University of Groningen Adipose tissue Nies, Vera

University of Groningen

Adipose tissue

Nies, Vera

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TUB gene expression in hypothalamus

and adipose tissue and its association

with obesity in humans

Vera J.M. Nies

Dicky Struik

Thomas P. van der Meer

Marcel G.M. Wolfs

Sander S. Rensen

Ewa Szalowska

Unga A. Unmehopa

Kees Fluiter

Ghazaleh Hajmousa

Wim A. Buurman

Jan Willem Greve

Ronit Shiri-Sverdlov

Roel J. Vonk

Dick F. Swaab

Bruce H.R. Wolffenbuttel

Johan W. Jonker

Jana V. van Vliet-Ostaptchouk

Submitted to Journal of Clinical Endocrinology & Metabolism

Chapter 6

Abstract

Mutations in the Tubby gene (TUB) cause late-onset obesity and insulin resistance in mice and syndromic obesity in humans. Although TUB gene function has not been fully elucidated yet, studies in rodents indicate the role of TUB in the hypothalamic pathways regulating food intake and adiposity. Aside from the central effects, TUB may also be involved in energy metabolism in adipose tissue in rodents. We aimed to clarify the expression and distribution patterns of TUB in man and its potential association with obesity by investigating the expression of the two major human TUB splice variants in the hypothalamus and adipose tissue, and by correlating TUB expression with parameters of obesity and metabolic health. Both TUB transcripts are expressed in the hypothalamus, while only the short TUB-isoform was found in visceral and subcutaneous adipose tissues. TUB mRNA was detected in several hypothalamic regions involved in body weight regulation, including the nucleus basalis of Meynert and the paraventricular, supraoptic, and tuberomammillary nuclei. In adipose tissue TUB expression was negatively correlated with indices of body weight and obesity in a fat-depot specific matter. Our findings support a role for TUB in human obesity and put TUB forward as an important obesity risk gene.

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Introduction

The TUB gene encodes the protein Tubby, which is found in a wide range of organisms across the animal and plant kingdom, suggesting an evolutionary conserved biological function (Boggon et al. 1999). TUB was initially discovered through positional cloning of the spontaneous mutation that lead to obesity in a mice breeding colony at the Jackson Laboratories (Coleman & Eicher 1990; Kleyn et al. 1996; Noben-Trauth et al. 1996; Stubdal et al. 2000). Mice carrying this mutation (later designated as tubby mice), are characterized by obesity, insulin resistance and neurosensory deficits (Coleman & Eicher 1990). Compared to other murine models of obesity such as leptin-deficient (ob/ob) and leptin receptor-deficient (db/ db) mice, the obesity of tubby mice is relatively mild, late in onset and associated with transient alterations in glucose homeostasis (Coleman & Eicher 1990). The similarity of the tubby phenotype with common obesity in the human population puts TUB forward as a potential target in the pathogenesis of obesity.

Despite its evident physiological relevance, the exact molecular function of TUB remains enigmatic. Based on the protein structure it has been hypothesized that Tubby is a transcription factor (Boggon et al. 1999), whereas functional data suggest that Tubby is an integrator of insulin and leptin signalling and/or of G-protein coupled receptor trafficking (Kapeller et al. 1999; Prada et al. 2013; Santagata et al. 2001). However, so far none of these studies have been completely conclusive. In rodents, TUB is abundantly expressed in several areas of the hypothalamus (Prada et al. 2013; Ikeda et al. 2002; Sahly et al. 1998), a brain region critically involved in the regulation of appetite and energy expenditure by the central nervous system (CNS) (Berthoud 2002; Williams et al. 2000). As previously reported, tubby mice display alterations in the hypothalamic pathways related to feeding behaviour (Bäckberg et al. 2004; Guan et al. 1998). Already at young age tubby mice show reductions in daily food intake and physical activity, which in the end result in a net positive energy balance and weight gain over time, while older obese mice develop hyperphagia (Coyle et al. 2008; Bäckberg et al. 2004). There are also indications of abnormal carbohydrate metabolism in tubby mice due to the defects in neuronal innervation of the liver (Bäckberg et al. 2004; Wang et al. 2006), resulting in increased fat deposition (Wang et al. 2006). Similarly, deletion of the Tubby ortholog tub-1 in C. elegans is also associated with fat accumulation (Mukhopadhyay et al. 2007). Taken together, these data indicate an evolutionary conserved role for TUB in the regulation of energy metabolism. Besides its role in the CNS, TUB is also expressed in adipocytes in rodents and may be important for control of peripheral insulin-sensitivity (Stretton et al. 2009). To what extent the latter contributes to the tubby phenotype is, however, still unclear.

