Elevated Neuropeptide Y1 Receptor Signaling Contributes to β-cell Dysfunction and Failure in Type 2 Diabetes

Loss of functional β-cell mass is a key factor contributing to the poor glycaemic control in type 2 diabetes. However, therapies that directly target these underlying processes remains lacking. Here we demonstrate that gene expression of neuropeptide Y1 receptor and its ligand, neuropeptide Y, was significantly upregulated in human islets from subjects with type 2 diabetes. Importantly, the reduced insulin secretion in type 2 diabetes was associated with increased neuropeptide Y and Y1 receptor expression in human islets. Consistently, pharmacological inhibition of Y1 receptors by BIBO3304 significantly protected β-cells from dysfunction and death under multiple diabetogenic conditions in islets. In a preclinical study, Y1 receptor antagonist BIBO3304 treatment improved β-cell function and preserved functional β-cell mass, thereby resulting in better glycaemic control in both high-fat-diet/multiple low-dose streptozotocin- and db/db type 2 diabetic mice. Collectively, our results uncovered a novel causal link of increased islet NPY-Y1 receptor signaling to β-cell dysfunction and failure in human type 2 diabetes. These results further demonstrate that inhibition of Y1 receptor by BIBO3304 represents a novel and effective β-cell protective therapy for improving functional β-cell mass and glycaemic control in type 2 diabetes.


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Increased NPY and Y1 receptor levels in T2D islets are associated with reduced insulin 118

