ghrelin inhibition restores glucose homeostasis in ......the specific mechanisms involved in this...

Ghrelin inhibition restores glucose homeostasis in hepatocyte nuclear factor-1alpha

(MODY3) deficient mice

François Brial,1 Carine R. Lussier,

1 Karine Belleville,

2 Philippe Sarret,

2 and François Boudreau

1

1 Department of Anatomy and Cell Biology and

2 Department of Pharmacology and Physiology,

Faculty of Medicine and Health Sciences, Université de Sherbrooke, Quebec, Canada.

Short running title: Anti-ghrelin therapy in MODY3-deficient mice

Correspondence: Pr. François Boudreau, Département d’Anatomie et de Biologie Cellulaire,

Faculté de Médecine et des Sciences de la Santé, Pavillon de recherche appliquée sur le cancer,

3201 rue Jean-Mignault, Sherbrooke, QC Canada, J1E 4K8. Tel: 819-821-8000 ext 72122. Fax:

819 820-6831. E-Mail: [email protected]

Word count: 2,000

Number of figures: 4

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Diabetes Publish Ahead of Print, published online May 15, 2015

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ABSTRACT

Hepatocyte nuclear factor-1α (HNF1α) is a transcription factor expressed in tissues of endoderm

origin. Mutations in HNF1A are associated with maturity-onset diabetes of the young 3

(MODY3). Mice deficient for Hnf1α are hyperglycemic with their pancreatic β-cells being

defective in glucose-sensing insulin secretion. The specific mechanisms involved in this defect

are unclear. Gut hormones control glucose homeostasis. Our objective was to explore whether

changes in these hormones play a role in glucose homeostasis in absence of Hnf1α. An increase

in ghrelin gene transcript and a decrease in glucose-dependent insulinotropic polypeptide (GIP)

gene transcripts were observed in the gut of Hnf1α null mice. These changes correlated with an

increase of ghrelin and a decrease of GIP labeled-cells. Ghrelin serological levels were

significantly induced in Hnf1α null mice. Paradoxically, GIP levels were also induced in these

mice. Treatment of Hnf1α null mice with a ghrelin antagonist led to a recovery of the diabetic

symptoms. We conclude that up-regulation of ghrelin in absence of Hnf1α impairs insulin

secretion and can be reversed by pharmacological inhibition of ghrelin/GHS-R interaction. These

observations open up on future strategies to counteract ghrelin action in a program that could

become beneficial in controlling non-insulin dependent diabetes.

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INTRODUCTION

Maturity-onset diabetes of the young (MODY) is a monogenic autosomal dominant form of

diabetes that first occurs during early adulthood and characterized by pancreatic β-cell

dysfunction (1). Subtypes of MODY have been classified based on the specific nature of the

mutated genes of which six have been identified (2). MODY3, the most common MODY

mutation in the population, encodes the transcription factor HNF1α (3; 4) involved in regulation

of a large subset of genes in the liver, pancreas, kidney and intestine. Although some pancreatic

HNF1α targets are suggested to impact the disease phenotypes, the exact nature of the molecular

links between loss of HNF1α function and manifestation of the disease is still unclear.

Mouse models with deletion of Hnf1α functions display hepatic and renal dysfunction coupled

to non-insulin-dependent diabetes and dwarfism (5; 6). While these mice still produce insulin,

their pancreatic β-cells are defective in glucose-sensing insulin secretion (7; 8). Simultaneous

Hnf1α reexpression in both liver and endocrine pancreas of Hnf1α null mice failed to restore

normal blood glucose and insulin levels suggesting that other tissues in which Hnf1α was deleted

could be participating in the diabetic phenotype of these mice (9).

Gut hormones are produced by enteroendocrine cells and are crucial regulators of glucose

homeostasis and pancreatic insulin secretion (10). Glucose-dependent insulinotropic polypeptide

(GIP) and glucagon-like peptide 1 (GLP-1) are incretins that stimulate insulin secretion while

ghrelin targets pancreatic β-cells to limit insulin production (10). Hnf1α null mice display

intestinal epithelium dysfunctions including altered enteroendocrine cell differentiation (11).