Chapter 6

To date, little is known about the function of TUB and its role in obesity in humans. We have previously shown that common variants in the human TUB gene are associated with body weight (Shiri-Sverdlov et al. 2006; Snieder et al. 2008; van Vliet-Ostaptchouk et al. 2008) and differences in macronutrient intake (i.e. higher carbohydrate consumption) (van Vliet-Ostaptchouk et al. 2008), whereas others described that the TUB mutation causes retinal dystrophy and syndromic obesity (Borman et al. 2014). The precise role of TUB in metabolic regulation in humans is still unexplored.

In an attempt to further specify a role for TUB in energy homeostasis and disease in humans, we investigated the expression and distribution patterns of TUB in the hypothalamus and adipose tissue, two tissues that are highly relevant in the context of metabolic regulation and obesity. We also examined how TUB expression correlates with several parameters of metabolic health and body weight in obese and healthy control individuals. Finally, we assessed TUB expression during adipogenesis in vitro and investigated whether TUB expression in human adipocytes can be modulated by metabolic hormones.

Materials and methods

Hypothalamus tissues from human subjects

Post-mortem hypothalamic material was obtained by autopsy from 24 individuals, of whom six subjects without any known neurological or psychiatric diseases were used to assess TUB mRNA distribution by in situ hybridization. Twelve pairs of obese and control subjects (BMI>30 and BMI<25, respectively), matched for sex, age, clinical diagnosis and Braak stage of Alzheimer progression (Braak & Braak 1991) were used to examine TUB mRNA expression level. The brain samples were obtained from The Netherlands Brain Bank, Netherlands Institute for Neuroscience, Amsterdam (open access: www.brainbank.nl). All Material has been collected from donors for or from whom a written informed consent for a brain autopsy and the use of the material and clinical information for research purposes had been obtained by the Netherlands Brain Bank.

TUB in situ hybridization

LNA probe. An LNA-2’-O-methyl-RNA probe specific for the human TUB mRNA

was designed. LNA-2’-O-methyl-RNA nucleic acid analogues were used because of their stability and high hybridization affinity, and because of their successful application in the human brain (26-28). An antisense probe was used (sequence: 5’-lTmUmAlCmUmAlTmUmUlAmGmClTmGmGlGmAmGlG-3’) complementary to

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and to bases 778–796 of the short TUB isoform, respectively (RefSeq NM_177972.2), wherein ‘m’=2’-O-methyl-RNA and ‘l’=LNA bases (Supplementary Fig. 1). Probe specificity was assessed using a scrambled probe containing the same nucleotides but in random order: 5’-lGmGmClTmUmUlAmGmAlTmGmClGmGmUlTmAmAlT-3’, and by testing of a concentration gradient ranging from 25nM to 500nM. Probes were FAM tagged at the 5’-end and custom ordered (Ribotask, Denmark).

In situ hybridization. Hypothalami were dissected during autopsy and fixed in

10% (v/v) phosphate-buffered formalin at room temperature (RT). After dehydration in graded ethanol series, tissues were cleared in toluene and embedded in paraffin. Coronal serial sections (6μm) were cut over the entire rostro-caudal axis. Each 100th section was collected, mounted on slides and subsequently dried for 2 d at 37C. The in situ hybridization procedure was performed as described in detail in Supplementary Text. In total, 120 sections were included in the experiment. Ten in situ hybridisation runs were performed, with twelve sections per run.

Gene expression analysis.

Frozen human hypothalami were homogenized in liquid nitrogen. Per hypothalamus, approximately 34.1±5.4mg of homogenized tissue was used for RNA isolation with the RNeasy Lipid Tissue Mini Kit (#74804, Qiagen, Germany) according to the manufacturer’s instructions. RNA quantity was measured on a NanoDrop, and the quality was determined with a 2100 BioAnalyzer (Agilent Technologies, USA). RNA integrity number (RIN) was used to assess the RNA quality (scale 1–10, with 1 being the lowest and 10 the highest). 1000ng of total RNA was used for cDNA synthesis. RNA was pre-incubated with 2.5uM Random Nonamers (Sigma-Aldrich) and 2mM dNTPs (Roche) at 60C for 5 min after which the samples were put on ice and subsequently supplemented with 5xFirst Strand Buffer, 2U·uL-1 RNaseOUT, 0.01M DTT, and 10U·uL-1 M-MLV reverse transcriptase (Invitrogen, USA). Samples were then incubated for 10min at 25C, 60min at 37C, and 15min at 70C. RT-PCR was performed using 5xSensiMix SYBR Hi-rox kit (Bioline, UK). RT-PCR reactions were performed on the StepOnePlus Real-Time PCR System (Applied Biosystems, USA). TUB expression was normalized to the U36B4 gene (also known as RPLP0), which was selected from our test of 5 different house keeping genes that have been reported to be most stable in human CNS post-mortem tissue (Durrenberger et al., 2012). Relative expression was calculated with Δ[Δ (CT)]–method (Livak et al., 2001).

