ABSTRACTFibrogenic and inflammatory processes in the prostate are linked to the development of lower urinary tract symptoms (LUTS) in men. Our previous studies identified that osteopontin (OPN), a pro-fibrotic cytokine, is abundant in the prostate of men with LUTS and its secretion is stimulated by inflammatory cytokines potentially to drive fibrosis. This study investigates whether the lack of OPN ameliorates inflammation and fibrosis in the mouse prostate.We instilled uropathogenic E. coli (UTI89) or saline (control) transurethrally to C57BL/6J (WT) or Spp1tm1Blh/J (OPN-KO) mice and collected the prostates one or 8 weeks later. We found that OPN mRNA and protein expression were significantly induced by E. coli-instillation in the dorsal prostate (DP) after one week in WT mice. Deficiency in OPN expression led to decreased inflammation and fibrosis and the prevention of urinary dysfunction after 8 weeks. RNAseq analysis identified that E. coli-instilled WT mice expressed increased levels of inflammatory and fibrotic marker RNAs compared to OPN-KO mice including Col3a1, Dpt, Lum and Mmp3 which were confirmed by RNAscope.Our results indicate that OPN is induced by inflammation and prolongs the inflammatory state; genetic blockade of OPN accelerates recovery after inflammation, including a resolution of prostate fibrosis.

Fibrogenic and inflammatory processes in the prostate are linked to the development of lower urinary tract symptoms (LUTS) in men. Our previous studies identified that osteopontin (OPN), a pro-fibrotic cytokine, is abundant in the prostate of men with LUTS, and its secretion is stimulated by inflammatory cytokines potentially to drive fibrosis. This study investigates whether the lack of OPN ameliorates inflammation and fibrosis in the mouse prostate. We instilled uropathogenic E. coli (UTI89) or saline (control) transurethrally to C57BL/6J (WT) or Spp1(tm1Blh)/J (OPN-KO) mice and collected the prostates one or 8 weeks later. We found that OPN mRNA and protein expression were significantly induced by E. coli-instillation in the dorsal prostate (DP) after one week in WT mice. Deficiency in OPN expression led to decreased inflammation and fibrosis and the prevention of urinary dysfunction after 8 weeks. RNAseq analysis identified that E. coli-instilled WT mice expressed increased levels of inflammatory and fibrotic marker RNAs compared to OPN-KO mice including Col3a1, Dpt, Lum and Mmp3 which were confirmed by RNAscope. Our results indicate that OPN is induced by inflammation and prolongs the inflammatory state; genetic blockade of OPN accelerates recovery after inflammation, including a resolution of prostate fibrosis.

