M Steriotis

Jouiad
email: mjouiad@masdar.ac.ae

E V Chatzidouros

A Traidia

R S Devarapalli

D I Pantelis

T A Steriotis

M Jouiad

Fracture Toughness Properties of HIC Susceptible Carbon Steels in Sour Service Conditions

Keywords: fracture toughness, pipeline steel, hydrogen, sour environment, embrittlement, Hydrogen Induced Cracking Nomenclature BS British standards

ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

1-Introduction

The presence of wet H2S (sour environment) in hydrocarbons is one of the most prominent causes for failures and defects formation in carbon steel pipelines . A significant part of the atomic hydrogen, produced as a result of the (electrochemical) corrosion reaction between H2S and iron in steels, penetrates the bulk material through an adsorptionabsorption-diffusion mechanism. At the interface of defects and imperfections in steels, such as elongated non-metallic inclusions (e.g., Manganese Sulfides MnS), the diffusing hydrogen is trapped and recombines to form high pressure hydrogen gas cavities [START_REF] Domizzi | Influence of sulphur content and inclusion distribution on the hydrogen induced blister cracking in pressure vessel and pipeline steels[END_REF][START_REF] Popov | Chapter 7 -Hydrogen Permeation and Hydrogen-Induced Cracking[END_REF][START_REF] Revie | Hydrogen-Induced Cracking of Linepipe Steels Part 1-Threshold Hydrogen Concentration and pH[END_REF][START_REF] Elboujdaini | Hydrogen-Induced Cracking and Effect of Non-Metallic Inclusions in Linepipe Steels[END_REF][START_REF] Moore | Factors influencing the hydrogen cracking sensitivity of pipeline steels[END_REF][START_REF] Park | Effect of microstructure on the hydrogen trapping efficiency and hydrogen induced cracking of linepipe steel[END_REF]. Increasing pressure build up within these cavities is maintained by the continuous hydrogen flux from the corroding steel surface, known to be the main driving force for the formation and the growth of small cracks parallel to the pipe wall, commonly called Hydrogen-Induced Cracks (HIC) or blisters [START_REF] Mcmahon | 6 -The role of grain boundaries in hydrogen induced cracking (HIC) of steels[END_REF][START_REF] Kittel | Hydrogen induced cracking (HIC) testing of low alloy steel in sour environment: Impact of time of exposure on the extent of damage[END_REF][START_REF] Kittel | Hydrogen Induced Cracking (Hic) -Laboratory Testing Assessment Of Low Alloy Steel Linepipe[END_REF][START_REF] Chattoraj | The effect of hydrogen induced cracking on the integrity of steel components. Sadhana[END_REF]. The latter can grow, coalesce to an advanced form of HIC known as Step-Wise Cracking (SWC) [START_REF] Sezgin | Modelling of hydrogen induced pressurization of internal cavities[END_REF][START_REF] Zakaria | Formation and analysis of stack cracks in a pipeline steel[END_REF][START_REF] Lino | The extension of hydrogen blister-crack array in linepipe steels[END_REF], which could lead to equipment failure [START_REF] Koyama | Overview of hydrogen embrittlement in high-Mn steels[END_REF][START_REF] Villalobos | Evaluation of mechanical properties of an experimental microalloyed steel subjected to tempering heat treatment and its effect on hydrogen embrittlement[END_REF][START_REF] Mcmahon | Hydrogen-induced intergranular fracture of steels[END_REF][START_REF] Kim | Relationship between hydrogen-induced cracking and type I sulfide stress cracking of high-strength linepipe steel[END_REF][START_REF] Suárez | Finite Element Analysis of the Interaction of Hydrogen Induced Stepwise Cracks[END_REF]. It is well admitted that steel microstructure [START_REF] Beidokhti | Effects of alloying elements and microstructure on the susceptibility of the welded HSLA steel to hydrogen-induced cracking and sulfide stress cracking[END_REF][START_REF] Shi | Effect of Microstructure on Hydrogen Induced Cracking Behavior of a High Deformability Pipeline Steel[END_REF][START_REF] Kim | Effect of environmental and metallurgical factors on hydrogen induced cracking of HSLA steels[END_REF] as well as shape, size, type and distribution of nonmetallic inclusions [START_REF] Beidokhti | Effects of alloying elements and microstructure on the susceptibility of the welded HSLA steel to hydrogen-induced cracking and sulfide stress cracking[END_REF][START_REF] Domizzi | Influence of sulphur content and inclusion distribution on the hydrogen induced blister cracking in pressure vessel and pipeline steels[END_REF][START_REF] Dong | Effects of hydrogen-charging on the susceptibility of X100 pipeline steel to hydrogen-induced cracking[END_REF][START_REF] Park | Effect of microstructure on the hydrogen trapping efficiency and hydrogen induced cracking of linepipe steel[END_REF][START_REF] Kim | Effect of environmental and metallurgical factors on hydrogen induced cracking of HSLA steels[END_REF][START_REF] Liu | Effect of inclusions on initiation of stress corrosion cracks in X70 pipeline steel in an acidic soil[END_REF][START_REF] Mendibide | Composition optimization of high-strength steels for sulfide stress cracking resistance improvement[END_REF][START_REF] Nayak | Microstructure and properties of low manganese and niobium containing HIC pipeline steel[END_REF][START_REF] Ren | The effects of inclusions and second phase particles on hydrogeninduced blistering in iron[END_REF][START_REF] Xue | Characterization of inclusions of X80 pipeline steel and its correlation with hydrogen-induced cracking[END_REF][START_REF] Zhao | Investigation on the H2S-resistant behaviors of acicular ferrite and ultrafine ferrite[END_REF][START_REF] Zheng | Effect of hydrogen and inclusions on the tensile properties and fracture behaviour of A350LF2 steels after exposure to wet H2S environments[END_REF][START_REF] Zhuo | Influence of Sulfur Content and Microstructure of Pipeline Steels on Mechanical property and H2S-Resistant Behavior[END_REF][START_REF] Brown | Hydrogen Induced Cracking in Pipeline Steels[END_REF][START_REF] Chou | Comparison of the Behavior of Hydrogen in Two Carbon Steels Differing in Sulfur Content[END_REF][START_REF] Wilde | Some Observations on the Role of Inclusions in the Hydrogen Induced Blister Cracking of Linepipe Steels in Sulfide[END_REF][START_REF] Garet | Hydrogen trapping on non metallic inclusions in Cr-Mo low alloy steels[END_REF][START_REF] Kobayashi | A study of the hydrogen-induced degradation of two steels differing in sulfur content[END_REF][START_REF] Koh | Effect of Microstructure on Hydrogen-Induced Cracking of Linepipe Steels[END_REF] are the key ingredients for the development of internal pressurized hydrogen cavities [START_REF] Jin | Effect of non-metallic inclusions on hydrogen-induced cracking of API5L X100 steel[END_REF][START_REF] Mohtadi-Bonab | A focus on different factors affecting hydrogen induced cracking in oil and natural gas pipeline steel[END_REF][START_REF] Mohtadi-Bonab | Effect of different microstructural parameters on hydrogen induced cracking in an API X70 pipeline steel[END_REF]. This degradation mechanism is especially acute in old pipeline steel grades, also referred to as "legacy/heritage steels" commissioned prior the mandate of NACE TM-0284 [START_REF] Nace Standard | Evaluation of Pipeline and Pressure Vessel Steels for Resistance to Hydrogen-Induced Cracking[END_REF] and ISO-15156 acceptance criteria for sour service material section [START_REF]Petroleum and natural gas industries --Materials for use in H2S-containing environments in oil and gas production --Part 2: Cracking-resistant carbon and low-alloy steels, and the use of cast irons[END_REF]. Such steel grades contain a high level of impurities and nonmetallic inclusions in addition to a banded microstructure which constitute the perfect ingredients for HIC initiation and growth. Operating equipment made of such non-HIC resistant steels in "mildly" sour environments [START_REF] Smith | HIC Resistance of Heritage Pipelines Exposed to Mildly Sour Environments[END_REF] will continue to be for the foreseeable future due to the associated replacement cost, provided that the stage of HIC damage is monitored in routine inspections and the fitness for service of the asset is assessed with respect to international standards, such as API-579 [START_REF]API 579-1, Fitness-For-Service[END_REF]. A typical case study from the field is presented in section 2. For such case, it is crucial to have accurate knowledge of their material properties, particularly fracture toughness data, measured in representative testing environments, which is the purpose of this work.

The review of the published literature shows that many works have focused on the causes of HIC formation in relation to microstructural features, such as the existence and role of nonmetallic inclusions or the microstructural morphology [START_REF] Mohtadi-Bonab | A focus on different factors affecting hydrogen induced cracking in oil and natural gas pipeline steel[END_REF]. These works have followed NACE TM 0284 [START_REF] Nace Standard | Evaluation of Pipeline and Pressure Vessel Steels for Resistance to Hydrogen-Induced Cracking[END_REF] or used custom electrolytic solutions [START_REF] Mohtadi-Bonab | The mechanism of failure by hydrogen induced cracking in an acidic environment for API 5L X70 pipeline steel[END_REF] for determining HIC resistance of studied steels and proceeded in identifying the microstructural features, which promote HIC formation and growth. In particular, the fracture toughness and crack propagation resistance were found to be reduced in wide nuances of carbon steels in the presence of high-pressure hydrogen gas, where KTH is degraded by as much as 50% in hydrogen gas of pressure as reported by Marchi and Somerday [START_REF] Marchi | Technical Reference for Hydrogen Compatibility of Materials[END_REF]. Similarly, Lam and co-workers [START_REF] Lam | Literature Survey of Gaseous Hydrogen Effects on the Mechanical Properties of Carbon and Low Alloy Steels[END_REF] provided with an interesting literature survey regarding the degradation of mechanical properties of carbon steels including the fracture toughness properties such as the threshold stress intensity and the crack propagation. Hence, there is an undisputed lack of published relevant fracture toughness data that can be used for assessing the fitness for service of pipelines and pressure vessels containing HIC damage. Besides the need for accurate fitness for service assessment of equipment containing HIC damage, relevant fracture toughness data are also critically needed for predicting their remaining lifetime [START_REF] Findley | Critical Assessment 17 : Mechanisms of hydrogen induced cracking in pipeline steels Critical A ssessment 17 : M echanisms of hydrogen induced cracking in pipeline steels[END_REF].

