Compensatory Response of the Somatotropic Axis from IGFBP-2b Gene Editing in Rainbow Trout (Oncorhynchus mykiss)

Abstract

Rainbow trout with gene editing-induced reductions in serum insulin-like growth factor binding protein (IGFBP)-2b exhibit similar growth performance compared to fish without IGFBP-2b gene disruption. The objective of this study is to determine how the components of the insulin-like growth factor (IGF)/IGFBP system respond to a reduction in serum IGFBP-2b abundance. Editing the IGFBP-2b genes in rainbow trout resulted in an 83% decrease in serum IGFBP-2b in mutants. This resulted in a 35% reduction in serum IGF-I, which was offset by reduced expression of hepatic igfbp-1a2 and increased muscle igfr-1a; these responses suggest that an increased IGF-I signaling capacity offset reductions in serum IGF-I. During feed deprivation, the differential expression of igfbp genes supports the attenuation of the growth inhibitory response, likely due to the further reduction in serum IGF-I that alleviated the need for an IGF-inhibitory response. Unique igfbp expression patterns occurred during refeeding, suggesting an enhanced IGF-I signaling capacity in controls. Collectively, these findings support that the role of IGFBP-2b is to regulate serum IGF-I concentrations. The compensatory regulation of IGF/IGFBP system genes indicates that adjustments in other IGFBP, both circulating and at the local level, maintain IGF-I signaling at a level appropriate for the nutritional state of the fish.

Keywords: CRISPR/Cas9; IGF; IGFBP; feed deprivation; gene editing; insulin-like growth factor; rainbow trout.

Figure 1

Panels a through c represent examples of chromatograms, indicating amplicons from (a) un-injected fish (a), injected controls (b), and high-rate IGFBP-2b mutants (c). Black asterisk indicates intact TYR2, red asterisk indicates intact IGFBP-2b1, and green asterisk indicates intact IGFBP-2b2. Panel (d) shows a representative Western ligand blot indicating IGFBP abundance in serum from control and IGFBP-2b mutant rainbow trout. Arrows on the left indicate known IGFBP in normal human serum (NHS). The arrows on the right indicate IGFBP-2b and a 32 kDa IGFBP in rainbow trout. Chi: Chinook salmon.

Figure 1

Panels a through c represent examples of chromatograms, indicating amplicons from (a) un-injected fish (a), injected controls (b), and high-rate IGFBP-2b mutants (c). Black asterisk indicates intact TYR2, red asterisk indicates intact IGFBP-2b1, and green asterisk indicates intact IGFBP-2b2. Panel (d) shows a representative Western ligand blot indicating IGFBP abundance in serum from control and IGFBP-2b mutant rainbow trout. Arrows on the left indicate known IGFBP in normal human serum (NHS). The arrows on the right indicate IGFBP-2b and a 32 kDa IGFBP in rainbow trout. Chi: Chinook salmon.

Figure 1

Panels a through c represent examples of chromatograms, indicating amplicons from (a) un-injected fish (a), injected controls (b), and high-rate IGFBP-2b mutants (c). Black asterisk indicates intact TYR2, red asterisk indicates intact IGFBP-2b1, and green asterisk indicates intact IGFBP-2b2. Panel (d) shows a representative Western ligand blot indicating IGFBP abundance in serum from control and IGFBP-2b mutant rainbow trout. Arrows on the left indicate known IGFBP in normal human serum (NHS). The arrows on the right indicate IGFBP-2b and a 32 kDa IGFBP in rainbow trout. Chi: Chinook salmon.

Figure 2

Body weight (a) and specific growth rate (SGR) (b) over the course of the trial. Differences between treatment groups within the same experimental week are indicated by different letters (p < 0.05, n = 21 or 42). FD: feed deprivation.

Figure 3

Differential regulation of somatotropic axis components. Panels indicate differentially regulated genes in (a) liver and (b) white muscle and (c) serum IGF-I in continuously fed control and mutant fish. Asterisks indicate significantly different means, p < 0.05 (gene expression: n = 21; plasma IGF-I: n = 8). AU: arbitrary units.

Figure 4

Abundance of serum (a) IGF-I, (b) IGFBP-2b, and (c) 32 kDa IGFBP during feed deprivation (FD, Wk 3) and refeeding (Wk 4). The horizontal dotted line is a visual reference for the mean of the continuously fed control fish at Wk 3. Asterisks indicate that the feed-deprived (dashed line) or refed groups (solid line) differ from continually fed fish within the same treatment group and week. The carrot (^) in panel (b) represents p = 0.06. The black diamond indicates that the magnitude of regulation from continually fed fish differs between the mutants and controls. Panels (d) through (i) represent associations between plasma variables in control (d–f) and mutant (g–i) fish. Trendlines indicate the regression of best fit; analysis data are provided when p < 0.05 (n = 8).

Figure 5

Expression of igf1, igf2, and igfr-1a in liver (a,b) and white muscle (c,d,e) during feed deprivation and refeeding. The y-axis represents gene expression values in arbitrary units normalized to the continuously fed control group from Wk 3 and expressed as log₂ (fold change). The horizontal dotted line is a visual reference for log₂ (fold change) = 0. Asterisks indicate that the feed-deprived (FD, Wk 3) or refed (Wk 4) groups (solid line) differ from continually fed fish (dashed line) within the same treatment group and week (p < 0.05, n = 21). The black diamond indicates that the fold change differs between the mutants and controls.

Figure 6

Hepatic expression of igfbp genes during feed deprivation (FD) and refeeding in controls (blue lines) and mutants (red lines). Genes are indicated in the top left corner of each panel (a–j). The y-axis represents gene expression values normalized to the continuously fed control group from week 3 and expressed as log₂ (fold change). The horizontal dashed line is a visual reference for log₂ (fold change) = 0. Asterisks indicate that the feed-deprived (FD, Wk 3) or refed (Wk 4) groups (solid line) differ from continually fed fish (dashed line) within the same treatment group and week (p < 0.05, n = 21). The black diamond indicates that the fold change differs between mutants and controls.

Figure 7

White muscle expression of igfbp genes during fed deprivation (FD) and refeeding in controls (blue lines) and mutants (red lines). Genes are indicated in the top left corner of each panel (a–k). The y-axis represents gene expression values normalized to the continuously fed control group from week 3 and expressed as log₂ (fold change). The horizontal dashed line is a visual reference for log₂ (fold change) = 0. Asterisks indicate that the feed deprived (FD, Wk 3) or refed (Wk 4) groups (solid line) differ from continually fed fish (dashed line) within the same treatment group and week (p < 0.05, n = 21). The black diamond indicates the fold change differs between mutants and controls.

Figure 7

White muscle expression of igfbp genes during fed deprivation (FD) and refeeding in controls (blue lines) and mutants (red lines). Genes are indicated in the top left corner of each panel (a–k). The y-axis represents gene expression values normalized to the continuously fed control group from week 3 and expressed as log₂ (fold change). The horizontal dashed line is a visual reference for log₂ (fold change) = 0. Asterisks indicate that the feed deprived (FD, Wk 3) or refed (Wk 4) groups (solid line) differ from continually fed fish (dashed line) within the same treatment group and week (p < 0.05, n = 21). The black diamond indicates the fold change differs between mutants and controls.