You have accessJournal of UrologyBenign Prostatic Hyperplasia: Basic Research & Pathophysiology (PD11)1 Sep 2021PD11-04 LOSS OF OSTEOPONTIN LEADS TO THE RESOLUTION OF E. COLI-INDUCED PROSTATIC INFLAMMATION AND FIBROSIS Petra Popovics, Asha Jain, Francesca Van, Hannah Ruetten, Mark Cadena, Kristen S. Uchtmann, Chad M. Vezina, and William A. Ricke Petra PopovicsPetra Popovics More articles by this author , Asha JainAsha Jain More articles by this author , Francesca VanFrancesca Van More articles by this author , Hannah RuettenHannah Ruetten More articles by this author , Mark CadenaMark Cadena More articles by this author , Kristen S. UchtmannKristen S. Uchtmann More articles by this author , Chad M. VezinaChad M. Vezina More articles by this author , and William A. RickeWilliam A. Ricke More articles by this author View All Author Informationhttps://doi.org/10.1097/JU.0000000000001986.04AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookLinked InTwitterEmail Abstract INTRODUCTION AND OBJECTIVE: Fibrogenic and inflammatory processes in the prostate are linked to the development of lower urinary tract symptoms (LUTS) in men, but the molecular mechanism is unknown. We previously established that osteopontin (OPN), a pro-fibrotic cytokine, is more abundant in the prostate of men with LUTS compared to prostate tissue with normal histology. We also identified that OPN secretion is stimulated by inflammatory cytokines in prostate stromal cells and hypothesized that OPN exacerbates the prostatic inflammatory environment. The current study investigates whether the lack of OPN ameliorates inflammation-induced prostatic fibrosis and urinary dysfunction in mice following transurethral instillation of E. coli. METHODS: Uropathogenic Escherichia coli (UTI89) or saline (control) was instilled via a transurethral catheter (OD 0.8, 100 ul) two times, 3 days apart to C57BL/6J (WT) or B6.129S6(Cg)-Spp1tm1Blh/J (OPN-KO) mice. Urinary function was assessed by weekly void spot assays (VSA). Prostatic collagen was visualized with picrosirius red (PSR) staining and inflammation by immunohistochemistry of the pan-leukocyte marker CD45. RNA-Seq analysis was performed from the ventral prostate lobe. RESULTS: Urinary frequency significantly increased (2.05-fold, p=0.0097) 33 days after E. coli instillation in WT but not in OPN-KO mice. One week after infection, the ventral and dorsal prostate lobes were significantly inflamed (more CD45 cells) and collagen fibers were thicker in both WT and OPN-KO mice. In contrast, 2 months after instillation, OPN-KO mouse prostates contained significantly less total collagen and inflammation compared to WT mice. RNAseq analysis identified that E. coli-instilled WT mice expressed more inflammatory and fibrotic marker RNAs, such as Col3a1, Il1b, MMP3, Mmp7 and Cxcl12 than OPN-KO mice. CONCLUSIONS: Our results show that by preventing the inflammation-related increase in OPN levels, mice undergo accelerated recovery from fibrosis and do not develop chronic inflammation. This suggests that drugs targeting OPN signaling could be beneficial for LUTS patients whose symptoms are primarily linked to fibrosis. Source of Funding: The study was supported by an NIH NIDDK K01 (K01DK127150) and a K12 award (K12DK100022-06, both to PP) and by U54 DK104310 (to WAR and CMV) © 2021 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 206Issue Supplement 3September 2021Page: e198-e199 Advertisement Copyright & Permissions© 2021 by American Urological Association Education and Research, Inc.MetricsAuthor Information Petra Popovics More articles by this author Asha Jain More articles by this author Francesca Van More articles by this author Hannah Ruetten More articles by this author Mark Cadena More articles by this author Kristen S. Uchtmann More articles by this author Chad M. Vezina More articles by this author William A. Ricke More articles by this author Expand All Advertisement PDF downloadLoading ...

Bacterial infection is one known etiology of prostatic inflammation. Prostatic inflammation is associated with prostatic collagen accumulation and both are linked to progressive lower urinary tract symptoms in men. We characterized a model of prostatic inflammation using transurethral instillations of Escherichia coli UTI89 in C57BL/6J male mice with the goal of determining the optimal instillation conditions, understanding the impact of instillation conditions on urinary physiology, and identifying ideal prostatic lobes and collagen 1a1 prostatic cell types for further analysis. The smallest instillation volume tested (50 µL) distributed exclusively to the bladder, 100- and 200-µL volumes distributed to the bladder and prostate, and a 500-µL volume distributed to the bladder, prostate, and ureter. A threshold optical density of 0.4 E. coli UTI89 in the instillation fluid was necessary for significant ( P < 0.05) prostate colonization. E. coli UTI89 infection resulted in a low frequency, high volume spontaneous voiding pattern. This phenotype was due to exposure to E. coli UTI89, not catheterization alone, and was minimally altered by a 50-µL increase in instillation volume and doubling of E. coli concentration. Prostate inflammation was isolated to the dorsal prostate and was accompanied by increased collagen density. This was partnered with increased density of protein tyrosine phosphatase receptor type C + , procollagen type I-α 1 + copositive cells and decreased density of α 2 -smooth muscle actin + , procollagen type I-α 1 + copositive cells. Overall, we determined that this model is effective in altering urinary phenotype and producing prostatic inflammation and collagen accumulation in mice.

