Abstract
Cpe(fat/fat) mice are obese, diabetic, and infertile. They have a mutation in carboxypeptidase E (CPE), an enzyme that converts prohormone intermediates to bioactive peptides. The Cpe(fat) mutation leads to rapid degradation of the enzyme. To test whether pro-thyrotropin-releasing hormone (TRH) conversion to TRH involves CPE, processing was examined in the Cpe(fat/fat) mouse. Hypothalamic TRH is depressed by at least 75% compared with wild-type controls. Concentrations of pro-TRH forms are increased in homozygotes. TRH-[Gly(4)-Lys(5)-Arg(6)] and TRH-[Gly(4)-Lys(5)] represent approximately 45% of the total TRH-like immunoreactivity in Cpe(fat/fat) mice; they constitute approximately 1% in controls. Levels of TRH-[Gly(4)] were depressed in homozygotes. Because the hypothalamus contains some TRH, another carboxypeptidase must be responsible for processing. Immunocytochemical studies indicate that TRH neurons contain CPE- and carboxypeptidase D-like immunoreactivity. Recombinant CPE or carboxypeptidase D can convert synthetic TRH-[Gly(4)-Lys(5)] and TRH-[Gly(4)-Lys(5)-Arg(6)] to TRH-[Gly(4)]. When Cpe(fat/fat) mice are exposed to cold, they cannot maintain their body temperatures, and this loss is associated with hypothalamic TRH depletion and reduction in thyroid hormone. These findings demonstrate that the Cpe(fat) mutation can affect not only carboxypeptidase activity but also endoproteolysis. Because Cpe(fat/fat) mice cannot sustain a cold challenge, and because alterations in the hypothalamic-pituitary-thyroid axis can affect metabolism, deficits in pro-TRH processing may contribute to the obese and diabetic phenotype in these mice.
Highlights
Neuropeptides and peptide hormones are first biosynthesized as precursors that must undergo a series of conversions to become biologically active [1, 2]
thyrotropin-releasing hormone (TRH) is released from median eminence nerve terminals into the hypophyseal blood where it is transported to the pituitary to stimulate the biosynthesis and secretion of thyroid-stimulating hormone (TSH; see Ref. 10)
Hypothalamic protein contents did not differ among the three genotypes. These data indicate clearly that whereas the amounts of TRH are substantially reduced in homozygote hypothalamus, some small amount of pro-TRH may be fully converted to TRH in these animals
Summary
Animals—The BKS.HRS-Cpefat/J mice were received as a gift from Dr Edward H. To determine the levels of TRH-[Gly4-Lys5] and TRH-[Gly4-Lys5Arg6] in hypothalami from WT and CPEfat/fat mice, samples were separated by HPLC, and fractions surrounding and including the elution positions for the synthetic standards were incubated with the recombinant CPD for 4 h at 37 °C, boiled for 5 min, and submitted to the TRH-[Gly4] RIA. The following day, slides were incubated for 2 h at room temperature with FITC conjugated secondary anti-rabbit sera (1:500 dilution; Vector Laboratories). In this case, the pro-TRH immunostaining was visualized with anti-pAV37 sera that had been conjugated previously to Texas Red. Co-localization studies were performed with sequential immunostaining for CPE and pro-TRH or CPD and pro-TRH as described above. A posteriori analyses were performed by Duncan and Newman-Keuls tests
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