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Here, we aimed to explore if specific changes in gastrointestinal hormones could functionally

relate to glucose homeostasis in Hnf1α null mice.

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RESEARCH DESIGN AND METHODS

Animals and Analytical procedures

Hnf1α null mice (5; 11) and control littermates were treated in accordance with the Institutional

Animal Research Review Committee of the Université de Sherbrooke (approval ID number 102-

14B). Hnf1α null mice were genotyped as described before (11). Blood glucose values were

determined from whole venous blood from mice fed ad libitum or 16-hr-fasted using a glucose

monitor (FreeStyle Lite, Abbott Diabetes Care). (D-Lys3)-GHRP6 (Bachem), a classical but not

highly selective GHS-R antagonist (12-14), was freshly diluted in 100 µl of saline and

intraperitoneal (IP) injections were performed every 12 hours during 5 consecutive days followed

by a 16-hr-fasting period before sacrifice or IP glucose tolerance tests (IPGTT) (2 g of D-

glucose/Kg). Optimal dose of GHS-R antagonist (200 nmoles/30 g) was determined accordingly

to previous published work (15; 16). For metabolic analyses, mice were individually placed in

metabolic cages, provided with the same quantities of food and water and housed on a reverse

light-dark cycle. All groups were fed ad libitum throughout the duration of the study. Following

a 5-day adaptation period after being transferred from group housed cages to single housed

metabolic cages, mice were treated with GHS-R antagonist or saline during 5 days. Body weight

(g), food intake (g), water intake (ml), urine (ml), and feces (g) were measured every morning

before injections. Urine glucose content was determined with Chemstrip 10® urine test strips

(Roche Diagnostics).

RNA isolation and RT-PCR

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Total RNA from the jejunum and stomach was isolated and qRT-PCR performed as previously

described (11). Results were calibrated with TATA box binding protein (TBP). Primer

sequences are available upon request.

Immunofluorescence

Jejunum segments and pancreas were fixed in 4% paraformaldehyde overnight at 4°C,

dehydrated, embedded in paraffin and cut to 5-µm sections. Immunofluorescences were

performed as previously described (17). The following affinity-purified antibodies (Santa Cruz

Biotechnology) were used: goat anti-Ghrelin (sc-10368; diluted 1/100), goat anti-GIP (sc-23554;

diluted 1/50) and mouse anti-Insulin (sc-8033; diluted 1/200).

ELISA

Blood was collected from the right heart ventricle of 16-hr-fasted mice and pretreated with

Pefabloc solution (Ghrelin) or dipeptidyl peptidase 4 (DPP4) inhibitor (GIP and GLP-1). After 30

min at room temperature, samples were centrifuged at 3,000 g for 15 min at 4°C. Acidification of

the serum samples with HCl to a final concentration of 0.05 N was performed. Total Ghrelin

(EZRGRT-91K), Active ghrelin (EZRGRA-90K), Total GIP (EZRMGIP-55K), Active GLP-1

(EGLP-35K) and Insulin (EZRMI-13K) were measured using ELISA kits from EMD Millipore.

Total Glucagon was assessed with the ELISA kit DGCG0 (R&D Systems). The Ultra Sensitive

Mouse Insulin ELISA kit 90080 (CrystalChem) was used during IPGTT procedures. Total DPP4

was measured with the DPP4 ELISA kit SEA884Mu (USCN Life Science).

Statistical analysis

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Statistical analyses were performed using the GraphPad Prism 6 software. Statistics were

calculated using the two-way Student’s two-tailed t test or two-way nested analysis of variance

(ANOVA). Differences were considered significant with a P value of < 0.05.