secretion. 119
To investigate whether the NPY system in pancreatic islets is associated with reduced -cell 120 function in the pathogenesis of T2D, we first determined the NPY system expression profiles 121 in human islets isolated from non-diabetic and T2D subjects as described Methods and 122 Supplementary Data. Interestingly, we found that in islets from diabetic donors, the 123 expression of NPY and its receptor NPY1R was increased by 2.7-and 2.5-fold, respectively, as 124 compared to the non-diabetic donors ( Figure 1A and 1B). Importantly, the increased NPY and 125 NPY1R mRNA expression in human islets correlated with reduced insulin secretion as 126 indicated by the insulin stimulation index (Spearman's r=0.7151,p=0.0376 and r=0.6524,127 p=0.0473) ( Figure 1C and 1E), whereas the differential expression of NPY and NPY1R was 128 not associated with HbA1c ( Figure 1D and 1F) or BMI (Figure S1A and S1B). These results 129 suggests that elevated NPY/Y1 receptor signaling may contribute to impaired insulin secretion 130 in human with T2D. 131 132 On the other hand, the basal levels of NPY2R, PPYR1 (also known as NPY4R) and NPY5R were 133 very low in pancreatic islets (Figure 1B), suggesting that these Y receptors are unlikely to play 134 a major role in mediating NPY function in human pancreatic islets. Despite the low level of 135 expression, NPY5R was also moderately upregulated in islets of T2D subjects ( Figure 1B), but 136 there was no significant correlation between NPY5R expression and insulin stimulation index, 137 HbA1c ( Figure 1G and 1H) or BMI ( Figure S1C). Importantly, the NPY/Y1 receptor axis 138 appeared to be exclusively up-regulated as there were no noticeable changes in other NPY 139 ligands such as PYY and PPY and their correlation with insulin stimulation index or HbA1c 140 compared to placebo ( Figure 4E). BIBO3304-treated mice also exhibited significantly lower 241 fasted plasma insulin levels ( Figure 4F), suggesting that the inhibition of Y1 receptor 242 signalling may improve glucose control via increasing insulin action. Consistently, insulin 243 tolerance tests revealed that BIBO3304-treated db/db mice exhibited a markedly improved 244 insulin responsiveness, as evidenced by lower blood glucose across the duration of 120-245 minutes and when quantified as area under the curve ( Figure 4G). Although the differences 246 were not statistically significant, BIBO3304-treated mice displayed modestly improved whole-247 body glucose tolerance (Figure S4B and S4C). The enhanced insulin responsiveness was 248 correlated with increased insulin induced Akt phosphorylation in muscle ( Figure 4H) but not 249 in the liver or adipose tissue ( Figure S4D). In line with this, insulin-induced 2DG glucose 250 uptake was significantly enhanced in extensor digitorum longus (EDL) muscle isolated from 251 db/db mice treated with BIBO3304 for 4 weeks, an effect that was impaired in the placebo 252 group ( Figure 4I). Strikingly, human muscle NPY1R expression was 3-fold higher in obese 253 compared to lean subjects ( Figure 4J). The increased NPY1R expression in human vastus 254 lateralis muscle also exhibited a positive correlation with BMI (Spearman's r=-0.6291, 255 p=0.005) as well as fasting blood glucose levels (Spearman's r=-0.5273, p=0.0245) ( Figure  256 4K and 4L). Consistently, we show in primary human myotubes that insulin-stimulated 257 glucose uptake was suppressed significantly by NPY, an effect that was diminished in the 258 presence of BIBO3304 ( Figure 4M). Taken together, these results suggest that on the early 259 onset of T2D, Y1 receptor antagonism attenuates hyperglycaemia which can be attributed to 260 improved insulin action as a consequence of reduced adiposity and/or directly due to inhibition 261 of Y1 receptor in muscle. 262 263 While BIBO3304 treatment of late stage diabetic db/db mice at 16 weeks of age did not show 264 any effects on body weights, lean mass, fat mass, fat pads mass and insulin response ( Figure  265 S4E-S4I), and the impaired -cell compensation in the 16-week-old db/db mice became 266 evident as indicated by a greater than 2.5-fold reduction in serum insulin level as compared 267 to10-week-old db/db mice (11.4±1.91 ng/ml in 10-week-old vs. 4.1±0.26 ng/ml in 16-week-268 old db/db mice, n=5-6) ( Figure 4N). It is of interest that hyperinsulinemia in the early 269 pathogenesis of T2D was associated with a greater than 60% reduction in Npy and Npy1r 270 expression in islets of 8-week-old db/db mice when compared to the non-diabetic db/+ mice 271 ( Figure S4J), which further supports an inhibitory role for NPY/Y1R signaling in the 272 regulation of insulin secretion. More importantly, BIBO3304 treatment led to a significant 273 enhancement of insulin secretion in response to re-feeding after an overnight fast in the  week-old db/db cohort when compared to the placebo group ( Figure 4N), suggesting an 275 increase in postprandial-induced insulin secretion. To further investigate whether Y1 receptor 276 antagonism has also effects on preserving -cell mass during the transition from β-cell 277 compensation to failure, pancreases from 16-week-old db/db mice treated daily for 6 weeks 278 with BIBO3304 or placebo were examined. Pancreatic histological analysis revealed that 279 pancreas weights, pancreatic islet area, islet number, and islet proportion were comparable 280 between BIBO3304 and placebo treated groups ( Figure S4K-S4N). However, a significant 281 increase in the intensity of insulin staining in -cells was observed in pancreases from 282 BIBO3304 treated db/db mice compared to placebo treated db/db mice ( Figure 4O). 283 Collevtively, these results suggest that Y1 antagonism preserves β-cell insulin content and 284 secretory capacity, thereby delaying diabetes progression in db/db mice. In this study, we demonstrated that the increased NPY and Y1 receptor expression in islets are 292 associated with reduced insulin secretion in human type 2 diabetes. In addition, 293 pharmacological inhibition of Y1 receptor signalling under diabetogenic conditions resulted in 294 improved glucose-stimulated insulin secretion and reduced β-cell death ex vivo. Y1 receptor 295 antagonism with BIBO3304 improved β-cell function and preserved functional β-cell mass, 296 thereby resulting in better glycaemic control in HFD/STZ-induced diabetic mouse models. 297 Furthermore, treatment of early-diabetic db/db mice with BIBO3304 resulted in reduced 298 adiposity accompanied by lower fasted and postprandial blood glucose levels due to enhanced 299 insulin sensitivity and muscle glucose uptake. Importantly, we also showed that administration 300 of BIBO3304 in severely diabetic db/db mice delays diabetes progression through preserving 301 functional β-cell mass during the transition from β-cell compensation to failure. These findings 302 extend our previous studies which revealed that inhibition of Y1 receptor signalling improves 303 β-cell function in both rodent and human islets which can be utlised to improve islet 304 transplantation outcomes and a delayed onset of T1D [20]. 305 306 Compared to the critical role of neuronal NPY and its receptor Y1 in the regulation of appetite 307 and energy metabolism, the role of NPY-Y1 receptor signalling in the regulation of β-cell 308 function and mass, in particular in human type 2 diabetes, is far less clear. In this study, we 309 demonstrate that in addition to NPY, its receptor Y1 expression was also upregulated in islets 310 from subjects with T2D and the augmented islet NPY and Y1 receptor expression is associated 311 with β-cell dysfunction and failure, thus representing a potential driver of diabetes onset. These 312 results are in line with studies that showed impaired glucose-stimulated insulin secretion in β-313 cell specific NPY overexpressed islets [21]. Indeed, our results further indicate that 314 pharmacological inhibition of Y1 receptor using BIBO3304 in mouse islets resulted in 315 decreased apoptosis as well as improved β-cell function under various diabetogenic stress 316 conditions. These results demonstrate the significance of NPY-Y1 receptor signalling 317 inhibition, which not only enhances insulin secretion but also protects β-cell against apoptosis. 318 319 More importantly, results from these preclinical proof-of-concept studies revealed that Y1 320 receptor antagonism with BIBO3304 can act as an insulin sensitiser when -cells remain 321 functioning (early pathogenesis of T2D) and prevents -cell loss at the late stage of T2D. Y1 322 receptors are G-protein coupled receptors which preferentially associate with Gi/o G-protein 323 and therefore act in an inhibitory fashion [22]. Intracellular cAMP levels are reduced in target 324 cells in response to Y1 receptor ligands, whereas cAMP is increased in response to Y1 325 antagonism [23]. In line with this, a previous study on islets isolated from Y1 receptor knockout 326 mice found up-regulated cAMP levels [20]. The cAMP signalling-dependent mechanisms have 327 been identified to play a critical role in improving insulin secretion and β-cell survival in 328 diabetes [24]. For instance, pharmacological cAMP inducers such as GLP-1 agonist exendin-329 4, decreases cytokine-and ER stress-induced impaired β-cell function and apoptosis via a 330 cAMP-dependent signalling pathway in both rodent and human β-cells [25,26]. Supporting 331 this notion, our findings showed that under diabetogenic conditions, islets treated with 332 BIBO3304 exhibited significant improvement in glucose-stimulated insulin secretion, 333 suggesting that this is attributed to enhanced intracellular cAMP levels. 334 335 Most of the known effects of the NPY system in the development of obesity arise from the 336 central activation of Y1 receptors, where it plays a critical role in the regulation of appetite and 337 energy homeostasis [9]. Inhibition of Y1 receptors or NPY deficiency in the brain have been 338 linked to decreased body weight gain and adiposity by decreasing energy intake and increasing 339 energy expenditure [27,28]. Our findings revealed that the administration of the non-brain 340 penetrable Y1 receptor antagonist BIBO3304 also resulted in decreased body weight and fat 341 mass in db/db mice in the absence of any alteration in food intake, suggesting that BIBO3304 342 reduces adiposity by acting on mechanisms other than regulation of appetite centrally. This is 343 consistent with results from a previous study conducted by Zhang et al., where it was revealed 344 that conditional knockdown of Y1 receptors in the periphery exhibited a phenotype of reduced 345 RER, indicating increased lipid oxidation [29]. The underlying mechanisms behind the 346 increased lipid oxidation under peripheral Y1 antagonism was reportedly associated with 347 increased levels of carnitine palmitoyltransferase-1 (CPT-1) and upregulation of key enzymes 348 involved in β-oxidation, consequently increasing the capacity of the mitochondria for lipid 349 oxidation and transport of fatty acids particularly in the liver and muscle [29]. 350 351 In addition to reduced adiposity, BIBO3304 treatment in db/db mice also significantly 352 enhanced insulin responsiveness as demonstrated by increased insulin induced AKT 353 phosphorylation and insulin-stimulated glucose uptake in skeletal muscle of db/db mice and in 354 primary human muscle cells. The insulin sensitising effect observed in db/db mice might be, 355 at least in part, due to reduced body weight and adiposity or muscle fat content. In addition, in 356 line with our finding in primary human myotubes, previous studies showed that deficiency of 357 peripheral Y1 receptor results in increased mitochondrial capacity in muscle [29], supporting 358 a role of Y1 receptor antagonism acting directly on muscle insulin receptor signalling. 359 Nonetheless, these results are consistent with the notion that increasing muscle glucose uptake 360 improves glycaemic control and suggesting that, in addition to reduced adiposity, these effects 361 may at least in part be responsible for the observed improvement in glucose homeostasis in 362 BIBO3304 treated db/db mice. 363

