Abcc5 Knockout Mice Have Lower Fat Mass and Increased Levels of Circulating GLP‐1

Objective A previous genome‐wide association study linked overexpression of an ATP‐binding cassette transporter, ABCC5, in humans with a susceptibility to developing type 2 diabetes with age. Specifically, ABCC5 gene overexpression was shown to be strongly associated with increased visceral fat mass and reduced peripheral insulin sensitivity. Currently, the role of ABCC5 in diabetes and obesity is unknown. This study reports the metabolic phenotyping of a global Abcc5 knockout mouse. Methods A global Abcc5‐/‐ mouse was generated by CRISPR/Cas9. Fat mass was determined by weekly EchoMRI and fat pads were dissected and weighed at week 18. Glucose homeostasis was ascertained by an oral glucose tolerance test, intraperitoneal glucose tolerance test, and intraperitoneal insulin tolerance test. Energy expenditure and locomotor activity were measured using PhenoMaster cages. Glucagon‐like peptide 1 (GLP‐1) levels in plasma, primary gut cell cultures, and GLUTag cells were determined by enzyme‐linked immunosorbent assay. Results Abcc5‐/‐ mice had decreased fat mass and increased plasma levels of GLP‐1, and they were more insulin sensitive and more active. Recombinant overexpression of ABCC5 protein in GLUTag cells decreased GLP‐1 release. Conclusions ABCC5 protein expression levels are inversely related to fat mass and appear to play a role in the regulation of GLP‐1 secretion from enteroendocrine cells.


Introduction
Hormones secreted by enteroendocrine cells, the endocrine cells of the gut, are central to the regulation of gastrointestinal physiology, energy metabolism, and appetite (1). In particular, the central role of gut hormone action on energy metabolism has been demonstrated by bariatric surgery outcomes, which show that increased gut hormone release contributes to the reversal of pathological insulin resistance in those with type 2 diabetes (T2D) within days following Roux-en-Y bypass surgery (2)(3)(4). The incretin hormone glucagon-like peptide 1 (GLP-1), which potentiates insulin release from pancreatic β-cells, plays a pivotal role in the changes observed in glucose handling post surgery (5,6). GLP-1 has multiple actions on peripheral tissues, such as driving cell proliferation in the pancreas and being both antiapoptotic and neuroprotective (7). GLP-1 receptor (GLP-1R) activation leads to enhanced satiety, weight loss, decreased glucose production in the liver, and enhanced insulin sensitivity in skeletal muscle; GLP-1R agonists are currently in clinical use for the treatment of T2D (7).
A genome-wide association study (GWAS) linked elevated expression of an ATP-binding cassette transporter, ABCC5, in subcutaneous adipose tissue to reduced peripheral insulin sensitivity in nondiabetic individuals with associated increased visceral fat accumulation and a threefold increased risk of developing T2D with age (8). This trend was observed in populations of disparate ancestry. The role of ABCC5 in diabetes and obesity remains unexplored, and any link between ABCC5 overexpression and increased fat stores is unknown.
The ABC transporters are a large family of membrane ATPases best known for their roles in multidrug resistance observed in chemotherapy-resistant tumors (9). However, these transporters fulfill many other essential functions, such as antigen presentation to the immune system (TAP1/ABCB2) (10,11), Clion permeability of the cell membrane (CFTR/ABCC7) (12), and the regulation of insulin release by adenosine nucleotides and sulfonylurea drugs from pancreatic β-cells by the sulfonylurea receptor (SUR1/ABCC8), which forms the K ATP channel complex along with inward rectifier K(+) channel Kir6.2 (13)(14)(15). The role of ABCC5 transporter activity in mammals is currently unknown, but knocking Abcc5 gene expression out in animal models indicated a role for this protein in heme transport in Caenorhabditis elegans and hind gut formation in sea urchins (16,17). Jansen et al. (18) demonstrated that ABCC5 is a glutamate conjugate transporter, and tissues of the knockout mouse were shown to accumulate up to eight different glutamate metabolites, including the inhibitory neuropeptides N-acetylaspartylglutamate (NAAG) and N-acetylaspartyldiglutamate (NAAG 2 ).
This study reports the metabolic phenotyping of Abcc5 knockout mice, Abcc5 -/-. Our work demonstrated that ABCC5 protein expression plays a central role in energy metabolism in mammals, with Abcc5 -/mice showing lower white and brown adipose tissue and increased GLP-1 release from enteroendocrine cells of the small intestine.

