Devil's Club Falcarinol-Type Polyacetylenes Inhibit Pancreatic Cancer Cell Proliferation.

Natural falcarinol-type (FC-type) polyacetylenes are known to show anticancer activities. We studied the bioactivity of synthetic FC, 1,2-dihydrofalcarinol (FCH) and 3-acetoxyfalcarinol (FCA) and compared them with the natural bioactive polyacetylene [9,17-octadecadiene-12,14-diyne-1,11,16-triol,1-acetate] (DCA) isolated from Devil's club (DC) Oplopanax horridus. Antiproliferation activity of these polyacetylenes, along with DC inner stem bark 70% ethanol and water extracts, was tested on human pancreatic ductal adenocarcinoma cell lines PANC-1 and BxPC-3. Chemically synthesized FC and FCA showed consistent IC50 (50% inhibition concentration) and higher potency than DCA. FC and DCA's mechanism of action investigated by antibody array on apoptosis-associated genes, and cellular features confirmed by microscopy demonstrated that both compounds modulated genes related to pro-apoptosis, antiapoptosis, apoptosis, cell cycle, stress related, and death receptors. FC-type polyacetylenes with a terminal double bond (FC, FCA, and DCA) are potent inhibitors of pancreatic cancer cell proliferation compared to FCH with a terminal single bond. Liquid chromatography mass spectrometry confirmed the presence of FC and FCH in the inner stem bark of DC. For potential applications of FC-type polyacetylenes as anticancer agents, preparing them by chemical synthesis may provide an advantage over the labor intensive extraction process from raw plant material.

Dual role of interleukin-1ß in islet amyloid formation and its ß-cell toxicity: Implications for type 2 diabetes and islet transplantation.

AIMS: Islet amyloid, formed by aggregation of human islet amyloid polypeptide (hIAPP), contributes to ß-cell failure in type 2 diabetes, cultured and transplanted islets. We previously showed that biosynthetic hIAPP aggregates induce ß-cell Fas upregulation and activation of the Fas apoptotic pathway. We used cultured human and hIAPP-expressing mouse islets to investigate: (1) the role of interleukin-1ß (IL-1ß) in amyloid-induced Fas upregulation; and (2) the effects of IL-1ß-induced ß-cell dysfunction on pro-islet amyloid polypeptide (proIAPP) processing and amyloid formation. RESEARCH DESIGN AND METHODS: Human and h IAPP -expressing mouse islets were cultured to form amyloid without or with the IL-1 receptor antagonist (IL-1Ra) anakinra, in the presence or absence of recombinant IL-1ß. Human islets in which amyloid formation was prevented (amyloid inhibitor or Ad-prohIAPP-siRNA) were cultured similarly. ß-cell function, apoptosis, Fas expression, caspase-8 activation, islet IL-1ß, ß-cell area, ß-/α-cell ratio, amyloid formation, and (pro)IAPP forms were assessed. RESULTS: hIAPP aggregates were found to increase IL-1ß levels in cultured human islets that correlated with ß-cell Fas upregulation, caspase-8 activation and apoptosis, all of which were reduced by IL-1Ra treatment or prevention of amyloid formation. Moreover, IL-1Ra improved culture-induced ß-cell dysfunction and restored impaired proIAPP processing, leading to lower amyloid formation. IL-1ß treatment potentiated impaired proIAPP processing and increased amyloid formation in cultured human and h IAPP -expressing mouse islets, which were prevented by IL-1Ra. CONCLUSIONS: IL-1ß plays a dual role by: (1) mediating amyloid-induced Fas upregulation and ß-cell apoptosis; (2) inducing impaired proIAPP processing thereby potentiating amyloid formation. Blocking IL-1ß may provide a new strategy to preserve ß cells in conditions associated with islet amyloid formation.

IDO-Expressing Fibroblasts Protect Islet Beta Cells From Immunological Attack and Reverse Hyperglycemia in Non-Obese Diabetic Mice.

