Loss of arginine vasopressin- and vasoactive intestinal polypeptide-containing neurons and glial cells in the suprachiasmatic nucleus of individuals with type 2 diabetes
The central pacemaker of the mammalian biological timing system is located within the suprachiasmatic nucleus (SCN) in the anterior hypothalamus. Together with the peripheral clocks, this central brain clock ensures a timely, up-to-date and proper behaviour for an individual throughout the day–night cycle. A mismatch between the central and peripheral clocks results in a disturbance of daily rhythms in physiology and behaviour. It is known that the number of rhythmically expressed genes is reduced in peripheral tissue of individuals with type 2 diabetes mellitus. However, it is not known whether the central SCN clock is also affected in the pathogenesis of type 2 diabetes. In the current study, we compared the profiles of the SCN neurons and glial cells between type 2 diabetic and control individuals.
We collected post-mortem hypothalamic tissues from 28 type 2 diabetic individuals and 12 non-diabetic control individuals. We performed immunohistochemical analysis for three SCN neuropeptides, arginine vasopressin (AVP), vasoactive intestinal polypeptide (VIP) and neurotensin (NT), and for two proteins expressed in glial cells, ionised calcium-binding adapter molecule 1 (IBA1, a marker of microglia) and glial fibrillary acidic protein (GFAP, a marker of astroglial cells).
The numbers of AVP immunoreactive (AVP-ir) and VIP-ir neurons and GFAP-ir astroglial cells in the SCN of type 2 diabetic individuals were significantly decreased compared with the numbers in the SCN of the control individuals. In addition, the relative intensity of AVP immunoreactivity was reduced in the individuals with type 2 diabetes. The number of NT-ir neurons and IBA1-ir microglial cells in the SCN was similar in the two groups.
Our data show that type 2 diabetes differentially affects the numbers of AVP- and VIP-expressing neurons and GFAP-ir astroglial cells in the SCN, each of which could affect the daily rhythmicity of the SCN biological clock machinery. Therefore, for effectively treating type 2 diabetes, lifestyle changes and/or medication to normalise central biological clock functioning might be helpful.
KeywordsAstroglial cells Biological clock Insulin resistance Microglia Neurotensin Rhythmicity Type 2 diabetes mellitus
Glial fibrillary acidic protein
Ionised calcium-binding adapter molecule 1
Vasoactive intestinal polypeptide
In mammals, the circadian timing system plays a critical role in coordinating the daily and seasonal rhythmicity of all physiological and behavioural processes in the body. The master pacemaker of this timing system is located in the suprachiasmatic nucleus (SCN) in the hypothalamus. Multiple types of neurons are involved in the SCN neuronal network . In rodents, these mainly include the vasoactive intestinal polypeptide (VIP)-producing and the arginine vasopressin (AVP)-producing neurons , and in addition to these, humans also possess neurotensin (NT)-containing neurons .
Type 2 diabetes mellitus is characterised by hyperglycaemia and insulin resistance. Glucose homeostasis and insulin sensitivity are tightly controlled by the circadian timing system, mainly through balancing sympathetic and parasympathetic outputs from the hypothalamus . Previous studies have shown that impaired insulin secretion in prediabetic animal models results in decreased insulin signalling in the hypothalamus, leading to decreased inhibition of glucose production in the liver and impaired glucose uptake [4, 5]. Evidence is accumulating for a link between circadian misalignment, for example, by sleep deprivation, and profound disruptions in blood glucose and insulin levels . Thus far, few studies have investigated peripheral clock machinery in individuals with type 2 diabetes , and it has never been studied whether the central clock in the SCN itself is affected by type 2 diabetes. The current study aimed to profile and compare SCN neurons, especially the ones producing AVP, VIP and NT, as well as the astroglial cells (using glial fibrillary acidic protein, GFAP, as a marker) and microglia (using ionised calcium-binding adapter molecule 1, IBA1, as a marker) in control and type 2 diabetic individuals.
Post-mortem hypothalamic tissues from 28 type 2 diabetic and 12 non-diabetic control individuals were obtained from the Netherlands Brain Bank, through autopsy approved by the Medical Ethic Committee of the VU Medical Center, the Netherlands. Individuals with Braak stage V–VI or clinically diagnosed severe dementia were excluded . Sex, age, time/month of death were similar between groups (Electronic supplementary material [ESM] Table 1). Data on the latest post-absorptive blood glucose and HbA1c, although not complete, as indications of glycaemic control are presented in ESM Table 1. Other donor details, including post-mortem delay, clinical diagnosis, diagnosed high blood pressure, insulin treatment and cause of death are provided in ESM Table 1.
