Loss of Liver Kinase B1 (LKB1) in Beta Cells Enhances Glucose-stimulated Insulin Secretion Despite Profound Mitochondrial Defects

The tumor suppressor liver kinase B1 (LKB1) is an important regulator of pancreatic β cell biology. LKB1-dependent phosphorylation of distinct AMPK (adenosine monophosphate-activated protein kinase) family members determines proper β cell polarity and restricts β cell size, total β cell mass, and glucose-stimulated insulin secretion (GSIS). However, the full spectrum of LKB1 effects and the mechanisms involved in the secretory phenotype remain incompletely understood. We report here that in the absence of LKB1 in β cells, GSIS is dramatically and persistently improved. The enhancement is seen both in vivo and in vitro and cannot be explained by altered cell polarity, increased β cell number, or increased insulin content. Increased secretion does require membrane depolarization and calcium influx but appears to rely mostly on a distal step in the secretion pathway. Surprisingly, enhanced GSIS is seen despite profound defects in mitochondrial structure and function in LKB1-deficient β cells, expected to greatly diminish insulin secretion via the classic triggering pathway. Thus LKB1 is essential for mitochondrial homeostasis in β cells and in parallel is a powerful negative regulator of insulin secretion. This study shows that β cells can be manipulated to enhance GSIS to supra-normal levels even in the face of defective mitochondria and without deterioration over months.     

Can CD44 Be a Mediator of Cell Destruction? The Challenge of Type 1 Diabetes

CD44 is a multi-functional receptor with multiple of isoforms engaged in modulation of cell trafficking and transmission of apoptotic signals. We have previously shown that injection of anti-CD44 antibody into NOD mice induced resistance to type 1 diabetes (T1D). In this communication we describe our efforts to understand the mechanism underlying this effect. We found that CD44-deficient NOD mice develop stronger resistance to T1D than wild-type littermates. This effect is not explained by the involvement of CD44 in cell migration, because CD44-deficient inflammatory cells surprisingly had greater invasive potential than the corresponding wild type cells, probably owing to molecular redundancy. We have previously reported and we show here again that CD44 expression and hyaluronic acid (HA, the principal ligand for CD44) accumulation are detected in pancreatic islets of diabetic NOD mice, but not of non-diabetic DBA/1 mice. Expression of CD44 on insulin-secreting β cells renders them susceptible to the autoimmune attack, and is associated with a diminution in β-cells function (e.g., less insulin production and/or insulin secretion) and possibly also with an enhanced apoptosis rate. The diabetes-supportive effect of CD44 expression on β cells was assessed by the TUNEL assay and further strengthened by functional assays exhibiting increased nitric oxide release, reduced insulin secretion after glucose stimulation and decreased insulin content in β cells. All these parameters could not be detected in CD44-deficient islets. We further suggest that HA-binding to CD44-expressing β cells is implicated in β-cell demise. Altogether, these data agree with the concept that CD44 is a receptor capable of modulating cell fate. This finding is important for other pathologies (e.g., cancer, neurodegenerative diseases) in which CD44 and HA appear to be implicated.     

Sirt1 Regulates Insulin Secretion by Repressing UCP2 in Pancreatic β Cells

Sir2 and insulin/IGF-1 are the major pathways that impinge upon aging in lower organisms. In Caenorhabditis elegans a possible genetic link between Sir2 and the insulin/IGF-1 pathway has been reported. Here we investigate such a link in mammals. We show that Sirt1 positively regulates insulin secretion in pancreatic β cells. Sirt1 represses the uncoupling protein (UCP) gene UCP2 by binding directly to the UCP2 promoter. In β cell lines in which Sirt1 is reduced by SiRNA, UCP2 levels are elevated and insulin secretion is blunted. The up-regulation of UCP2 is associated with a failure of cells to increase ATP levels after glucose stimulation. Knockdown of UCP2 restores the ability to secrete insulin in cells with reduced Sirt1, showing that UCP2 causes the defect in glucose-stimulated insulin secretion. Food deprivation induces UCP2 in mouse pancreas, which may occur via a reduction in NAD (a derivative of niacin) levels in the pancreas and down-regulation of Sirt1. Sirt1 knockout mice display constitutively high UCP2 expression. Our findings show that Sirt1 regulates UCP2 in β cells to affect insulin secretion.     