Indoleamine 2,3-dioxygenase (IDO) induces immunological tolerance in physiological and pathological conditions. Therefore, we used dermal fibroblasts with stable IDO expression as a cell therapy to: (i) Investigate the factors determining the efficacy of this cell therapy for autoimmune diabetes in non-obese diabetic (NOD) mice; (ii) Scrutinize the potential immunological mechanisms. Newly diabetic NOD mice were randomly injected with either 10 × 10(6) (10M) or 15 × 10(6) (15M) IDO-expressing dermal fibroblasts. Blood glucose levels (BGLs), body weight, plasma kynurenine levels, insulitis severity, islet beta cell function, autoreactive CD8(+) T cells, Th17 cells and regulatory T cells (Tregs) were then investigated in these mice. IL-1ß and cleaved caspase-3 levels were assessed in islets co-cultured with IDO-expressing fibroblasts. BGLs in 83% mice treated with 15M IDO-expressing fibroblasts recovered to normal up to 120 days. However, only 17% mice treated with 10M IDO-expressing cells were reversed to normoglycemia. A 15M IDO-expressing fibroblasts significantly reduced infiltrated immune cells in islets and recovered the functionality of remaining islet beta cells in NOD mice. Additionally, they successfully inhibited autoreactive CD8(+) T cells and Th17 cells as well as increased Tregs in different organs of NOD mice. Islet beta cells co-cultured with IDO-expressing fibroblasts had reduced IL-1ß levels and cell apoptosis. Both cell number and IDO enzymatic activity contributes to the efficiency of IDO cell therapy. Optimized IDO-expressing fibroblasts successfully reverse the progression of diabetes in NOD mice through induction of Tregs as well as inhibition of beta cell specific autoreactive CD8(+) T cells and Th17 cells. J. Cell. Physiol. 231: 1964-1973, 2016. © 2016 Wiley Periodicals, Inc.

Human islet function following 20 years of cryogenic biobanking.

AIMS/HYPOTHESIS: There are potential advantages to the low-temperature (-196 °C) banking of isolated islets, including the maintenance of viable islets for future research. We therefore assessed the in vitro and in vivo function of islets cryopreserved for nearly 20 years. METHODS: Human islets were cryopreserved from 1991 to 2001 and thawed between 2012 and 2014. These were characterised by immunostaining, patch-clamp electrophysiology, insulin secretion, transcriptome analysis and transplantation into a streptozotocin (STZ)-induced mouse model of diabetes. RESULTS: The cryopreservation time was 17.6 ± 0.4 years (n = 43). The thawed islets stained positive with dithizone, contained insulin-positive and glucagon-positive cells, and displayed levels of apoptosis and transcriptome profiles similar to those of freshly isolated islets, although their insulin content was lower. The cryopreserved beta cells possessed ion channels and exocytotic responses identical to those of freshly isolated beta cells. Cells from a subset of five donors demonstrated similar perifusion insulin secretion profiles pre- and post-cryopreservation. The transplantation of cryopreserved islets into the diabetic mice improved their glucose tolerance but did not completely normalise their blood glucose levels. Circulating human insulin and insulin-positive grafts were detectable at 10 weeks post-transplantation. CONCLUSIONS/INTERPRETATION: We have demonstrated the potential for long-term banking of human islets for research, which could enable the use of tissue from a large number of donors with future technologies to gain new insight into diabetes.

Inhibition of Human Pancreatic Cancer Cell Proliferation by Devil's Club Oplopanax horridus and Its Polyacetylene Bioactive Compound.