Although HIC is well documented and relevant standards [START_REF] Nace Standard | Evaluation of Pipeline and Pressure Vessel Steels for Resistance to Hydrogen-Induced Cracking[END_REF] are in place to asses a steel's performance, the prediction of HIC growth rate, as well as the critical hydrogen pressure in the HIC cavity and its relation to the existing H2S environment have only been studied using analytical [START_REF] Balueva | Modeling of Hydrogen Embrittlement Cracking in Pipe-lines under High Pressures[END_REF] or numerical tools [START_REF] Gonzalez | Hydrogen-Induced Crack Growth Rate in Steel Plates Exposed to Sour Environments[END_REF], without using experimental data which fit the case. In particular, if we assume that an isolated HIC defect is a cavity, which has an elliptical shape with two singularities (crack tips) at each end, the HIC defect will grow in size when the fracture toughness (K or CTOD) at the crack tip will reach the steel fracture toughness in hydrogen environment [START_REF] Traidia | An effective finite element model for the prediction of hydrogen induced cracking in steel pipelines[END_REF][START_REF] Gonzalez | Hydrogen-Induced Crack Growth Rate in Steel Plates Exposed to Sour Environments[END_REF]. The intrinsic effect of H2S, i.e. atomic hydrogen accumulation and subsequent hydrogen pressure build up in the cavity leading to eventual growth of the HIC, can be assessed, through a numerical modeling scheme such as the work of Gonzalez and co-workers [START_REF] Gonzalez | Hydrogen-Induced Crack Growth Rate in Steel Plates Exposed to Sour Environments[END_REF]. This requires using input data of the fracture toughness properties which are relevant to the direction of the growth of HIC cavity and measured in environment representative of the in-service sour environment. In this case, fracture toughness properties measured in the SL direction i.e. parallel to the pipeline/vessel wall and in various H2S environments, are the most applicable since preferential growth path of the HIC is along the SL direction. Using the aforementioned inputs, a reliable integrity assessment tool able to predict HIC growth rates was proposed by Traidia and co-workers [START_REF] Traidia | An effective finite element model for the prediction of hydrogen induced cracking in steel pipelines[END_REF][START_REF] Traidia | Hydrogen-induced-cracking prediction software[END_REF].

The present work focuses on the measurement of fracture toughness of an API X42 non-HIC resistant pipeline steel in simulated sour service environment along two different directions, namely TL and SL directions. Correlation with the total hydrogen concentration absorbed in the steel (noted CH) measured using Thermal Desorption Spectroscopy (TDS) is also established. While it is a straightforward process to extract fracture toughness specimens in the TL direction, this is not the case for the SL direction, especially for thin pipelines. This is because there is not enough material in the SL direction to extract a full novel single edgenotch bending specimen (SEB) or compact tension specimen (CT) for evaluation of fracture toughness properties. Hence, novel SEB geometry was developed to overcome the pipeline thickness constrains and perform fracture toughness tests in the SL direction as per ASTM1820 standards [START_REF] Traidia | Method and Device for Testing a Material Sample in Standard test for In-Plane Fracture Toughness Evaluation US-Patent # US[END_REF][START_REF] Chatzidouros | Effect of hydrogen on fracture toughness properties of a pipeline steel under simulated sour service conditions[END_REF]. The fracture toughness properties in the SL direction, along with the CH measurements in the metal lattice of the steel, can be used for subsequent work on estimating HIC growth rates at critical hydrogen pressure, which in turn can be fitted in the existing or future developed numerical and integrity assessment tools. Moreover, the role of the X42 microstructure in promoting the initiation and growth of HIC is also discussed and compared with relevant available literature without tackling the well-established HIC mechanism as per the aforementioned investigations

2-Typical case study from the field

This section highlights a specific case study from the field where accurate knowledge of relevant material fracture toughness data is critically needed to conduct a reliable Engineering Critical Assessment (ECA), also referred to as Fitness-For-Service (FFS) assessment procedure. ECA or FFS are standard calculations, often based on fracture mechanic principles, meant to check the integrity of an asset with respect to flaws found inservice and provide recommendations on possible pressure de-rating and/or repairs. Such level-based assessments follow the well-established industry standards developed since the late 1960s [START_REF] Wintle | Which procedures for fitness-for-service assessment: API 579 or BS[END_REF], where API-579 [68] and BS-7910 [START_REF]Guide to methods for assessing the acceptability of flaws in metallic structures[END_REF] are the most commonly used ones.

Detailed discussions and comparisons of both standards are available elsewhere [START_REF] Hadley | 2013 in brief[END_REF][START_REF] Larrosa | Comparisons of the solutions of common FFS standard procedures to benchmark problems[END_REF][START_REF] Hadley | Materials properties for Engineering Critical Assessment : Background to the advice given in BS 7910 : 2013[END_REF].

The case study described in the present work, considers a 20-inch nominal diameter sour service transmission pipeline (11.4 mm wall thickness) carrying out sour condensate (crude/gas mixture), with 3 mol % H2S and 0.5 mol% CO2, operating at 480 psi and 70°F.

The pipeline is made of API X60 steel, commissioned prior to the mandate of HIC resistant steels for sour service applications. Magnetic Flux Leakage In-Line Inspections (MFL-ILI) runs carried out on this pipeline revealed the presence of areas featured as "laminations" with relatively large size. Given the commissioning date of the pipeline and the relatively high H2S content in the carried hydrocarbons, decision was made to excavate these locations and carry out advanced ultrasonic testing (corrosion mapping) to confirm/infirm the presence of HIC or SWC. Figure 1, shows a typical section of the pipe (approximately 14 m long) which has been excavated for this purpose. Four different locations on this section, located between 3'oclock to 9'oclock positions, have been selected for advanced UT scanning. The results clearly show the presence of large penny-shaped HIC cavities, as large as 200 mm in diameter. In some cases, the HIC cavities, located approximately at mid-wall thickness (6 mm from the outer surface), are either isolated (scan 1), have coalesced with nearby HICs (scan 2) or are about to coalesce (scan 3 and 4). Linear HIC damage (i.e., without SWC), far from welds and major structural discontinuities, is not harmful to the integrity of the pipeline as it does not reduce the overall load bearing capacity, or remaining strength. However, the presence of SWC is always alarming since it directly impacts the remaining strength and can lead to unstable local failure (crack growth) or local plastic rupture.

Carrying proper FFS assessment, using the scans shown in Figure 2, it is particularly challenging, for such study case, SWC has been confirmed with height ranging from 1.8 mm to 3.1 mm (that is almost 30% of the corroded wall thickness), and therefore API 579 (Part 7, Level 2) requires a crack-like flaw assessment (as per Part 9) to be carried out to demonstrate the integrity against unstable crack growth and fracture. Such assessment involves the use of the Failure Assessment Diagram (FAD) procedure which requires knowledge of the steel fracture toughness in the direction of crack growth, and also in representative testing environment (i.e., sour service). The experimental data generated in the present work will constitute the seed towards the development of a representative fracture toughness database to be used for reliable FFS assessments.

3-Experimental procedure 3-1-Test sample orientations

Evaluation of fracture toughness properties KQ and CTOD were performed following the approach described in a previous published work [START_REF] Traidia | Method and Device for Testing a Material Sample in Standard test for In-Plane Fracture Toughness Evaluation US-Patent # US[END_REF] as per the ASTM E1820 standard [START_REF] Astm E | Standard test method for measurement of fracture toughness[END_REF].

It is here worth noting that measured KQ values reported in this work do not satisfy the conditions for valid linear elastic plane strain fracture toughness KIC.

3-2-Material: API X42

API X42 microstructure was analyzed using scanning electron microscope (SEM) Quanta3D FEI ™ and Electron Back Scattered Diffraction (EBSD) from EDAX ™ . The steel samples used in this work have been machined from a 10 mm thick pipeline sample extracted from a field pipeline. HIC qualification tests were carried out as per NACE TM0284 [START_REF] Nace Standard | Evaluation of Pipeline and Pressure Vessel Steels for Resistance to Hydrogen-Induced Cracking[END_REF] and the results

showed that the pipeline steel used in this study is not HIC resistant. The chemical composition and the mechanical properties of this steel are provided respectively in Table 1 and Table 2.

3-3-Hydrogen charging

In a typical process, the X42 steel sample is electrochemically charged with hydrogen using TDS as described elsewhere [START_REF] Chatzidouros | Effect of hydrogen on fracture toughness properties of a pipeline steel under simulated sour service conditions[END_REF]. The corresponding targeted values of CH to achieve at steady state using cathodic charging are presented in Table 3. CH was measured in notched X42 samples as shown in Figure 3, namely 10 mm (length) × 10mm (width) × 5 mm (thickness) X42 notched samples, with notch depth of 2.5 mm. Precharging for 48 hours was found to be sufficient to achieve a stable CH in the metal lattice of the steel as shown in previous works [START_REF] Chatzidouros | Effect of hydrogen on fracture toughness properties of a pipeline steel under simulated sour service conditions[END_REF][START_REF] Capelle | The effect of hydrogen concentration on fracture of pipeline steels in presence of a notch[END_REF]. The notion of ex-situ means that the SEB specimens were hydrogen charged for 48 hours, prior to being fracture toughness tested, i.e. the SEB specimens are removed from the charging arrangement after the charging procedure and they are immediately fracture toughness tested in air. Following the aforementioned rationale, the current densities which can produce the desired CHs in the X42 samples are identified, in the first set of charging tests and in the second set the actual SEB specimens are charged using the evaluated current densities.

4-Experimental results

4-1-Hydrogen measurement

The three levels of CH described in section 3.3 were achieved by hydrogen charging X42 samples, with various values of current densities. Current densities were varied between 0.4 and 4 mA/cm 2 . The CH in the X42 samples were evaluated after integrating the TDS curves in desorption vs. time and are given in Table 4. Furthermore, the TDS results in terms of desorption vs. temperature are shown in Figure 4.

As depicted in Table 4, a current density of 4mA/cm 2 was used for pre-charging the fracture toughness specimens with CH of 1.5 ppmw, similarly a current density of 1mA/cm 2 and 0.4mA/cm 2 for CH of 1ppmw and CH for 0.5 pppmw, respectively.

4-2-Microstructural analyses

The microstructure and the fracture characteristics of the X42 micro alloyed steel, in the form of sheets and pipes were carried out to study the effect of the morphology of secondary phase, in addition to the matrix phase. were not observed here on the surface of non-tested sample (Figure 5), however for the fractured samples, the dimples observed are sign of their presence in X42 (Figure 6).