BACKGROUND:Castration-insensitive epithelial progenitors capable of regenerating the prostate have been proposed to be concentrated in the proximal region based on facultative assays. Functional characterization of prostate epithelial populations isolated with individual cell surface markers has failed to provide a consensus on the anatomical and transcriptional identity of proximal prostate progenitors. METHODS:Here, we use single-cell RNA sequencing to obtain a complete transcriptomic profile of all epithelial cells in the mouse prostate and urethra to objectively identify cellular subtypes. Pan-transcriptomic comparison to human prostate cell types identified a mouse equivalent of human urethral luminal cells, which highly expressed putative prostate progenitor markers. Validation of the urethral luminal cell cluster was performed using immunostaining and flow cytometry. RESULTS:Our data reveal that previously identified facultative progenitors marked by Trop2, Sca-1, KRT4, and PSCA are actually luminal epithelial cells of the urethra that extend into the proximal region of the prostate, and are resistant to castration-induced androgen deprivation. Mouse urethral luminal cells were identified to be the equivalent of previously identified human club and hillock cells that similarly extend into proximal prostate ducts. Benign prostatic hyperplasia (BPH) has long been considered an "embryonic reawakening," but the cellular origin of the hyperplastic growth concentrated in the periurethral region is unclear. We demonstrate an increase in urethral luminal cells within glandular nodules from BPH patients. Urethral luminal cells are further increased in patients treated with a 5-α reductase inhibitor. CONCLUSIONS:Our data demonstrate that cells of the proximal prostate that express putative progenitor markers, and are enriched by castration in the proximal prostate, are urethral luminal cells and that these cells may play an important role in the etiology of human BPH.

NBCe1 is an electrogenic Na + bicarbonate cotransporter expressed as three isoforms. NBCe1A is mainly found in the kidney (100% activity), NBCe1B is a general isoform (~25% activity), and NBCe1C is found in the nervous system (20% activity). This project was to characterize mice with loss of NBCe1 in the kidney and urogenital tract and to test isoform activity ± IRBIT. Previous work has shown that NBCe1‐B is activated when IRBIT is present and interacts with the N‐terminus (Nt). Even though NBCe1C shares this Nt, the effects of IRBIT on human NBCe1C are unknown. Interstitial Cells of Cajal (ICC)pacemaker cells in the gastrointestinal tract ( c‐kit + ) have NBCe1C, IRBIT and require bicarbonate for normal gut activity. Developmentally similar ICC cells are found in the urogenital tract; however, NBCe1C locations within the kidney and urogenital tract are unknown. Using c‐kit ‐copGFP and NBCe1A‐knockout mice, kidneys, ureters, prostate, bladder, and urethra were dissected and analyzed via direct imaging, sectioning, and immunofluorescence. Oocytes injected with NBCe1C ± IRBIT were voltage clamped to determine the cotransporter's activity. “Strings” of c‐kit + ‐cells were found in the copGFP & nbce1A kidneys. In nbce1 (+/−) ‐(Het) mice (Figure), prostate ducts appeared enlarged, and in nbce1A − / − kidney, c‐kit protein was broadly expressed, showing cluster colocalization with NBCe1C. NBCe1C is not obviously expressed in WT or copGFP mice. Electrophysiology data showed that IRBIT activates NBCe1C by ~10‐fold. As c‐kit is also a marker of stem cells, NBCe1C/c‐kit association and c‐kit increase in nbce1A − / − mice, could suggest that NBCe1A loss elicits kidney repair itself (proliferation) and causes expression of other NBCe1‐isoforms. Support or Funding Information R25‐DK101405, R01‐DK057061, U54‐DK100227 (Mayo) and U54‐DK104310 (UW‐Madison) This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .

Prostate autonomic and sensory axons control glandular growth, fluid secretion, and smooth muscle contraction and are remodeled during cancer and inflammation. Morphogenetic signaling pathways reawakened during disease progression may drive this axon remodeling. These pathways are linked to proliferative activities in prostate cancer and benign prostate hyperplasia. However, little is known about which developmental signaling pathways guide axon investment into prostate. The first step in defining these pathways is pinpointing when axon subtypes first appear in prostate. We accomplished this by immunohistochemically mapping three axon subtypes (noradrenergic, cholinergic, and peptidergic) during fetal, neonatal, and adult stages of mouse prostate development. We devised a method for peri-prostatic axon density quantification and tested whether innervation is uniform across the proximo-distal axis of dorsal and ventral adult mouse prostate. Many axons directly interact with or innervate neuroendocrine cells in other organs, so we examined whether sensory or autonomic axons innervate neuroendocrine cells in prostate. We first detected noradrenergic, cholinergic, and peptidergic axons in prostate at embryonic day (E) 14.5. Noradrenergic and cholinergic axon densities are uniform across the proximal-distal axis of adult mouse prostate while peptidergic axons are denser in the periurethral and proximal regions. Peptidergic and cholinergic axons are closely associated with prostate neuroendocrine cells whereas noradrenergic axons are not. These results provide a foundation for understanding mouse prostatic axon development and organization and, provide strategies for quantifying axons during progression of prostate disease.

The α-subunit of the heterotrimeric Gz protein, Gαz, promotes β-cell death and inhibits β-cell replication when pancreatic islets are challenged by stressors. Thus, we hypothesized that loss of Gαz protein would preserve functional β-cell mass in the nonobese diabetic (NOD) model, protecting from overt diabetes. We saw that protection from diabetes was robust and durable up to 35 weeks of age in Gαz knockout mice. By 17 weeks of age, Gαz-null NOD mice had significantly higher diabetes-free survival than wild-type littermates. Islets from these mice had reduced markers of proinflammatory immune cell infiltration on both the histological and transcript levels and secreted more insulin in response to glucose. Further analyses of pancreas sections revealed significantly fewer terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive β-cells in Gαz-null islets despite similar immune infiltration in control mice. Islets from Gαz-null mice also exhibited a higher percentage of Ki-67-positive β-cells, a measure of proliferation, even in the presence of immune infiltration. Finally, β-cell-specific Gαz-null mice phenocopy whole-body Gαz-null mice in their protection from developing hyperglycemia after streptozotocin administration, supporting a β-cell-centric role for Gαz in diabetes pathophysiology. We propose that Gαz plays a key role in β-cell signaling that becomes dysfunctional in the type 1 diabetes setting, accelerating the death of β-cells, which promotes further accumulation of immune cells in the pancreatic islets, and inhibiting a restorative proliferative response.

The alpha-subunit of the heterotrimeric G(z) protein, G alpha(z), promotes beta-cell death and inhibits beta-cell replication when pancreatic islets are challenged by stressors. Thus, we hypothesized that loss of Gaz protein would preserve functional beta-cell mass in the nonobese diabetic (NOD) model, protecting from overt diabetes. We saw that protection from diabetes was robust and durable up to 35 weeks of age in G alpha(z) knockout mice. By 17 weeks of age, G alpha(z)-null NOD mice had significantly higher diabetes-free survival than wild-type littermates. Islets from these mice had reduced markers of proinflammatory immune cell infiltration on both the histological and transcript levels and secreted more insulin in response to glucose. Further analyses of pancreas sections revealed significantly fewer terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling (TUNEL)-positive beta-cells in G alpha(z)-null islets despite similar immune infiltration in control mice. Islets from G alpha(z)-null mice also exhibited a higher percentage of Ki-67-positive beta-cells, a measure of proliferation, even in the presence of immune infiltration. Finally, beta-cell-specific G alpha(z)-nullmice phenocopy whole-body G alpha(z)-null mice in their protection from developing hyperglycemia after streptozotocin administration, supporting a beta-cell-centric role for G alpha(z) in diabetes pathophysiology. We propose that G alpha(z) plays a key role in beta-cell signaling that becomes dysfunctional in the type 1 diabetes setting, accelerating the death of beta-cells, which promotes further accumulation of immune cells in the pancreatic islets, and inhibiting a restorative proliferative response.