Devil's club Oplopanax horridus (DC) is a close relative of ginseng; its inner root and stem bark extract showed antiproliferation activity on human leukemia, ovarian, breast and colon cancer cells. We study here the effects of DC 70% ethanol extract alone, or in combination with cisplatin, gemcitabine, and paclitaxel on pancreatic endocrine HP62 and pancreatic ductal carcinoma PANC-1 and BxPC-3 cells. Antiproliferation activity assay, cell cycle analysis by flow cytometry, apoptosis-related markers by antibody array, and RT-PCR assay were used for this study. DC extract inhibited proliferation of HP62 with IC50 (50% inhibition concentration) at 0.037±0.002% (v/v), PANC-1 at 0.0058 ± 0.0004% and BxPC-3 at 0.021 ± 0.003%. DC at 0.0033% combined with 1 nM of paclitaxel showed inhibition synergy on PANC-1 cells with a combination index of 0.44. Apoptosis focused antibody array profile indicated upregulation of cytochrome C, claspin, cIAP-2 and HTRA2/Omi apoptosis-related markers in DC-treated HP62 and PANC-1. Our data suggest that DC acts through targeting the intrinsic mitochondrial apoptosis pathway in the pancreatic cancer cells. The high antiproliferation potency of DC on PANC-1 is potentially useful as an adjunct therapy for treating pancreatic cancer, which is known for developing resistance to conventional chemotherapeutics.

Why Canada needs networks to provide rural surgical care, including family doctors with essential surgical skills.

SUMMARY: Time is long overdue for action to improve rural surgical services in Canada. In this issue of CJS, a proposed curriculum for the provision of enhanced surgical skills (ESS) to rural family physicians offers an opportunity to fortify a seamless network of high-quality surgical care for rural Canada. It is supported and enhanced by the best available evidence and measured advice from specialists and generalists alike. Publication of this curriculum proposal provides for essential dialogue with general surgeons. We discuss why we must play an active role in the development, teaching and evaluation of ESS, or we will have minimal influence and limited grounds on which to criticize its outcome or celebrate the opportunity of success it promises.

The role of caspase-8 in amyloid-induced beta cell death in human and mouse islets.

AIMS/HYPOTHESIS: Reduced beta cell mass due to increased beta cell apoptosis is a key defect in type 2 diabetes. Islet amyloid, formed by the aggregation of human islet amyloid polypeptide (hIAPP), contributes to beta cell death in type 2 diabetes and in islet grafts in patients with type 1 diabetes. In this study, we used human islets and hIAPP-expressing mouse islets with beta cell Casp8 deletion to (1) investigate the role of caspase-8 in amyloid-induced beta cell apoptosis and (2) test whether caspase-8 inhibition protects beta cells from amyloid toxicity. METHODS: Human islet cells were cultured with hIAPP alone, or with caspase-8, Fas or amyloid inhibitors. Human islets and wild-type or hIAPP-expressing mouse islets with or without caspase-8 expression (generated using a Cre/loxP system) were cultured to form amyloid. Caspase-8 and -3 activation, Fas and FLICE inhibitory protein (FLIP) expression, islet beta cell and amyloid area, IL-1ß levels, and the beta:alpha cell ratio were assessed. RESULTS: hIAPP treatment induced activation of caspase-8 and -3 in islet beta cells (via Fas upregulation), resulting in apoptosis, which was markedly reduced by blocking caspase-8, Fas or amyloid. Amyloid formation in cultured human and hIAPP-expressing mouse islets induced caspase-8 activation, which was associated with Fas upregulation and elevated islet IL-1ß levels. hIAPP-expressing mouse islets with Casp8 deletion had comparable amyloid, IL-1ß and Fas levels with those expressing hIAPP and Casp8, but markedly lower beta cell apoptosis, higher beta:alpha cell ratio, greater beta cell area, and enhanced beta cell function. CONCLUSIONS/INTERPRETATION: Beta cell Fas upregulation by endogenously produced and exogenously applied hIAPP aggregates promotes caspase-8 activation, resulting in beta cell apoptosis. The prevention of amyloid-induced caspase-8 activation enhances beta cell survival and function in islets.

Intravital biobank and personalized cancer therapy: the correlation with omics.