β-cell Smad2 null mice have improved β-cell function and are protected from diet-induced hyperglycemia

Understanding signaling pathways that regulate pancreatic β-cell function to produce, store, and release insulin, as well as pathways that control β-cell proliferation, is vital to find new treatments for diabetes mellitus. Transforming growth factor-beta (TGF-β) signaling is involved in a broad range of β-cell functions. The canonical TGF-β signaling pathway functions through intracellular smads, including smad2 and smad3, to regulate cell development, proliferation, differentiation, and function in many organs. Here, we demonstrate the role of TGF-β/smad2 signaling in regulating mature β-cell proliferation and function using β-cell-specific smad2 null mutant mice. β-cell-specific smad2-deficient mice exhibited improved glucose clearance as demonstrated by glucose tolerance testing, enhanced in vivo and ex vivo glucose-stimulated insulin secretion, and increased β-cell mass and proliferation. Furthermore, when these mice were fed a high-fat diet to induce hyperglycemia, they again showed improved glucose tolerance, insulin secretion, and insulin sensitivity. In addition, ex vivo analysis of smad2-deficient islets showed that they displayed increased glucose-stimulated insulin secretion and upregulation of genes involved in insulin synthesis and insulin secretion. Thus, we conclude that smad2 could represent an attractive therapeutic target for type 2 diabetes mellitus.     

Pancreatic β-Cell Death due to Pdx-1 Deficiency Requires Multi-BH Domain Protein Bax but Not Bak

Diabetes develops in Pdx1-haploinsufficient mice due to an increase in β-cell death leading to reduced β-cell mass and decreased insulin secretion. Knockdown of Pdx1 gene expression in mouse MIN6 insulinoma cells induced apoptotic cell death with an increase in Bax activation and knockdown of Bax reduced apoptotic β-cell death. In Pdx1 haploinsufficient mice, Bax ablation in β-cells increased β-cell mass, decreased the number of TUNEL positive cells and improved glucose tolerance after glucose challenge. These changes were not observed with Bak ablation in Pdx1-haploinsufficient mice. These results suggest that Bax mediates β-cell apoptosis in Pdx1-deficient diabetes.     

Autophagy plays a protective role in endoplasmic reticulum stress-mediated pancreatic β cell death

There is a growing evidence of the role of autophagy in pancreatic β cell homeostasis. During development of type 2 diabetes, β cells are required to supply the increased demand of insulin. In such a stage, β cells have to address high ER stress conditions that could lead to abnormal insulin secretion, and ultimately, β cell death and overt diabetes. In this study, we used insulin secretion-deficient β cells derived from fetal mice. These cells present an increased accumulation of polyubiquitinated protein aggregates and LC3B-positive puncta, when compared with insulinoma-derived β cell lines. We found that insulin secretion deficiency renders these cells hypersensitive to endoplasmic reticulum (ER) stress-mediated cell death. Chemical or shRNA-mediated inhibition of autophagy increased β cell death under ER stress. On the other hand, rapamycin treatment increased both autophagy and cell survival under ER stress. Insulin secretion-deficient β cells showed a marked reduction of the antiapoptotic protein BCL2, together with increased BAX expression and ERN1 hyperactivation upon ER stress induction. These results showed how insulin secretion deficiency in β cells may be contributing to ER stress-mediated cell death, and in this regard, we showed how the autophagic response plays a prosurvival role.     

Insulin Receptor-Overexpressing β-Cells Ameliorate Hyperglycemia in Diabetic Rats through Wnt Signaling Activation

To investigate the therapeutic efficacy and mechanism of β-cells with insulin receptor (IR) overexpression on diabetes mellitus (DM), rat insulinoma (INS-1) cells were engineered to stably express human insulin receptor (INS-IR cells), and subsequently transplanted into streptozotocin- induced diabetic rats. Compared with INS-1 cells, INS-IR cells showed improved β-cell function, including the increase in glucose utilization, calcium mobilization, and insulin secretion, and exhibited a higher rate of cell proliferation, and maintained lower levels of blood glucose in diabetic rats. These results were attributed to the increase of β-catenin/PPARγ complex bindings to peroxisome proliferator response elements in rat glucokinase (GK) promoter and the prolongation of S-phase of cell cycle by cyclin D1. These events resulted from more rapid and higher phosphorylation levels of insulin-signaling intermediates, including insulin receptor substrate (IRS)-1/IRS-2/phosphotylinositol 3 kinase/v-akt murine thymoma viral oncogene homolog (AKT) 1, and the consequent enhancement of β-catenin nuclear translocation and Wnt responsive genes including GK and cyclin D1. Indeed, the higher functionality and proliferation shown in INS-IR cells were offset by β-catenin, cyclin D1, GK, AKT1, and IRS-2 gene depletion. In addition, the promotion of cell proliferation and insulin secretion by Wnt signaling activation was shown by 100 nM insulin treatment, and to a similar degree, was shown in INS-IR cells. In this regard, this study suggests that transferring INS-IR cells into diabetic animals is an effective and feasible DM treatment. Accordingly, the method might be a promising alternative strategy for treatment of DM given the adverse effects of insulin among patients, including the increased risk of modest weight gain and hypoglycemia. Additionally, this study demonstrates that the novel mechanism of cross-talk between insulin and Wnt signaling plays a primary role in the higher therapeutic efficacy of IR-overexpressing β-cells.     

