3.3.3. IGFBP-4

As an effect of age, IGFBP-4 was significantly decreased between weeks 2 and 7 (*p* < 0.05), increased between week 7 and 11 (*p* < 0.05) and decreased between week 11 and 42 (*p* < 0.001). In all male mice, IGFBP-4 was increased between weeks 4 and 11 (*p* < 0.01) and decreased between weeks 11 and 29 (*p* < 0.001). Again, significant differences present in isolated mouse lines are depicted in Figures 3 and 4.

#### *3.4. E*ff*ects of Growth Selection on the Concentrations of IGFBP-2 to -4*

As a main effect of growth selection and irrespective of age, obese female mice had significantly higher levels of IGFBP-3 in serum (*p* < 0.001). Independent of age, selection for high protein accretion increased IGFBP-3 and IGFBP-2 (*p* < 0.001). In males, selection for high body mass and selection for high protein mass had an effect on the concentrations of IGFBP-2 and IGFBP-3 in serum (*p* < 0.05) over all age groups.

As an interaction of genetic group and age, in growth-selected obese and lean male mice (Figure 4), levels of IGFBP-3 at the age of 11 and 16 weeks were increased if compared to age-matched unselected controls (*p* < 0.05). In lean male mice, IGFBP-3 remained on a higher level also at later time points, with significant differences if compared to obese male mice and unselected controls at the age of 29 and 54 weeks (*p* < 0.05). Growth selection further stimulated the increase of IGFBP-2 from younger ages to week 16, observed in unselected controls, resulting in about 3-fold increased levels of IGFBP-2 in serum from lean male mice (*p* < 0.001). Between 4 and 11 weeks of age, a significant increase of IGFBP-4 was observed (*p* < 0.01) in all males independent of line. In all genetic groups and in both genders, the postnatal increase of IGFBP-3 in serum is lagging behind the increases of IGF-1.

#### *3.5. Longitudinal Molar Ratio of IGF-1 and IGFBP Concentrations in Serum*

In order to estimate the longitudinal molar ratio of IGF-1 with respect to the IGFBPs detected in serum by Western ligand blotting, the concentrations of IGF-1 and IGFBP-2 to -4 were corrected for their respective molecular weights (IGF-1: 7.5 kDa, IGFBP-2: 32 kDa, IGFBP-3: 41 kDa, IGFBP-4: 24 kDa). The longitudinal molar ratio of IGF-1 versus the sum of IGFBP-2, IGFBP-3, and IGFBP-4 is presented in Figure 5. Neither in female nor in male mice of all genetic groups, IGF-1 was in molar excess over the sum of IGFBP-2 to -4. As an effect of age and genetic group, between weeks 2 and 4, there was a significant increase in all groups and genders (*p* < 0.001) with the exception of female controls (*p* < 0.05). At the age of 4 weeks, obese male and female mice have higher ratios of IGF-1/IGFBPs if compared to unselected controls (*p* < 0.05).

**Figure 5.** Molar ratio of IGF-1 to the sum of IGFBP-2, -3, and -4 (left Y-axis) present in serum from mice selected for high body weight (obese), for high protein amount (lean), and in unselected controls at an age of 2, 4, 7, 11, 16, 29, 42, and 54 weeks (*n* ≥ 7 with the exception of obese male at 54 weeks with *n* = 4; different letters indicate significant effects of age; identical letters indicate no statistically significant difference; \*: *p* < 0.05; \*\*\*: *p* < 0.001; b+: significantly different if compared to unselected controls, with *p* < 0.001 for males and *p* < 0.05 for females). On the right Y-axis, daily weight increases, intrapolated from the body weight data in Figure 1 by the Gompertz function, were included.