Primers. mRNA reference sequences were obtained from the NCBI website (http://

www.ncbi.nlm.nih.gov.), and the mRNA levels for long and short isoforms of TUB gene were determined using a primer set designed by Primer3 software (http://frodo.wi.mit.

Chapter 6

edu/cgi-bin/primer3/primer3.cgi): forward: 5’-CCAGGAGGAAGTACTGGAAGG-3’ for the long isoform and forward: 5’-ATGACTTCCAAGCCGCATT-3’ for the short isoform, reverse: 5’-TTCTGCTGCCTCAGGTTTCT-3’ for both isoforms.

Human adipose tissue

Control samples. Human adipose tissue was obtained and prepared as described

previously (Szalowska et al., 2009). In short, subcutaneous adipose tissue (SAT) and visceral adipose tissue (VAT) biopsies were obtained from 24 Caucasian women who had to undergo surgery due to benign gynecological problems (Szalowska et al., 2009). From 15 individuals both VAT and SAT samples were collected. Research subjects were in good health without history or symptoms of type 2 diabetes or inflammatory diseases. Subjects were between 30 and 45 years of age, with BMI ranging from 23 to 29 kg/m2_{. The research protocols were approved by the Medical} Ethical Review Committee of the University Medical Centre Groningen.

Samples from obese individuals. In this study, 53 severely obese individuals were included with BMI ranging from 30 to 74 kg/m2 _{who underwent elective bariatric} surgery at the Department of General Surgery, Maastricht University Medical Centre (Wolfs et al. 2010). Subjects with acute or chronic inflammatory diseases, degenerative diseases and those reporting an alcohol intake exceeding 10g per day or the use of anti-inflammatory drugs were excluded. The sampling of VAT and SAT tissues obtained during bariatric surgery has been described before (Wolfs et al. 2010). Sample collection was approved by the Medical Ethics Board of Maastricht University Medical Centre, in line with the ethical guidelines of the Declaration of Helsinki. Written informed consent was obtained from each individual from both control and obese groups.

Gene expression analysis. RNA was isolated with the Qiagen Lipid Tissue Mini

Kit (#74804, Qiagen). RNA concentration was determined with NanoDrop. cDNA synthesis was performed from total RNA with QuantiTect Reverse Transcription Kit (Qiagen) according to the manufacturer’s instructions. 20ng cDNA was used for subsequent RT-PCR analysis using SYBR Green (Biorad, The Netherlands) and 7900HT real-rime PCR System (ThermoFisher, The Netherlands). Data were analyzed with SDS 2.0 software (Applied Biosystems). TUB mRNA levels were expressed relative to those of the beta-2 microglobulin housekeeping gene (B2M), since the geNorm VBA applet for Microsoft Excel (Vandesompele et al. 2002) determined B2M as the most stable housekeeping gene compared to other common housekeeping genes. Adipose derived stromal cell (ASC) experiments

ASC isolation, culture and differentiation. Human subcutaneous lipoaspirate samples were obtained from healthy human subjects (BMI<30, non-diabetic)

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written informed consent; all procedures were performed in accordance with the national and institutional guidelines and with the ethical guidelines of the Declaration of Helsinki. For the isolation of ASCs the lipoaspirates were digested with 0.1% Collagenase A in PBS+1% FBS at 37C for 90 min. The stromal cell fraction was separated from adipocytes and lipids by centrifugation (300g, 4C, 10 min), and subsequently subjected to Lymphoprep (Axis-Shield PoC, Norway) density gradient centrifugation. Cells from the interface were seeded in culture flasks at 10000 cells/ cm2 and cultured in DMEM (Lonza #BE12-707F, The Netherlands) supplemented with 10% FBS, 100U/ml penicillin, 100µg/ml streptomycin and 2mM L-glutamine at 37C, 5% CO2 and 95% humidity. ASCs in passage 1 were harvested and checked for mesenchymal cell surface markers as previously described (Bourin et al. 2013). To differentiate the ASCs to adipocytes, ASCs in passage 4-6 were seeded at a density of 20.000 cells/cm2_{, grown to confluence and incubated for another 48hr.} Cells were subsequently treated with differentiation medium, consisting of normal culture medium plus 0.1µM dexamethasone, 0.5mM IBMX, 20µM rosiglitazone and 100nM insulin. Differentiation medium was replaced 2 times a week. Differentiation efficiency was assessed by checking for the expression of the adipogenic marker aP2 (FABP4) by RT-qPCR. Before experiments, the differentiated or undifferentiated ASCs were incubated for 3hr in serum-free media. When applicable, the cells were treated with vehicle control, 100nM insulin or 100nM triiodothyronine (T3).