Proglucagon Promoter Cre-Mediated AMPK Deletion in Mice Increases Circulating GLP-1 Levels and Oral Glucose Tolerance

Background

Enteroendocrine L-cells synthesise and release the gut hormone glucagon-like peptide-1 (GLP-1) in response to food transit. Deletion of the tumour suppressor kinase LKB1 from proglucagon-expressing cells leads to the generation of intestinal polyps but no change in circulating GLP-1 levels. Here, we explore the role of the downstream kinase AMP-activated protein kinase (AMPK) in these cells.

Method

Loss of AMPK from proglucagon-expressing cells was achieved using a preproglucagon promoter-driven Cre (iGluCre) to catalyse recombination of floxed alleles of AMPKα1 and α2. Oral and intraperitoneal glucose tolerance were measured using standard protocols. L-cell mass was measured by immunocytochemistry. Hormone and peptide levels were measured by electrochemical-based luminescence detection or radioimmunoassay.

Results

Recombination with iGluCre led to efficient deletion of AMPK from intestinal L- and pancreatic alpha-cells. In contrast to mice rendered null for LKB1 using the same strategy, mice deleted for AMPK displayed an increase (WT: 0.05 ± 0.01, KO: 0.09±0.02%, p<0.01) in L-cell mass and elevated plasma fasting (WT: 5.62 ± 0.800 pg/ml, KO: 14.5 ± 1.870, p<0.01) and fed (WT: 15.7 ± 1.48pg/ml, KO: 22.0 ± 6.62, p<0.01) GLP-1 levels. Oral, but not intraperitoneal, glucose tolerance was significantly improved by AMPK deletion, whilst insulin and glucagon levels were unchanged despite an increase in alpha to beta cell ratio (WT: 0.23 ± 0.02, KO: 0.33 ± 0.03, p<0.01).

Conclusion

AMPK restricts L-cell growth and GLP-1 secretion to suppress glucose tolerance. Targeted inhibition of AMPK in L-cells may thus provide a new therapeutic strategy in some forms of type 2 diabetes.     

LKB1 Represses Focal Adhesion Kinase (FAK) Signaling via a FAK-LKB1 Complex to Regulate FAK Site Maturation and Directional Persistence

Liver kinase β1 (LKB1, also known as STK11) is a serine/threonine kinase that has multiple cellular functions including the regulation of cell polarity and motility. Murine proteomic studies show that LKB1 loss causes aberrant adhesion signaling; however, the mechanistic underpinnings of this relationship are unknown. We show that cells stably depleted of LKB1 or its co-activator STRADα have increased phosphorylation of focal adhesion kinase (FAK) at Tyr³⁹⁷/Tyr⁸⁶¹ and enhanced adhesion to fibronectin. LKB1 associates in a complex with FAK and LKB1 accumulation at the cellular leading edge is mutually excluded from regions of activated Tyr³⁹⁷-FAK. LKB1-compromised cells lack directional persistence compared with wild-type cells, but this is restored through subsequent pharmacological FAK inhibition or depletion, showing that cell directionality is mediated through LKB1-FAK signaling. Live cell confocal imaging reveals that LKB1-compromised cells lack normal FAK site maturation and turnover, suggesting that defects in adhesion and directional persistence are caused by aberrant adhesion dynamics. Furthermore, re-expression of full-length wild-type or the LKB1 N-terminal domain repressed FAK activity, whereas the kinase domain or C-terminal domain alone did not, indicating that FAK suppression is potentially regulated through the LKB1 N-terminal domain. Based upon these results, we conclude that LKB1 serves as a FAK repressor to stabilize focal adhesion sites, and when LKB1 function is compromised, aberrant FAK signaling ensues, resulting in rapid FAK site maturation and poor directional persistence.     

