Expression of the ob (obese) gene during lactation in mice.

Abstract

The mutant gene responsible for the development of obesity in the ob/ob mouse has recently been localised and sequenced [l]. The identification of the ob (obese) gene provides an opportunity to make rapid progress in unravelling the critical factors involved in the regulation of body weight. The ob gene appears to be expressed exclusively in adipose tissue [ 1-31. where it codes for an 18,000 A4, protein which is secreted from the adipocyte as a 16,000 Mr product [I], termed leptin [4]. Leptin may act as a satiety factor [1,4-61, signalling the size of the white adipose tissue depots, but there is also evidence that it stimulates energy expenditure [4,5]. Increased expression of the ob gene has been observed following the administration of glucocorticoids [7], while fasting leads to a marked fall in gene expression which is reversed on subsequent re-feeding [3]. Cold exposure induces a substantial increase in energy expenditure and substrate flux in an animal, and we have recently found that expression of the ob gene is rapidly suppressed in the cold, a response which appears to be mediated by the sympathetic nervous system [8]. In the present study we have examined the effects of lactation, a physiological state which also induces major changes in nutrient flux and food intake in small animals [9], on the expression of the ob gene. Female mice of the 'Aston' variety were mated at 6-8 weeks of age. The main studies focused on mid-lactation (10-12 days postpartum), but analyses were also performed at early and late lactation (1-2 days and 18-20 days post-partum, respectively). The effects of abrupt weaning were examined in mid-lactation, tissue being taken 24 h after removal of the pups. Virgin mice, agematched to the lactating animals, were used as controls. The mice were killed by cervical dislocation and gonadal fat (and other tissues in pilot studies) rapidly removed and frozen in liquid N2. Total RNA was extracted and hctionated by agarose gel electrophoresis [3,8]. After vacuum blotting onto a charged nylon membrane (Boehringer Mannheim), the RNA was fixed with W light. The blots were probed with a 33-mer antisense oligonucleotide (5'-GGTCTGAGGCAGGGAGCAGCTCTTGGAGAAGGC) targeted to the mRNA encoding mouse ob mRNA [3,8]. The oligonucleotide was synthesised commercially, and labelled with DIG at the 5' end (R & D Systems Europe). Following post-hybridization washes, the membranes were processed essentially according to the protocols provided in the DIG detection kit (Boehringer), as described previously [3,8]. The membranes were immersed in 250 pM CDP-Stur (Tropix), a new chemiluminescence substrate. After incubation at 37°C for 15 min, the membranes were exposed to film at room temperature for up to 90 min, and bands on the film quantitated by densitometry. The blots were then stripped and sequentially reprobed for lipoprotein lipase mRNA and 18s rRNA using 30-mer and 31-mer antisense oligonucleotides, respectively [8]. The mRNA encoding ob was readily detected by chemiluminescence in white fat of female mice, but not in other tissues. using the 33-mer antisense oligonucleotide as a probe. ob mRNA was evident in gonadal adipose tissue throughout lactation. In three separate experiments the level of ob mRNA was