Astrocyte leptin receptor (ObR) and leptin transport in adult-onset obese mice - PubMed (original) (raw)

Astrocyte leptin receptor (ObR) and leptin transport in adult-onset obese mice

Weihong Pan et al. Endocrinology. 2008 Jun.

Abstract

The agouti viable yellow (A vy) spontaneous mutation generates an unusual mouse phenotype of agouti-colored coat and adult-onset obesity with metabolic syndrome. Persistent production of agouti signaling protein in A vy mice antagonizes melanocortin receptors in the hypothalamus. To determine how this disruption of neuroendocrine circuits affects leptin transport across the blood-brain barrier (BBB), we measured leptin influx in A vy and B6 control mice after the development of obesity, hyperleptinemia, and increased adiposity. After iv bolus injection, (125)I-leptin crossed the BBB significantly faster in young (2 month old) B6 mice than in young A vy mice or in older (8 month old) mice of either strain. This difference was not observed by in situ brain perfusion studies, indicating the cause being circulating factors, such as elevated leptin levels or soluble receptors. Thus, A vy mice showed peripheral leptin resistance. ObRa, the main transporting receptor for leptin at the BBB, showed no change in mRNA expression in the cerebral microvessels between the age-matched (2 month old) A vy and B6 mice. Higher ObRb mRNA was seen in the A vy microvasculature with unknown significance. Immunofluorescent staining unexpectedly revealed that many of the ObR(+) cells were astrocytes and that the A vy mice showed significantly more ObR(+) astrocytes in the hypothalamus than the B6 mice. Although leptin permeation from the circulation was slower in the A vy mice, the increased ObR expression in astrocytes and increased ObRb mRNA in microvessels suggest the possibility of heightened central nervous system sensitivity to circulating leptin.

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Figures

Figure 1

Body weight and metabolic indices of the B6 and Avy mice. A, Growth curve of the Avy and age-matched B6 control mice (n = 4 per group). Repeated measures ANOVA showed a significant (P < 0.01) overall increase of body weight in the Avy group. Post hoc analysis indicated that the difference was significant at 8 wk of age and persisted until the end of the study at 24 wk of age. **, P < 0.01; ***, P < 0.005. B, Body fat of the Avy and B6 mice was determined by nuclear spin resonance. The 8-wk-old (n = 3), 15-wk-old (n = 6), and 24-wk-old B6 mice had 9.47, 8.32, and 10.44% fat, respectively. The 8-wk-old (n = 6), 15-wk-old (n = 7), and 24-wk-old (n = 4) Avy mice had 25.24, 24.82, and 28.60% fat, respectively. The increase in the Avy mice was significant at all three time points. ***, P < 0.005. C, Serum leptin concentrations were measured at 8 wk of age by ELISA. The Avy mice had significantly higher concentrations of circulating leptin. ***, P < 0.005. n = 4 per group.

Figure 2

Permeation of blood-borne leptin across the BBB in the Avy mice. A, The rate of 125I-leptin crossing the BBB was determined in four groups of mice (n = 7 per group) by multiple-time regression analysis. The influx rate of leptin, shown by the slope of the regression line, was significantly higher in the 2-month-old B6 mice than in the rest of the groups (P < 0.05). The dashed, unmarked lines represent the influx of 131I-albumin. Permeation of albumin showed no difference in any of the groups, being significantly lower than that of 125I-leptin. B, In the hypothalamus, the influx rate Ki of 125I-leptin in the 2-month-old Avy mice showed a trend of increase compared with the age-matched B6 mice or the older age groups. This pattern differed from that for the rest of the brain, where a significant increase was seen in the 2-month-old B6 group compared with others. +, P < 0.1; *, P < 0.05; **, P < 0.01; ***, P < 0.005.

Figure 3

Leptin permeation across the BBB after delivery by in situ brain perfusion for 5 min. Groups of B6 (n = 4) and Avy (n = 6) mice were studied at 2 months old. The brain uptake of 125I-leptin from the blood-free perfusate did not differ between the two groups.

Figure 4

The level of mRNA expression of ObR isoforms in cerebral microvessels and hypothalamic homogenate. A, ObRa mRNA was unchanged between the 2-month-old B6 and Avy mice, in both the microvessels (n = 8 per group) and hypothalamus (n = 10 per group). B, ObRb mRNA in cerebral microvessels was significantly higher in the Avy mice. ObRb mRNA in the hypothalamus showed no change between the two strains at 2 months of age (n = 11 for B6 and n = 10 for Avy). *, P < 0.05.

Figure 5

Regional distribution and cellular phenotypes of ObR(+) cells by immunohistochemistry. A, Specificity of the ObR immunofluorescence was shown by the absence of signals with inclusion of a blocking peptide. Left panel, ObR immunofluorescence was seen in a hypothalamic section from a B6 mouse. The signal was present in the cytoplasm of neurons and occasional astrocytes (arrowheads). Right panel, A blocking peptide (sc-1834p) was included at 5-fold

excess during the overnight incubation with the ObR antibody. This preabsorption step completely abolished the immunofluorescent signal (objective, ×20; scale bar, 50 μm). B, ObR expression between the B6 (A) and Avy (B) mice differed in both neurons and astrocytes in the arcuate nucleus. The confocal microscopical images were taken with a 20× objective (scale bar, 50 μm). Seen at higher magnification (40× objective, scale bar, 30 μm), the B6 mouse (C) had fewer ObR(+) astrocytes (arrowheads) than the Avy mouse (D). C, In the dorsomedial nucleus of hypothalamus (DMH), more cells and higher staining intensity of ObR(+) astrocytes were seen in the Avy mouse (right panel) compared with the B6 control (left panel). D, The relative amount of ObR(+) astrocytes in the arcuate nucleus of the Avy mice was significantly higher than that in the B6 mice. This was inversely correlated with the relative amount of neurons. ***, P < 0.005, comparison of the same cell type between B6 and Avy; +++, P < 0.005, comparison of astrocytes and neurons of the same mouse strain (n = 3).

Figure 6

Confocal microscopical analysis of the type of cells expressing ObR. A, Colocalization of ObR (green) and GFAP (red) in the arcuate nucleus region of a B6 mouse. Essentially, all GFAP(+) astrocytes (arrowheads) coexpress ObR either at the cell body or processes. Not all ObR(+) cells are GFAP(+); the GFAP(−) cells resemble neurons (arrows). The yellow coloring indicates colocalization of ObR and GFAP in the same cells. B, Lack of colocalization of ObR (green) and CD11b (red), the marker for microglia, is shown in the Avy arcuate nucleus (scale bar, 50 μm).

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Astrocyte leptin receptor (ObR) and leptin transport in adult-onset obese mice - PubMed (original) (raw)