Growth hormone: a newly identified developmental organizer

Abstract

The sexually dimorphic expression of cytochromes P450 (CYP) drug-metabolizing enzymes has been reported in all species examined. These sex differences are only expressed during adulthood and are solely regulated by sex differences in circulating growth hormone (GH) profiles. Once established, however, the different male- and female-dependent CYP isoform profiles are permanent and immutable, suggesting that adult CYP expression requires imprinting. As the hormone that regulates an adult function is likely the same hormone that imprints the function, we selectively blocked GH secretion in some newborn male rats, whereas others received concurrent physiologic replacement of rat GH. The results demonstrate that adult male GH activation of the signal transduction pathway regulating expression of the principal CYP2C11 isoform is obligatorily dependent on perinatal GH imprinting, without which CYP2C11 and drug metabolism would be permanently and profoundly suppressed. As there are other adult metabolic functions also regulated by GH, pediatric drug therapy known to disrupt GH secretion could unintentionally impair adult health.

Keywords: CYP2C11; JAK/STAT; growth hormone; imprinting; miRNA.

Figure 1. Plasma growth hormone (GH) of male rat pups treated with diluents (CONTROL), MSG and/or GH

Neonatally treated pups were injected with either MSG (4mg/g b wt) or an equivalent dose of diluent on postnatal days 1, 3, 5, 7 and 9; rat GH (20μg/100g b wt) and/or the GH diluent, twice/d for the first 12 days of life. Results are presented as the means ± SD of at least 7 pups/group. *P<0.01 vs CONTROL; ^†P<0.01 vs MSG treatment alone.

Figure 2. Adult plasma growth hormone (GH) profiles in growth hormone depleted neonates

Plasma levels of circulating GH were obtained from individual, undisturbed catheterized adult rats neonatally treated with diluents (CONTROL), MSG and/or GH for 8 continuous hours at 15 min intervals. Similar results were obtained from 4 to 5 additional animals in each treatment group. See Figure 1 for details of neonatal treatment.

Figure 3. Growth hormone (GH) imprinting affects post-weaning maturational Lee index profiles (A) and body weight gains (B)

Results are presented as the means ± SD of at least 15 rats/group. CONTROL, MSG and/or GH treatments are described in detail in Figure 1. *P<0.01 vs CONTROL; ^†P<0.01 vs MSG treatment alone.

Figure 4. Neonatal growth hormone (GH) imprints adult hepatic CYP2C11 expression

(A) CYP2C11-dependent hexobarbital-induced sleeping times in adult rats neonatally administered diluents (CONTROL), MSG and/or GH determined following an ip injection of hexobarbital (150mg/kg b wt). Results are presented as the means ± SD of at least 10 rats/group. *P<0.01 vs CONTROL; ^†P<0.01 vs MSG treatment alone. (B1) Relative expression levels (mRNA and protein) of the paramount male-specific CYP2C11 isoform in freshly isolated preplated hepatocytes from adult male rats neonatally administered diluent (CONTROL), MSG and/or GH. Results are presented as a percentage of mRNA or protein in hepatocytes from CONTROL rats arbitrarily designated as 100%. Values are means ± SD of at least 6 rats/data point. *P<0.01 vs CONTROL; ^†P<0.01 vs MSG treatment alone. (B2) Hepatocytes from the same rats as (B1) above were exposed to either the episodic male like GH profile or the hormone’s vehicle for 6 days in culture. Results are presented as a percentage of mRNA or protein in hepatocytes exposed to vehicle arbitrarily designated 100%. Values are means ± SD of at least 6 rats/data point. *P<0.01 vs hepatocytes exposed to the episodic vehicle profile from rats of the same neonatal treatment, i.e. CONTROL, MSG, GH or MSG+GH as described in Figure 1.

Figure 5. Neonatal growth hormone (GH) imprints adult growth hormone regulation of STAT5b activation, nuclear translocation and binding to the CYP2C11 promoter

(Left Panel) Nuclear levels of activated STAT5b (pSTAT5b) in cultured primary hepatocytes exposed to either the episodic male-like GH profile or the episodic profile containing GH vehicle alone for 6 days. Results are presented as a percentage of nuclear pSTAT5b in hepatocytes exposed to vehicle arbitrarily designated as 100%. Values are means ± SD of at least 6 rats/data point. *P<0.01 vs hepatocytes exposed to the episodic vehicle GH profile from rats of the same neonatal treatment, i.e. CONTROL, MSG, GH or MSG+GH as described in Figure 1. (Right Panel) Representative ChIP blots (2 of 5/group) demonstrating episodic GH regulation (♂GH) described above, of pSTAT5b binding to the Cyp2c11 promoter in hepatocytes derived from adult male rats neonatally treated with either diluents (CONTROL), MSG, GH or MSG+GH as described in Figure 1.

Figure 6. Neonatal growth hormone (GH) imprints adult growth hormone regulation of CIS/SOCSs expression

(A1, B1, C1, D1) Levels of 4 members of the CIS/SOCS family (i.e. CIS, SOCS2, SOCS1 and SOCS3) were measured in freshly isolated preplated hepatocytes from adult male rats neonatally administered diluents (CONTROL), MSG, GH or MSG+GH as described and statistically analyzed in Figure 1. (A2, B2, C2, D2) Concentrations of the same CIS/SOCS family members in hepatocytes from the same rats (A1, B1, C1, D1) were exposed to either the episodic male-like GH profile or the hormone vehicle for 6 days in culture. Results are presented and statistically analyzed as described in Figure 4-B2.

Figure 7. Adult expression of 3 rat-specific hepatic microRNAs (miR) neonatally imprinted by growth hormone (GH)

(A1, B1, C1) Levels of 3 microRNAs (rno-miR-29b-3p, rno-miR-381a-3p, rno-miR-130a-3p) were measured in freshly isolated preplated hepatocytes from adult male rats neonatally administered diluents (CONTROL), MSG, GH or MSG+GH as described and statistically analyzed in Figure 1. (A2, B2, C2) Levels of the same hepatic microRNAs from the same rats (A1, B1, C1) were exposed to either the episodic male-like GH profile or the hormone vehicle for 6 days in culture. Results are presented and statistically analyzed as described in Figure 4-B2. (D) Sequence of the 3 microRNAs and their complementary sites in the 3′-UTR of CYP2C11, SOCS2 and IGF1.