Metabolic phenotyping
All animal studies were approved by the MRC Harwell Institute Ethical Review Committee, and all procedures were carried out within license restrictions (PPL 30/3146) under the Animal (Scientific Procedures) Act 1986, issued by the UK Government Home Office Department. Abcc5 -/mice and wild-type littermate controls were kept in accordance with Home Office welfare guidance (12 hours of light and dark cycles; temperature 21°C ± 2°C and humidity 55% ± 10% at the Mary Lyon Centre animal facility, MRC Harwell, Oxfordshire, UK). Mice had free access to water (10 parts per million chlorine) and were fed ad libitum on standard chow (RM3; Special Diet Services, Essex, UK). All in vivo studies were performed on mice aged 4 to 18 weeks.

Body mass and composition
Body mass was measured for two independent cohorts of mice at baseline (week 4) and weekly thereafter on scales calibrated to 0.01 g. Whole body composition (fat and lean mass) was determined using an EchoMRI-136 Body Composition Analyzer for Live Small Animals (Echo Medical Systems, Houston, Texas) at baseline (week 4) and weekly thereafter for Abcc5 -/mice and wild-type littermate controls.

Glucose tolerance tests
Oral glucose tolerance test. Twelve-week-old Abcc5 -/mice and wild-type littermate controls were fasted overnight (16 hours). The mice were weighed and fasting glucose levels measured from whole blood via tail bleed under local anesthesia (5% EMLA cream (lidocaine/prilocaine), AstraZeneca, Cambridge, UK). An oral gavage of 20% glucose solution in 0.9% NaCl at 2 g/kg of body mass was administered and whole blood glucose measurements taken at 15 minutes, 30 minutes, 60 minutes, and 120 minutes after the gavage. The glucose measurements were performed using a handheld AlphaTRAK glucometer for pets (Abbott Laboratories, Lake Bluff, Illinois).
Intraperitoneal glucose tolerance test. Thirteen-week-old Abcc5 -/mice and wild-type littermate controls were processed as for the oral glucose tolerance test. Intraperitoneal injections of 20% glucose solution in 0.9% NaCl at 2 g/kg of body mass were administered and whole blood glucose samples taken at 15 minutes, 30 minutes, 60 minutes, and 120 minutes via tail bleed.

Insulin tolerance test
At week 14, Abcc5 -/mice and wild-type littermate controls were fasted for 4 hours during the light phase. The mice were weighed, and whole blood samples were collected at time 0 via tail vein for baseline glucose measurements. Intraperitoneal injections of 0.5 IU/kg per mouse (females) or 1.0 IU/kg per mouse (males) of insulin diluted in 0.9% NaCl in sterile water were made, and subsequent blood samples were taken at 15 minutes, 30 minutes, 45 minutes, 60 minutes, and 90 minutes. Blood glucose uptake measurements were taken using an AlphaTRAK glucometer.
Indirect calorimetry (energy expenditure, locomotor activity) At week 12, Abcc5 -/mice and wild-type littermate controls were individually housed in PhenoMaster cages (TSE Systems, Bad Homburg, Germany) for collection of energy intake-and expenditure-related data over 24 hours. The cage system included photobeam-based activity monitoring that records ambulatory movements in the horizontal and vertical planes. An indirect gas calorimetry system simultaneously measured oxygen consumption (VO 2 ), carbon dioxide production (VCO 2 ), and respiratory exchange ratio (RER).

Adipose tissue harvest and Western blots
Epididymal white adipose tissue (epiWAT), periovarian white adipose tissue (periWAT), and interscapular brown adipose tissue (iBAT) were collected as described previously (19) and immediately weighed. All tissues were snap frozen in liquid nitrogen and stored at −80°C. Tissue processing is detailed in the online Supporting Information.

Isolation and culture of gut primary cells from
Abcc5 -/mice Mixed primary cultures of murine intestine isolated from Abcc5 -/mice and wild-type littermate controls were prepared as previously described (20) and detailed in online Supporting Information.