Gene expression analysis. Total RNA was isolated using Trizol according to the manufacturer’s protocol. cDNA was synthesized and RT-PCR was performed as described for the hypothalami. Gene expression levels were normalized to U36B4. Statistics

Continuous variable data are presented as mean ± SE unless stated otherwise. Before statistical analysis, non-normally distributed parameters were logarithmically transformed to approach a normal distribution. The differences between the groups were statistically evaluated by the nonparametric Mann-Whitney U-test. Spearman’s rho test was used to examine correlations. Expression differences between SAT and VAT were assessed using the paired Student’s test. Box and whisker plots were computed with GraphPad Prism software for Windows, Version 5. Statistical analyses were conducted with SPSS (version 22, SPSS corporation). Tests were two-tailed. The level of significance was set at P<0.05.

Chapter 6

Results

Distribution of TUB mRNA in the human hypothalamus

The expression pattern of TUB in the human hypothalamus was determined by in situ hybridization using an LNA-2’-O-methyl-RNA probe detecting both the short and the long transcripts of TUB. Probe specificity was confirmed by the absence of staining with the control-scrambled probe (Fig. 1). To localize regions and cells expressing TUB mRNA, consecutive sections of six hypothalami (three men, three women) were analyzed (for subject characteristics see: Supplementary table 1). Although considerable inter-individual differences were observed in TUB mRNA staining intensity, there was a similar staining distribution in the hypothalamus between individuals. No obvious effects of sex, age, fixation time (e.g. up to 130 days) and post-mortem delay (up to 19:35 hours) were observed (data not shown). The distribution of TUB mRNA expression in the hypothalamus was widespread with strongest staining in the nucleus basalis of Meynert (NBM), neurons of the paraventricular nucleus (PVN), supraoptic nucleus (SON) and tuberomammillary nucleus (TMN) and less intense staining in the infundibular nucleus (IFN) (Fig. 1, Supplementary Fig. 2).

Hypothalamic expression of TUB in obesity and controls

We were not able to distinguish the expression patterns between the long and the short TUB isoform using in situ hybridization approach. Therefore, the expression of the two individual TUB isoforms was determined by RT-PCR using hypothalamic tissue from six normal-weight and six obese subjects (for subject characteristics see Supplementary Table 1). Both TUB transcripts were expressed in the hypothalamus with the level of the short TUB isoform being markedly higher than that for the long TUB isoform (Fig. 2A). In addition, there was a high correlation between the expression of the long and short TUB isoforms (r=0.78, p=0.003).

Because central defects in TUB function have been associated with obesity in mice, we next examined the association of TUB mRNA levels in the hypothalamus with obesity in humans. For both TUB isoforms, however, there were no significant differences in expression between lean and obese subjects (Fig. 2B and 2C). Since RNA quality from the post-mortem material may influence gene expression profile, we assessed whether the RIN values were affected by confounding factors. No difference in the mean RIN values between controls and obese individuals were found, indicating that the quality of the RNA was good and similar in both groups (6.47±0.24 vs. 6.37±0.24, respectively) (Supplementary Fig. 3).

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Figure 1. Representative images of TUB mRNA expression in the human hypothalamus.

Tuberomammillary nucleus (TMN) shows intense staining while there is no specific staining with the control scrambled probe. Staining in neurons in the infundibular nucleus (IFN), nucleus basalis of Meynert (NBM), paraventricular nucleus (PVN), supraoptic nucleus (SON), and suprachiasmatic nucleus (SCN). Control male (patient #97157) Scale bar = 25 μm.

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Chapter 6

TUB expression in adipose tissue

Because it is unknown whether TUB is expressed in human adipose tissue, we assessed TUB mRNA levels in VAT and SAT in two study populations (control and obese; clinical characteristics see Table 1). In both fat depots and study populations, only the short isoform of TUB was detected. Expression of TUB was slightly higher in the SAT than in VAT, but only statistically significant in the obese study population and when control and obese samples were pooled (Fig 3A-C). In obese individuals expression was not different between men and women in either fat depot (data not shown). We also found lower TUB expression in obese subjects as compared to non-obese individuals in both SAT and VAT (Fig. 4 A,B).