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In summary, one unmet clinical need in treating T2D is the availability of therapeutics that 365 improves glycaemic control by targeting the underlying β-cell dysfunction and failure. As such 366 the reduced adiposity and improved insulin action seen by the inhibition of NPY/Y1 signalling 367 in vivo highlights a potential therapy of targeting these peripheral Y1 receptor pathways to 368 preserve functional β-cell mass, which may ultimately provide greater therapeutic benefits in 369 controlling glucose levels in T2D.  Australia) for all in vivo studies. To induce T2D, C57BL/6 mice were fed a high fat diet (SF04-419 027; Specialty Feeds) for 4 weeks from 8 weeks of age, followed by multiple intraperitoneal 420 injections of low-dose streptozotocin (35 mg/kg) (Sigma Aldrich). Streptozotocin was prepared 421 fresh each time in 0.1 M sodium citrate buffer (pH 4.5) and filter sterilised prior to use. Blood 422 glucose level was measured twice a week until blood glucose reached 15 mmol/L and above. 423 C57BL/6 mice used in islets experiments were bred in house at BioResources Centre (St 424 Vincent's Hospital). All mice were housed in a temperature-controlled room of 22 ֯ C on a 12 425 h/12 h light/dark cycle (lights on from 0700-1900 hours) with free access to water and food. 426 427