For fracture surface analyses, two types of samples were selected corresponding to the two extreme conditions of the mechanical testing, namely the one tested in air and the other one tested in hydrogen environment at 1.5 ppmw of CH. The fractured surfaces were systematically protected by coating to preserve fine fracture details after the mechanical testing. Figure 6 shows the low and high magnification micrographs of the fractured surface from X42 sample in parallel direction (SL), respectively in air Figure 6 To further comprehend the mechanism of the X42 steel failure in air, and under hydrogen environment, EBSD mapping was conducted at the edge region of the notched sample at the vicinity of the crack propagation path. The tested samples in air for both directions (SL and TL) are showing relatively similar crack propagation features, confirming the fact that the primary crack is propagating through the grains with random orientations. The presence of fine grain structures could due to the recrystallization at the edge of notch region. Figure 7 for instance, illustrate respectively, the general view of the crack path as captured by optical microscope (Figure 7(a)) and EBSD maps on X42 sample tested in SL direction in air (Figure 7(b)) and at 1.5 ppmw of CH (Figure 7(c)), respectively. In the case of the sample tested in air, there is neither a clear preference for all the cracks to propagate crossing the grains with various orientations (transgranular cracks) nor to make random transitions between the grains. No evidence either for a propagation of the cracks along grain boundaries (intergranular cracks) was confirmed by our experiments. For the specimen tested at 1.5 ppmw of CH, the zoom-in at the crack paths depicted in Figure 8 shows the presence of multiple cracks compared to single fatal crack observed for the sample tested in air. Here also the cracks seem to propagate through the grains and along the grain boundaries mixture between transgranular and intergranular cracks as highlighted in

4-3-Fracture toughness results

In this work, the FT parameter KQ0 will be the maximum stress intensity factor for hydrogen free SEB specimens and KQ-H the maximum value of the stress intensity factor of hydrogen pre-charged SEB specimens. KQ-H0.5, KQ-H1 and KQ-H1.5 will be used, respectively for the case of hydrogen pre-charged specimens with 0.5 ppmw, 1ppmw and 1.5ppmw of CH using the following formula.

= ⁄ × ( )

Where:

PQ is the force at the point where the secant line with a slope 0.95 (P/v)0 intersects the forcedisplacement record. The Secant line is constructed from the tangent line OA to the initial portion of the data record (Figure S1 in supplementary information).

S is the span of supports B is the specimen thickness W is the specimen width a0 is the initial crack length (including notch)

Following the same rationale, maximum CTOD will be referred as CTOD0 for hydrogen free specimens, CTODH0.5, CTODH1 and CTODH1.5 for the case of pre-charged specimens with 0.5 ppmw, 1.5 ppmw and 1.5 ppmw of CH, respectively using the following formula.

= (1 -) 2 + ( -)! " [ ( -) + + $]
Where: K is the stress intensity factor for a = a0 ν is the Poisson's ratio σys is the yield stress or 0.2% offset yield strength at room temperature E is the elastic modulus at room temperature Vpl is the plastic component of crack mouth opening displacement at the point of evaluation on the force-displacement curve (see Figure S2 in supplementary information). z is the distance of knife edge measurement point from the notched edge on the SEB specimen rp is the plastic rotation factor given here as 0.44.

Fracture toughness results of X42 specimens in TL direction, are depicted in Tables 5 and6 and in Figure 9. The results show that ( 1 (1/2) ] KQ-H0.5 [MPa m (1/2) ]

KQ-H1

[MPa m (1/2) ] KQ-H1.5 [MPa m (1/2)

[MPa m (1/2) ] KQ-H0.5 [MPa m (1/2) ] KQ-H1 [MPa m (1/2) ] KQ-H1.5 [MPa m (1/2) ]

5-Discussion

Table 7 and 8 show that some of the test results at 1.5 ppmw hydrogen charging are reported as "failed". Indeed, during the FT tests in the SL direction, several failed tests were reported due to HIC features, which manifested during the 48 hours hydrogen pre-charging. This can be clearly observed in Figure 11, where the fatigue pre-crack growing in the perpendicular direction during the FT test is diverted when meeting a HIC feature at mid-wall thickness. This is resulting in crack branching and failure of the FT test. Indeed, recall that the X42 samples used in this study are non-HIC resistant and therefore 48 hours hydrogen precharging at relatively high concentration (1.5 ppmw) led to macroscopic HICs, preventing the completion of all FT tests at 1.5 ppmw (all spare samples were used). This problem was not observed at lower CH levels. The results of the fracture toughness in the TL direction (Table 5, Figure 9(a)), show mixed values of KQ at 0.5 ppmw (KQ-H0.5 ) and 1 ppmw (KQ-H1 ) hydrogen pre-charging concentrations and a 25% reduction in KQ-H1.5 compared to KQ0 at higher CH of 1.5 ppmw.

The maximum CTOD values are reduced for CH values of 0.5ppmw and 1ppmw, but they are almost not affected for the case of 1.5 ppmw (Table 6, Figure 9b) compared to CTOD measured in air. These observations along with the fact that the standard deviation (SD) values of KQ-H1.5 and CTODH1.5 measured for the case at 1.5 ppmw are quite low, requiring further investigations of the effect of hydrogen. Indeed, the X42 is known to have two major characteristics: 1) presence of inclusion MnS type and 2) the lamellar structure of the pearlite phase. The inclusions generate voids as no bond is possible between the inclusions and the matrix [START_REF] Tsunekage | Effects of sulfur content and sulfide-forming elements addition on impact properties of ferrite-pearlitic microalloyed steels[END_REF]. During the mechanical tests, the voids coalesce to promote early failure, this phenomenon is more pronounced in the hydrogen environment especially at high level of CH. Actually, the dimples observed in Figure 6 (c) and (d) for the sample tested under hydrogen environment were also observed for TL direction. These dimples are known to be connected to voids coalescence due to the presence of inclusions. Despite the dimples observed in this study are originating from the inclusions 'coalescence, the shape and the size of these inclusions were not captured by our SEM observations to be directly compared to the elongated MnS inclusions reported elsewhere [START_REF] Hejazi | Effect of manganese content and microstructure on the susceptibility of X70 pipeline steel to hydrogen cracking[END_REF]. In addition, the pearlite as lamellar phase contributes significantly in hardening the steel by hindering the dislocations motion.

Nevertheless, when the orientation of the lamella is perpendicular to the mechanical solicitation, the dislocations could propagate easily along the space between the lamella. This propagation is increased in the presence of hydrogen known to promote the dislocations motion [START_REF] Traidia | Review of hydrogen-assisted cracking models for application to service lifetime prediction and challenges in the oil and gas industry[END_REF][START_REF] Robertson | The effect of hydrogen on dislocation dynamics[END_REF]. For comparison, the case of CH content of 1.5ppmw, the SD of KQ-H1.5 is 15% of the average value and the SD of CTOD is 3%. Furthermore, the CTODH1 values measured for CH of 1ppmw show high SD, which might be attributed to the nature of the X42 steel (high inclusion content for a non-HIC resistant steel thus high probability of HIC initiation).

The results of fracture toughness in the SL direction (Table 7, Figure 10), show that KQ is not affected by increasing hydrogen content (CH) absorbed in the steel considering the SD. On the other hand, the maximum CTOD values are reduced with increasing hydrogen content (Table 8, Figure 10(b)), even if we consider the large SD observed in the case of 1ppmw hydrogen content and CTOD stabilized showing no effect of increasing CH considering the SD. The failure of several specimens but also the scatter of results indicates that several specimens might had internal cracks forming due to inclusions or defects and the cracks propagated non-uniformly, i.e. in the absence of inclusions, the fatal cracks are expected to propagate slowly compared to the cracks encountering inclusions in their path. In the latter case, HIC may develop and the cracks could propagate and spread rapidly, taking advantage from the inclusions 'coalescence.

In terms of the impact of HIC in fracture toughness properties of pipeline steels, very few studies are available. In particular, a decrease of fracture toughness has been observed by Domizzi and co-workers [START_REF] Domizzi | Influence of sulphur content and inclusion distribution on the hydrogen induced blister cracking in pressure vessel and pipeline steels[END_REF]. It took place in the presence of hard phases, inclusions and precipitates similar to X42 steel. Those authors reported that the presence of the pearlite phase which is harder than the ferrite provides an easy path for HIC growth, which was observed in this work. In addition, for a pearlite-ferrite steel, Hejazi and co-workers [START_REF] Hejazi | Effect of manganese content and microstructure on the susceptibility of X70 pipeline steel to hydrogen cracking[END_REF] reported lower fracture toughness values, in terms of JQ,. Nevertheless, MnS inclusions, seemed to not play a governing role in the void formation, growth and coalescence but rather the density of carbonitrides and Ca and Al oxides. In contrast to the findings in [START_REF] Hejazi | Effect of manganese content and microstructure on the susceptibility of X70 pipeline steel to hydrogen cracking[END_REF], the MnS inclusions in the X42 steel play a dominant role in the void formation and growth of HICs in our case and a reduction in fracture toughness above the elastic region, in terms of CTOD, was recorded, which is more pronounced in the SL direction. Lastly in [START_REF] Chong | Effects of wet H2S containing environment on mechanical properties of NACE grade C-Mn steel pipeline girth welds[END_REF], when pipeline steel was tested in H2S environment, it was observed at least a 40% reduction in fracture resistance as expressed in terms of JR curve. This is again in agreement with the findings of this work.

5-1-Comparison between HIC resistant (X65) and HIC susceptible (X42) steels

The aforementioned conclusion can be generalized for the CTOD results observed in the TL direction. It is assumed that in several cases, hydrogen charging of X42 specimens has produced HICs, in the direction of the propagating crack. Although, this is more pronounced in the case of FT testing along the SL direction, with the large SD of CTOD results observed and the failure of individual specimens, it is also considered applicable for the TL direction.

In particular, for TL direction and specimen 1 at CH = 1 ppmw, it is assumed that an HIC developed directly on the path of the propagating crack, which affected the overall CTOD results.

It can be observed that the X42 fracture toughness test results show a reduction of KQ of about 25% when the CH reaches 1.5 ppmw, while the CTOD remains unaffected, considering that no HICs are formed in the path of the propagating crack. On the other hand, in the SL direction the KQ is largely unaffected; while the CTOD is reduced by 74.5% at CH of 1.5 ppmw (CTODH1.5) compared to CTOD in air (CTOD0). The differences, mainly in CTOD results, between TL and SL, can be attributed to the formation of HIC defects along the path of the cracks in the direction parallel to the wall thickness. This is in full accordance with other works which show that HIC defects initiate and propagate parallel to the pipeline wall [START_REF] Mohtadi-Bonab | Hydrogen induced cracking susceptibility in different layers of a hot rolled X70 pipeline steel[END_REF][START_REF] Hejazi | Effect of manganese content and microstructure on the susceptibility of X70 pipeline steel to hydrogen cracking[END_REF][START_REF] Wang | Tensile and impact properties of X70 pipeline steel exposed to wet H2S environments[END_REF].