Though many methods can be used to identify cell types contained in complex tissues, most require cell disaggregation and destroy information about where cells reside in relation to their microenvironment. Here, we describe a polytomous key for cell type identification in intact sections of adult mouse prostate and prostatic urethra. The key is organized as a decision tree and initiates with one round of immunostaining for nerve, epithelial, fibromuscular/hematolymphoid, or vascular associated cells. Cell identities are recursively eliminated by subsequent staining events until the remaining pool of potential cell types can be distinguished by direct comparison to other cells. We validated our identification key using wild type adult mouse prostate and urethra tissue sections and it currently resolves sixteen distinct cell populations which include three nerve fiber types as well as four epithelial, five fibromuscular/hematolymphoid, one nerve-associated, and three vascular-associated cell types. We demonstrate two uses of this novel identification methodology. We first used the identification key to characterize prostate stromal cell type changes in response to constitutive phosphatidylinositide-3-kinase activation in prostate epithelium. We then used the key to map cell lineages in a new reporter mouse strain driven by Wnt10aem1(cre/ERT2)Amc. The identification key facilitates rigorous and reproducible cell identification in prostate tissue sections and can be expanded to resolve additional cell types as new antibodies and other resources become available.

A defining characteristic of type 1 diabetes mellitus (T1DM) pathophysiology is pancreatic β-cell death and dysfunction, resulting in insufficient insulin secretion to properly control blood glucose levels. Treatments that promote β-cell replication and survival, thus reversing the loss of β-cell mass, while also preserving β-cell function, could lead to a real cure for T1DM. The α-subunit of the heterotrimeric Gz protein, Gαz, is a tonic negative regulator of adenylate cyclase and downstream cAMP production. cAMP is one of a few identified signaling molecules that can simultaneously have a positive impact on pancreatic islet β-cell proliferation, survival, and function. The purpose of our study was to determine whether mice lacking Gαz might be protected, at least partially, from β-cell loss and dysfunction after streptozotocin treatment. We also aimed to determine whether Gαz might act in concert with an activator of the cAMP-stimulatory glucagon-like peptide 1 receptor, exendin-4 (Ex4). Without Ex4 treatment, Gαz-null mice still developed hyperglycemia, albeit delayed. The same finding held true for wild-type mice treated with Ex4. With Ex4 treatment, Gαz-null mice were protected from developing severe hyperglycemia. Immunohistological studies performed on pancreas sections and in vitro apoptosis, cytotoxicity, and survival assays demonstrated a clear effect of Gαz signaling on pancreatic β-cell replication and death; β-cell function was also improved in Gαz-null islets. These data support our hypothesis that a combination of therapies targeting both stimulatory and inhibitory pathways will be more effective than either alone at protecting, preserving, and possibly regenerating β-cell mass and function in T1DM.