Treatment with Y1 receptor antagonist BIBO3304 and metformin 429
A non-brain penetrable Y1 receptor antagonist BIBO3304 (Tocris Bioscience) was prepared 430 in Milli Q water at a concentration of 1 mg/ml. C57BL/6 mice (average weight 27.4 ± 0.3 g) 431 were received 0.5 mg BIBO3304 daily in jelly containing 4.9% (wt/v) gelatine and 7.5% (v/v) 432 chocolate flavouring essence as described previously [13]. The obese db/db mice at 4-(average 433 weight 20.3 ± 0.9 g) or 12-weeks of age (average weight 42 ± 1.3 g) were given 2.5 mg 434 BIBO3304 once daily via oral gavage, while control mice on placebo treatment received the 435 same volume of Milli Q water. Metformin (Sigma Aldrich) was prepared in Milli Q water, and 436 0.25 g/kg was given daily via oral gavage. The duration of treatment is as stated in the text for 437 each procedure. 438 439

Metabolic assessment and body composition measures 440
The effect of Y1 receptor antagonist BIBO3304 on blood glucose control and body weight 441 were monitored weekly on the same day of the week between 09:00 hours and 10:00 hours. 442 Random blood glucose was taken from tail tipping and measured on an Accu-Check Performa 443 glucometer (Roche, Switzerland). For the fast-refeeding experiment, food was removed from 444 the mice at the dark cycle before the experiment. Blood was collected by retro-orbital bleed 445 after a 16 h fast as well as 30 minutes after refeeding to determine blood glucose and plasma 446 insulin levels. Food intake was measured at the same time points (n = 8 per group). Whole 447 body lean mass and fat mass were measured at the end of study using the whole-body 448 composition analyzer, EchoMRI (Houston, USA). 449

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In vivo assessment of glucose, insulin and pyruvate tolerance tests 451 Glucose tolerance tests were performed on 6 h-fasted HFD/STZ mice or overnight fasted db/db 452 mice by intraperitoneal injection of 1 g/kg and 0.5 g/kg glucose, respectively. Insulin tolerance 453 was measured by intraperitoneal injection of 0.75 i.u./kg and 2.5 i.u./kg human insulin 454 (Actrapid, Novo Nordisk Pharmaceuticals) on HFD/STZ mice and db/db mice after a 6h-fast. 455 Pyruvate tolerance tests were conducted on mice after an overnight fast with intraperitoneal 456 injection of 1 g/kg sodium pyruvate. Blood glucose was measured at basal and, 15, 30, 60, 90 457 and 120 minutes following glucose, insulin or pyruvate administration. The in vivo glucose-458 stimulated insulin secretion was determined by intravenous glucose tolerance test using 1 g/kg 459 glucose on overnight fasted HFD/STZ mice as previously described [ref]. Briefly, mice were 460 anaesthetised and jugular venous catheters were inserted. Mice were allowed to recover for 20 461 minutes after surgery. A bolus of glucose was given via catheter and blood glucose was 462 measured at 2, 5, 10, 15 and 30 minutes post glucose administration. 463 464

