protein than caloric restriction.

    Dietary protein requirements in adult cats have been extrapolated from growth studies in kittens and may therefore overestimate maintenance levels.

    Feline serum IGF-II concentrations were unaffected by short-term dietary restriction, as also demonstrated in humans Davenport et al.

    The reason for this is unclear but may be related to decreased serum IGF-I concentrations. Serum IGFBP concentrations by WLB analysis and RIA were also unchanged with short-term dietary restriction, which differs from other species Oster et al.

    In common with adult dogs, adult cats have greater concentrations of serum IGFBP-2 when nutritionally replete compared with humans, rats and guinea pigs.

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