Blood chemistry analysis
Total cholesterol, HDL, LDL, glycerol, and triglyceride levels were determined in terminal lithium-heparin plasma samples collected at 5 minutes, following an oral glucose gavage of overnight-fasted Abcc5 -/mice and wild-type littermate controls, using a Beckman Coulter AU680 clinical chemistry analyzer (Beckman Coulter, Inc., Brea, California) with reagents and settings recommended by the manufacturer.
Abcc5 gene expression analysis ABC transporter expression profiles were determined by RNA sequencing as previously described (21). Briefly, fluorescent and nonfluorescent cells were isolated in triplicate by fluorescenceactivated cell sorting from the duodenum/jejunum (top 10 cm of small intestine), ileum (bottom 10 cm of small intestine), and colon of NeuroD1-CrexRosa26EYFP or GLU-Venus mice (Gribble/Reimann laboratory, Cambridge, UK), labeling all enteroendocrine cells or only proglucagon-expressing cells, respectively. Total RNA (isolated with RNeasy Plus Micro Kit [Qiagen, Hilden, Germany] and amplified using Ovation RNA-Seq System V2 [NuGEN Technologies, Inc., Redwood City, California]) was used to create barcoded libraries, which were sequenced using an Illumina HiSeq 2500 System (Illumina, Inc., San Diego, California) at the Genomics Core Facility of the Cancer Research UK Cambridge Institute. Sequence reads were demultiplexed using the Casava pipeline (Illumina) and then aligned to the mouse genome (GRCm38) using TopHat version 2.1.0 (Johns Hopkins University, Baltimore, Maryland). Gene expression (fragments per kilobase per million read FPKM) was determined using Cufflinks version 2.2.1 (http://cole-trapn ell-lab. github.io/cuffl inks/), and differential gene expression was assessed by DESeq2 (https ://bioco nduct or.org/packa ges/relea se/bioc/html/ DESeq2.html), excluding one GLU-Venus duodenal fluorescently labeled data set because of apparent contamination.

Metabolomics
Metabolites were extracted from approximately 5 × 10 6 cells (grown in cell culture dishes) by the addition of 500 µL of ice-cold 80% aqueous methanol. Supernatants were combined and filtered using a 3-kDa ultrafilter (Millipore), dried in a SpeedVac (Thermo Fisher Scientific), and subsequently stored at −80°C. On the day of analysis, the dried extracts were reconstituted in 60 μL of ice-cold 80% aqueous methanol. A quality control sample was made by combining 5 µL of each sample. Sample analysis was performed using anion-exchange chromatography (Thermo UltiMate 3000 UHPLC, Thermo Fisher Scientific) coupled directly to a high-resolution Orbitrap mass spectrometer (Q Exactive HF Hybrid Quadrupole-Orbitrap, Thermo Fisher Scientific) as previously described (22) and detailed in online Supporting Information.

Statistics
Data are presented as mean ± SEM. Simple pairwise comparisons were made using unpaired two-tailed t tests. For sample numbers ≥ 10 to 15, a Student t test was used. For sample numbers < 10 or where unequal numbers of two groups were compared, the more stringent Welch's unequal variances t test was used. Multiple comparisons were made using one-or two-way ANOVA with a Bonferroni post hoc test. P < 0.05 for a 95% CI was regarded as statistically significant. Statistics were performed using GraphPad Prism 6 (Graphpad Software, La Jolla, California).