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RESULTS

Assessment of circulating levels of glucose (Fig.1A), insulin (Fig.1B) and glucagon (Fig.1C) in

Hnf1α mutant and control mice confirmed hyperglycaemic state of the mutants with reduced

circulating insulin levels without alteration of glucagon levels. Immunofluorescence detection of

insulin in the pancreas of Hnf1α mutant (Fig.1D) and control mice (Fig.1E) suggested a

comparable potential of pancreatic β-cells in expressing insulin peptide. Since the intestinal

endocrine system plays a crucial role in regulating glucose metabolism, expression of relevant

hormones in the intestine of Hnf1α mutant mice was monitored. Analysis of gene transcript

expression for ghrelin, GIP and GLP-1 was determined by RT-qPCR in the jejunum of newborn

and adult Hnf1α mutant and control mice. While ghrelin transcripts were significantly increased

in the jejunum of mutant as compared to control mice (1.79 fold-increase at day 1, P < 0.01; 4.30

fold-increase at 4 months, P < 0.01, Fig.1F), GIP transcripts were significantly decreased (4.41

fold-decrease at day 1, P < 0.05; 3.48 fold-decrease at 4 months, P < 0.01, Fig.1G). GLP-1

transcripts were not affected under these conditions (Fig.1H). Immunofluorescences were

performed to monitor the distribution of corresponding enteroendocrine cells. The number of

ghrelin-positive cells was significantly increased in the jejunum of adult Hnf1α mutant when

compared to control mice (3.72 fold-increase, P < 0.0001, Fig.1I-J) while the number of GIP-

positive cells decreased (2.23 fold-decrease, P < 0.0001, Fig.1K-L).

ELISA was next performed to measure circulating levels of gastrointestinal hormones in Hnf1α

mutant and control mice. Total circulating ghrelin was significantly increased in Hnf1α mutant

as compared to control mice (6.57 fold-increase at 1 month, P < 0.001 and 4.16 fold-increase at 4

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months, P < 0.01, Fig.2A). These increases were also reflected at the level of active ghrelin form

(4.92 fold-increase at 1 month, P < 0.01 and 4.72 fold-increase at 4 months, P < 0.05, Fig.2B).

As opposed to gene transcripts level, total circulating GIP was significantly up-regulated in

Hnf1α mutant as compared to control mice (14.51 fold-increase at 1 month, P < 0.05 and 4.03

fold-increase at 4 months, P < 0.01, Fig.2C). Basal active GLP-1 circulating levels were

undetectable in both Hnf1α mutant and control mice under these conditions. Since GIP peptide

stability is dependent on DPP4 activity (18) and Hnf1α activates transcription of DPP4 (19),

DPP4 circulating levels were measured. A reduction of circulating DPP4 was observed in Hnf1α

mutant as compared to control mice (5.07 fold-decrease, P < 0.01, Fig.2D). Since ghrelin is

mostly secreted from the stomach and the jejunum, the relative ratio of active ghrelin in each of

these tissues was monitored in Hnf1α mutant and control mice. ELISA revealed a significant

2.55 fold-increase (P < 0.05) in active ghrelin per gram of jejunum of Hnf1α mutant when

compared to control mice, while no significant change was observed in the stomach of these

animals (Fig.2E). Coincidently, ghrelin transcripts were not significantly modulated in the

stomach of Hnf1α mutant as compared to control mice (P = 0.49, n = 4).

Ghrelin can limit insulin release by interacting with the GHS-R1a receptor on β-pancreatic cells

(20). To test whether increases in active ghrelin were functionally related to the hypoinsulinemia

state of Hnf1α mutant mice, the GHS-R antagonist [D-Lys-3]-GHRP-6 was administrated IP to

mice. Single injections of the GHS-R antagonist every 12 hours progressively led to a decrease in

blood glucose level in Hnf1α mutant mice to reach statistically undistinguishable levels from the

controls after 5 days of treatment (Fig.3A). This effect was reversible with a progressive return

to hyperglycaemia steady state 1 week after stopping injections (Fig.3B). Hypoinsulinemia of

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Hnf1α mutant mice was corrected after 5 days of ghrelin antagonist treatment (Fig.3C). IPGTT

were further performed to monitor glucose clearance of treated and non-treated mice. Glucose

levels in fasted non-treated Hnf1α mutant mice rose above 23 mmol/L after 15 min and failed to

significantly decline at 120 min (Fig.3D). Glucose levels in fasted Hnf1α mutant mice pre-

treated with GHS-R antagonist rose above non-treated and treated control mice at 15 min but

rapidly declined to reach comparable values with the control groups at 120 min (Fig.3D). AUC

calculations revealed a significant recovery for Hnf1α mutant mice pre-treated with the GHS-R

antagonist in blood glucose clearance (Fig.3E). Circulating insulin levels during IPGTT were

significantly increased between Hnf1α mutant mice pre-treated with GHS-R antagonist versus

non-treated Hnf1α mutant mice (Fig.3F) while GIP levels were significantly decreased with

GHS-R antagonist pre-treatment (Fig.3G).