Regarding reduction of mechanical properties, other works using relevant charging techniques on pipeline steels, have shown similar effect of hydrogen on the loss of ductility and tensile strength [START_REF] Qi | Comparison of tensile and impact behavior of carbon steel in H2S environments[END_REF][START_REF] Chong | Effects of wet H2S containing environment on mechanical properties of NACE grade C-Mn steel pipeline girth welds[END_REF][START_REF] Zheng | Effect of hydrogen and inclusions on the tensile properties and fracture behaviour of A350LF2 steels after exposure to wet H2S environments[END_REF][START_REF] Zhuo | Influence of Sulfur Content and Microstructure of Pipeline Steels on Mechanical property and H2S-Resistant Behavior[END_REF][START_REF] Wang | Tensile and impact properties of X70 pipeline steel exposed to wet H2S environments[END_REF][START_REF] Wang | Effect of H2S/CO2 partial pressure ratio on the tensile properties of X80 pipeline steel[END_REF], as well as the fracture toughness [START_REF] Qi | Comparison of tensile and impact behavior of carbon steel in H2S environments[END_REF][START_REF] Wang | Tensile and impact properties of X70 pipeline steel exposed to wet H2S environments[END_REF]. In these works, microstructure, inclusion content and inclusion shape have been identified as the main factors controlling HIC initiation and propagation. Furthermore, microstructure morphologies play an important role in HIC formation and propagation, with banded microstructures [START_REF] Elboujdaîni | Studies on Inhibition of Hydrogen-Induced Cracking of Linepipe Steels[END_REF] providing a low fracture resistance path for crack propagation. It has also been shown that there is no clear preference on HIC propagation path, i.e. intergranular or transgranular which has also been shown in this work through the EBSD measurements. The HIC defects do not have a preferential propagation path, rather they grow through the grains (Figure 8). In this sense, it has been shown that high angle grain boundaries are preferential sites for crack propagation, while lower indices grains are prone to intergranular HIC propagation [START_REF] Mohtadi-Bonab | The mechanism of failure by hydrogen induced cracking in an acidic environment for API 5L X70 pipeline steel[END_REF][START_REF] Mohtadi-Bonab | Evaluation of hydrogen induced cracking behavior of API X70 pipeline steel at different heat treatments[END_REF][START_REF] Mohtadi-Bonab | Hydrogen induced cracking susceptibility in different layers of a hot rolled X70 pipeline steel[END_REF].

The aforementioned observations are clearly in line with the findings in this paper where most grains are oriented along <001> direction (Figure 5), while the HIC defects clearly cross, in several instances, grains with <001> orientation (Figure 8).

In elsewhere [START_REF] Qi | Comparison of tensile and impact behavior of carbon steel in H2S environments[END_REF], it is reported that HIC is formed near MnS inclusions, this result is also corroborated by many others [START_REF] Mohtadi-Bonab | The mechanism of failure by hydrogen induced cracking in an acidic environment for API 5L X70 pipeline steel[END_REF][START_REF] Mohtadi-Bonab | Hydrogen induced cracking susceptibility in different layers of a hot rolled X70 pipeline steel[END_REF][START_REF] Hejazi | Effect of manganese content and microstructure on the susceptibility of X70 pipeline steel to hydrogen cracking[END_REF][START_REF] Zheng | Effect of hydrogen and inclusions on the tensile properties and fracture behaviour of A350LF2 steels after exposure to wet H2S environments[END_REF][START_REF] Elboujdaîni | Studies on Inhibition of Hydrogen-Induced Cracking of Linepipe Steels[END_REF][START_REF] Hardie | Hydrogen embrittlement of high strength pipeline steels[END_REF][START_REF] Zhang | Study of correlation between hydrogen-induced stress and hydrogen embrittlement[END_REF]. Our findings suggest that these inclusions play dominant role in HIC formation and propagation as shown by dimples major features observed in fracture surface (Figure 6).

5-2-Impact of current findings and Engineering Critical Assessment practices

Current FFS/ECA calculations and codes are using K derived values, such as KIC or KC for assessing the fitness for service of a pipeline under a specific hydrogen environment (H2S).

Nevertheless, it has been shown in this work that non-HIC resistant steels, such as the X42 steel under this study, show considerable resistance to maximum KQ reduction, in hydrogen environment, as observed in both TL and SL direction. It is reminded that the biggest reduction in KQ (25%) is observed in the TL direction, which is not the direction of HIC initiation and propagation. On the other hand, there is a considerable reduction in the CTOD with hydrogen uptake, in the SL direction, i.e. the direction of HIC growth. To this end, it is proposed that CTOD is a more applicable parameter for HIC assessment using FFS/ECA tools, in hydrogen environments. The same conclusion has been shown for HIC resistant steels in a previous work [START_REF] Chatzidouros | Effect of hydrogen on fracture toughness properties of a pipeline steel under simulated sour service conditions[END_REF].

It is also suggested to use the CTOD as a fracture toughness parameter when evaluating critical fracture toughness criteria for growth of cavity under uniform hydrogen pressure using numerical modeling schemes [START_REF] Gonzalez | Hydrogen-Induced Crack Growth Rate in Steel Plates Exposed to Sour Environments[END_REF]. These fracture toughness properties should be evaluated and modeled in the SL direction to be consistent with the direction of growth of HIC defects. To that extent, CTOD fracture toughness properties measured in the SL direction and at different levels of hydrogen uptake, are of the utmost importance.

Figure 1 :

 1 Figure 1: Illustration of specific locations on a pipeline selected for advanced ultrasonic inspection with respect to HIC and SWC. All measurements are in millimeters, scans width is 500 mm and scans are stopped at 300 mm

Figure 2 :

 2 Figure 2: Results of the advanced ultrasonic inspections on scan 1 to scan 4, respectively (a) to (d).

Figure 3 :

 3 Figure 3: (a) Sketch of the W-shaped assembled SEB specimen, (b) samples machined from a pipeline steel before final assembly and (c) illustration of the region exposed to cathodic hydrogen charging and TDS measurement (red zone).

Figure 4 :

 4 Figure 4: X42 TDS desorption curves vs. temperature

Figure 5 (

 5 a), (b) and (c) show the low and high magnification micrograph from polished and etched surface by SEM in backscattered electrons (BSE) mode. The high magnification micrograph shows the presence of fine pearlite colonies (indicated by P) along with ferritic equiaxed grain. Figure 5 (d) shows the EBSD micrograph highlighting the presence of equiaxed grains with random orientation without the presence of casting or rolling texture.

Figure 5 :

 5 Figure 5: (a) SEM-BSE image at low magnification: X42 sample was polished and etched, (b) and (c) SEM-BSE images at high magnification: Ferrite and Pearlite regions are marked as F and P, (d) EBSD inverse pole figure, indicating no textured equiaxed grains X42 steel strength can be modified by altering ferrite grain size and Pearlitic lamella spacing

  (a) and 6(b) and at 1.5 ppmw CH Figure 6(c) and 6(d).

Figure 6 :

 6 Figure 6: SEM micrographs of fractured surface of X42 along the SL direction in different environments: Air at low and high magnification (a) and (b), respectively. And at 1.5 ppmw of CH at low and high magnification (c) and (d), respectively. Green (G), red (R) and blue (B) arrows indicate, respectively microcracks, beach marks and dimples.

Figure 7 :

 7 Figure 7: (a) Optical micrograph from sample X42 tested in air (SL direction), shows presence of no cracks, (b) and (c) EBSD inverse pole figure map.

Figure 8 (

 8 Figure 8 (d) and (e).

Figure 8 :

 8 Figure 8: a) Optical micrograph of X42 sample tested at 1.5 ppmw of CH (SL direction), b) Low magnification EBSD map c) crack region and d), e) Zoom-in at crack region.

  ) the hydrogen free SEB specimens exhibit an average of KQ0 = 27.26 MPa m 1/2 and CTOD0 = 0.32 mm, (2) hydrogen pre-charged SEB specimens with 0.5ppmw CH, show an average of KQ-H0.5 = 23.4 MPa m 1/2 and CTODH0.5 = 0.27 mm, (3) hydrogen pre-charged SEB specimens with 1 ppmw CH, show an average of KQ-H1 = 27.58 MPa m 1/2 and CTODH1 = 0.15 mm and lastly (4) hydrogen pre-charged SEB specimens with 1.5 ppmw CH, show an average of KQ-H1.5 = 20.44 MPa m 1/2 and CTODH1.5 = 0.31 mm.

Figure 9 :

 9 Figure 9: Tested X42 samples in TL direction: (a) KQ variance as function of CH, (b) CTOD variance as function of CH.

Figure 10 :

 10 Figure 10: Tested X42 samples in SL direction: (a) KQ variance as function of CH, (b) CTOD variance as function of CH

Figure 11 :

 11 Figure 11: Typical failed test in the SL direction, showing horizontal HIC feature encountered by vertical fatigue pre-crack during the FT test.

Table 1 .

 1 X42 chemical composition obtained following ASTM E415 standard[START_REF]Standard Test Method for Optical Emission Vacuum Spectrometric Analysis of Carbon and Low-Alloy Steel[END_REF]

					Chemical Analysis ASTM E415
	C	S	P	Mn	Si	Ni	Cr	Mo	Cu	V	B	Ti
	X42 0.100 0.006 0.027 1.100 0.230 0.020 0.120 0.001 <0.01 0.0007 0.001 <0.01

Table 2 .

 2 X42 mechanical properties obtained following ASTM E370[START_REF]Standard Test Methods and Definitions for Mechanical Testing of Steel Products[END_REF]

			Mechanical testing ASTM A370
		Yield strength (MPa) Tensile strength (MPa)	Elongation (%)	Rockwell hardness (HRB)
	X42	316.00	437.67	25.83	68.96

Table 3 :

 3 Calculated steady state bulk CH for three levels of sour environment

	Level	CH	CH corrected	pH	pH2S
		(ppmw)	(ppmw)		(mbar)
	1	0.48	0.54	6.5	20
	2	0.92	1.02	4.5	30
	3	1.3	1.44	4	1000

Table 4 :

 4 CH obtained from TDS measurements for three different charging currents after 48 hours charging period

	Current density (mA/cm 2 )	Measured CH (ppmw)
	4	1.48
	1	1.05
	0.4	0.55

Table 5 :

 5 Maximum stress intensity fracture toughness of X42 samples in TL direction

		Air	0.5 ppmw	1 ppmw	1.5 ppmw
	X42 TL	KQ0
	Specimens	[MPa
		m

  ]

	Test 1	27.62	23.34	27.05	20.57
	Test 2	28.31	23.43	25.73	17.18
	Test 3	25.87	23.42	29.97	23.58
	Average	27.26	23.40	27.58	20.44
	SD	1.26	0.05	2.17	3.20

Table 6 :

 6 Maximum CTOD of X42 samples in TL direction

		Air	0.5 ppmw	1 ppmw	1.5 ppmw
	X42 TL	CTOD0	CTODH0.5	CTODH1	CTODH1.5
	Specimens	(mm)	(mm)	(mm)	(mm)
	Test 1	0.29	0.31	0.07	0.30
	Test 2	0.33	0.21	0.20	0.31
	Test 3	0.34	0.30	0.18	0.31
	Average	0.32	0.27	0.15	0.31
	SD	0.03	0.05	0.07	0.01

Table 7 :

 7 Maximum stress intensity fracture toughness of X42 samples in SL direction

		Air	0.5ppmw	1ppmw	1.5ppmw
	X42 SL	KQ
	Specimens

Table 8 :

 8 Maximum CTOD of X42 samples in SL direction

	Test 1	26.73	26.04	27.14	25.42
	Test 2	28.30	27.29	22.15	Failed
	Test 3	22.89	25.41	21.65	25.81
	Average	24.81	26.25	23.65	25.62
	SD	2.72	0.96	3.04	0.28

Acknowledgments

The authors are grateful to Saudi Aramco for their approval to publishing this work carried out in collaboration with Masdar Institute under contract # 8443000172.