Cask methylation involved in the injury of insulin secretion function caused by interleukin1‐β

Islet inflammation severely impairs pancreatic β‐cell function, but the specific mechanisms are still unclear. Interleukin1‐β (IL‐1β), an essential inflammatory factor, exerts a vital role in multiple physio‐pathologic processes, including diabetes. Calcium/calmodulin‐dependent serine protein kinase (CASK) is an important regulator especially in insulin secretion process. This study aims to unveil the function of CASK in IL‐1β–induced insulin secretion dysfunction and the possible mechanism thereof. Islets of Sprague‐Dawley (SD) rats and INS‐1 cells stimulated with IL‐1β were utilized as models of chronic inflammation. Insulin secretion function associated with Cask and DNA methyltransferases (DNMT) expression were assessed. The possible mechanisms of IL‐1β‐induced pancreatic β‐cell dysfunction were also explored. In this study, CASK overexpression effectively improved IL‐1β‐induced islet β‐cells dysfunction, increased insulin secretion. DNA methyltransferases and the level of methylation in the promoter region of Cask were elevated after IL‐1β administration. Methyltransferase inhibitor 5‐Aza‐2’‐deoxycytidine (5‐Aza‐dC) and si‐DNMTs partially up‐regulated CASK expression and reversed potassium stimulated insulin secretion (KSIS) and glucose‐stimulated insulin secretion (GSIS) function under IL‐1β treatment in INS‐1 and rat islets. These results reveal a previously unknown effect of IL‐1β on insulin secretion dysfunction and demonstrate a novel pathway for Cask silencing based on activation of DNA methyltransferases via inducible nitric oxide synthase (iNOS) and modification of gene promoter methylation.     

Liver Kinase B1 Expression Promotes Phosphatase Activity and Abrogation of Receptor Tyrosine Kinase Phosphorylation in Human Cancer Cells

Aberrant receptor tyrosine kinase phosphorylation (pRTK) has been associated with diverse pathological conditions, including human neoplasms. In lung cancer, frequent liver kinase B1 (LKB1) mutations correlate with tumor progression, but potential links with pRTK remain unknown. Heightened and sustained receptor activation was demonstrated by LKB1-deficient A549 (lung) and HeLaS3 (cervical) cancer cell lines. Depletion (siRNA) of endogenous LKB1 expression in H1792 lung cancer cells also correlated with increased pRTK. However, ectopic LKB1 expression in A549 and HeLaS3 cell lines, as well as H1975 activating-EGF receptor mutant lung cancer cell resulted in dephosphorylation of several tumor-enhancing RTKs, including EGF receptor, ErbB2, hepatocyte growth factor receptor (c-Met), EphA2, rearranged during transfection (RET), and insulin-like growth factor I receptor. Receptor abrogation correlated with attenuation of phospho-Akt and increased apoptosis. Global phosphatase inhibition by orthovanadate or depletion of protein tyrosine phosphatases (PTPs) resulted in the recovery of receptor phosphorylation. Specifically, the activity of SHP-2, PTP-1β, and PTP-PEST was enhanced by LKB1-expressing cells. Our findings provide novel insight on how LKB1 loss of expression or function promotes aberrant RTK signaling and rapid growth of cancer cells.     

Possible Protective Effect of Membrane Lipid Rafts against Interleukin-1β-Mediated Anti-Proliferative Effect in INS-1 Cells