Since ghrelin can impact on appetite and metabolism, solid and liquid metabolism was

investigated among the various mouse groups using metabolic cages. Analysis of solid

metabolism indicated that food-intake ratios were increased in Hnf1α mutant compared to control

mice (145%) and GHS-R antagonist treatment did not significantly influence this tendency

(Fig.4A). This observation was consistent with fecal ratios that were increased in Hnf1α mutant

mice (178%, Fig.4B). Analysis of liquid metabolism revealed that water intake ratios were

increased by 182% in Hnf1α mutant compared to control mice (Fig.4C) and GHS-R antagonist

treatment significantly reduced this ratio in Hnf1α mutant mice (Fig.4C). Urine ratios were not

significantly affected when Hnf1α mutant mice were compared to controls (Fig.4D). However,

GHS-R antagonist treatment significantly reduced this ratio in both Hnf1α mutant and control

mice (Fig.4D). Detection of glucose in the urine of these Hnf1α mutant mice revealed an

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important eradication of glucose content after GHS-R antagonist treatment (Fig.4E) while control

mice remained negative under these treatments (not shown).

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DISCUSSION

MODY3 is characterized by a loss of insulin secretory capacity. Past efforts to better define

molecular links between HNF1α function and disease phenotypes have focused on the pancreas

(21). Using Hnf1α mutant mice, we identified a novel functional regulatory loop between

deregulated production of intestinal ghrelin, restricted potential of insulin secretion and control of

blood glucose homeostasis.

Our data suggest that sustained increases of circulating ghrelin in Hnf1α mutants are dependent

on defects from the intestine. This assumption is reasonable given that intestine size is larger

than stomach and that Hnf1α mutants display intestinalomegaly (11). Although studies support

that pancreatic epsilon cells can produce ghrelin (22), attempts to detect ghrelin in the pancreas of

Hnf1α mutants was unsuccessful. These observations suggest that specific regulatory

mechanisms must occur to differentially regulate expression and/or ghrelin cells commitment in

the intestine as compared to other tissues.

The regulatory mechanisms connecting Hnf1α with ghrelin and GIP expression are likely to be

complex. Loss of Hnf1α could mechanistically impact enteroendocrine cells fate including GIP

and ghrelin cells. It is also possible that Hnf1α regulates ghrelin and GIP transcription.

Bioinformatic analysis of murine ghrelin and GIP gene promoters predicted several

Hnf1α elements. In contrast to GIP, this transcriptional connection would imply a negative

regulatory loop for ghrelin as it has been suggested for Pax4 transcriptional regulator (23).

However, assessment of such mechanisms remain challenging since the population of intestinal

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ghrelin and GIP cells represent a tiny portion of this epithelium and no normal enteroendocrine

cellular models are yet available for such studies.

Although the number of GIP positive cells and gene transcripts are reduced in the small intestine

of Hnf1α mutants, GIP circulating levels are paradoxically increased. Similar observations were

reported in type 2 diabetic patients with exaggerated GIP secretion and dissociated insulin

response (24; 25). GIP peptide stability could be increased due to the reduction of circulating

DPP4 in Hnf1α mutant mice.

In conclusion, pharmacological blockade of ghrelin/GHS-R interaction corrected diabetic

features in a MODY3 mouse model. This opens up on pre-clinical studies targeting MODY3

patients in a program designed to limit ghrelin action and better control blood glucose

homeostasis.

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ACKNOWLEDGMENTS

The authors thank the Electron Microscopy & Histology Research Core of the FMSS at the

Université de Sherbrooke for their histology and phenotyping services. This study was supported

by a grant from CIHR (MOP-126147 to F.Bo.). C.R.L was a recipient of a NSERC fellowship.

F.Bo. and P.S. are members of the FRQS-funded « Centre de Recherche du CHUS».