6-Conclusions

The behavior of an X42 pipeline steel was investigated in terms of fracture toughness response in TL and SL directions, in air and under hydrogen environment mimicking the sour service conditions. The main findings are summarized as follow:

• In both directions TL and SL, there is no significant effect on KQ with increasing CH, especially in SL direction. However, there is mixed values of KQ at 0.5ppmw and 1ppmw and 20 to 25% reduction in KQ in TL direction at 1.5 ppmw compared to tested samples in air.

• Although CTOD seems to drop at 0.5 ppmw compared to the CTOD in air in TL direction, the CTOD values measured at 1ppmw of CH, show high standard deviation, which might be attributed to the nature of the X42 steel (high inclusion content for a non-HIC resistant steel thus high probability of HIC initiation during hydrogen pre-charging)

• In the SL direction, the CTOD shows clear signs of reduction with increasing CH, in the SL direction and a saturation was observed at 1 ppmw of CH.

• The most probable cause is the presence of dimples indicating voids coalescence due to the presence of inclusions.

• CTOD of hydrogen pre-charged FT specimens, measured in the SL direction, is a more suitable fracture toughness parameter for ECA or FFS assessment of HICaffected equipment.

• CTOD is also recommended as the most suitable parameter for use in modeling schemes of critical fracture toughness criteria of HIC initiation and growth.


Chiara Saponaro

Valéry Gmyr

Julien Thévenet

Ericka Moerman

Nathalie Delalleau

Gianni Pasquetti

Anais Coddeville

Audrey Quenon

Mehdi Daoudi

Thomas Hubert

Marie-Christine Vantyghem

Corinne Bousquet

Yvan Martineau

Julie Kerr-Conte

Bart Staels

François Pattou

PhD Caroline Bonner
email: caroline.bonner@univ-lille.fr

The GLP1R Agonist Liraglutide Reduces Hyperglucagonemia Induced by the SGLT2 Inhibitor Dapagliflozin via Somatostatin Release

Keywords: Dapagliflozin, Liraglutide, GLP1R, Glucagon, Insulin, Somatostatin, Type 2 Diabetes

de niveau recherche, publiés ou non, émanant des établissements d'enseignement et de recherche français ou étrangers, des laboratoires publics ou privés.

Introduction

Glucotoxicity is a major determinant of pancreatic islet cell demise seen in type 2 diabetic (T2D) individuals [START_REF] Prentki | Islet beta cell failure in type 2 diabetes[END_REF]. In addition, postprandial hyperglucagonemia and endogenous glucose production (EGP) are also key contributors to exaggerated postprandial glucose excursions [START_REF] Defronzo | Fasting hyperglycemia in non-insulin-dependent diabetes mellitus: contributions of excessive hepatic glucose production and impaired tissue glucose uptake[END_REF][START_REF] Dunning | The role of alpha-cell dysregulation in fasting and postprandial hyperglycemia in type 2 diabetes and therapeutic implications[END_REF][START_REF] Muller | Abnormal alpha-cell function in diabetes. Response to carbohydrate and protein ingestion[END_REF][START_REF] Rizza | Pathogenesis of fasting and postprandial hyperglycemia in type 2 diabetes: implications for therapy[END_REF]. How these combined abnormalities exacerbate T2D is unclear.

Clearly, postprandial insulin secretion is decreased and delayed, and glucagon is not suppressed [START_REF] Muller | Abnormal alpha-cell function in diabetes. Response to carbohydrate and protein ingestion[END_REF]. Therefore, agents that can reduce glucotoxicity and postprandial hyperglucagonemia will most likely improve glycemic control. The newest classes of anti-diabetic agents include sodium-glucose-cotransporter-2 (SGLT2) inhibitors and glucagon-like-peptide-1-receptor (GLP1R) agonists. Both agents are associated with weight-loss, a decrease in A1c levels, a low-rate of hypoglycemia and decreased risk of heart failure [START_REF] Bailey | Effect of dapagliflozin in patients with type 2 diabetes who have inadequate glycaemic control with metformin: a randomised, double-blind, placebocontrolled trial[END_REF][START_REF] Bajaj | Lowest Glucose Variability and Hypoglycemia Are Observed With the Combination of a GLP-1 Receptor Agonist and Basal Insulin (VARIATION Study)[END_REF][START_REF] Chen | The efficacy and safety of SGLT2 inhibitors for adjunctive treatment of type 1 diabetes: a systematic review and meta-analysis[END_REF][START_REF] Ferrannini | Energy Balance After Sodium-Glucose Cotransporter 2 Inhibition[END_REF][START_REF] Frias | Exenatide once weekly plus dapagliflozin once daily versus exenatide or dapagliflozin alone in patients with type 2 diabetes inadequately controlled with metformin monotherapy (DURATION-8): a 28 week, multicentre, double-blind, phase 3, randomised controlled trial. The lancet[END_REF][START_REF] Frias | Effects of exenatide once weekly plus dapagliflozin, exenatide once weekly alone, or dapagliflozin alone added to metformin monotherapy in subgroups of patients with type 2 diabetes in the DURATION-8 randomized controlled trial[END_REF][START_REF] Jabbour | Effects of exenatide once weekly plus dapagliflozin, exenatide once weekly, or dapagliflozin, added to metformin monotherapy, on body weight, systolic blood pressure, and triglycerides in patients with type 2 diabetes in the DURATION-8 study[END_REF][START_REF] Ludvik | Dulaglutide as add-on therapy to SGLT2 inhibitors in patients with inadequately controlled type 2 diabetes (AWARD-10): a 24-week, randomised, double-blind, placebo-controlled trial[END_REF][START_REF] Lundkvist | Dapagliflozin once-daily and exenatide once-weekly dual therapy: A 24-week randomized, placebo-controlled, phase II study examining effects on body weight and prediabetes in obese adults without diabetes[END_REF][START_REF] Marso | Liraglutide and Cardiovascular Outcomes in Type 2 Diabetes[END_REF][START_REF] Merovci | Dapagliflozin lowers plasma glucose concentration and improves beta-cell function[END_REF][START_REF] Zinman | Empagliflozin, Cardiovascular Outcomes, and Mortality in Type 2 Diabetes[END_REF].

The primary mechanism of SGLT2 inhibitors is to inhibit renal glucose reabsorption by glycosuria, thus reducing glucotoxicity and improving insulin sensitivity and beta cell function [START_REF] Abdul-Ghani | Renal sodium-glucose cotransporter inhibition in the management of type 2 diabetes mellitus[END_REF][START_REF] Merovci | Dapagliflozin lowers plasma glucose concentration and improves beta-cell function[END_REF][START_REF] Merovci | Dapagliflozin improves muscle insulin sensitivity but enhances endogenous glucose production[END_REF][START_REF] Rossetti | Glucose toxicity[END_REF]. Secondary to this, they also elevate glucagon secretion and EGP [START_REF] Ferrannini | Metabolic response to sodium-glucose cotransporter 2 inhibition in type 2 diabetic patients[END_REF][START_REF] Merovci | Dapagliflozin improves muscle insulin sensitivity but enhances endogenous glucose production[END_REF], a phenomenon that was later explained at least in part, by the specific expression of SGLT2 in human pancreatic alpha cells [START_REF] Bonner | Inhibition of the glucose transporter SGLT2 with dapagliflozin in pancreatic alpha cells triggers glucagon secretion[END_REF]. It was later suggested that blocking SGLT2 in alpha cells partly relieves glucose suppression of glucagon secretion by allowing full-scale action potentials to develop [START_REF] Pedersen | Dapagliflozin stimulates glucagon secretion at high glucose: experiments and mathematical simulations of human A-cells[END_REF].

Recent clinical trials such as AWARD-10 and DURATION-8 have tested the efficacy SGLT2 inhibitors with GLP1R agonists for the treatment of T2D individuals, and have found that this combination significantly improved glycemic control better than each drug alone [START_REF] Frias | Exenatide once weekly plus dapagliflozin once daily versus exenatide or dapagliflozin alone in patients with type 2 diabetes inadequately controlled with metformin monotherapy (DURATION-8): a 28 week, multicentre, double-blind, phase 3, randomised controlled trial. The lancet[END_REF][START_REF] Frias | Effects of exenatide once weekly plus dapagliflozin, exenatide once weekly alone, or dapagliflozin alone added to metformin monotherapy in subgroups of patients with type 2 diabetes in the DURATION-8 randomized controlled trial[END_REF][START_REF] Ludvik | Dulaglutide as add-on therapy to SGLT2 inhibitors in patients with inadequately controlled type 2 diabetes (AWARD-10): a 24-week, randomised, double-blind, placebo-controlled trial[END_REF]. This is of great interest for clinical diabetology as reviewed recently by DeFronzo [START_REF] Defronzo | Combination therapy with GLP-1 receptor agonist and SGLT2 inhibitor[END_REF], however, the mechanism of action of the combination on human pancreatic hormone secretion was not established. A recent study by Ørgaard and colleagues have shown that the glucagon-lowering effect of GLP-1 is mediated through the paracrine actions of somatostatin in a perfused mouse pancreas model [START_REF] Orgaard | The role of somatostatin in GLP-1-induced inhibition of glucagon secretion in mice[END_REF], however, many aspects of GLP1 signaling in the human islet remain unresolved. This prompted us to investigate if the glucagon-lowering actions of the GLP1R agonist, liraglutide could also reduce the glucagon over-secretion induced by the SGLT2 inhibitor dapagliflozin, using human islet cultures and mouse models of diabetes. Our pre-clinical study was designed to (1) compare the efficacy of liraglutide and dapagliflozin as mono-therapies or in combination, for the long-term treatment of T2D, and (2) to provide a mechanistic insight for the combination of SGLT2 inhibitors and GLP1R agonists.

Results

A single administration of liraglutide alone or in combination with dapagliflozin suppresses glycemia and glucagon levels in diabetic mice

We first studied the effect of liraglutide alone, dapagliflozin alone or both drugs in combination in db/db mice. A single administration of liraglutide alone or in combination with dapagliflozin significantly reduced glycemia (Figure 1A). Liraglutide reduced plasma glucagon levels alone and in combination with dapagliflozin in fasted diabetic mice, which have elevated fasting glycemia and hyperglucagonemia (Figure 1B). The same profile for glycemia was observed after an intra-peritoneal (i.p.) injection of glucose (Figure S1A). In addition, healthy mice had normal beta-hydroxybutrate levels compared to db/db mice.

Dapagliflozin potently increased beta-hydroxybutrate levels, but liraglutide had no effect (Figure 1C), despite lowering plasma glucagon levels. After an oral glucose challenge, db/db mice treated with dapagliflozin had significantly higher glucagon levels, which were reduced in mice treated with dapagliflozin in combination with liraglutide (Figure 1D). A similar profile for glucagon was observed after i.p. glucose administration in db/db mice treated with dapagliflozin alone or in combination with liraglutide (Figure S1B).

