A Stereological Analysis of NPY, POMC, Orexin, GFAP Astrocyte, and Iba1 Microglia Cell Number and Volume in Diet-Induced Obese Male Mice

Abstract

The hypothalamic arcuate nucleus (ARC) contains 2 key neural populations, neuropeptide Y (NPY) and proopiomelanocortin (POMC), and, together with orexin neurons in the lateral hypothalamus, plays an integral role in energy homeostasis. However, no studies have examined total neuronal number and volume after high-fat diet (HFD) exposure using sophisticated stereology. We used design-based stereology to estimate NPY and POMC neuronal number and volume, as well as glial fibrillary acidic protein (astrocyte marker) and ionized calcium-binding adapter molecule 1 (microglia marker) cell number in the ARC; as well as orexin neurons in the lateral hypothalamus. Stereological analysis indicated approximately 8000 NPY and approximately 9000 POMC neurons in the ARC, and approximately 7500 orexin neurons in the lateral hypothalamus. HFD exposure did not affect total neuronal number in any population. However, HFD significantly increased average NPY cell volume and affected NPY and POMC cell volume distribution. HFD reduced orexin cell volume but had a bimodal effect on volume distribution with increased cells at relatively small volumes and decreased cells with relatively large volumes. ARC glial fibrillary acidic protein cells increased after 2 months on a HFD, although no significant difference after 6 months on chow diet or HFD was observed. No differences in ARC ionized calcium-binding adapter molecule 1 cell number were observed in any group. Thus, HFD affects ARC NPY or POMC neuronal cell volume number not cell number. Our results demonstrate the importance of stereology to perform robust unbiased analysis of cell number and volume. These data should be an empirical baseline reference to which future studies are compared.

Hypothalamic inflammation and gliosis in obesity

“…Conversely, neuronal peroxisome proliferator–activated receptor-δ and growth hormone receptor knockout mice are resistant to HFD-induced hypothalamic inflammation and gliosis yet more susceptible to DIO than wild-type controls (71). Finally, a stereological analysis of the hypothalamus in HFD-fed mice revealed minimal evidence of neuronal loss and sustained gliosis though the diet composition and duration were different than prior studies (72•). Thus, the evidence supporting a direct role of hypothalamic inflammation and gliosis in promoting weight gain is clearly mixed.…”

One-day high-fat diet induces inflammation in the nodose ganglion and hypothalamus of mice

“…The consumption of a HFD activates microglia and the expression of inflammatory cytokines in the hypothalamus, thereby disrupting the hypothalamic neuronal routes that maintain energy homeostasis [8,16]. We observed that the inflammatory effects of HFD were rapid, appearing in the nodose ganglion and hypothalamus one day after exposure to a HFD.…”

60 YEARS OF POMC: Regulation of feeding and energy homeostasis by α-MSH

“…Earlier studies quantifying POMC neurons by β-endorphin immunohistochemistry (Huo et al 2006) or using mice expressing green fluorescent protein (GFP) under the POMC promoter (Cowley et al 2001) have reported around 3000–3500 POMC neurons in rodent ARC. However, a recent report quantifying POMC neurons using an antibody specific to the POMC precursor, estimated around 9000 immunoreactive cells (Lemus et al 2015). On the other hand, the number of NPY/AgRP neurons was estimated to be between 8000 and 10,000 (Betley et al 2013, Lemus et al 2015), analyzed using mice expressing GFP in an AgRP promoter-dependent manner.…”

“…However, a recent report quantifying POMC neurons using an antibody specific to the POMC precursor, estimated around 9000 immunoreactive cells (Lemus et al 2015). On the other hand, the number of NPY/AgRP neurons was estimated to be between 8000 and 10,000 (Betley et al 2013, Lemus et al 2015), analyzed using mice expressing GFP in an AgRP promoter-dependent manner.…”