Rapid communication R1 Journal of Endocrinology (1999) 163, R1–R4 Accepted 20 August 1999 0022-0795/99/0163-00R1 © 1999 Society for Endocrinology Printed in Great Britain Online version via http://www.endocrinology.org Abundance of leptin mRNA in fetal adipose tissue is related to fetal body weight S J Bernard, I Yuen 1 , C McMillen 1 , M E Symonds 3 and P C Owens 2 1 Department of Physiology, University of Adelaide, Adelaide 5005, Australia 2 Department of Obstetrics and Gynaecology, University of Adelaide, Adelaide 5005, Australia 3 Academic Division of Child Health, School of Human Development, Queen’s Medical Centre, Nottingham, UK Abstract Leptin mRNA was measured in adipose tissue of fetal sheep by reverse transcription polymerase chain reaction (RTPCR). Abundance of leptin mRNA relative to β-actin mRNA in fetal perirenal adipose tissue increased (P<0.02) with gestation, being higher at 144 d (0.73 ± 0.10, n=5) than at 90-91 d (0.40 ± 0.08, n=6) or 125 d (0.40 ± 0.04, n=5) gestation (term ~147- 150 d). There was a positive relationship between relative abundance of leptin mRNA (y) and fetal body weight (x) between 90 and 144 d gestation (r 2 =0.27, P<0.01). The slope of the linear dependence of leptin mRNA on fetal weight was 15-fold greater (P<0.001) at 90-91d (y = 2.81x - 1.1, n=6, r 2 =0.71, P<0.025) than between 125-144 d gestation (y = 0.195x - 0.15, n=16, r 2 =0.39, P<0.01). Thus the leptin synthetic capacity of fetal adipose tissue appears to increase in late gestation but this is accompanied by constraint of its sensitivity to fetal body weight. We hypothesise that leptin synthesis in fetal adipose tissue is related to fetal nutrient supply and growth rate. Introduction Leptin is a 16 kDa polypeptide hormone synthesised and secreted by adipocytes that acts to suppress appetite and increase energy expenditure in adults (Friedman & Halaas 1998, Houseknecht et al. 1998). Abundance of leptin mRNA in adipose tissue and concentrations of leptin protein in blood correlate positively with body weight and adiposity in human adults (Maffei et al. 1995, Considine et al. 1996). In rodents leptin mRNA is expressed in a number of fetal tissues including cartilage, hair follicles and placenta (Senaris et al. 1997, Hoggard et al. 1997, Masuzaki et al. 1997) and in humans there is a positive association between leptin levels in cord blood and body weight at birth (Schubring et al. 1997, Koistinen et al. 1997, Ong et al. 1999). These data suggest there is a relationship between growth and leptin synthesis before birth, but there have been no reports relating fetal expression of leptin to fetal growth. We have measured leptin mRNA in perirenal fat, the major adipose tissue in the sheep fetus. We also investigated the relationship between leptin mRNA and fetal weight before (90-91 d) and after (125-144 d gestation) the development of sympathetic innervation of the perirenal fat depot (Gemmell & Alexander 1978, Clarke et al. 1997). Materials and Methods Animals The study design was approved by the Animal Ethics Committee of the University of Adelaide. Merino ewes (n=51) were mated and provided unrestricted access to feed and water. They were killed between 90 and 145 d of pregnancy (term ~147-150 d) with an overdose of sodium pentobarbitone (6.5 g i.v.) and the fetuses were removed and weighed. In one group (n=29, 12 (male) and 17 (female)) of singletons (n=12) and twins (n=17 fetuses from 12 ewes), both left and right fetal perirenal fat depots were collected between 90-99 d (n=9) and 137-145 d (n=20) and weighed. In another group consisting entirely of twins a biopsy of fetal perirenal adipose tissue was obtained from one twin at 90-91 d (n=6), 125 d (n=5), 139-141 d (n=6) and 144 d (n=5), quickly frozen in liquid N2 and stored at -80 °C for measurement of leptin and β-actin mRNA. Reverse transcription polymerase chain reactions RNA was extracted from ~100 mg adipose tissue (Tri Reagent, Prod T9424, Sigma) as recommended. cDNA was obtained by reverse transcription of 2 μg total RNA with random sequence hexanucleotides (Cat RP-6, GeneWorks, Adelaide, Australia) using Super-Script RNase H - (Cat 18053- 017, GIBCOBRL). A fragment of ovine leptin cDNA was amplified through 26 cycles of 60 sec at 94 °C, 15 sec at 53 °C and 60 sec at 72 °C (Hybaid PCR Express, Teddington, UK) from 5 μl of reverse transcription product using Taq* DNA polymerase (Biotech International, Bently, Australia) as recommended by the manufacturer with 5'-GACAT- CTCACACACGCAG-3' and 5'-GAGGTTCTCCAGG- TCATT-3' (GeneWorks) as primers. This produced 183 bp of ovine leptin ds cDNA (nucleotides 67-249 of the 441