Impaired neonatal survival of pro-opiomelanocortin null mutants

Abstract

Intercrosses of heterozygous pro-opiomelanocortin (POMC) mice result in homozygous null progeny at lower frequencies than expected. Genotyping offspring at pre-, peri-, and postnatal stages revealed that over half of homozygous null mutants die in the early postnatal stages. To investigate the reasons for this early postnatal lethality, we analyzed in detail different parameters in the initial hours after birth. POMC null mutants born to heterozygous dams presented at birth with corticosterone levels no different from wildtype littermates, were euglycemic, and had normal liver glycogen stores. However, already 30 min after birth corticosterone levels dropped by 80% and were undetectable thereafter, while corticosterone levels in wildtype animals increased during postnatal hours. Circulating adrenaline was almost below detection 1 h after birth. Blood glucose levels fell sharply in all genotypes within 30 min after birth; however, wildtype and heterozygous pups overcame hypoglycemia within an hour, while mutant pups stayed hypoglycemic. The depletion of liver glycogen stores in mutant pups was significantly less efficient compared to their littermates in the hours after birth. POMC null mutant mice born to POMC null mutant dams completely lack corticosterone and die of the expected respiratory dysfunction. In contrast, POMC null mutant mice born to heterozygous dams do not die of respiratory problems, but rather due to hypoglycemia. Our studies confirm an essential involvement of POMC peptides and of adrenal glucocorticoids and catecholamines on glucose homeostasis critical for early postnatal survival.

Introduction

In crosses of POMC heterozygous mutants less than half of the expected homozygous null mutants are found at weaning age (Yaswen et al., 1999). The “missing” half of POMC-deficient mutant mice might die pre-, peri-, or postnatally. Furthermore, this lethality may result from the lack of all POMC-derived peptides as well as the lack of downstream factors regulated by POMC-peptides.

During pregnancy transcripts for POMC and for its receptors (specifically delta-, kappa-, and mu-opioid receptors) are found in the uterus, the placenta, and the developing embryo (Liotta et al., 1977, Loh et al., 1991, Zhu et al., 1998, Zhu and Pintar, 1998). Relatively little is known about the specific roles of POMC peptides for the developing fetus during pregnancy. In expression studies of pregnant mouse uterus and placenta, POMC transcripts were detected in the uterine environment soon after implantation and expression was retained until late gestation (Zhu et al., 1998). There is no POMC expression in the uterus before implantation. In the placenta POMC is expressed in trophoblast giant cells. Taken together, these results suggest roles for POMC peptides in maternal adaptation to pregnancy and in supporting embryo growth. In addition to maternal supplies there are at least three potential sources of peptides of the POMC family available to the embryo during intrauterine development: the fetal pituitary, the fetal brain, and the placenta (Genazzani et al., 1981, Pintar and Lugo, 1987, Loh et al., 1991). In the embryonic brain, the POMC system, among opioid peptides, is one of the earliest peptidergic systems to appear (Khachaturian et al., 1985, Elkabes et al., 1989, Khachaturian et al., 1991, Rius et al., 1991); POMC mRNA is first expressed in the mouse pituitary gland on embryonic day 12.5 (E12.5) (Pintar and Lugo, 1987, Loh et al., 1991). In the developing embryo expression of POMC outside the CNS and pituitary was demonstrated by in situ hybridization and RT/PCR at low levels in the basal layer of E10.5 neural tube, in several discrete areas around the dorsal aorta, and in developing limb buds (De Angelis et al., 1993).

In the transition of fetal to neonatal life the adrenal gland is of central importance by supplying glucocorticoids and catecholamines needed in the adaptation of neonates to the sudden lack of maternal supplies in nutrients and oxygen. Adrenal structure and function are dependent on the presence of POMC peptides, and especially ACTH, which binds to its receptor on cells of the adrenal gland, the melanocortin receptor 2 (MC2R), resulting in the release of corticosterone. Plasma corticosterone levels are high at delivery and rapidly decline during the first hours after birth. Epinephrine and norepinephrine levels are increased several fold in newborns in response to the stresses of birth, such as transient hypoxia, cold exposure, and cord cutting (Girard et al., 1992).

Glucocorticoids are important during embryonic lung development, specifically with respect to cell proliferation and architectural maturation (Muglia et al., 1999). In addition, they help promote surfactant synthesis in alveolar epithelial cells of the developing lung (Mendelson and Boggaram, 1991) and are used effectively in the treatment of the respiratory distress syndrome of prematurely born children.

Glucocorticoids play a major role in glucose homeostasis. Immediately after birth, the newborn mouse has to withstand a brief period of starvation before being fed with milk that is high in fat and low in carbohydrates. The adaptation of neonates to these changes in nutrition requires modification of glucose and fatty acid metabolism (Girard et al., 1992). Defective gluconeogenesis leads to neonatal death (Wang et al., 1995).

We undertook a more detailed analysis into the phenotypic expression of this partial lethality of POMC null mutants to get a better understanding of the role of POMC in fetal development and fetal to neonatal transition.