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Insulin Synthesized in the Paraventricular Nucleus of the Hypothalamus Regulates Pituitary Growth Hormone Production

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Insulin Synthesized in the Paraventricular Nucleus of the Hypothalamus Regulates Pituitary Growth Hormone Production Insulin synthesized in the paraventricular nucleus of the hypothalamus regulates pituitary growth hormone production Jaemeun Lee, … , Yong Chul Bae, Eun-Kyoung Kim JCI Insight. 2020. https://doi.org/10.1172/jci.insight.135412. Research In-Press Preview Endocrinology Neuroscience Graphical abstract Find the latest version: https://jci.me/135412/pdf 1 Insulin synthesized in the paraventricular nucleus of the hypothalamus regulates 2 pituitary growth hormone production 3 4 Jaemeun Lee1†, Kyungchan Kim1†, Jae Hyun Cho1, Jin Young Bae2, Timothy P. O’Leary3, 5 James D. Johnson3, Yong Chul Bae2, and Eun-Kyoung Kim1, 4* 6 7 1Department of Brain and Cognitive Sciences, Daegu Gyeongbuk Institute of Science and 8 Technology, Daegu, Republic of Korea 9 2Department of Anatomy and Neurobiology, School of Dentistry, Kyungpook National 10 University, Daegu, Republic of Korea 11 3Department of Cellular and Physiological Sciences, Diabetes Research Group, Life 12 Sciences Institute, University of British Columbia, Vancouver, BC, Canada 13 4Neurometabolomics Research Center, Daegu Gyeongbuk Institute of Science and 14 Technology, Daegu, Republic of Korea 15 †JL and KK contributed equally to this work. 16 *Correspondence: 17 Eun-Kyoung Kim, Ph.D. 18 Department of Brain & Cognitive Sciences, 19 Daegu Gyeongbuk Institute of Science and Technology, 20 333, Techno Jungang-daero, Hyeonpung-myeon, Dalseong-gun, 21 Daegu 42988, Republic of Korea 22 Phone: +82-53-785-6111 23 Fax: +82-53-785-6109 24 Email: [email protected] 25 26 Conflict of interest statement: The authors have declared that no conflict of interest exists. 1 27 Abstract 28 29 Evidence has mounted that insulin can be synthesized in various brain regions including 30 the hypothalamus. However, the distribution and functions of insulin-expressing cells in the 31 hypothalamus remain elusive. Herein, we show that in the mouse hypothalamus, the perikarya 32 of insulin-positive neurons are located in the paraventricular nucleus (PVN) and their axons 33 project to the median eminence; these findings define parvocellular neurosecretory PVN 34 insulin neurons. Contrary to corticotrophin-releasing hormone expression, insulin expression 35 in the PVN was inhibited by restraint stress (RS) in both adult and young mice. Acute RS– 36 induced inhibition of PVN insulin expression in adult mice decreased both pituitary growth 37 hormone (GH) mRNA level and serum GH concentration, which were attenuated by 38 overexpression of PVN insulin. Notably, PVN insulin knockdown or chronic RS in young mice 39 hindered normal growth via the down-regulation of GH gene expression and secretion, 40 whereas PVN insulin overexpression in young mice prevented chronic RS–induced growth 41 retardation by elevating GH production. Our results suggest that in both normal and stressful 42 conditions, insulin synthesized in the parvocellular PVN neurons plays an important role in the 43 regulation of pituitary GH production and body length, unveiling a physiological function of 44 brain-derived insulin. 2 45 Introduction 46 47 Insulin secreted from pancreatic β-cells can cross the blood–brain barrier and therefore 48 dominates the overall insulin content in the brain (1). Surprisingly, however, insulin expression 49 has been also discovered in various brain regions such as the choroid plexus, olfactory bulb, 50 cerebellum, cerebral cortex, hippocampus, and hypothalamus (2-6). A single-cell quantitative 51 reverse transcription–polymerase chain reaction (qRT-PCR) study demonstrated that Insulin 52 2 (Ins2) mRNA is present in GABAergic neurogliaform cells in the cerebral cortex of the rat 53 (7). In situ hybridization and immunohistochemistry studies revealed that insulin is produced 54 in the CA1 and CA3 pyramidal neurons, and dentate granule neurons in the adult mammalian 55 hippocampus (8-10). These findings suggest that local synthesis might be an alternative 56 source of brain insulin, but the physiological role of brain-derived insulin has not been clarified. 