Biobanks have played a decisive role in all aspects of the field of cancer, including pathogenesis, diagnosis, prognosis and treatment. The significance of cancer biobanks is epitomized through the appropriate application of various "-omic" techniques (omics). The mutually motivated relationship between biobanks and omics has intensified the development of cancer research. Human cancer tissues that are maintained in intravital biobanks (or living tissue banks) retain native tumor microenvironment, tissue architecture, hormone responsiveness and cell-to-cell signalling properties. Intravital biobanks replicate the structural complexity and heterogeneity of human cancers, making them an ideal platform for preclinical studies. The application of omics with intravital biobanks renders them more active, which makes it possible for the cancer-related evaluations to be dynamically monitored on a real-time basis. Integrating intravital biobank and modern omics will provide a useful tool for the discovery and development of new drugs or novel therapeutic strategies. More importantly, intravital biobanks may play an essential role in the creation of meaningful patient-tailored therapies as for personalized medicine.

Effects of insulin on human pancreatic cancer progression modeled in vitro.

BACKGROUND: Pancreatic adenocarcinoma is one of the most lethal cancers, yet it remains understudied and poorly understood. Hyperinsulinemia has been reported to be a risk factor of pancreatic cancer, and the rapid rise of hyperinsulinemia associated with obesity and type 2 diabetes foreshadows a rise in cancer incidence. However, the actions of insulin at the various stages of pancreatic cancer progression remain poorly defined. METHODS: Here, we examined the effects of a range of insulin doses on signalling, proliferation and survival in three human cell models meant to represent three stages in pancreatic cancer progression: primary pancreatic duct cells, the HPDE immortalized pancreatic ductal cell line, and the PANC1 metastatic pancreatic cancer cell line. Cells were treated with a range of insulin doses, and their proliferation/viability were tracked via live cell imaging and XTT assays. Signal transduction was assessed through the AKT and ERK signalling pathways via immunoblotting. Inhibitors of AKT and ERK signalling were used to determine the relative contribution of these pathways to the survival of each cell model. RESULTS: While all three cell types responded to insulin, as indicated by phosphorylation of AKT and ERK, we found that there were stark differences in insulin-dependent proliferation, cell viability and cell survival among the cell types. High concentrations of insulin increased PANC1 and HPDE cell number, but did not alter primary duct cell proliferation in vitro. Cell survival was enhanced by insulin in both primary duct cells and HPDE cells. Moreover, we found that primary cells were more dependent on AKT signalling, while HPDE cells and PANC1 cells were more dependent on RAF/ERK signalling. CONCLUSIONS: Our data suggest that excessive insulin signalling may contribute to proliferation and survival in human immortalized pancreatic ductal cells and metastatic pancreatic cancer cells, but not in normal adult human pancreatic ductal cells. These data suggest that signalling pathways involved in cell survival may be rewired during pancreatic cancer progression.

Incretin-stimulated interaction between ß-cell Kv1.5 and Kvß2 channel proteins involves acetylation/deacetylation by CBP/SirT1.

The incretins, GIP (glucose-dependent insulinotropic polypeptide) and GLP-1 (glucagon-like peptide-1) are gastrointestinal hormones conferring a number of beneficial effects on ß-cell secretion, survival and proliferation. In a previous study, it was demonstrated that delayed rectifier channel protein Kv2.1 contributes to ß-cell apoptosis and that the prosurvival effects of incretins involve Kv2.1 PTMs (post-translational modifications), including phosphorylation and acetylation. Since Kv1.5 overexpression was also shown to stimulate ß-cell death, the present study was initiated in order to determine whether incretins modulate Kv1.5α-Kvß2 interaction via PTM and the mechanisms involved. GIP and GLP-1 reduced apoptosis in INS-1 ß-cells (clone 832/13) overexpressing Kv1.5, and RNAi (RNA interference)-mediated knockdown of endogenous Kv1.5 attenuated apoptotic ß-cell death. Both GIP and GLP-1 increased phosphorylation and acetylation of Kv1.5 and its Kvß2 protein subunit, leading to their enhanced interaction. Further studies demonstrated that CBP [CREB (cAMP-response-element-binding protein)-binding protein]/SirT1 mediated acetylation/deacetylation and interaction between Kvß2 and Kv1.5 in response to GIP or GLP-1. Incretin regulation of ß-cell function therefore involves the acetylation of multiple Kvα and Kvß subunits.