In control subjects, we further observed negative correlations between VAT TUB mRNA and weight (r=-0.64, p=0.002), BMI (r=-0.56, p=0.009), waist circumference (r=-0.66, p=0.002) and hip circumference (r=-0.54, p=0.017) (Fig. 5A-D and Table 2). Similar correlations, but to a lesser extent, were found for SAT TUB mRNA and the same anthropometric phenotypes (Table 2). In contrast, in obese subjects a strong correlation was detected between BMI and SAT TUB mRNA (Fig. 5G) but not VAT TUB mRNA (r=-0.44, p=0.004 and r=-0.15, p=0.32, respectively; Table 2). The analysis also revealed significant relationships between TUB expression and different metabolic traits (glucose-, insulin- and HDL cholesterol levels) in severely obese individuals, but not in controls (Figure 5F-H and Table 2). The level of SAT TUB mRNA was negatively correlated with insulin (r=-0.31, p=0.047) and positively correlated with HDL-cholesterol (r=0.48, p=0.002) (Fig. 5H, F). We also observed opposite trends in the direction of the correlations between TUB mRNA in VAT and blood glucose in severely obese patients (Fig. 5E) and controls (r=-0.30, p=0.046 and r=0.16, p=0.50, respectively) (Table 2).

Figure 2. Characterisation of TUB gene expression in the human hypothalamus. A) Expression of the

long and short isoform in the hypothalamus of both control and obese people (n = 24, mean±SD: 0.0021±0.0012 vs. 0.1880±0.065, P< 0.0001. Expression of B) the long (n = 12, mean±SE: 0.0020±0.001

vs. 0.0022±0.0014) and C) the short isoform (n = 12, mean±SD: 0.1934±0.0713 vs. 0.1827±0.0604) in

the hypothalami from control and obese people. Data plotted as mean ± SEM.                                                      

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Table 1. Anthropometric and biochemical parameters of study population for TUB in adipose tissue

Trait Controls

(females) (females n=41 (77.4%))Obese

N Mean ± SD N Mean ± SD Age (years) 24 45.2 ± 10.8 53 42.4 ± 9.0 Weight (kg) 24 74.1 ± 11.2 - -BMI (kg/m2_{) 24 25.5 ± 3.3 53 46.8 ± 10.4} Waist (cm) 21 85.1 ± 9.6 - -Hip (cm) 22 103.1 ± 8.5 - -Glucose (mmol/L) 23 5.7 ± 0.8 53 6.2 ± 1.7 Insulin (pg/ml) 23 304.5 ± 129.6 52 138.2 ± 80.6 HbA1C (%) - - 52 6.4 ± 1.2

Total cholesterol (mmol/L) 23 2.6 ± 0.7 50 4.9 ± 0.9 HDL cholesterol (mmol/L) 23 1.2 ± 0.4 51 1.0 ± 0.3 LDL cholesterol (mmol/L) 23 4.2 ± 0.9 51 3.1 ± 0.8 Triglycerides (mmol/L) 23 1.5 ± 0.9 51 1.9 ± 1.2

Figure 3. Expression of the TUB short isoform in paired samples from VAT and SAT: A) In controls,

Mean±SD for VAT: 0.72±0.69; for SAT: 0.96±0.63 (n = 15). B) In obese individuals, mean±SD for VAT: 0.42±0.46; for SAT: 0.62±0.35; p=0.049 (n = 34). C) In pooled samples of controls and obese individuals (n = 49), mean±SD for VAT: 0.51±0.55; for SAT: 0.72±0.47; p=0.018. Data plotted as mean ± SEM.

Figure 4. TUB expression (short isoform) in the adipose depots of control and obese individuals. A)

TUB expression in the SAT, mean±SD: 0.99±0.67 vs. 0.64±0.36 (n = 18, 41) B) TUB expression in the VAT, mean±SD: 1.10±1.3 vs. 0.45±0.45 (n = 21, 46). Data plotted as mean ± SEM.

                         

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Figure 5. Scatter plots showing significant correlations between TUB expression in SAT and VAT

adipose depots and metabolic traits in control and severely obese people. Correlations between

TUB mRNA expression in the VAT and A) weight (r=-0.64, p=0.002) B) BMI (r=-0.56, p=0.009) C) waist

circumference (r=-0.66, p=0.002) and D) hip circumference (r=-0.54, p=0.017) of control subjects. Correlation between TUB mRNA expression the VAT and E) blood glucose levels (r=-0.30, p=0.046),

TUB mRNA in the SAT and F) plasma HDL cholesterol levels (r=0.48, p=0.002), G) BMI (r=-0.44, p=0.004)

F) plasma insulin levels (r=-0.31, p=0.047) of severely obese subjects.