The chronic treatment with liraglutide and dapagliflozin potently reduces glycemia and glucagon levels compared to each drug alone

In order to investigate if the combination of liraglutide and dapagliflozin has an enhanced therapeutic potential for the treatment of T2D, we next studied the effect of these drugs alone or in combination in db/db mice over 45 days. Glycemia during fasting was not significantly lowered by liraglutide alone. Dapagliflozin decreased fasting glycemia until day 30, an effect that was not maintained. However, when liraglutide was used in combination with dapagliflozin, there was a sustainable reduction in fasting glycemia (Figure 1E). After a glucose challenge, the combination therapy was more effective in reducing glycemia compared to each drug alone (Figure 1F). Moreover, the glucagon lowering effect of liraglutide when combined with dapagliflozin in the acute experiment was maintained in the chronic treatment (Figure 1G).

Liraglutide reduces glucagon over-secretion induced by dapagliflozin in human islets

To determine whether GLP1R activation may be directly related to its intra-islet localization, we first examined its distribution in healthy human pancreatic sections. We show that GLP1R is predominantly expressed on the membrane in human islet cells and to a lesser extent in exocrine tissue. Double-label immunofluorescence reveals that GLP1R is highly expressed on the membrane of insulin-positive beta cells, and weakly expressed in alpha cells, but moderately expressed in somatostatin-positive delta cells (Figure S2A). Of note, the GLP1R is localized on the membrane of the islet (indicated in red), whereas insulin, glucagon and somatostatin are cytoplasmic (green), hence precluding a complete yellow merged staining.

Western blot analysis showed a strong signal for GLP1R protein at the expected size (≈ 62 kDa) in healthy human islets and to a lesser extent in exocrine tissue (Figure S2B). Since liraglutide reduces glucagon over-secretion induced by dapagliflozin in vivo, we investigated whether it was due to a direct effect on isolated islets: human islet preparations from normoglycemic donors were treated with liraglutide alone, dapagliflozin alone or both drugs in combination in the presence of 6 mM glucose for 1h, the concentration associated with maximal inhibition of glucagon release [START_REF] Walker | Regulation of glucagon secretion by glucose: paracrine, intrinsic or both? Diabetes[END_REF]. Under these conditions glucagon secretion was lower compared to islets treated with 1 mM glucose. At 6mM glucose liraglutide did not further reduce glucagon secretion. Consistent with our previous results [START_REF] Bonner | Inhibition of the glucose transporter SGLT2 with dapagliflozin in pancreatic alpha cells triggers glucagon secretion[END_REF], dapagliflozin treatment increased glucagon secretion at 6 mM glucose.

This effect was significantly reduced by co-treatment with liraglutide (Figure 2A), without any significant change in insulin secretion under these experimental conditions (Figure 2B).

Liraglutide inhibits glucagon secretion in a glucose-dependent manner

To further demonstrate the pharmacological relevance of our findings in vivo, we next studied the effect of each drug alone or in combination in healthy C57BL/6J mice. Under fasting conditions dapagliflozin and liraglutide alone or in combination reduced glycemia. However, after a glucose challenge, only the mice treated with liraglutide showed a reduction in glycemia. The effect of dapagliflozin in reducing glycemia was negligible (Figure 2C), possibly because of the time needed for glucose clearance. In healthy mice, under fasting conditions, basal glucagon levels were elevated and further potentiated by dapagliflozin.

However, liraglutide alone or in combination with dapagliflozin was unable to reduce glucagon levels (Figure 2D). After a glucose challenge, where insulin is increased and glucagon is suppressed [START_REF] Unger | Glucagon physiology and pathophysiology[END_REF][START_REF] Van Dijk | Effects of glucagon-like peptide-I on glucose turnover in rats[END_REF], liraglutide alone has no additional lowering effect on plasma glucagon concentrations. However, dapagliflozin induced glucagon secretion, an effect that was reduced by the combination with liraglutide (Figure 2E), thus confirming that the action of liraglutide is glucose dependent. Moreover, we demonstrate that the glucagon-lowering action of liraglutide is more effective when glucagon levels are elevated beyond the physiological range, for example dapagliflozin-induced glucagon secretion or during hyperglucagonemia. Similar profiles for glycemia and glucagon were observed in healthy mice during fasting and after refeeding (Figure S2C-D). In healthy mice after a glucose challenge, insulin secretion was induced by a single administration of liraglutide, whereas dapagliflozin alone or in combination with liraglutide had no effect on insulin secretion (Figure 2F). Of note, mice administered dapagliflozin and liraglutide in combination increased somatostatin secretion compared to each drug alone (Figure 2G).

Blockade of SSTR2 in human islets blunts the inhibitory effect of somatostatin on glucagon secretion

Since the combination therapy showed an increase in somatostatin levels in vivo, we tested whether its glucagon-lowering effect is mediated via a direct action on pancreatic delta cells.

Using a cross-sectional cohort we found SSTR2 gene expression to be elevated in islets from T2D individuals compared to obese (non-diabetic) and lean individuals (Figure S3A). We also found that GLP1R gene expression was induced in the islets of obese non-diabetic and T2D individuals compared to healthy individuals (Figure S3B). We confirmed by confocal immunofluorescence imaging techniques that SSTR2 colocalized with insulin and glucagon in human pancreatic tissue (Figure S3C). We next demonstrated that incubation of human islets with the selective SSTR2 inhibitor CYN154806 blunted the inhibitory effect of liraglutide on dapagliflozin-induced glucagon secretion (Figure 3A). Since glucagon secretion was already decreased at 6 mM glucose, CYN154806 alone had no further effect (Figure 3B).

Liraglutide inhibits glucagon over-secretion in vivo via somatostatin release

We next investigated the effects of the pharmacological treatment of dapagliflozin and liraglutide in the presence of the SSTR2 inhibitor CYN154806 in DIO mice. Each drug alone or in combination reduced glycemia after a glucose challenge (Figure 4A). Liraglutide decreased the dapagliflozin-induced glucagon secretion, but when CYN154806 was coadministered with the combination therapy, it reduced the inhibitory effect of somatostatin on glucagon secretion (Figure 4B). Although, the plasma insulin levels were higher in mice treated with liraglutide, these changes were not significant (Figure 4C), most likely because somatostatin secretion was increased (Figure 4D).

Somatostatin requires SSTR2 to exert its glucagon-lowering effect

We used SSTR2 deficient mice [START_REF] Zheng | Somatostatin receptor subtype 2 knockout mice are refractory to growth hormone-negative feedback on arcuate neurons[END_REF] to further demonstrate that the inhibition of glucagon secretion by liraglutide in alpha cells requires SSTR2. Firstly, we validated the specificity of SSTR2 antibody by showing the absence of its expression in the pancreatic islets of the SSTR2 KO mice compared to littermate control mice (Figure S3D). SSTR2 KO mice showed decreased glycemia after a glucose challenge compared to their littermate control mice (Figure S3E), possibly due to the mild hyperinsulinemia in these mice (Figure S3F). The acute glycemic response to the drugs in SSTR2 KO mice (Figure S3G) was similar to that found in healthy mice. However, SSTR2 KO mice had a marked hyperglucagonemia compared to control mice, which was not reduced by liraglutide alone or the combination treatment (Figure 4E). These data confirm in vivo that the effect of combination of liraglutide and dapagliflozin is mediated by the action of somatostatin via SSTR2 to reduce glucagon release.

Discussion

Glucagon is a strong stimulator of hepatic glucose production. In T2D patients, glucagon secretion is not anymore suppressed after a glucose challenge [START_REF] Meier | Postprandial suppression of glucagon secretion depends on intact pulsatile insulin secretion: further evidence for the intraislet insulin hypothesis[END_REF][START_REF] Muller | Abnormal alpha-cell function in diabetes. Response to carbohydrate and protein ingestion[END_REF] and is further exacerbated by SGLT2 inhibitors [START_REF] Ferrannini | Metabolic response to sodium-glucose cotransporter 2 inhibition in type 2 diabetic patients[END_REF][START_REF] Martinez | Endogenous Glucose Production and Hormonal Changes in Response to Canagliflozin and Liraglutide Combination Therapy[END_REF][START_REF] Merovci | Dapagliflozin lowers plasma glucose concentration and improves beta-cell function[END_REF]. In light of these emerging issues, a drug combination that can reduce hyperglucagonemia and EGP independent of insulin therapy could be an attractive adjunct therapy for the treatment of diabetes. Our pre-clinical data demonstrate that the combination of GLP1R agonists and SGLT2 inhibitors attenuates glucagon over-secretion in diabetic mice to produce sustainable glycemic control both during fasting and after a glucose challenge in the long-term, better than each agent alone. These findings are in line with recent clinical trials in patients with T2D, which have demonstrated that, the combination of GLP1R agonists and SGLT2 inhibitors reduce A1c levels more effectively than GLP1R agonist alone [START_REF] Frias | Exenatide once weekly plus dapagliflozin once daily versus exenatide or dapagliflozin alone in patients with type 2 diabetes inadequately controlled with metformin monotherapy (DURATION-8): a 28 week, multicentre, double-blind, phase 3, randomised controlled trial. The lancet[END_REF][START_REF] Goncalves | Glucagon-like peptide-1 receptor agonists and sodium-glucose cotransporter-2 inhibitors: Sequential or simultaneous start?[END_REF][START_REF] Ludvik | Dulaglutide as add-on therapy to SGLT2 inhibitors in patients with inadequately controlled type 2 diabetes (AWARD-10): a 24-week, randomised, double-blind, placebo-controlled trial[END_REF]. We also observed that a single administration of dapagliflozin induced circulating beta-hydroxybutrate levels, and these levels were maintained when co-administrated with liraglutide, despite reduced plasma glucagon concentrations. These data indicate that the decreased glucagon upon SGLT2 inhibition and GLP1R agonist treatment does not impede the production of ketone bodies upon prolonged fasting. The increase in ketone body metabolism after SGLT2 inhibitor treatment was shown to improve cardiac fuel utilization, thus reducing the risk of heart failure; however this is only one of a multitude of plausible mechanisms involved in the beneficial cardiac effects of SGLT2 inhibitors [START_REF] Staels | Cardiovascular Protection by Sodium Glucose Cotransporter 2 Inhibitors: Potential Mechanisms[END_REF]. Moreover, a recent study showed that the combination of GLP1R agonists and SGLT2 inhibitors reduced plasma glucagon levels in T2D patients, but EGP remained elevated [START_REF] Martinez | Endogenous Glucose Production and Hormonal Changes in Response to Canagliflozin and Liraglutide Combination Therapy[END_REF]. These findings together suggest that factors other than glucagon secretion can participate to increase EGP and ketone body production by the liver. Indeed, ketone body production is also under control of other hormones such as insulin, thyroid hormones and other factors such free fatty acids and catecholamines whose production are increased during prolonged fasting [START_REF] Keller | Human ketone body production and utilization studied using tracer techniques: regulation by free fatty acids, insulin, catecholamines, and thyroid hormones[END_REF], as was the case in our overnight fasting protocol. Further studies are needed to clarify these sequential events. We also observed in healthy mice under fasting conditions in which glucagon is elevated, that liraglutide did not reduce glucagon secretion induced by dapagliflozin; thus confirming that the effect of liraglutide is glucose dependent. These findings explain the low-risk of developing hypoglycemia during treatment with these agents [START_REF] Trujillo | GLP-1 receptor agonists: a review of head-to-head clinical studies[END_REF]. Notably, we found in human islet cultures and in healthy mice in vivo that the glucagon-lowering effect of liraglutide was more profound when combined with dapagliflozin. Whether this is solely mediated by a direct action of GLP1R on glucagonsecreting alpha cells or whether dapagliflozin is also inducing endogenous GLP1 production from the pancreatic islets [START_REF] Dunning | Alpha cell function in health and disease: influence of glucagon-like peptide-1[END_REF][START_REF] Ferrannini | Metabolic response to sodium-glucose cotransporter 2 inhibition in type 2 diabetic patients[END_REF][START_REF] Millar | Metabolic and neuroprotective effects of dapagliflozin and liraglutide in diabetic mice[END_REF][START_REF] Timper | Glucose-Dependent Insulinotropic Peptide Stimulates Glucagon-Like Peptide 1 Production by Pancreatic Islets via Interleukin 6, Produced by alpha Cells[END_REF] remains unclear.