Thyroid Hormone Levels Correlate With the Maturation of Implanted Pancreatic Endoderm Cells in Patients With Type 1 Diabetes.

BACKGROUND: Macroencapsulated pancreatic endoderm cells (PECs) can reverse diabetes in rodents and preclinical studies revealed that thyroid hormones in vitro and in vivo bias PECs to differentiate into insulin-producing cells. In an ongoing clinical trial, PECs implanted in macroencapsulation devices into patients with type 1 diabetes were safe but yielded heterogeneous outcomes. Though most patients developed meal responsive C-peptide, levels were heterogeneous and explanted grafts had variable numbers of surviving cells with variable distribution of endocrine cells. METHODS: We measured circulating triiodothyronine and thyroxine levels in all patients treated at 1 of the 7 sites of the ongoing clinical trial and determined if thyroid hormone levels were associated with the C-peptide or glucagon levels and cell fate of implanted PECs. RESULTS: Both triiodothyronine and thyroxine levels were significantly associated with the proportion of cells that adopted an insulin-producing fate with a mature phenotype. Thyroid hormone levels were inversely correlated to circulating glucagon levels after implantation, suggesting that thyroid hormones lead PECs to favor an insulin-producing fate over a glucagon-producing fate. In mice, hyperthyroidism led to more rapid maturation of PECs into insulin-producing cells similar in phenotype to PECs in euthyroid mice. CONCLUSION: These data highlight the relevance of thyroid hormones in the context of PEC therapy in patients with type 1 diabetes and suggest that a thyroid hormone adjuvant therapy may optimize cell outcomes in some PEC recipients.

B7-H4.Ig inhibits the development of type 1 diabetes by regulating Th17 cells in NOD mice.

Type 1 diabetes (T1D) is an autoimmune disease characterized by immunological destruction of insulin-producing pancreatic ß-cells and subsequent hyperglycemia. The non-obese diabetic (NOD) mouse strain spontaneously develops a disease similar to human T1D and is commonly used as an animal model for studying this disease. We have previously shown that the administration of B7-H4-immunoglobulin fusion protein (B7-H4.Ig), a newly identified T-cell co-inhibitory signaling molecule, blocks the onset of diabetes in NOD mice. However, the mechanism(s) by which B7-H4 protects NOD mice from T1D is not fully understood. IL-17 is a pro-inflammatory cytokine, produced by Th17 cells, that activates T cells and other immune cells to produce a variety of cytokines and chemokines. Increasing evidence has shown that therapeutic agents targeting the IL-17 molecule or directly inhibiting IL-17-producing cells regulate autoimmune diabetes in NOD mice, suggesting that IL-17 is involved in the pathogenesis of this disease. In this study, we investigate whether B7-H4.Ig treatment inhibits the generation of Th17 cells which subsequently decreases IL-17 production and prevents the onset of T1D in NOD mice. Pre-diabetic female NOD mice were injected intraperitoneally with control mouse IgG or B7-H4.Ig starting at 4 weeks of age for 12 weeks. Our data showed that the frequency of Th17 cells in B7-H4.Ig-treated mice was significantly decreased. In addition, our data showed that B7-H4.Ig-treated mice had decreased levels of pro-inflammatory cytokines and Th17-associated cytokines, and an increased level of the potent Th17 inhibitor IFN-γ. To further investigate the effect of B7-H4.Ig on differentiation of Th17 cells, we co-cultured splenocytes with Th17-polarizing cytokines in the absence or presence of B7-H4.Ig. Our results indicated that splenocytes, under the Th17 driving conditions in the presence of B7-H4.Ig, had significantly decreased the numbers of Th17 cells compared to cells co-cultured in the absence of B7-H4.Ig. Together, this study suggests that blocking the generation of Th17 cells with the administration of B7-H4.Ig effectively inhibits the development of T1D in NOD mice.

Three-dimensional scaffolds reduce islet amyloid formation and enhance survival and function of cultured human islets.