                                                                                                                                                                                                                                            

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6

To establish whether the expression of TUB in human adipocytes is regulated by adiposity and/or adipose development, adipose-derived stromal cells (ASCs) were cultured in vitro and differentiated to adipocytes. The expression of both TUB isoforms was determined, as well as TUB expression over time during differentiation. To assess the putative effects of prolonged culture time on gene expression patterns, undifferentiated cells were cultured alongside the differentiated cells. Differentiation was assessed by visual assessment of lipid droplets and quantified by assessing the expression of the adipogenesis marker adipocyte P2 (aP2, FABP4) (Supplementary Fig. 4A, B). In line with the expression of TUB in primary adipose tissue, ASCs (both differentiated and undifferentiated) only expressed the short variant of TUB (Fig. 6A). Differentiation of ASCs into mature adipocytes, or treatment with the metabolic hormones insulin and triiodothyronine (T3), however, did not affect the expression levels of TUB in undifferentiated or differentiated ASCs (Fig. 6A, B, C).

Table 2. The correlation between anthropometric and biochemical parameters and the TUB mRNA

expression in visceral and subcutaneous adipose tissues.

Controls Severely obese individuals

VAT SAT VAT SAT

n r_s p n r_s p n r_s p n r_s p Weight 21 -0.64 0.002 21 -0.42 0.08 - - - -BMI 21 -0.56 0.009 18 -0.37 0.13 46 -0.15 0.32 41 -0.44 0.004 Waist 19 -0.66 0.002 16 -0.48 0.06 - - - -Hip 19 -0.54 0.017 17 -0.38 0.13 - - - -Glucose 20 0.16 0.50 17 -0.02 0.93 46 -0.30 0.046 41 -0.08 0.64 Insulin 20 -0.26 0.27 17 0.09 0.72 45 -0.16 0.29 41 -0.31 0.047 HbA1C - - - 45 -0.26 0.08 40 -0.10 0.56 Total cholesterol 20 0.20 0.40 17 0.06 0.83 43 0.08 0.62 39 0.16 0.34 HDL cholesterol 20 0.11 0.65 17 -0.01 0.96 44 0.28 0.07 40 0.48 0.002 LDL cholesterol 20 0.06 0.79 17 0.00 0.99 44 0.19 0.21 40 0.11 0.48 Triglycerides 20 -0.31 0.19 17 -0.23 0.37 44 -0.28 0.07 40 -0.26 0.11

Chapter 6

Discussion

Defects in TUB gene function are known to cause metabolic disturbances and development of obesity in mice (Coleman & Eicher 1990; Kleyn et al. 1996; Noben-Trauth et al. 1996), yet the relevance of TUB in human obesity is unclear. Here we investigated the expression and distribution patterns of human TUB in hypothalamus and adipose tissue, two tissues that play a role in the development of obesity and metabolic disease (Williams et al. 2000; Scherer 2006).

TUB expression in hypothalamus

Most evidence on the mechanisms underlying the phenotype of the tubby mouse point towards distortion in the control of body weight by the CNS (Berthoud 2002; Wynne et al. 2005). In rodents, TUB is abundantly expressed in several areas of the hypothalamus, particularly in the ARC, PVN, and VMH nuclei, which are involved in

          _{}                



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Figure 6. Expression of TUB in ASCs. A) ASCs only express the short TUB isoform. TUB expression is

not affected by differentiation (n = 6-11). TUB expression is not affected by insulin (100nM) or thyroid hormone (100nM) in B) undifferentiated (n = 3-6) and C) differentiated (n = 5-7) ASCs. Data plotted as mean±SEM.

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1998). Here we found that TUB expression in the human hypothalamus has a similar distribution pattern as in mice. High levels of TUB mRNA were detected especially in the IFN, which is the human ortholog of the rodent ARC (Andra et al. 2015), and in the PVN. Both IFN/ARC and PVN are important hypothalamic regions for the integration of homeostatic signals that regulate energy balance (Berthoud 2002; Wynne et al. 2005). We also detected expression of TUB in SCN and TMN, two nuclei involved in the circadian control of energy metabolism in humans; in the NBM, which is important for memory; and the SON, which regulates water reabsorption (Andra et al. 2015). The observed expression patterns for human TUB are similar to those reported for neuropeptide Y (NPY), agouti-related protein (AGRP) and the melanocortin 4 receptor (MC4R), which are two major neuropeptides and a crucial receptor for the central regulation of energy homeostasis (Siljee et al. 2013; Alkemade et al. 2005; Alkemade et al. 2012). Our findings thus support the hypothesis that human TUB is involved in the central regulation of energy metabolism. The expression of TUB in NBM, SON, SCN and TMN, which was not described for the mouse orthologues of these nuclei, suggests species-specific differences in hypothalamic TUB localization between mice and men (Sahly et al. 1998).