F.B., C.R.L and K.B. designed, researched data, and reviewed, edited and approved the final

version of the manuscript. P.S. designed and approved the final version of the manuscript. F.Bo.

designed, researched data, wrote the manuscript, and reviewed, edited, and approved the final

version of the manuscript. F.Bo. is the guarantor of this work and, as such, had full access to all

the data in the study and takes responsibility for the integrity of the data and the accuracy of the

data analysis.

Data from this study were presented in part at the Canadian Digestive Disease Week 2013 in

Victoria (Canada) and at the Digestive Disease Week 2013 in Orlando (USA).

The authors have no conflict of interest to declare.

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FIGURE LEGENDS

Figure 1. Loss of Hnf1αααα affects expression of gastrointestinal hormones. Blood glucose (A),

insulin (B) and glucagon (C) levels were determined from 16-hr fasted adult control and Hnf1α

null mice (n=5-9). Representative immunofluorescence for insulin was performed on sections of

pancreas of both Hnf1α mutant (D) and control (E) mice. qRT-PCR detection of Ghrelin (F), GIP

(G) and GLP-1 (H) mRNA was performed on total small intestinal RNA extracts from newborn

and adults control and Hnf1α null mice and calibrated in comparison to TBP mRNA detection

(n=4-7). The proximal small intestine of both control and Hnf1α null mice was labeled for

ghrelin (I) or GIP (K) by immunofluorescence. Total numbers of positively stained cells for

ghrelin (J) and GIP (L) were calculated on an average of 40 crypt-villus axis per animal (n = 6); *

P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data were analysed with the unpaired t

test and error bars represent SE.

Figure 2. Loss of Hnf1αααα impacts ghrelin and GIP circulating levels. Total ghrelin (A), active

ghrelin (B), GIP (C) and DPP4 (D) circulating levels were assessed from 16-hr fasted adult

control and Hnf1α null mice by ELISAs (n = 3-6). (E) Total protein extracts were isolated from

whole stomach or jejunum of 16-hr fasted adult control and Hnf1α null mice and active ghrelin

was assessed by ELISA (n=5-6); * P < 0.05, ** P < 0.01, *** P < 0.001. Data were analysed

with the unpaired t test and error bars represent SE.

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Figure 3. Impact of Hnf1αααα mutant mice treatment with the GHS-R antagonist on glucose

homeostasis. (A) Adult control and Hnf1α null mice were IP injected with saline (upper panel)

or GHS-R antgonist (lower panel) for 5 days. Blood glucose levels were assessed every morning

of each day (n=6 for each group). (B) Hnf1α null mice were IP injected with GHS-R antagonist

for 5 days and left to recover. Blood glucose levels were assessed at each indicated days (n=10).

(C) Adult control and Hnf1α null mice were IP injected with saline or GHS-R antagonist for 5

days. Mice were fasted for 16-hr and blood insulin levels assessed by ELISA (n=10 for each

group). (D) Adult control and Hnf1α null mice were IP injected with saline or GHS-R antagonist

for 5 days. Mice were fasted for 16-hr and IPGTT was performed. Blood glucose levels were

measured at each indicated time (n=5 for each group). Glucose AUC was calculated over the 120

min period (E) and insulin levels (F) and GIP levels (G) measured by ELISAs at 30 and 120 min;

* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001. Data were analysed with the unpaired t

test except for AUC where ANOVA was performed. Error bars represent SE.

Figure 4. Impact of Hnf1αααα mutant mice treatment with the GHS-R antagonist on liquid and

solid metabolisms. Hnf1α mutant (n=6) and control mice (n=6) metabolism was evaluated at

the beginning (day 0) and the end (day 5) of saline or GHS-R antagonist IP treatment. Solid

metabolism was measured by calculating food ratios (grams of chow per gram weight) (A) and

fecal excretion (grams of feces per gram weight) (B). Liquid metabolism was measured by

calculating water ratios (milliliters of water per gram weight) (C) and urine ratios (milliliters of

urine per gram weight) (D). (E) Urine glucose content was determined in Hnf1α mutant mice at

the beginning (day 0) and the end (day 5) of GHS-R antagonist IP treatment; *P < 0.05, **P <

0.01. Data were analysed with the unpaired t test and error bars represent SE.

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