Islet transplantation provides a promising approach for treatment of type 1 diabetes mellitus. Amyloid formation and loss of extracellular matrix are two nonimmune factors contributing to death of isolated human islets. We tested the effects of two types of three-dimensional scaffolds, collagen matrix (CM) and fibroblast-populated collagen matrix (FPCM), on amyloid formation, viability, and function of isolated islets. Islets from cadaveric donors were cultured in FPCM, CM, or two-dimensional plate (2D) for 7 days. After 7 days, compared with the 2D culture condition, CM and FPCM markedly reduced amyloid formation of cultured islets and decreased apoptotic ß-cell rate by ∼75%. IL-1ß and Fas levels were also reduced in scaffold-embedded islets. Furthermore, ß/α cell ratios were increased by ∼18% and ∼36% in CM- and FPCM-embedded islets, respectively. Insulin content and insulin response to elevated glucose were also enhanced by both three-dimensional scaffolds. Moreover, culture in CM and FPCM (but not 2D) preserved insulin, GLUT-2, and PDX-1 mRNA expression. FPCM-embedded islets had significantly higher insulin response and lower amyloid formation than CM-embedded islets. These findings suggest that three-dimensional scaffolds reduce amyloid formation and improve viability and function of human islets in vitro, and that CM and fibroblasts have additive effects in enhancing islet function and reducing amyloid formation. Using this strategy is likely to improve outcome in human islet transplantation.

Effect of bariatric surgery on oncologic outcomes: a systematic review and meta-analysis.

BACKGROUND: Obesity is a major public health issue and is associated with increased risk of several cancers, currently a leading cause of mortality. Obese patients undergoing bariatric surgery may allow for evaluation of the effect of intentional excess weight loss on subsequent risk of cancer. We aimed to evaluate cancer risk, incidence, and mortality after bariatric surgery. METHODS: A comprehensive literature search was conducted using PubMed/MEDLINE and Embase with literature published from the inception of both databases to January 2012. Inclusion criteria incorporated all human studies examining oncologic outcomes after bariatric surgery. Two authors independently reviewed selected studies and relevant articles from their bibliographies for data extraction, quality appraisal, and meta-analysis. RESULTS: Six observational studies (n = 51,740) comparing relative risk (RR) of cancer in obese patients undergoing bariatric surgery versus obese control subjects were analyzed. Overall, the RR of cancer in obese patients after undergoing bariatric surgery was 0.55 [95% confidence interval (CI) 0.41-0.73, p < 0.0001, I(2) = 83%]. The effect of bariatric surgery on cancer risk was modified by gender (p = 0.021). The pooled RR in women was 0.68 (95% CI 0.60-0.77, p < 0.0001, I(2) < 0.1%) and in men was 0.99 (95% CI 0.74-1.32, p = 0.937, I(2) < 0.1%). CONCLUSIONS: Bariatric surgery reduces cancer risk and mortality in formerly obese patients. When stratifying the meta-analysis by gender, the effect of bariatric surgery on oncologic outcomes is protective in women but not in men.

Determination of factors predictive of outcome for patients undergoing a pancreaticoduodenectomy of pancreatic head ductal adenocarcinomas.

INTRODUCTION: A pancreaticoduodenectomy is the reference treatment for a resectable pancreatic head ductal adenocarcinoma. The probability of 5-year survival in patients undergoing such treatment is 5-25% and is associated with relatively high peri-operative morbidity and mortality. The objective of the present study was to evaluate risk factors predictive of outcome for patients undergoing a pancreaticoduodenectomy for a pancreatic adenocarcinoma. METHODS: This retrospective analysis incorporated data from the Vancouver General Hospital and the British Columbia Cancer Agency (BCCA) from 1999-2007. RESULTS: The 5-year survival of 100 patients was 12% with a median survival of 16.5 months. Ninety-day mortality was 7%. Predictors of 90-day mortality included age ≥ 80 years (P < 0.001) and an American Society of Anesthesiologists (ASA) score = 3 (P= 0.012) by univariate analysis and age ≥80 years (P < 0.001) by multivariate analysis. The identifiable predictive factor for poor 5-year survival was an ASA score = 3 (P= 0.043) whereas a Dindo-Clavien surgical complication grade ≥ 3 was associated with a worse outcome (P= 0.013). Referral to the BCCA was associated with a favourable 5-year survival (P= 0.001). CONCLUSIONS: The present study identifies risk factors for patient selection to enhance survival benefit in this patient population.