Immunohistochemistry and image analysis
Immunohistochemistry for AVP-ir, VIP-ir, NT-ir, GFAP-ir and IBA1-ir cells in the SCN was performed (see ESM Methods). Images were analysed by the Fiji image processing program, an ImageJ distribution (Madison, WI, USA). The soma number and relative intensity of immunoreactivity for AVP-ir, VIP-ir and NT-ir neurons; the number of GFAP-ir astroglial cells and the soma number/soma size for IBA1-ir microglia (per section) were quantified by a blinded investigator (R. Hogenboom) (see ESM Methods for further details).
The numbers of AVP-ir and VIP-ir cells at each level of the SCN were plotted along the rostral–caudal axis for all control individuals. To profile the other cells, for each individual we selected consecutive sections next to the one that contained the highest number of AVP-ir cells (ESM Fig. 1).
All data are presented as means ± SEM. Comparisons between control and type 2 diabetic individuals were analysed by Student’s t test. A p value of <0.05 was considered to be significant. Daily rhythmicity and monthly variation in the number of AVP-ir, VIP-ir, NT-ir, GFAP-ir and IBA1-ir cells in the SCN was assessed using cosinor analysis SigmaPlot 14.0 software (SPSS, Chicago, IL, USA) (see ESM Methods for further details).
In the current study, we performed an analysis of SCN AVP-ir, VIP-ir and NT-ir neurons and IBA1-ir and GFAP-ir glial cells in post-mortem human brain tissue obtained from non-diabetic and type 2 diabetic individuals. Our analysis revealed that the numbers of AVP-ir neurons, VIP-ir neurons and GFAP-ir astroglial cells is significantly decreased in the SCN of type 2 diabetic individuals.
Some major obstacles currently hamper translational studies on brain dysfunction in type 2 diabetic individuals at the molecular level. First, there is no perfect animal model that fully mimics the pathogenesis of type 2 diabetes in humans. Second, although non-invasive brain imaging techniques have provided data on overall changes in brain metabolism in type 2 diabetes, it is poorly understood what these changes mean for specific brain regions and individual cells. In the current study, the unique collection of the Netherlands Brain Bank, with fully informative medical records, gave us the opportunity to retrogradely analyse the medical characteristics of type 2 diabetic individuals and control subjects, and systemically study differences in their brains at the molecular level.
One of the major targets of the SCN projections is the hypothalamic pre-autonomic neurons [12, 13]. The loss of AVP-ir and VIP-ir SCN neurons, therefore, could result in a disbalanced autonomic hypothalamic output, as often observed in type 2 diabetes . Intriguingly, the number of GFAP-ir astroglial cells was reduced in the SCN of type 2 diabetic donors, suggesting that astroglial cells play an important role in maintaining SCN function. Indeed, a recent study demonstrated that in the absence of other cellular clocks, the cell-autonomous astroglial intracellular transcription–translation negative feedback loops alone could drive molecular oscillations in the SCN and circadian behaviour in mice .
Previous studies have shown that individuals with type 2 diabetes have a more irregular sleep/wake cycle than the general population . The ‘cause–effect’ question is whether the reduced number of AVP-ir and VIP-ir neurons and GFAP-ir astroglial cells is responsible for the disturbed sleep/wake rhythms or whether the disturbed sleep/wake rhythms affected the SCN. Observations in elderly people and ageing rats suggest the former, since increasing daytime light exposure not only improved sleep/wake rhythms but also increased AVP-ir in the SCN . Nevertheless, whether modifying light exposure can improve the sleep/wake rhythm of individuals with type 2 diabetes and eventually add benefits to their treatment remains to be explored.
In summary, to start to understand the association between circadian clockwork perturbations and the metabolic syndrome in humans, we took advantage of the unique collection of type 2 diabetes human brain tissue in the Netherlands Brain Bank, and systematically analysed SCN cells. Our data indicate that besides regular glucose-lowering medication, normalising circadian rhythms by pharmacological or behavioural approaches might be helpful to treat type 2 diabetes more effectively.
We thank E. Fliers and S. E. la Fleur (Department of Endocrinology and Metabolism, Amsterdam University Medical Centers [UMC], location AMC, Amsterdam, the Netherlands) for intellectual input.
Data acquisition was performed by RH, MJK, NLK and MK. Data analysis and interpretation was performed by RH, PdG, RMB, JAR, DFS, AK and CXY. CXY and AK conceived and designed the study, RH, MJK, PdG, CXY and AK drafted the manuscript. NLK, MK, RMB, JAR and DFS revised the manuscript. All authors approved the final version of the manuscript. CXY is the guarantor of this work.
This work was supported by an AMC fellowship (CXY, 2014, Amsterdam University Medical Center), the Dutch Diabetes Research Foundation (CXY, Diabetes Fonds, 2015.82.1826), and the ZonMW TOP grant (MJK, PdeG and AK, no. 91214047).
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
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