Results
Abcc5 -/mice have lower body mass because of decreased adiposity At 16 weeks, both female and male Abcc5 -/mice weighed ~10% less than wild-type littermates, with a difference in body mass at 16 weeks of 2.5 g ± 0.8 g for Abcc5 -/females and 3.6 g ± 0.8 g for Abcc5 -/males ( Figure 1A-1B). Weekly EchoMRI results showed that Abcc5 -/mice had proportionally less fat (expressed as a fraction of total body mass, Figure 1C-1D) and therefore more lean mass (expressed as a fraction of total body mass, Figure 1E-1F), with changes more pronounced in female mice than males. Differences in weight were statistically significant from 5 weeks of age and for all weeks onward, as analyzed by a Student t test for wild-type compared with Abcc5 -/mice for that  week (Figure 1A-1B). Changes in body composition over time shown in Figure 1 between wild-type and Abcc5 -/mice were analyzed by two-way ANOVA with a Bonferroni post hoc test, and significance values are indicated by asterisks shown at the end of the two curves.
Notably, mice were of equal weight at weaning. Dual-energy x-ray absorptiometry scans performed at week 14 showed no difference in bone mineral density or bone mineral content, and x-rays confirmed that there were no changes in femur length, showing that growth was not stunted in Abcc5 -/mice (data not shown). Both female and male Abcc5 -/mice had significantly lower masses of white adipose tissues, including periWAT depots in females and epiWAT depots in male mice ( Figures 2E and 3E). Cholesterol and triglyceride plasma profiles showed decreased levels of total cholesterol, LDL, glycerol, and triglycerides in both female and male Abcc5 -/mice, which would suggest that Abcc5 -/mice did not have dyslipidemia ( Figures 2H and 3H).

Abcc5 -/mice are more active
Indirect calorimetry analysis found that female Abcc5 -/mice had increased VO 2 , CO 2 release, and energy expenditure in both dark and light cycles, while male mice did not ( Figure 4A-4F). Both sexes of Abcc5 -/mice were more active in both dark and light cycles, showing increased total activity (the sum of ambulatory movement and fine movement) ( Figure 4G-4H). No changes in respiratory exchange ratio (RER) were observed for either sex (data not shown), which would indicate that the energy source used by Abcc5 -/mice was not switched from carbohydrate (standard chow RER = 0.9-1.0) to fat (RER = 0.7). Abcc5 -/mice did not eat less, with female mice having slightly elevated food and water intake, while male Abcc5 -/mice showed no change ( Figure 5). The decreased fat depots in Abcc5 -/mice can therefore not be explained by hypophagia in Abcc5 -/mice.

Abcc5 -/mice are more insulin sensitive
Female Abcc5 -/mice were able to lower plasma glucose more efficiently than wild-type littermates in response to an intraperitoneal insulin bolus following a 4-hour fast ( Figure 6A-6B), and the same trend was observed in male mice ( Figure 6C-6D). Changes in plasma glucose in response to intraperitoneal insulin over time for Abcc5 -/versus wild-type mice were analyzed by two-way ANOVA with a Bonferroni post hoc test ( Figure 6A and 6C). The area under the curve were analyzed by an unpaired two-tailed Student t test ( Figure 6B  and 6D). An intraperitoneal glucose tolerance test showed no phenotype-dependent differences in response to glucose for either sex ( Figure 6E-6F). However, a small but significant increase in plasma glucose levels was observed at 15 minutes post glucose administration in female ( Figure 6E) but not male Abcc5 -/mice ( Figure 6F) when analyzed by multiple comparison two-way ANOVA with a Bonferroni post hoc test. By contrast, no differences were observed in oral glucose tolerance in either sex (Supporting Information Figure S2). It is important to note that the increased insulin sensitivity observed in Abcc5 -/mice may therefore be secondary to the decreased adiposity of Abcc5 -/mice. By extension, it would appear that glucose-stimulated insulin secretion in Abcc5 -/mice is not greatly affected, as plasma glucose levels following both intraperitoneal glucose tolerance test and oral glucose tolerance test showed little or no change when compared with wild-type littermates. Future studies using a euglycemic hyperinsulinemic clamp with stable isotopic glucose and water tracers will be required to delineate the basis of increased insulin sensitivity in Abcc5 -/mice. Histological analysis of hematoxylin-and eosin-stained gut, liver, and pancreas samples showed no discernible changes in tissue morphology of Abcc5 -/mice when compared with wild-type littermates (data not shown).

Abcc5 is expressed in mouse enteroendocrine cells
Transcriptional profiling of the enteroendocrine cells of mouse duodenum, ileum, and colon showed increased gene expression of Abcc5 in the enteroendocrine cells of the duodenum and in preproglucagonexpressing L-cells of the ileum when compared with nonendocrine cells (Table 1). GLUTag cells, a model L-cell line, expressed levels of Abcc5 mRNA similar to that observed in the enteroendocrine cells of the duodenum and ileum (Table 1). Interestingly, low-resolution fluorescent microscopy images indicated that ABCC5 was not expressed in the apical or basolateral membranes of enteroendocrine cells, and protein expression was shown to be intracellular in both GLUTag cells (Supporting Information Figure S3A) and gut L-cells from GLU-Venus mice (Supporting Information Figure S3B).