Fibroblast populated collagen matrix promotes islet survival and reduces the number of islets required for diabetes reversal.

Islet transplantation represents a viable treatment for type 1 diabetes. However, due to loss of substantial mass of islets early after transplantation, islets from two or more donors are required to achieve insulin independence. Islet-extracellular matrix disengagement, which occurs during islet isolation process, leads to subsequent islet cell apoptosis and is an important contributing factor to early islet loss. In this study, we developed a fibroblast populated collagen matrix (FPCM) as a novel scaffold to improve islet cell viability and function post-transplantation. FPCM was developed by embedding fibroblasts within type-I collagen and used as scaffold for islet grafts. Viability and insulin secretory function of islets embedded within FPCM was evaluated in vitro and in a syngeneic murine islet transplantation model. Islets embedded within acellular matrix or naked islets were used as control. Islet cell survival and function was markedly improved particularly after embedding within FPCM. The composite scaffold significantly promoted islet isograft survival and reduced the critical islet mass required for diabetes reversal by half (from 200 to 100 islets per recipient). Fibroblast embedded within FPCM produced fibronectin and growth factors and induced islet cell proliferation. No evidence of fibroblast over-growth within composite grafts was noticed. These results confirm that FPCM significantly promotes islet viability and functionality, enhances engraftment of islet grafts and decreases the critical islet mass needed to reverse hyperglycemia. This promising finding offers a new approach to reducing the number of islet donors per recipient and improving islet transplant outcome.

Glucose-dependent insulinotropic polypeptide is expressed in pancreatic islet alpha-cells and promotes insulin secretion.

BACKGROUND & AIMS: Glucose-dependent insulinotropic polypeptide (GIP) and the proglucagon product glucagon-like peptide-1 (GLP-1) are gastrointestinal hormones that are released in response to nutrient intake and promote insulin secretion. Interestingly, a subset of enteroendocrine cells express both GIP and GLP-1. We sought to determine whether GIP also might be co-expressed with proglucagon in pancreatic alpha-cells. METHODS: We assessed GIP expression via reverse-transcription polymerase chain reaction, in situ hybridization, and immunohistochemistry. We developed a novel bioassay to measure GIP release from isolated islets, compared the biological activities of full-length and truncated GIP, and assessed the impact of immunoneutralization of islet GIP on glucose-stimulated insulin secretion in isolated islets. RESULTS: GIP messenger RNA was present in mouse islets; GIP protein localized to islet alpha-cells of mouse, human, and snake pancreas, based on immunohistochemical analyses. However, using a C-terminal GIP antibody, immunoreactivity was detected in islets from prohormone convertase (PC) 2 knockout but not wild-type mice. Bioactive GIP was secreted from mouse and human islets after arginine stimulation. In the perfused mouse pancreas, GIP(1-42) and amidated GIP(1-30) had equipotent insulinotropic actions. Finally, immunoneutralization of GIP secreted by isolated islets decreased glucose-stimulated insulin secretion. CONCLUSIONS: GIP is expressed in and secreted from pancreatic islets; in alpha-cells, PC2 processes proGIP to yield a truncated but bioactive form of GIP that differs from the PC1/3-derived form from K-cells. Islet-derived GIP promotes islet glucose competence and also could support islet development and/or survival.

The role of mitochondrial apoptotic pathway in islet amyloid-induced ß-cell death.