57 58 Although several brain regions have been suggested to possess the capacity for insulin 59 production, the existence of insulin-expressing cells in the hypothalamus remains 60 controversial. Insulin mRNA of rodents exists in 2 non-allelic forms, Ins1 and Ins2, and only 61 Ins2 is expressed in the brain (5, 11, 12). Li et al claimed that the Ins2 promoter is inactive in 62 the hypothalamus of Ins2-Cre knock-in mice (13). On the contrary, Mehran et al observed Ins2 63 mRNA expression in the hypothalamus of Ins2+/+ mice, which was significantly higher than the 64 background level seen in Ins2−/− hypothalamus, as well as patterns of histone methylation that 65 differed from those seen in islets (5). In terms of the mechanisms regulating insulin gene 66 expression in the mouse hypothalamus, glucose was found to up-regulate Ins2 mRNA levels, 67 while glucagon-like peptide 1 and forskolin biphasically regulated Ins2 mRNA levels in a 68 mouse hypothalamic cell line in a time-dependent manner (14). We have previously reported 69 that acute Wnt3a injection into the third ventricle of the mouse brain increases hypothalamic 70 Ins2 mRNA levels by triggering the expression of NeuroD1, which is a transcription factor for 71 insulin (15). These findings reinforce the notion that insulin-expressing cells exist within the 3 72 hypothalamus, but the regional distribution of the populations of these cells has not yet been 73 sufficiently explored. 74 75 The hypothalamus consists of several sub-regions with different nuclei including the 76 paraventricular nucleus (PVN). The PVN consists of 3 types of neurons: parvocellular 77 neurosecretory, parvocellular pre-autonomic, and magnocellular neurosecretory neurons. The 78 parvocellular neurosecretory neurons produce several neurohormones including thyrotropin- 79 releasing hormone (TRH), corticotropin-releasing hormone (CRH), and somatostatin (SST), 80 and release these neurohormones through their neurosecretory nerve terminals in the external 81 zone of the median eminence (ME) (16-18). The neurohormones are then transported to the 82 anterior pituitary via hypophyseal portal vessels adjacent to the ME and regulate the 83 production of a wide array of anterior pituitary hormones including growth hormone (GH) (16, 84 19-21). The hypothalamo–pituitary regulation mediates the body’s responses to physiological 85 stimuli or challenges, especially stress, by regulating hormone levels (22, 23). 86 87 In this study, we identify novel parvocellular neurosecretory neurons as the PVN insulin- 88 expressing neurons. We discovered a physiological role of PVN insulin in regulating pituitary 89 GH gene expression and secretion, leading to a change in body length of young mice under 90 both normal and stress conditions. This study will help better understanding of the regulatory 91 roles of brain-derived insulin in hormone production, growth, and other bodily functions. 4 92 Results 93 94 Hypothalamic insulin is synthesized mainly in the PVN neurons 95 To determine which hypothalamic sub-regions express insulin, we first conducted in situ 96 hybridization on serial coronal sections of the hypothalamus. Clear Ins2 mRNA-positive 97 signals were observed in PVN sections incubated with the Ins2 antisense probe (Figures 1A, 98 1B, 1C, and Supplemental Figure 1A) but not with the sense probe (Figure 1D). Consistent 99 with previous studies (8, 10), Ins2 mRNA expression was also found in the hippocampal 100 granule cell layer (Supplemental Figure 1B). Furthermore, immunostaining using an antibody 101 specific for proinsulin, a biosynthetic precursor of insulin, showed that insulin-expressing cells 102 were clearly located within the PVN (Figure 1E). Immunostaining using another antibody which 103 recognizes both proinsulin and mature insulin also confirmed that insulin-expressing cells 104 existed in the PVN (Supplemental Figure 1C). PVN sections from Ins2 KO (Ins1+/+:Ins2−/−) 105 mice showed no immunoreactivity to each antibody (Figure 1F and Supplemental Figure 1D), 106 confirming not only the specificity of the antibodies but also the expression of PVN insulin. The 107 specificity of the antibodies was further validated with the pancreas sections of WT and Ins2 108 KO mice (Supplemental Figures 2A and 2B). To verify the co-localization of Ins2 mRNA and 109 proinsulin protein within the same PVN cells, we next monitored β-gal expression in the 110 nucleus as a surrogate marker of Ins2 gene expression, since the coding sequencing of the 111 Ins2 gene was replaced with LacZ gene in the Ins2 KO mice used in our study (5). As expected, 112 all proinsulin-positive cells contained β-gal-positive nuclei in the PVN sections from Ins2+/β-gal 113 (Ins1+/+:Ins2+/−(β-gal)) mice (Supplemental Figure 3). Lastly, immunoblot analysis detected 114 proinsulin-positive bands in the hypothalamus and micro-dissected PVN of WT mice but not 115 in Ins2 KO mice (Supplemental Figure 4). Collectively, these data demonstrate that insulin- 116 expressing cells are present in the hypothalamic PVN. 117 5 118 To identify the cell types that express insulin in the PVN, we performed double 119 immunostaining of proinsulin with NeuN (a neuronal nuclear marker), microtubule-associated 120 protein 2 (MAP2; a neuronal soma and dendrite marker), glial fibrillary acidic protein (GFAP; 121 an astrocyte marker), or Iba-1 (a microglial marker). Proinsulin-positive cells in the PVN 122 contained mainly NeuN-positive nuclei (Figure 1G). Co-staining with MAP2 revealed that 123 proinsulin was prominently
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