Reversal of physiological deficits caused by diminished levels of peptidylglycine alpha-amidating monooxygenase by dietary copper

Abstract

Amidated peptides are critically involved in many physiological functions. Genetic deletion of peptidylglycine alpha-amidating monooxygenase (PAM), the only enzyme that can synthesize these peptides, is embryonically lethal. The goal of the present study was the identification of physiological functions impaired by haploinsufficiency of PAM. Regulation of the hypothalamic-pituitary-thyroid axis and body temperature, functions requiring contributions from multiple amidated peptides, were selected for evaluation. Based on serum T(4) and pituitary TSH-beta mRNA levels, mice heterozygous for PAM (PAM(+/-)) were euthyroid at baseline. Feedback within the hypothalamic-pituitary-thyroid axis was impaired in PAM(+/-) mice made hypothyroid using a low iodine/propylthiouracil diet. Despite their normal endocrine response to cold, PAM(+/-) mice were unable to maintain body temperature as well as wild-type littermates when kept in a 4 C environment. When provided with additional dietary copper, PAM(+/-) mice maintained body temperature as well as wild-type mice. Pharmacological activation of vasoconstriction or shivering also allowed PAM(+/-) mice to maintain body temperature. Cold-induced vasoconstriction was deficient in PAM(+/-) mice. This deficit was eliminated in PAM(+/-) mice receiving a diet with supplemental copper. These results suggest that dietary deficiency of copper, coupled with genetic deficits in PAM, could result in physiological deficits in humans.

Figure 1

Verification of animal model of PAM haploinsufficiency. Extracts from individual mice were used to quantify PAM enzymatic activity (A) in the POA, PVN, and pituitary gland of PAM^+/− (n = 4–6) and WT (n = 4–6) mice as described in Materials and Methods; error bars, sem. One-way ANOVAs demonstrated the following levels of significance: POA, P = 0.002; PVN, P = 0.001; pituitary, P < 0.0005. PAM levels in individual PVN extracts (30 μg protein) from PAM^+/− and WT mice (n/group = 4) were determined by immunoblot analysis using a rabbit polyclonal antibody to exon A, which detects full-length PAM-1 and cleaved PHM and peptidyl-α-hydroxyglycine α-amidating lyase (PAL) (B). Data for PAM were normalized to levels of β3-tubulin, which were determined after stripping the blot; error bars, sem. One-way ANOVAs revealed a significant difference between genotypes: PHM, P = 0.008; PAM-1 P = 0.023. Levels of TRH-Gly and amidated TRH were assessed in POA and PVN extracts from PAM^+/− (n = 7 or 8) and WT (n = 7 or 8) mice (C). Raw data (nanograms peptide per milligram protein) are presented in supplementary Table 2. Percent TRH-Gly was calculated using the sum of TRH-Gly plus TRH as the total; error bars, sem. One-way ANOVAs revealed the following levels of statistical significance: POA, P = 0.001; PVN, P = 0.002. Asterisk, See Statistical analysis for P-values.

Figure 2

HPT axis regulation is altered in PAM^+/− mice. Pituitary levels of TSH-β (A) and PAM (B) mRNA (normalized to GAPDH) were determined in euthyroid and hypothyroid animals by quantitative PCR. PAM^+/− (n/group = 8–9) and WT (n/group = 8 each) mice were kept on the low-iodine/PTU diet or a normal diet for 14 d; error bars, sem. Two independent sets of animals were examined; data shown are a combination of both experiments (total = 42; n/group = 7–9). A two-way ANOVA revealed a significant effect of low-iodine/PTU diet on the levels of TSH (P < 0.0005) and a significant interaction (i.e. a difference in the effect of the diet) between genotype and the low-iodine/PTU diet (P < 0.0005; A). WT mice showed a more substantial increase in TSH mRNA to hypothyroidism than PAM^+/− mice. The pituitary levels of PAM mRNA increased in response to hypothyroidism in WT mice but not PAM^+/− mice. A second two-way ANOVA revealed a significant effect of low-iodine/PTU diet on the levels of PAM (P = 0.023) and a significant interaction between genotype and the low-iodine/PTU diet (P = 0.05; B). Asterisk, See Statistical analysis for P-values.