Islet amyloid, formed by aggregation of human islet amyloid polypeptide (hIAPP), contributes to ß-cell death in type 2 diabetes. We previously showed that extracellular hIAPP aggregates promote Fas-mediated ß-cell apoptosis. Here, we tested if hIAPP aggregates can trigger the mitochondrial apoptotic pathway (MAP). hIAPP aggregation in Ad-hIAPP transduced INS-1 and human islet ß-cells promoted cytochrome c release, caspase-9 activation and apoptosis, which were reduced by Bax inhibitor. Amyloid formation in hIAPP-expressing mouse islets during culture increased caspase-9 activation in ß-cells. Ad-hIAPP transduced islets from CytcKA/KA and BaxBak ßDKO mice (models of blocked MAP), had lower caspase-9-positive and apoptotic ß-cells than transduced wild-type islets, despite comparable amyloid formation. Blocking Fas (markedly) and Bax or caspase-9 (modestly) reduced ß-cell death induced by extracellular hIAPP aggregates. These findings suggest a role for MAP in amyloid-induced ß-cell death and a potential strategy to reduce intracellular amyloid ß-cell toxicity by blocking cytochrome c apoptotic function.

Implanted pluripotent stem-cell-derived pancreatic endoderm cells secrete glucose-responsive C-peptide in patients with type 1 diabetes.

An open-label, first-in-human phase 1/2 study is being conducted to evaluate the safety and efficacy of pancreatic endoderm cells (PECs) implanted in non-immunoprotective macroencapsulation devices for the treatment of type 1 diabetes. We report an analysis on 1 year of data from the first cohort of 15 patients from a single trial site that received subcutaneous implantation of cell products combined with an immunosuppressive regimen. Implants were well tolerated with no teratoma formation or severe graft-related adverse events. After implantation, patients had increased fasting C-peptide levels and increased glucose-responsive C-peptide levels and developed mixed meal-stimulated C-peptide secretion. There were immunosuppression-related transient increases in circulating regulatory T cells, PD1high T cells, and IL17A+CD4+ T cells. Explanted grafts contained cells with a mature ß cell phenotype that were immunoreactive for insulin, islet amyloid polypeptide, and MAFA. These data, and associated findings (Shapiro et al., 2021), are the first reported evidence of meal-regulated insulin secretion by differentiated stem cells in patients.

Suppression of p38 MAPK and JNK via Akt-mediated inhibition of apoptosis signal-regulating kinase 1 constitutes a core component of the beta-cell pro-survival effects of glucose-dependent insulinotropic polypeptide.

Glucose-dependent insulinotropic polypeptide (GIP) potentiates glucose-stimulated insulin secretion, insulin biosynthesis, and beta-cell proliferation and survival. In previous studies GIP was shown to promote beta-cell survival by modulating the activity of multiple signaling modules and regulating gene transcription of pro- and anti-apoptotic bcl-2 family proteins. We have now evaluated the mechanisms by which GIP regulates the dynamic interactions between cytoplasmic bcl-2 family members and the mitochondria in INS-1 cells during apoptosis induced by treatment with staurosporine (STS), an activator of the mitochondria-mediated apoptotic pathway. STS induced translocation of bad and bimEL, activation of mitochondrial bax, release of mitochondrial cytochrome c, cleavage of caspase-3, and apoptosis. Each response was significantly diminished by GIP. Using selective enzyme inhibitors, overexpression of dominant-negative Akt, and Akt siRNA, it was demonstrated that GIP promoted beta-cell survival via Akt-dependent suppression of p38 MAPK and JNK and that combined inhibition was sufficient to explain the entire pro-survival responses to GIP during STS treatment. This signaling pathway also explained the pro-survival effects of GIP on INS-1 cells exposed to two other promoters of stress: thapsigargin (endoplasmic reticulum stress) and etoposide (genotoxic stress). Importantly, we discovered that GIP suppressed p38 MAPK and JNK via Akt-mediated changes in the phosphorylation state of the apoptosis signal-regulating kinase 1 in INS-1 cells and human islets, resulting in inhibition of its activity. Inhibition of apoptosis by GIP is therefore mediated via a key pathway involving Akt-dependent inhibition of apoptosis signal-regulating kinase 1, which subsequently prevents the pro-apoptotic actions of p38 MAPK and JNK.