GLP-1 exocytosis from gut enteroendocrine cells is inversely dependent on ABCC5 protein expression levels
In order to test a direct link between ABCC5 protein expression and GLP-1 secretion, we used a well-characterized model L-cell line, GLUTag cells, which secrete GLP-1 in response to stimulation by nutrients (23,24). siRNA knockdown of Abcc5 gene expression in GLUTag cells resulted in a ~60% increase in active GLP-1 release ( Figure 7C), while recombinant overexpression of ABCC5 attenuated GLP-1 release below the exocytosis levels observed in the untreated control. ABCC5 protein expression levels in GLUTag cells used for GLP-1 secretion assays were confirmed by Western blot ( Figure 7D). Western blot analysis of wild-type GLUTag cells showed robust ABCC5 protein expression ( Figure 7C, lanes 1 and 2), while siRNA knockdown of Abcc5 gene expression in GLUTag cells reduced ABCC5 protein expression levels substantially ( Figure 7C, lanes 3 and 4); recombinant overexpression of ABCC5 protein was also confirmed ( Figure 7C, lanes 5 and 6).
Using metabolomics, we identified a known ABCC5 substrate, NAAG, as an abundant glutamate metabolite in GLUTag cells. In order to confirm that the cellular levels of NAAG were also inversely related to ABCC5 expression in GLUTag cells, similar to that previously observed in human embryonic kidney (HEK) cells, ABCC5 was overexpressed in GLUTag cells and the levels of NAAG analyzed by comparative metabolomics (18). The intracellular levels of NAAG were decreased in the ABCC5-overexpressing GLUTag cells when compared with sham-transfected GLUTag cells ( Figure 7D). To investigate a potential role of this inhibitory neuropeptide in ABCC5-mediated regulation of gut hormone release, the effects of exogenous NAAG on GLP-1 levels were measured. The exogenous addition of 2mM NAAG to both GLUTag cells and ex vivo gut crypt primary cell cultures, generated from Abcc5 -/mice, inhibited the release of GLP-1 ( Figure 7E-7F). Taken together, this data would suggest that ABCC5 activity modulates GLP-1 release from gut endocrine cells through a NAAG-dependent mechanism.