Pancreatic islet isolation and culture ex vivo 465
Mouse islets were isolated from C57BL/6 and db/db mice as previously described [30]. Briefly, 466 Collagenase P (0.45 mg/mL) (Sigma Aldrich) was injected into the bile duct to distend the 467 pancreas. After perfusion, pancreas was excised and incubated at 37°C for 15 minutes. Islets 468 was further purified using Histopaque-1077 gradient (Sigma Aldrich and 2 mmol/l l-glutamine. All islets were incubated in a 37°C, 5% CO2 humidified incubator. 481 Insulin stimulation index was determined and presented as the ratio of insulin secretion at 28 482 mmol/l to that of at 2.8 mmol/l from the same islets.

Glucose-stimulated insulin secretion in isolated islets 493
Wild-type C57BL/6 islets were incubated in the respective diabetogenic stressors for the 494 indicative duration: Inflammation: islets were incubated with proinflammatory cytokine 495 cocktail (50 ng/ml TNFa, 250 ng/ml IFNg and 25 ng/ml IL1b) for 48 hours; oxidative stress: 496 10 mM H2O2 for 16 hours; ER stress: 1 mM thapsigargin for 24 hours. Following culture, islets 497 were handpicked and pre-incubated for 1 hour in HEPES-buffered-KREBS buffer containing 498 0.2% BSA and 2.8 mmol/L D-glucose. Subsequently, 15 size matched islets were incubated at 499 37°C for another 1 hour in KREBS buffer containing either 2.8 mmol/L or 20 mmol/L D-500 glucose, treated with or without 1 M BIBO3304. Culture medium was collected, and insulin 501 secretion was assayed using a mouse insulin ELISA kit (ALPCO Diagnostics, Salem, NH, 502 USA). 503 504 DNA fragmentation assay 505 To induced islet cell death, wild-type C57BL/6 islets were incubated in the respective 506 diabetogenic stressors: Inflammation: islets were incubated with proinflammatory cytokine 507 cocktail (50 ng/ml TNFa, 250 ng/ml IFNg and 25 ng/ml IL1b) for 72 hours; oxidative stress: 508 70 mM H2O2 for 18 hours; ER stress: 5 mM thapsigargin for 72 hours, glucolipotoxicity: 25 509 mmol/L glucose plus 0.5 mM palmitate for 96 hours. Cell apoptosis was measured by analysis 510 of DNA fragmentation as described previously [32]. Briefly, islets in uniform size were 511 handpicked into 3.5 cm Petri dishes containing the appropriate stimuli to induce apoptosis in 512 1.5 ml complete CMRL medium. At the end of the culture period, islets were dispersed by 513 trypsin digestion for 5 min at 37°C, followed by mechanical disruption by pipetting up and 514 down for 10 times. The dispersed islet cells were then resuspended in 150 ml of Nicoletti buffer 515 containing 50 mg/ml propidium iodide (Miltenyi Biotec), 0.1% (wt/v) sodium citrate and 0.1% 516  Technologies) diluted in 10% FBS for 1 hour at room temperature. The resulting slides were 595 then mounted in a mounting medium containing DAPI. Slides were scanned at 20x 596 magnification using 3D Histech Panoramic SCAN II slide Scanner (Phenomics Australia 597 Histopathology and Slide Scanning Service, University of Melbourne). For β-cell mass 598 measurement, Islets were outlined manually on the digital images. Islet area and islet number 599 were analysed using digital image processing software Image Scope (Aperio). Two sections 600 separated by at least 150 m was used for each mouse (n=8 per treatment). Cell mass of 601 pancreatic -cells was determined as the product of wet pancreas weight and the ratio of insulin 602 positive/total pancreas area. 603 604 Hepatic glucose production assay 605 Primary hepatocytes were isolated from C57BL/6 mice at 8 weeks of age and plated at a density 606 of 1 x10 6 cells in 6-well plates with the plating medium (Williams' E medium supplemented 607 with 10% fetal bovine serum, 1% penicillin-streptomycin and 1% of L-glutamine) for 4 hours 608 followed by starvation overnight in low glucose DMEM supplemented 1% L-glutamine and 609 1% Penicillin-Streptomycin. The following day, cells were pre-treated treated with/without 0.5 610 mM NPY and/or 1 mM BIBO3304 for 1 hour. Subsequently, the cells were washed once with 611 PBS, and the assay medium (DMEM without glucose, 1% penicillin-streptomycin, 2 mM of 612 sodium pyruvate, 20 mM sodium lactate, pH 7.4) was added with/without 0.5 mM NPY and/or 613 1 mM BIBO3304 for 6 hours. Glucose production was assayed with the Amplex Red glucose 614 assay kit (Invitrogen), and cell lysate was used in protein assay for normalisation. 615 616