Despite the numerous studies exploring the effects of GLP1R agonists, the precise mechanism by which GLP1 reduces glucagon remains controversial. Some suggest that GLP1 could act via an intrinsic mechanism [START_REF] De Marinis | GLP-1 inhibits and adrenaline stimulates glucagon release by differential modulation of N-and L-type Ca2+ channel-dependent exocytosis[END_REF], while others propose it works via the paracrine actions of insulin [START_REF] Samols | Intra-islet insulin-glucagon-somatostatin relationships[END_REF] or somatostatin [START_REF] Orgaard | The role of somatostatin in GLP-1-induced inhibition of glucagon secretion in mice[END_REF].

Recent studies suggest a direct effect on alpha cells because the inhibition was not reversed by the insulin receptor antagonist S961 or the SSTR2 inhibitor CYN154806 [START_REF] Ramracheya | GLP-1 suppresses glucagon secretion in human pancreatic alpha-cells by inhibition of P/Q-type Ca(2+) channels[END_REF].

Somatostatin potently inhibits insulin and glucagon release from pancreatic islets via two distinct receptors (SSTR2 and SSTR5). SSTR2 is the functionally dominant somatostatin receptor expressed in both human pancreatic alpha and beta cells [START_REF] Kailey | SSTR2 is the functionally dominant somatostatin receptor in human pancreatic beta-and alpha-cells[END_REF].

Therefore, in human pancreatic islets, somatostatin induced by liraglutide should exert its inhibitory effect on glucagon and insulin secretion. Using human islet cultures, we demonstrated that liraglutide reduces glucagon secretion induced by dapagliflozin via intraislet somatostatin release, because blockade of SSTR2 present on alpha cells blunted its inhibitory effect. In healthy subjects, the postprandial glucose rise reduces glucagon, unlike in diabetic subjects [START_REF] Meier | Postprandial suppression of glucagon secretion depends on intact pulsatile insulin secretion: further evidence for the intraislet insulin hypothesis[END_REF]. In this study, we used islets isolated from normoglycemic, lean individuals, and therefore, liraglutide would not further reduce glucagon or induce insulin secretion. We believe that this experimental setting is the most appropriate to demonstrate a crosstalk between dapagliflozin, which induces glucagon secretion, and liraglutide, which enhances somatostatin secretion to suppress the glucagon induction. The proposed mechanism of action in vitro is summarized in the graphical abstract. However, we acknowledge that further studies on islets isolated from obese diabetic individuals would be of interest to assess the direct impact of our findings in the pathological condition of diabetes.

We also assessed the metabolic actions of dapagliflozin in combination with liraglutide in db/db mice and DIO diabetic mice. The combination therapy showed an improvement of glycemia and hyperglucagonemia. Although plasma insulin levels were elevated by liraglutide treatment, these values did not reach significance. The attenuation of the increase of insulin secretion by liraglutide could be due to the paracrine inhibitory action of somatostatin, which was significantly elevated in these mice. In general, clinical studies on T2D patients report an increase of insulin or c-peptide secretion in response to liraglutide treatment [START_REF] Matsumoto | Effects of liraglutide on postprandial insulin and glucagon responses in Japanese patients with type 2 diabetes[END_REF][START_REF] Shankar | Native Oxyntomodulin Has Significant Glucoregulatory Effects Independent of Weight Loss in Obese Humans With and Without Type 2 Diabetes[END_REF][START_REF] Vanderheiden | Mechanisms of Action of Liraglutide in Patients With Type 2 Diabetes Treated With High-Dose Insulin[END_REF][START_REF] Vilsboll | Liraglutide, a once-daily human GLP-1 analogue, improves pancreatic B-cell function and arginine-stimulated insulin secretion during hyperglycaemia in patients with Type 2 diabetes mellitus[END_REF], but a more recent study has reported the contrary [START_REF] Anholm | Liraglutide improves the beta-cell function without increasing insulin secretion during a mixed meal in patients, who exhibit well-controlled type 2 diabetes and coronary artery disease[END_REF].

In summary, our findings demonstrate that liraglutide reduces hyperglucagonemia induced by dapagliflozin via somatostatin release. Moreover, the chronic dual-treatment of dapagliflozin and liraglutide produce a sustainable improvement in glycemia and hyperglucagonemia, better than each agent alone, and thus provide mechanistic insights to the ongoing clinical trials with the combination of SGLT-2 inhibitors and GLP1R agonists for the treatment of diabetes. However, our study has several limitations. Since, due to limited material availability, we could not measure somatostatin secretion in vitro in parallel to glucagon and insulin by the same donor islet preparations, we demonstrated the action of liraglutide to reduce glucagon secretion, indirectly via blockade of the SSTR2 present on alpha cells.

Moreover, the circulating forms of somatostatin, SST-14 (pancreatic) and are both detected by the EIA kit used in this study. Nevertheless, we believe our findings are of interest to be further explored in future clinical studies and or clinical trials.

STAR Methods

LEAD CONTACT AND MATERIALS AVAILABILITY

This study did not generate new unique reagents. Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Caroline Bonner (caroline.bonner@univ-lille.fr).

EXPERIMENTAL MODEL AND SUBJECT DETAILS

All experimental procedures were performed by at least two investigators, and some but not all experiments were performed in a blinded fashion. No data points were excluded from analyses in mice or human islet studies. Human pancreatic tissue and islets was harvested from brain-dead adult human donors in the context of the traceability requirements for our clinical islet transplantation program (clinicaltrials.gov, NCT01123187, NCT00446264, NCT01148680). Age/sex and metabolic phenotype were provided for all donors (Table S2).

Animals were not randomized for vehicle, dapagliflozin alone, liraglutide alone or for the combination of dapagliflozin and liraglutide treatments for the acute experiments, but were randomized for the chronic experiments. Sample sizes were chosen with adequate power based on the literature. In vivo experiments were conducted independently with at least six mice per group, and repeated at least two times, when applicable, except for the chronic treatments which was performed once over 45 days. The experimental design was approved in agreement with French regulations, our Institutional Ethical Committee of the University of Lille and the Centre Hospitalier Régional Universitaire de Lille.

In vivo animal studies

Control male C57BL/6J mice (aged 10-12 weeks) and db/db male mice (aged 10-12 weeks) were purchased from Elevage Janvier laboratories (Le Genest-Saint-Isle, France), were housed at 22±2oC with 12:12 h light/dark cycle. We chose C57BL/KsJ-db/db mice at 12 weeks of age because they exhibit many of the early metabolic disturbances of human T2D, including obesity, hyperglycemia, early hyperinsulinemia, hyperglucagonemia and renal pathology [START_REF] Berglund | Development of the insulin secretory defect in genetically diabetic (db/db) mouse[END_REF][START_REF] Hummel | Diabetes, a new mutation in the mouse[END_REF]. SSTR2 KO mice were obtained from L'Equipe labellisee Ligue Contre le Cancer Toulouse, France. All animals had free access to standard chow diet (Safe -A04 16% Protein Rodent Diet) and water. A group of male C57BL/6J mice had free access to high fat diet (HFD) (Research diet -D12492i, 60% fat;

Research Diet Brogaarden, Denmark) for 3 months. All animal procedures were conducted in accordance with the institutional guidelines approved by the institutional Animal Care and Ethical Use Committee of the University of Lille, France. Control healthy mice, db/db mice and mice receiving a HFD were fasted and split into 4 groups and treated acutely with the different drugs (Table S1). Glycemia was monitored before and after drug administration (time 0, 30, 60 and 90 min). After 90 min from drug administration all mice received an i.p. injection of glucose (2g/kg) and glycemia was measured after 30 or 60 min. An i.p. route was chosen to avoid the stimulation of endogenous GLP1 and to bypass the effect of liraglutide on reducing food intake. Blood samples were collected and pancreatic hormones were measured during fasting and after a glucose challenge. For the chronic experiment db/db mice were split into 4 groups as above and received the drug treatments for 45 days (dapagliflozin 5 mg/Kg and liraglutide 0,15 mg/Kg). Glycemia was monitored during fasting and after a glucose challenge at days 10, 20, 30 and 45. The same experimental protocol for acute treatment was followed and blood samples were collected.

METHOD DETAILS

Hormone measurements

Glucagon concentrations were measured using the Glucagon Quantikine ELISA Kit (R&D Systems Europe, Lille, France) according to the manufacturer's instructions. Insulin concentrations were measured using the mouse Insulin Elisa kit (Mercodia AB; Uppsala Sweden), and Somatostatin was measured using the Enzyme Immunoassay kit for Somatostatin (Phoenix Pharmaceuticals, Inc, Achen Germany).

Beta-hydroxybutrate measurements

Beta-hydroxybutrate concentrations were measured using the beta-hydroxybutrate colorimetric kit (Abcam ab83390, France) according to the manufacturer's guidelines.

Tissue processing and sectioning

Human pancreatic tissue was harvested from brain-dead adult human donors in the context of the traceability requirements for our clinical islet transplantation program (clinicaltrials.gov, NCT01123187, NCT00446264, NCT01148680), fixed in 4% (w/v) paraformaldehyde and embedded in paraffin. Sections (10 µm) were cut on a Leica RM 2165 microtome (Leica, Copenhagen, Denmark). The experimental design and protocols used in this study was approved in agreement with French regulations, our Institutional Ethical Committee of the University of Lille and the Centre Hospitalier Régional Universitaire de Lille.