We did not find evidence for an association between TUB expression levels in the hypothalamus and obesity. This could be due to the fact that we used whole hypothalami to assess gene expression levels, which eliminates the possibility to assess spatial differences in expression. Finally, it is known that obesity is a complex condition with a multifactorial background. Whereas the mutations in TUB cause syndromic obesity (Borman et al. 2014), moderate alterations in TUB function are not necessarily a common feature of obesity per se. In agreement with this hypothesis is the observation that the expression of TUB is not significantly different from normal weight controls in three different mouse models of obesity (Stubdal et al. 2000). So even though TUB is expressed in brain areas involved in the central regulation of metabolism, it remains to be elucidated if and to what extent hypothalamic TUB contributes to the development of obesity in a subpopulation of patients.

TUB expression in adipose tissue

In mice, TUB is expressed in a variety of insulin-sensitive peripheral tissues (Noben-Trauth et al. 1996; Prada et al. 2013; He et al. 2000). This suggests that Tubby, besides its role in the CNS, may also have a peripheral function in energy homeostasis. Therefore we analyzed TUB expression in adipose tissue, which is the main depot for energy storage and the largest endocrine organ of the body (Scherer 2006). While both TUB splice variants were expressed in the hypothalamus, only the

Chapter 6

short isoform of TUB was found in adipose tissue, suggesting that TUB expression might be regulated in a tissue-specific manner. Previously, it has been suggested that the two splice forms of TUB differ in their ability to activate transcription (Boggon et al. 1999). Taken together, these findings could indicate a differential regulation of transcription by the isoforms in the hypothalamus and in the adipose tissue respectively.

We also observed fat-depot specific differences in TUB expression, with TUB being expressed at higher levels in SAT than in VAT. A similar expression pattern in both adipose tissues has also been reported for the FTO gene, a major gene implicated in human obesity (Klöting et al. 2008). Notably, we found a negative correlation between TUB mRNA levels and BMI in both obese and non-obese individuals. However, in control subjects the levels of TUB mRNA in the VAT correlated the strongest with parameters of body weight, whereas in obese individuals a stronger relationship was found in the SAT. More detailed analysis revealed a significant reduction in TUB expression in the SAT and VAT of severely obese individuals as compared to controls.

In addition to fat-depot specific differences in TUB mRNA levels and its correlation with indices of body weight and morbid obesity, we found significant directionally consistent correlations between TUB mRNA levels and blood glucose and lipid-related traits, observed only in severely obese patients. This may indicate a peripheral role for TUB in the regulation of metabolic homeostasis. It should be noted, however, that from the present study design it is not possible to determine whether the detected relationships contribute to the causes of these metabolic changes or represent the obesity–driven consequences. Taking into account our observations and previously reported associations of the TUB common variants and increased risk for obesity (Shiri-Sverdlov et al. 2006; Snieder et al. 2008; van Vliet-Ostaptchouk et al. 2008), and the obesity phenotypes linked to the loss-of-function mutation in tubby mice or in C.elegans (Kleyn et al. 1996; Noben-Trauth et al. 1996; Stubdal et al. 2000; Mukhopadhyay et al. 2007), we hypothesize that the peripheral alterations in the function of TUB also influence several metabolic aspects of obesity.

Our experiments in human ASC-derived adipocytes showed that TUB mRNA levels remain stable during differentiation. These results suggest that adipocyte development is not a driver of TUB expression, and therefore TUB expression is unlikely to be modulated by the obese state, which is in agreement with in vivo mouse studies (Stubdal et al. 2000). Conversely, we cannot exclude that a decrease in TUB expression increases adiposity, because of the observed negative correlation between TUB mRNA levels and body weight parameters in our study. Previously, the regulation of TUB expression by insulin and T3, two important hormones in

6

al. 2009) and neuronal cells (Koritschoner et al. 2001). However, we were unable to reproduce these results in human ASC-derived adipocytes. This discrepancy possibly reflects a complex and distinct obesity aetiology between humans and rodents observed previously in similar functional studies (Lee et al. 2006; Yang et al. 2008).

Conclusions

Here we show for the first time the expression and localization of the human TUB gene in areas of the hypothalamus important for the central regulation of energy metabolism. We also show that TUB is expressed in human adipose tissues, and that adipose tissue TUB expression negatively correlates with indices of body weight and obesity in a fat-depot specific matter. In addition, we observed an organ-specific expression pattern of the TUB splice variants. Together, our findings are consistent with a role for TUB in energy metabolism and support the involvement of TUB in the development of obesity in humans, providing insights into the enigmatic function of Tubby. Further molecular studies should reveal how TUB affects body weight regulation, which will aid in the development of new drugs, identification of subgroups of patients with increased risk for the development of obesity, and improved preventive therapies.