Statistical analysis 617
All data are presented as mean ± SEM. A Student's t-test was conducted to test difference 618 between two groups of mice. Restricted randomisation was used to achieve treatment group 619 with similar numbers of mice. Sample size was estimated on previously published studies of 620 our and other's research groups [13,14,35,36]. Differences among groups of mice were 621 assessed by two-way ANOVA or repeated-measures ANOVA. Correlation coefficient was 622 calculated using Spearman's rank correlation coefficient. Statistical analyses were assessed 623 using Prism software 8.0. All experiments requiring the use of animals or animals to derive 624 cells were subject to randomization based on litter. Differences were regarded as statistically 625 significant if *P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001. Loss of functional β-cell mass is a key factor contributing to poor glycaemic control in 675 advanced type 2 diabetes. Hence, one of the most pressing unmet medical needs in type 2 676 diabetes is the development of new therapeutics that provide β-cell protective effects, such as 677 improvement of β-cell function, mass and survival. Improved understanding of diabetes 678 pathophysiology and the identification of a new biochemical pathway that regulates β-cell 679 function and mass will be extremely valuable for the development of more effective therapeutic 680 approaches for diabetes. 681

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In this study, we demonstrated that the increased NPY and Y1 receptor expression in islets 683 from patient with type 2 diabetes correlated with reduced β-cell function. Importantly, in a 684 preclinical study, pharmacological inhibition of neuropeptide Y1 receptors by BIBO3304, a 685 selective orally bioavailable neuropeptide Y1 receptor antagonist, significantly improved β-686 cell function and preserved β-cell mass, thereby resulting in better glycaemic control. 687 Furthermore, Y1 receptor antagonist BIBO3304 exhibited similar efficacy to attenuate 688 hyperglycaemia when compared with a first-line oral anti-diabetic drug, metformin. 689 Collectively, these results demonstrate that inhibition of Y1 receptor by BIBO3304 represents 690 a potential β-cell protective therapy for improving functional β-cell mass and glycaemic control 691 in type 2 diabetes. 692

Impacts 693
This research is the first to uncover a novel causal link of increased islet NPY-Y1 receptor 694 signaling to β-cell dysfunction and failure in human type 2 diabetes, contributing to the 695 understanding of the pathophysiology of type 2 diabetes. These novel findings provide 696 preclinical proof-of-concept for improving functional β-cell mass and resulting in better 697 glycaemic control by targeting the NPY-Y1 receptor pathway. Findings from the current 698 studies provide a significant conceptual advance that could have translational potential for 699 improving treatment of type 2 diabetes. 700 Four-and ten-week-old leptin receptor deficient db/db mice were randomized to receive 882 placebo or oral Y1 antagonist BIBO3304 for 6 weeks. Fasted and re-fed serum insulin levels 883 were measured (n = 5-8 per group). (O) Pancreases from placebo or BIBO3304 treated mice at 884 16 weeks of age were weighed and fixed in formalin and processed for immunostaining of 885 insulin (green) and nuclear counterstained with DAPI (blue). Insulin intensity was determined 886 by screening 138 and 172 islets on placebo and BIBO3304 treated pancreatic sections, 887 respectively. Insulin intensity was presented as insulin positive pixel normalized to the islet 888 area. Data are means ± SEM *P < 0.05, **P < 0.01; calculated by unpaired Student's t-test or 889 two-way ANOVA analysis.