Developmental changes of leptin receptors in cerebral microvessels: unexpected relation to leptin transport - PubMed (original) (raw)

Developmental changes of leptin receptors in cerebral microvessels: unexpected relation to leptin transport

Weihong Pan et al. Endocrinology. 2008 Mar.

Abstract

The adipokine leptin participates not only in the regulation of feeding and obesity in adults but also in neonatal development. It crosses the blood-brain barrier (BBB) by receptor-mediated transport. Leptin concentrations in blood differ between neonates and adults. We determined the developmental changes of leptin receptor subtypes in the cerebral microvessels composing the BBB and examined their expected correlation with leptin transport across the BBB. Total RNA was extracted from enriched cerebral microvessels of mice 1, 7, 14, and 60 d of age for real-time RT-PCR analysis of leptin receptor subtypes. In cerebral microvessels from neonates, ObRa, ObRb, ObRc, and ObRe mRNA were all higher than in adults, but ObRd was not detectable. Hypothalamus showed similar age-related changes except for ObRb, which was higher in adults. The homologous receptor gp130 did not show significant age-related changes in either region. Despite the increase of leptin receptors, leptin permeation across the BBB after iv injection was less in the neonates. In situ brain perfusion with blood-free buffer showed no significant difference in the brain uptake of leptin between neonates and adults, indicating an antagonistic role of leptin-binding proteins in the circulation, especially the soluble receptor ObRe. The results are consistent with our previous finding that ObRe antagonizes leptin endocytosis in cultured endothelia and transport from blood to brain in mice. Overall, the developmental changes observed for leptin receptors unexpectedly failed to correlate with the entry of leptin into brain, and this may indicate different functions of the receptors in neonates and adults.

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Figures

Figure 1

Figure 1

Expression of leptin receptors in cerebral microvessels composing the BBB and in the hypothalamus, shown by TaqMan real-time PCR. Compared with the 7-d-old neonates, ObR mRNA in the 2-month-old adults showed a significant decrease in the microvessels but not in the hypothalamic area. The homologous receptor gp130 had no age-related changes in mRNA in either region. *, P < 0.05 (n = 6 per group).

Figure 2

Figure 2

TaqMan real-time PCR on ObRa mRNA expression. In both microvessels and hypothalamus, ObRa was significantly lower in the 2-month-old adult mice than in the 7-d-old neonates. ***, P < 0.005 (n = 6 per group).

Figure 3

Figure 3

TaqMan real-time PCR on ObRb mRNA expression. ObRb was significantly lower in the microvessels but higher in the hypothalamus in the adult mice (2 months old) than in the neonates (7 d old). ***, P < 0.005 (n = 6 per group).

Figure 4

Figure 4

The reduction of ObRa with maturation was confirmed by SYBR Green real-time PCR. In both microvessels and hypothalamus, the neonates (7 d old, n = 5) had significantly higher ObRa than the adults (2 months old, n = 6). ***, P < 0.005.

Figure 5

Figure 5

SYBR Green real-time PCR showed that ObRb was lower in 2-month-old adults (n = 6) than in the 7-d-old neonates (n = 5) in the microvessels. The hypothalamus showed opposite changes, with a significant increase in the adult group. +, P = 0.08; ***, P < 0.005.

Figure 6

Figure 6

SYBR Green real-time PCR showed significant reduction of ObRc in both microvessels and hypothalamus in the adult mice (2 months old, n = 6) when compared with the 7-d-old neonates (n = 5). *, P < 0.05; **, P < 0.01.

Figure 7

Figure 7

SYBR Green real-time PCR showed that adults (2 months old, n = 6) had significantly lower ObRe than neonates (7 d old, n = 5). This was seen in both microvessels and hypothalamus.

Figure 8

Figure 8

Confirmation of ObR subtypes in cerebral microvessels and hypothalamus by PCR: lane 1, no-template control; lane 2, microvessels from 7-d-old neonate; lane 3, microvessels from 2-month-old adult; lane 4, hypothalamus from 7-d-old neonate; lane 5, hypothalamus from 2-month-old adult. Note the absence of amplification of ObRd, in contrast to the positive products of ObRa, ObRb, ObRc, and ObRe. β-Actin was amplified as an internal control.

Figure 9

Figure 9

Dynamic changes of ObRa expression from 1-d-old to 2-month-old mice shown by TaqMan PCR. Cerebral microvascular ObRa mRNA was highest at d 7 after birth and decreased by d 14. Both were higher than at d 1 and 2 months. Hypothalamic ObRa was highest at d 1, decreased during the neonatal period, and was lowest at 2 months. Asterisks above the bar indicate comparison with 1-d-old group. Asterisks connected by a line indicate comparison between other groups. ***, P < 0.005 (n = 2 for the 1-d group; n = 4 for the rest).

Figure 10

Figure 10

Dynamic changes in ObRb expression shown by TaqMan real-time PCR. Cerebral microvascular ObRb mRNA was highest at d 7 and increased again at 2 months. Hypothalamic ObRb was highest in the adults. Asterisks above the bar indicate comparison with 1-d-old group. Asterisks connected by a line indicate comparison between other groups. *, P < 0.05; ***, P < 0.005 (n = 2 for the 1-d group; n = 4 for the rest).

Figure 11

Figure 11

The influx rate of [125I]leptin (corrected for vascular space by subtraction of the brain/blood ratio of [131I]albumin) was significantly higher (P < 0.005) in the adult mice (2 months old, n = 12) than in the neonates (11 d old, n = 11).

Figure 12

Figure 12

The brain/perfusate ratio of [125I]leptin and [131I]albumin was not significantly different in the adult (2 months old, n = 6) and neonatal (7 d old, n = 7) groups 10 min after perfusion. The corrected values ([125I]leptin and [131I]albumin) also showed no difference.

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