Type I Diabetes (TID) is fundamentally characterized by insulitis and islet inflammation which, ultimately, leads to β‐cell death. The non‐obese diabetic (NOD) mouse is a well‐accepted model of TID as they exhibit insulitis, hyperglycemia and ultimately β‐cell failure. We have previously shown that loss of the catalytic alpha subunit of the inhibitory heterotrimeric G‐protein, G z , Gα z , ameliorates streptozotocin (STZ)‐induced hyperglycemia, a chemically‐induced model of TID. We have gone on to confirm this protection in the NOD model and delineate the mechanisms by which this protection is conferred. After backcrossing the Gα z ‐null mutation into the NOD background we tracked blood glucose and body weight for 17 weeks. Our results show that Gα z ‐null mice were nearly completely protected from developing diabetes during this timeframe, whether expressed as mean blood glucose or disease‐free survival. At 4–5 weeks, when we expect apoptosis to be high, Gα z ‐null mice exhibit significantly reduced terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)‐positive β‐cells. In correspondence, Gα z ‐null mice have significantly increased insulin positive pancreas area as demonstrated by immunohistochemistry (IHC). We hypothesized that decreased apoptosis would result in decreased immune infiltration. Indeed, we found that islets from Gα z ‐null mice exhibited a significant reduction in immune cell presence as analyzed by hematoxylin and eosin staining. Furthermore, Gα z ‐null islets demonstrate significantly reduced pro‐inflammatory immune cell markers and cytokines at the transcript level as compared to wild‐type controls at 16 weeks of age. We also observed a significant increase in glucose‐stimulated insulin secretion compared to wild‐type controls at 16 weeks of age. Our current work is to delineate the mechanisms by which Gα z exerts a tonic inhibition on β‐cell function and survival. The canonical role for Gα z , upon activation, is inhibition of adenylyl cyclase, thereby reducing intracellular levels of the ubiquitous second messenger molecule cyclic adenosine monophosphate (cAMP). We, and others, have already shown that expression of the islet autocrine/paracrine peptide hormone, cholecystokinin (CCK) is regulated by cAMP‐responsive transcription factors, and that CCK promotes β‐cell function and survival. Interestingly, CCK mRNA expression is increased four‐fold in Gα z ‐null NOD islets, whereas the mRNA expression of its receptor is increased 14‐fold. Our hypothesis is that Gα z regulation of cAMP interferes with the entire CCK synthesis and signaling pathway, negatively regulating β‐cell function and growth in the diabetic condition. Overall, our results confirm loss of Gα z protects NOD mice from the development of TID‐like islet pathogenesis and suggests that the Gα z signaling pathway may be a potential therapeutic target due to its multifaceted effects on β‐cell function and survival.

Stimulation of digestive organs by enteric peptides is lost during total parental nutrition (PN). Here we examine the role of the enteric peptide bombesin (BBS) in stimulation of the exocrine and endocrine pancreas during PN. BBS protects against exocrine pancreas atrophy and dysfunction caused by PN. BBS also augments circulating insulin levels, suggesting an endocrine pancreas phenotype. While no significant changes in gross endocrine pancreas morphology were observed, pancreatic islets isolated from BBS-treated PN mice showed a significantly enhanced insulin secretion response to the glucagon-like peptide-1 (GLP-1) agonist exendin-4, correlating with enhanced GLP-1 receptor expression. BBS itself had no effect on islet function, as reflected in low expression of BBS receptors in islet samples. Intestinal BBS receptor expression was enhanced in PN with BBS, and circulating active GLP-1 levels were significantly enhanced in BBS-treated PN mice. We hypothesized that BBS preserved islet function indirectly, through the enteroendocrine cell-pancreas axis. We confirmed the ability of BBS to directly stimulate intestinal enteroid cells to express the GLP-1 precursor preproglucagon. In conclusion, BBS preserves the exocrine and endocrine pancreas functions during PN; however, the endocrine stimulation is likely indirect, through the enteroendocrine cell-pancreas axis.