We recently reported that pancreatic islets from pre-diabetic rats undergo an inflammatory process in which IL-1β takes part and controls β-cell function. In the present study, using the INS-1 rat pancreatic β-cell line, we investigated the potential involvement of membrane-associated cholesterol-enriched lipid rafts in IL-1β signaling and biological effects on insulin secretion, β-cell proliferation and apoptosis. We show that, INS-1 cells exposure to increasing concentrations of IL-1β leads to a progressive inhibition of insulin release, an increase in the number of apoptotic cells and a dose-dependent decrease in pancreatic β-cell proliferation. Disruption of membrane lipid rafts markedly reduced glucose-stimulated insulin secretion but did not affect either cell apoptosis or proliferation rate, demonstrating that membrane lipid raft integrity is essential for β-cell secretory function. In the same conditions, IL-1β treatment of INS-1 cells led to a slight further decrease in insulin secretion for low concentrations of the cytokine, and a more marked one, similar to that observed in normal cells for higher concentrations. These effects occurred together with an increase in iNOS expression and surprisingly with an upregulation of tryptophane hydroxylase and protein Kinase C in membrane lipid rafts suggesting that compensatory mechanisms develop to counteract IL-1β inhibitory effects. We also demonstrate that disruption of membrane lipid rafts did not prevent cytokine-induced cell death recorded after exposure to high IL-1β concentrations. Finally, concerning cell proliferation, we bring strong evidence that membrane lipid rafts exert a protective effect against IL-1β anti-proliferative effect, possibly mediated at least partly by modifications in ERK and PKB expression/activities. Our results 1) demonstrate that IL-1β deleterious effects do not require a cholesterol-dependent plasma membrane compartmentalization of IL-1R1 signaling and 2) confer to membrane lipid rafts integrity a possible protective function that deserves to be considered in the context of inflammation and especially T2D pathogenesis.     

Kin of IRRE-like Protein 2 Is a Phosphorylated Glycoprotein That Regulates Basal Insulin Secretion

Direct interactions among pancreatic β-cells via cell surface proteins inhibit basal and enhance stimulated insulin secretion. Here, we functionally and biochemically characterized Kirrel2, an immunoglobulin superfamily protein with β-cell-specific expression in the pancreas. Our results show that Kirrel2 is a phosphorylated glycoprotein that co-localizes and interacts with the adherens junction proteins E-cadherin and β-catenin in MIN6 cells. We further demonstrate that the phosphosites Tyr^595–596 are functionally relevant for the regulation of Kirrel2 stability and localization. Analysis of the extracellular and intracellular domains of Kirrel2 revealed that it is cleaved and shed from MIN6 cells and that the remaining membrane spanning cytoplasmic domain is processed by γ-secretase complex. Kirrel2 knockdown with RNA interference in MIN6 cells and ablation of Kirrel2 from mice with genetic deletion resulted in increased basal insulin secretion from β-cells, with no immediate influence on stimulated insulin secretion, total insulin content, or whole body glucose metabolism. Our results show that in pancreatic β-cells Kirrel2 localizes to adherens junctions, is regulated by multiple post-translational events, including glycosylation, extracellular cleavage, and phosphorylation, and engages in the regulation of basal insulin secretion.     

Sperm-Associated Antigen 6 (SPAG6) Deficiency and Defects in Ciliogenesis and Cilia Function: Polarity,Density, and Beat

SPAG6, an axoneme central apparatus protein, is essential for function of ependymal cell cilia and sperm flagella. A significant number of Spag6-deficient mice die with hydrocephalus, and surviving males are sterile because of sperm motility defects. In further exploring the ciliary dysfunction in Spag6-null mice, we discovered that cilia beat frequency was significantly reduced in tracheal epithelial cells, and that the beat was not synchronized. There was also a significant reduction in cilia density in both brain ependymal and trachea epithelial cells, and cilia arrays were disorganized. The orientation of basal feet, which determines the direction of axoneme orientation, was apparently random in Spag6-deficient mice, and there were reduced numbers of basal feet, consistent with reduced cilia density. The polarized epithelial cell morphology and distribution of intracellular mucin, α-tubulin, and the planar cell polarity protein, Vangl2, were lost in Spag6-deficient tracheal epithelial cells. Polarized epithelial cell morphology and polarized distribution of α-tubulin in tracheal epithelial cells was observed in one-week old wild-type mice, but not in the Spag6-deficient mice of the same age. Thus, the cilia and polarity defects appear prior to 7 days post-partum. These findings suggest that SPAG6 not only regulates cilia/flagellar motility, but that in its absence, ciliogenesis, axoneme orientation, and tracheal epithelial cell polarity are altered.     

Identification of a small molecule activator of novel PKCs for promoting glucose-dependent insulin secretion

Using an image-based screen for small molecules that can affect Golgi morphology, we identify a small molecule, Sioc145, which can enlarge the Golgi compartments and promote protein secretion. More importantly, Sioc145 potentiates insulin secretion in a glucose-dependent manner. We show that Sioc145 selectively activates novel protein kinase Cs (nPKCs; δ and ɛ) but not conventional PKCs (cPKCs; α, βI and βII) in INS-1E insulinoma cells. In contrast, PMA, a non-selective activator of cPKCs and nPKCs, promotes insulin secretion independent of glucose concentrations. Furthermore, we demonstrate that Sioc145 and PMA show differential abilities in depolarizing the cell membrane, and suggest that Sioc145 promotes insulin secretion in the amplifying pathway downstream of K_ATP channels. In pancreatic islets, the treatment with Sioc145 enhances the second phase of insulin secretion. Increased insulin granules close to the plasma membrane are observed after Sioc145 treatment. Finally, the administration of Sioc145 to diabetic GK rats increases their serum insulin levels and improves glucose tolerance. Collectively, our studies identify Sioc145 as a novel glucose-dependent insulinotropic compound via selectively activating nPKCs.     

