Differential acute and chronic effects of leptin on hypothalamic astrocyte morphology and synaptic protein levels

Abstract

Astrocytes participate in neuroendocrine functions partially through modulation of synaptic input density in the hypothalamus. Indeed, glial ensheathing of neurons is modified by specific hormones, thus determining the availability of neuronal membrane space for synaptic inputs, with the loss of this plasticity possibly being involved in pathological processes. Leptin modulates synaptic inputs in the hypothalamus, but whether astrocytes participate in this action is unknown. Here we report that astrocyte structural proteins, such as glial fibrillary acidic protein (GFAP) and vimentin, are induced and astrocyte morphology modified by chronic leptin administration (intracerebroventricular, 2 wk), with these changes being inversely related to modifications in synaptic protein densities. Similar changes in glial structural proteins were observed in adult male rats that had increased body weight and circulating leptin levels due to neonatal overnutrition (overnutrition: four pups/litter vs. control: 12 pups/litter). However, acute leptin treatment reduced hypothalamic GFAP levels and induced synaptic protein levels 1 h after administration, with no effect on vimentin. In primary hypothalamic astrocyte cultures leptin also reduced GFAP levels at 1 h, with an induction at 24 h, indicating a possible direct effect of leptin. Hence, one mechanism by which leptin may affect metabolism is by modifying hypothalamic astrocyte morphology, which in turn could alter synaptic inputs to hypothalamic neurons. Furthermore, the responses to acute and chronic leptin exposure are inverse, raising the possibility that increased glial activation in response to chronic leptin exposure could be involved in central leptin resistance.

Fig. 1.

Effect of early overnutrition on the levels of structural and synaptic proteins in the adult hypothalamus. Relative levels of GFAP (A), vimentin (B), actin (C), and synaptotagmin (D) in the hypothalamus of 70-d-old male Wistar rats that were raised in litters of either four pups (SmL) or 12 pups (CtL) until weaning and then fed ad libitum with normal rat chow (n = 6/group). Representative Western blots are shown for each protein. #, P < 0.05; *, P < 0.01; ***, P < 0.0001. Results are represented as mean ± sem.

Fig. 2.

Effect of chronic central leptin exposure on glial proteins and activation in the hypothalamus. Relative levels of GFAP (A), vimentin (B), and actin (C) in the hypothalamus of adult male Wistar rats treated icv for 14 d with either leptin (Lep; 15 μg/d) or vehicle (Ct; n = 6/group). Representative Western blots are shown for each protein. #, P < 0.05; *, P < 0.01. Results are represented as mean ± sem. Immunohistochemistry for vimentin in the arcuate nucleus of control (D and F) and leptin-treated rats (E and G) are shown. Quantitative analysis demonstrated a significant increase (***, P < 0.0001) in the number of vimentin fibers in the arcuate nucleus of leptin-treated rats (H). Vimentin-labeled astrocytes are clearly visible in leptin-treated rats (arrows). Scale bar, 40 μm (D and E); 20 μm (F and G).

Fig. 3.

Effect of chronic central leptin exposure on astrocyte morphology. Photomicrographs of GFAP-immunopositive cells in the arcuate nucleus of control (A) and rats treated icv with leptin for 14 d (B). Quantitative analysis demonstrated that the mean length of the primary projections/astrocyte was increased in leptin-treated rats (C; icv, 15 μg/d for 14 d), with no significant change in the number of projections/astrocyte (D). **, P < 0.001. NS, Not significant. Scale bar, 20 μm.

Fig. 4.

Effect of chronic central leptin exposure on hypothalamic synaptic protein levels. The relative levels of synapsin I (A), synaptotagmin (B), syntaxin (C), and PSD-95 (D) in the hypothalamus of adult male Wistar rats treated with either leptin (icv, 15 μg/d for 14 d) or vehicle. Representative Western blots are shown for each protein. #, P < 0.05; **, P < 0.01, NS, Not significant. Results are represented as mean ± sem. Ct, Control, Lep, leptin treated (n = 6/group).

Fig. 5.

The effect of acute central leptin treatment on glial structural proteins and synaptic protein levels in the hypothalamus. Relative levels of GFAP (A), synapsin 1 (B), and syntaxin (C) in the hypothalamus of adult male rats receiving an icv bolus of either leptin (L; 3 μg; ) or saline (C; ■) and being killed 1 or 6 h later (n = 4/group). Representative Western blots are shown for each protein. ***, P < 0.0001. Results are represented as mean ± sem.

Fig. 6.

Effect of leptin on leptin receptor and glial structural proteins in vitro. A, mRNA levels of leptin receptor (ObR; A) in primary astrocyte cultures treated with vehicle (control;■) or leptin (L; 10 or 100 ng/ml; ) for 24 h. B, Relative levels of GFAP in primary astrocyte cultures treated with vehicle (C; ■) or leptin (L; 100 ng/ml, ) for 1, 6, or 24 h. A representative Western blot is shown. Results are represented as mean ± sem (n = 3–7). #, P < 0.05; ***, P < 0.0001.