Figure 3

Inability of PAM^+/− mice to regulate body temperature is reversed by dietary copper. Core body temperatures of PAM^+/− (n = 8, 9) and WT (n = 9, 11) mice with and without dietary copper supplementation (Cu²⁺ Suppl.; n/group = 8–11) were monitored every 40 min in response to a 120-min exposure to 4 C; error bars, sem. An ANOVA of this mixed design revealed significant main effects of both genotype and copper supplementation (P < 0.0005) and a significant interaction between genotype and copper supplementation (P < 0.0005). Only the PAM^+/− mice showed a significant change in their body temperature in response to supplementation of dietary copper. Asterisk, See Statistical analysis for P-values.

Figure 4

Endocrine responses to cold exposure. Plasma harvested from PAM^+/− and WT mice (n/group = 8–11) housed at room temperature or at 4 C for 2 h was assayed for T₄ (A), norepinephrine (B), and corticosterone (C); error bars, sem. Univariate analysis of variance revealed the following significance of cold exposure: T₄, P = 0.005; norepinephrine, P = 0.032; corticosterone, P < 0.0005. One-way ANOVAs revealed the following PAM^+/− responses to cold: T₄, P = 0.031; norepinephrine, P = 0.013. An additional one-way ANOVA revealed a significant increase in baseline corticosterone in PAM^+/− mice, P = 0.039. D, UCP1 mRNA levels (normalized to GAPDH) were quantified in brown adipose tissue harvested from PAM^+/− (n = 6) and WT (n = 6) mice kept at room temperature or 4 C for 120 min. The average level in WT mice housed at room temperature was normalized to 1.0; error bars, sem. In PAM^+/− mice, one-way ANOVAs revealed a significant decrease in baseline UCP1 expression (P = 0.004) and a significant increase in cold-stimulated UCP1 expression (P = 0.01). Asterisk, See Statistical analysis for P-values.

Figure 5

Pharmacological manipulation of shivering and vasoconstriction. A, PAM^+/− and WT mice received a single ip injection of the serotonin antagonist WAY-100635 (1 mg/kg) at the onset of a 2-h exposure to 4 C (n/group = 4–8). Average core body temperatures are shown; error bars, sem. An ANOVA of this mixed design revealed a significant interaction between genotype and pharmacological treatment (P < 0.0005), demonstrating that this pharmacological treatment had a significant effect on the body temperature of PAM^+/− mice but not WT mice. B, PAM^+/− (n = 4–8) and WT (n = 4–8) mice received a single ip injection of ketamine (10 mg/100 g) 3 min before a 5-min exposure to ice packs. PAM^+/− and WT mice experienced equivalent levels of hypothermia. C, PAM^+/− and WT mice received a single ip injection of the noradrenergic agonist phenylephrine (2 mg/kg) at the onset of a 2-h cold exposure. Average core body temperatures are reported (n/group = 4–8). An ANOVA of this mixed design revealed a significant main effect of genotype (P = 0.029) and a significant interaction between genotype and pharmacological treatment (P < 0.0005). Asterisk, See Statistical analysis for P-values.

Figure 6

A, PAM^+/− (total n = 15; n/group = 7–8, in white) and WT (total n = 14; n/group = 6– 7, in black) mice were anesthetized with isoflurane and a laser Doppler probe was used to monitor blood flow through their tail veins before and during a 5-min cold exposure (direct contact with ice packs stored at −20 C). The averaged data are presented as a percentage of baseline blood flow, and the error bars indicate the sem. A one-way ANOVA of repeated measures revealed a significant difference separating the genotypes (P < 0.0005). Additional analysis with a two-way ANOVA of repeated measures indicated that 14 d of supplementation (Suppl.) of dietary copper fully rescued the PAM^+/− deficit in cold-induced vasoconstriction without altering the WT level of cold-induced vasoconstriction. Specifically, a significant overall three-way interaction between blood flow, genotype, and dietary copper treatment (P = 0.012) was seen. Moreover, statistical analysis of the between subject factors revealed significant interaction between genotype and dietary copper treatment (P = 0.031). B, Core body temperature at baseline and after 5 min of cold exposure are shown.