LKB1 Mediates the Development of Conventional and Innate T Cells via AMP-Dependent Kinase Autonomous Pathways

The present study has examined the role of the serine/threonine kinase LKB1 in the survival and differentiation of CD4/8 double positive thymocytes. LKB1-null DPs can respond to signals from the mature α/β T-cell-antigen receptor and initiate positive selection. However, in the absence of LKB1, thymocytes fail to mature to conventional single positive cells causing severe lymphopenia in the peripheral lymphoid tissues. LKB1 thus appears to be dispensable for positive selection but important for the maturation of positively selected thymocytes. LKB1 also strikingly prevented the development of invariant Vα14 NKT cells and innate TCR αβ gut lymphocytes. Previous studies with gain of function mutants have suggested that the role of LKB1 in T cell development is mediated by its substrate the AMP-activated protein kinase (AMPK). The present study now analyses the impact of AMPK deletion in DP thymocytes and shows that the role of LKB1 during the development of both conventional and innate T cells is mediated by AMPK-independent pathways.     

BIG3 inhibits insulin granule biogenesis and insulin secretion

While molecular regulation of insulin granule exocytosis is relatively well understood, insulin granule biogenesis and maturation and its influence on glucose homeostasis are relatively unclear. Here, we identify a novel protein highly expressed in insulin-secreting cells and name it BIG3 due to its similarity to BIG/GBF of the Arf-GTP exchange factor (GEF) family. BIG3 is predominantly localized to insulin- and clathrin-positive trans-Golgi network (TGN) compartments. BIG3-deficient insulin-secreting cells display increased insulin content and granule number and elevated insulin secretion upon stimulation. Moreover, BIG3 deficiency results in faster processing of proinsulin to insulin and chromogranin A to β-granin in β-cells. BIG3-knockout mice exhibit postprandial hyperinsulinemia, hyperglycemia, impaired glucose tolerance, and insulin resistance. Collectively, these results demonstrate that BIG3 negatively modulates insulin granule biogenesis and insulin secretion and participates in the regulation of systemic glucose homeostasis.     

A Novel Antidiabetic Drug,Fasiglifam/TAK-875, Acts as an Ago-Allosteric Modulator of FFAR1

Selective free fatty acid receptor 1 (FFAR1)/GPR40 agonist fasiglifam (TAK-875), an antidiabetic drug under phase 3 development, potentiates insulin secretion in a glucose-dependent manner by activating FFAR1 expressed in pancreatic β cells. Although fasiglifam significantly improved glycemic control in type 2 diabetes patients with a minimum risk of hypoglycemia in a phase 2 study, the precise mechanisms of its potent pharmacological effects are not fully understood. Here we demonstrate that fasiglifam acts as an ago-allosteric modulator with a partial agonistic activity for FFAR1. In both Ca²⁺ influx and insulin secretion assays using cell lines and mouse islets, fasiglifam showed positive cooperativity with the FFAR1 ligand γ-linolenic acid (γ-LA). Augmentation of glucose-induced insulin secretion by fasiglifam, γ-LA, or their combination was completely abolished in pancreatic islets of FFAR1-knockout mice. In diabetic rats, the insulinotropic effect of fasiglifam was suppressed by pharmacological reduction of plasma free fatty acid (FFA) levels using a lipolysis inhibitor, suggesting that fasiglifam potentiates insulin release in conjunction with plasma FFAs in vivo. Point mutations of FFAR1 differentially affected Ca²⁺ influx activities of fasiglifam and γ-LA, further indicating that these agonists may bind to distinct binding sites. Our results strongly suggest that fasiglifam is an ago-allosteric modulator of FFAR1 that exerts its effects by acting cooperatively with endogenous plasma FFAs in human patients as well as diabetic animals. These findings contribute to our understanding of fasiglifam as an attractive antidiabetic drug with a novel mechanism of action.