Immunostaining techniques

To study the expression of GLP1R proteins in the pancreas, two antibodies were tested. The first antibody was sourced from Abcam, rabbit polyclonal GLP1R (1:100; Abcam Cat# ab39072, RRID: AB_880213) and was found to be non-specific. The second was a monoclonal GLP1R hybridoma (undiluted; DSHB Cat# Mab 3F52, RRID: AB_2618100).

The specificity of the anti-sera for GLP1R has been validated previously by Waser and colleagues [START_REF] Waser | Glucagon-like-peptide-1 receptor expression in normal and diseased human thyroid and pancreas[END_REF]. The specific expression of Somatostatin Receptor 2 secondary antibodies were omitted.

Western blot analysis

Human islets were harvested in a lysis buffer containing 40 mM HEPES (pH 7.5), 120 mM NaCl, 1 mM EDTA, 10 mM pyrophosphate, 10 mM glycerophosphate, 50 mM NaF, 1.5 mM sodium orthovanadate, 10 µM okadaic acid, 10 µg/ml aprotinin and 10 µg/ml leupeptin. After lysis, insoluble material was removed by centrifugation at 12,000 r.p.m. for 10 min at 4 °C. Protein concentration was determined using the Bradford protein assay (Bio-Rad, Marnes-la-Coquette, France). Samples were supplemented with 2-mercaptoethanol (Sigma-Aldrich, St Louis, USA), and denatured at 85°C for 10 min. An equal amount of protein (30 µg) was separated with 12% SDS-PAGE and blotted to nitrocellulose membranes (Protean BA 85, Schleicher & Schuell, Dassel, Germany). The membranes were blocked with 5% nonfat dry milk in PBS containing 0.1% (v/v) Tween 20 for 60 min at 22 °C. The mouse monoclonal antibody for GLP1R (DSHB Cat# Mab 3F52, RRID: AB_2618100) was diluted in 5% milk to a final concentration of 1,78 µg/mL [START_REF] Waser | Glucagon-like-peptide-1 receptor expression in normal and diseased human thyroid and pancreas[END_REF], and incubated overnight at 4 °C. The mouse anti-SSTR2 (Santa Cruz Biotechnology Cat# sc-365502, RRID: AB_10859216) was diluted 1:1000 and incubated overnight. The membranes were washed in Tris-buffered saline containing 0.05% Tween 20. The antimouse HRP-conjugated antibody was diluted 1:1000 in in 5% milk and incubated for 60 min at room temperature. The membranes were washed 3 times for 15 min in Tris-buffered saline containing 0.1% Tween 20 and blots were developed with ECL Plus, Amersham (GE Healthcare Life Science, Velizy-Villacoublay, France).

Human islet isolation and culture

For pancreatic hormone secretion experiments in response to glucose and drug treatments, human islets were cultured from normoglycemic individuals with a BMI < 30 and > 90% viability and > 80% purity (endocrine versus exocrine tissue) in glucose-free medium (Gibco, Life Technologies, Paris, France) supplemented with 0.625% HSA, 1% P/S and 5 mM glucose. For glucose responsive experiments, human islets were exposed to 1 mM or 6 mM glucose in the presence or absence of 12 µM of dapagliflozin alone [START_REF] Bonner | Inhibition of the glucose transporter SGLT2 with dapagliflozin in pancreatic alpha cells triggers glucagon secretion[END_REF] or (10 pmol/L) of liraglutide alone [START_REF] Shigeto | GLP-1 stimulates insulin secretion by PKC-dependent TRPM4 and TRPM5 activation[END_REF] or in combination. Using static incubation techniques, drug treatment was performed for 1h incubation and hormone secretion was normalized to intra-islet protein (hormone) content. Each condition was performed in quadruple from each donor islet preparation and hormone secretion was normalized to % of intracellular content. Dapagliflozin was obtained from AstraZeneca (Mölndal, Sweden), liraglutide (stylo Victoza) was purchased from Novo nordisk® (Bagsvaerd, Denmark) and SSTR2 inhibitor CYN154806 was obtained from (Sigma Aldrich, Paris, France).

Real-time quantitative RT-PCR (qPCR)

Human islets were isolated from pancreata harvested from lean, obese and T2DM adult brain-dead individuals as described previously [START_REF] Kerr-Conte | Upgrading pretransplant human islet culture technology requires human serum combined with media renewal[END_REF]. As recently suggested by American Diabetes Association and European Association for the Study of Diabetes [START_REF] Hart | Use of human islets to understand islet biology and diabetes: progress, challenges and suggestions[END_REF], we provide a standardized table titled key characteristics of human islets and their use Table S2.

Total RNA was extracted using the RNeasy Mini Kit (Qiagen, Courtaboeuf, France). Firststrand cDNA synthesis was performed using 0,5 µg total RNA as a template and Superscript IV reverse transcriptase (Life Technologies, Saint Aubin, France) primed with 50 pmol of random hexamers (New England BioLabs, Ipswich, MA, USA). Quantitative real-time PCR was performed using the Bio-Rad MyiQ Single-Color Real-Time PCR Detection System and Table S2. Key characteristics of human islets and their use. Related to Figure 2

Figure 1 .

 1 Figure legends:

Figure 2 .

 2 Figure 2. Single drug administrations in vitro in human islets and in vivo in C57Bl/6J

Figure 3 .

 3 Figure 3. A single dose of liraglutide and dapagliflozin with and without the SSTR2

Figure 4 .

 4 Figure 4. Acute treatments of liraglutide and dapagliflozin with and without the SSTR2

  Figure1

  Figure2

Acknowledgements

We thank the Biotherapy Plateform, the DHURE, Histology Department, Lille, France, and L'Equipe labellisee Ligue Contre le Cancer Toulouse, France for providing the SSTR2 KO mice. We thank Mr Yannick Carbonnaux (makitbe.com) for the Graphical abstract. This study was funded by AstraZeneca, le Conseil Régional Nord-Pas de Calais, Société Francophone du Diabète, the European commission (FEDER 12003944), the European Genomic Institute for Diabetes (ANR-10-LABX-46), the European Consortium for Islet Transplantation funded by the Juvenile Diabetes Research Foundation.

Author contributions

CS and CB designed and performed most experiments, analyzed the data, and wrote the manuscript. JT, VG, EM, ND, GP, AC and AQ performed specific experiments. C.Bousquet and YM provided the SSTR2 KO mice. JKC, MD, TH and MCV participated in experimental design of specific experiments. CB, FP and BS supervised the project and edited the manuscript. All authors approved the final version of the paper.

Declaration of interest

The authors have no conflict of interest to declare.

(SSTR2) in the pancreas was determined by using mouse anti-SSTR2 (1:100; Santa Cruz Biotechnology Cat# sc-365502, RRID: AB_10859216). To study the specific localization of GLP1R and SSTR2 in the pancreas, we performed dual immunofluorescence staining analysis with a mixture of antibodies against islet specific hormones, which included rabbit polyclonal anti-glucagon (1:800; Abcam Cat# ab8055, RRID: AB_2247265), rabbit polyclonal anti-insulin (1:800; Abcam Cat# ab63820, RRID: AB_1925116), rabbit polyclonal anti-somatostatin (1:800; Dako Cat# A0566, RRID: AB_2688022) for colocalization studies. For single-label immunohistochemistry DAB (Diaminobenzidine) or immunofluorescence staining we followed the exact protocol by Waser and colleagues [START_REF] Waser | Glucagon-like-peptide-1 receptor expression in normal and diseased human thyroid and pancreas[END_REF]. In brief, paraffin sections from the human pancreas, kidney and intestine were dewaxed and rehydrated to H2O. Sections were microwave treated for 8 min at 900W and 30 min at 360W in Tris-EDTA buffer, pH 9.0, were allowed to cool for 15 min, rinsed in Tris-buffered saline (TBS), pH 7.5, and blocked in 3% H2O2 with 0.15M NaN3.

Slides were incubated overnight with the primary GLP1R monoclonal mouse antibody 7 mg/ml MAb 3F5 diluted in antibody dilution buffer (S0809 Dako, Courtabouef, France).

The following day, the slides were washed in 5 times in TBS for 5 mins, followed by Flex-Linker (Dako) for 20 minutes. The slides were again washed before the addition of Flex/HRP for 15 minutes. Another wash was performed, and the slides were developed with DAB (Dako). For immunofluorescence co-staining experiments, secondary antibodies used include Alexa Fluor 488 or 594 nm, anti-rabbit, or anti-mouse IgG (Thermo Fisher Scientific Cat# A-21206, RRID: AB_2535792, Cat# A-11001, RRID: AB_2534069, Cat# A-11012, RRID: AB_2534079, and Cat# A-11032, RRID: AB_2534091), and DAPI (used as nuclei counterstain) (Vector Laboratories Cat# H-1200, RRID: AB_2336790). Images were taken with a Nikon ECLIPSE Ti microscope using NIS ELEMENTS AR 3.2 software (Nikon, Champigny-sur-Marne, France). No immunolabeling was observed when the primary or the Bio-Rad iQ SYBR Green Supermix (Bio-Rad Laboratories, CA, USA) as previously described [START_REF] Bonner | Inhibition of the glucose transporter SGLT2 with dapagliflozin in pancreatic alpha cells triggers glucagon secretion[END_REF]. Gene expression was normalized to the housekeeping gene RPL27, in agreement with a previously described panel of house-keeping genes consistently used for this normalization [START_REF] De Jonge | Evidence based selection of housekeeping genes[END_REF]. The primers for genes GLP1R and SSTR2 were designed with the website Primer3; the final concentration used for all the primers was 500 nM (250 forward + 250 reverse). The cDNA was diluted 1:10 and 1uL was used in 10uL final volume for each well. The reaction was: 95°C for 3 min (priming) followed by 40 cycles, 1 cycle at 95°C for 10 sec (denaturation) and 60°C for 30 sec (annealing). A melt curve analysis was performed for quality of primers and qPCR reaction.

QUANTIFICATION AND STATISTICAL ANALYSIS

Statistical Analysis

Data are expressed as means ± s.e.m. and two-way ANOVA, with appropriate corrections such as Tukey's and Bonferroni's post hoc tests were used for multiple comparisons. For the in vivo experiments, the Shapiro-Wilk normality test was performed to test if values had a Gaussian distribution and according to this, results between groups were analyzed using ordinary one-way ANOVA with Tukey's multiple comparisons test or non-parametric Kruskal-Wallis analysis with Dunn's multiple comparison test. Statistical analyses were performed using GraphPad PRISM 7.0 (GraphPad Software, La Jolla, California, USA).

Differences were considered significant at p< 0.05.

Further information and requests for resources and reagents should be directed to and will be fulfilled by the Lead Contact, Dr Caroline Bonner (caroline.bonner@univ-lille.fr).

DATA AND CODE AVAILABILITY

This study did not generate/analyze datasets/code.