Chapter 6

Acknowledgements

We thank Elinda J Bruin-Van Dijk of University of Groningen, University Medical Center Groningen, for the expression profiling in adipose tissues and Bahram Sanjabi of University of Groningen, University Medical Center Groningen, for measuring the RIN values. JV van Vliet-Ostaptchouk is supported by a Diabetes Funds Junior Fellowship from the Dutch Diabetes Research Foundation (project no. 2013.81.1673). This work was supported by the National Consortium for Healthy Ageing, and the European Union’s Seventh Framework programme (FP7/2007-2013) through the BioSHaRE-EU (Biobank Standardisation and Harmonisation for Research Excellence in the European Union) project, grant agreement 261433 and by grants from the Dutch Diabetes Foundation (grant 2012.00.1537 to J.W.J.) and The Netherlands Organization for Scientific Research (VIDI grant 016.126.338 to J.W.J.).

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Supplementary figure 1. LNA probe design, showing the probe binding location in the mRNA of both

TUB isoforms

(Next page) Supplementary figure 2. High and low magnification staining showing TUB mRNA in situ

hybridization. A) Low magnification (20x) of neurons in TMN, IFN, NBM, PVN, and SON nuclei control male (patient #97157). B) High magnification (63x) of neurons in TMN, NBM, PVN, and SON nuclei. Control female (patient #01009).

          

Chapter 6    

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Supplementary figure 3. RIN values of hypothalamic mRNA for gene expression experiments. RIN

values show that there is no difference in RNA integrity between non-obese controls and obese people (n=12).

Supplementary figure 4. Differentiation markers of ASCs. Differentiation of ASCs is characterized by A)

the accumulation of lipid droplets (left = undifferentiated, right = differentiated) and B) the expression of adipocyte marker aP2.

           _{}                 

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Chapter 6

Supplementary Table 1. Clinicopathological details of the subjects NBB

number Age(years) Sex BMI PMDhr:min Fix (days) Braak stage* Clinical diagnosis &Cause of death Subjects used in in situ hybridization study

94040 20 M ND 8:00 82 0 Heart failure, lymphoma pneumonia 01009 21 F ND 19:35 65 0 Myocardial infarction

99071 39 M ND <16:30 130 0 Myocardial infarction 96239 32 F ND < 8:15 390 0 AIDS, pneumonia

97157 69 M ND 5:55 45 0 Prostate cancer with metastases 01004 64 F ND 8:35 36 0 Ileus, perforation

Subjects used for hypothalamic gene expression analysis Obese

2007-077 49 M 31.2 04:55 NA 3 Fronto-temporal dementia tauopathy 2000-138 84 F 33.9 05:15 NA 5 Alzheimer’s disease

1998-180 81 F 31.1 04:00 NA 5 Alzheimer’s disease 2012-005 84 F 31.2 05:36 NA 2 Non-demented control

1999-028 73 M 33.2 05:30 NA 4 Alzheimer’s disease with congophilic angiopathy

2011-066 81 F 36.2 05:55 NA 4 Parkinson’s disease 2013-019 53 F 31.2 07:15 NA Multiple sclerosis 2009-071 77 F 35.3 04:30 NA 0 Fronto-temporal dementia

2002-067 84 F 36.6 05:10 NA 1 Dementia with s.i.c.c. and argyrophilic grains 2002-102 88 F 31.6 03:15 NA 5 Alzheimer’s disease, type 2 diabetes 2013-015 56 M 30.4 09:35 NA 0 Multiple sclerosis

2009-091 84 M 31.6 07:20 NA 1 Non-demented control with Lewy bodies, type 2 diabetes

Controls

2013-065 47 M 22.7 05:25 NA 0 Fronto-temporal dementia FUS 2012-021 84 F 24.6 06:33 NA 5 Alzheimer’s disease

2009-105 82 F 22.9 05:25 NA 5 Alzheimer’s disease

2010-072 83 F 24.0 04:05 NA 1 Control with ischemic changes

2010-120 71 M 20.1 04:00 NA 6 Alzheimer’s disease with congophilic angiopathy

2011-113 81 F 20.3 03:55 NA 2 Parkinson’s disease 2007-069 47 F 21.3 04:25 NA 1 Multiple sclerosis 2002-078 75 F 24.7 04:50 NA 1 Non-Alzheimer dementia 2012-058 84 F 23.4 06:45 NA 2 Vascular dementia

2013-075 86 F 24 05:15 NA 6 Alzheimer’s disease, type 2 diabetes 2008-021 55 M 22.6 06:20 NA Multiple sclerosis

2010-056 79 M 20.6 05:00 NA 3 Lewy bodies variant, type 2 diabetes

AIDS: acquired immune deficiency syndrome; BMI: body mass index, F: female; fix: fixation time; hr: hour; M: male; min: minutes; NBB: Netherlands Brain Bank; PMD: postmortem delay

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