Discussion
The most prominent metabolic phenotype of Abcc5 -/mice is a decrease in total levels of fat mass. Transcriptional profiling of human subcutaneous adipose tissue by Direk et al. (8) showed high levels of  ABCC5 gene expression and demonstrated that elevated expression of ABCC5 in subcutaneous adipose tissue confers an increased risk for developing T2D with age in populations of disparate ancestry. The prevalence of T2D was reported to be three times higher in subjects with high ABCC5 expression in subcutaneous adipose tissue compared with those with low expression, and overexpression was most strongly associated with increased visceral white adipose tissue accumulation and reduced peripheral insulin sensitivity in nondiabetic individuals. Interestingly, the human overexpression phenotype is the opposite of our global Abcc5 -/mice, in which both sexes displayed decreased fat mass and increased insulin sensitivity. Adipose tissue is regulated by multiple endocrine and neurocrine inputs, and Abcc5 -/mice showed decreases in both white adipose tissue (periWAT in females and epiWAT in males) and iBAT in the absence of hypophagia (i.e., Abcc5 -/mice did not eat less). Abcc5 -/mice were not burning fat in response to adaptive thermogenesis, as there was no difference between the RER of Abcc5 -/and wildtype littermates and no browning was observed in the white adipose deposits of Abcc5 -/mice (data not shown). Both male and female mice were more active overall, but only in the females was increased activity coupled to increased energy expenditure as reflected in increased VO 2 . Increased energy expenditure could therefore contribute to the decreased fat deposits observed in female mice. On the other hand, male mice appeared to have increased activity and decreased fat mass in the absence of changes in energy consumption (VO 2 ), food intake, or adaptive thermogenesis. It was previously shown that more-active mice did not expend more energy under standard laboratory conditions (i.e., below thermoneutrality) (25). The decreased fat deposits in male Abcc5 -/mice would therefore suggest an additional role for ABCC5 in adipocyte physiology.
In addition to decreased fat mass, raised circulating plasma levels of GLP-1 were also observed in Abcc5 -/mice. Raised plasma GLP-1 appears to be the result of increased GLP-1 release from gut endocrine cells, as ex vivo primary gut crypt cultures from Abcc5 -/mice also showed increased GLP-1 release, both at rest and in response to stimulation by glucose. Furthermore, an inverse relationship between ABCC5 protein expression and GLP-1 release was also confirmed in a model L-cell line, GLUTag cells, in which recombinant overexpression of ABCC5 suppressed GLP-1 secretion. ABCC5 has previously been identified as an amino acid conjugate transporter and it exports both N-lactoyl amino acids and glutamate-aspartate conjugates from stably transfected HEK cells (18,26). Jansen et al. (18) demonstrated the accumulation of eight glutamate conjugates in the tissues of Abcc5 -/mice using untargeted metabolomics, some of which are inhibitory neurotransmitters. Notably, the most abundant metabolite, NAAG, is a glutamate neurotransmission antagonist, and NAAG action downregulates excitatory glutamatergic neurons (27,28). Glutamate also stimulates GLP-1 release from gut endocrine cells; it has been previously shown that glutamate and GLP-1 are loaded into the same vesicles in intestinal L-cells, and exogenous glutamate administration stimulates GLP-1 release from GLUTag cells (24,29).
Here, we were able to duplicate the HEK cell work done by Jansen et al. (18) in GLUTag cells and demonstrated that ABCC5 also acts as a NAAG exporter in enteroendocrine cells. Furthermore, exogenous addition of NAAG inhibits GLP-1 release from both GLUTag cells and primary gut crypts. Intriguingly, as native ABCC5 protein expression in both GLUTag cells and L-cells of the ileum appears to be intracellular, and ABCC5 is a NAAG exporter, a possible explanation could be that ABCC5 is involved in loading this neuropeptide into synaptic-like vesicles of enteroendocrine cells, which are released upon exocytosis. The transport of glutamate and neuropeptides into both distinct and overlapping vesicle pools is well described in neurons, but little is known about how NAAG is loaded into the synaptic vesicles in the brain (30). Therefore, if ABCC5 transporter activity is indeed involved in the export of inhibitory neurotransmitters such as NAAG, loss of this transporter could lead to a general increase in glutamatergic activation, such as is observed in the increase in total activity of both male and female mice as well as increased GLP-1 release from enteroendocrine cells. However, it has been shown that when recombinantly overexpressed in vitro, in addition to glutamate metabolites, ABCC5 may also transport organic anions (such as 6-mercaptopurine and thioguanine), pyrimidine-based antivirals such as 2'-3'-dideoxynucleotides, folates, various cyclic nucleotides, and N-lactoyl amino acids. We therefore cannot exclude the possibility that any of these other substrates may also be involved in the regulation of GLP-1 secretion from L-cells (26,(31)(32)(33)(34)(35)(36).
In the brain, ABCC5 protein expression was localized to astrocytes of the subcortical white matter as well as to pyramidal neurons (37). Interestingly, a recent report attributed the weight loss observed following the administration of the GLP-1R agonist liraglutide to increased glutamatergic signaling in the brain (38). GLP-1R activation in Abcc5 -/mice could therefore be upregulated through both increased circulating levels of GLP-1 and increased glutamatergic signaling caused by a loss of NAAG inhibition.
In summary, Abcc5 -/mice have a surprisingly complex metabolic phenotype, are lean, have increased circulating plasma levels of GLP-1, and are more insulin sensitive. Abcc5 -/mice are the opposite of the observed human overexpression phenotype, which is associated with increased visceral fat, insulin resistance, and a susceptibility to T2D with age. This study confirmed an important role for ABCC5 in adipocyte physiology in mammals. Future studies using inducible tissue-specific Abcc5 -/mice housed at thermoneutrality are now needed to dissect the metabolic implications of ABCC5 protein loss in the gut, the brain, the pancreas, and adipose tissue. O