In type 1 diabetes (T1D), an autoimmune insulitis leads to β‐cell death. The non‐obese diabetic (NOD) mouse model mimics these facets of the human disease. Our laboratory has previously shown that mice deficient in the alpha subunit of the heterotrimeric G protein, Gz (Gαz), were protected from developing chemically induced diabetes via decreased β‐cell apoptosis and increased replication. We aimed to confirm this in the NOD model, as well as delineate the mechanisms of any protection. Between 4‐16 weeks of age, weekly blood glucose levels were monitored in wild‐type and Gαz‐null NOD mice. As compared to wild‐type mice, Gαz‐null mice were completely protected from developing hyperglycemia. Pancreas histologic analysis showed that Gαz‐null pancreata had significantly lower insulitis scores at 16 weeks as compared to wild‐type mice. We hypothesized that decreased immune infiltration increased β‐cell survival. To test our hypothesis, we calculated beta‐cell fractional area, and found it was increased in Gαz‐null NOD mice. New cohorts of mice were sacrificed at 4, 8, and 12 weeks of age. At 4 weeks, immune infiltration was already observed in the wild‐type islets, correlating with apoptotic beta‐cells in islet regions immediately surrounding the invading immune cells. As the first step in delineating mechanism, we showed that islets from Gαz‐null NOD mice have significantly less IL‐1β and IL‐6 cytokine expression; pro‐inflammatory cytokines that have been linked with β‐cell death. Overall, our results further strengthen our claim that Gαz has a protective effect in maintaining β‐cell regeneration and survival, and that the Gαz signaling pathway may be a valid therapeutic target for T1D.

Parenteral nutrition (PN) is the IV delivery of nutrients to individuals unable intake nutrients via the gut. During PN, intestinal and pancreatic physiology is compromised, leading to tissue atrophy and perturbed immune responses. In addition, PN is often associated with poor glucose control. We showed previously the gastrin releasing peptide analog, bombesin (BBS), enhanced gut immune response and architecture when administered during PN. Yet, the impact of BBS on pancreatic structure and function remained unclear. We hypothesized BBS treatment during PN would mitigate loss of exocrine pancreas structure and function and augment islet function. As compared to control mice, infusion of BBS during PN prevented exocrine tissue atrophy and preserved digestive enzyme secretion. Although there were no significant changes in gross islet morphology, we did observe increased β‐cell replication and function. We also found glucagon‐like peptide 1 (GLP‐1), a gut‐derived hormone with islet actions, was significantly elevated in plasma of mice BBS‐treated PN mice. Further, islets from BBS‐treated PN mice were significantly more responsive to a GLP‐1 receptor agonist. Finally, BBS increased intestinal cell proglucagon gene expression, suggesting it acts on the gut to promote GLP‐1 secretion in vivo. In sum, our data suggest that BBS preserves both exocrine and endocrine pancreas function during PN, with islet effects involving the gut‐pancreas axis.

EP3, a receptor for prostaglandin E2 (PGE2), plays a key role in the β‐cell dysfunction of diabetes. We demonstrated EP3 expression and PGE2 production are increased in islets isolated from diabetic mice and humans, and targeting this pathway in vitro restores proper β‐cell function. Yet, the mechanisms involved in the pathogenesis of β‐cell dysfunction upstream and downstream of EP3 remain elusive. The INS‐1 832/3 cell line has high EP3 expression, just as in diabetic islets. Interestingly, even though EP3 expression is high, PGE2 production is not; thus, an EP3 antagonist has no effect on β‐cell function. We hypothesized one or more of the multitude of insults β‐cells encounter in the diabetic state are necessary to stimulate PGE2 production and resulting β‐cell dysfunction. We mimicked diet by enriching INS‐1 cells with arachidonic acid (AA), an ω‐6 polyunsaturated fatty acid and PGE2 precursor, and inflammation by IL‐1β treatment. This combination significantly increased PGE2 production, blunting insulin secretion. Further, β‐cell function could be restored by an EP3 antagonist. In islets isolated from non‐diabetic mice and humans, AA and IL‐1β had a strong effect on PGE2 synthetic enzyme expression, but EP3 itself was relatively unchanged, and EP3‐mediated β‐cell dysfunction minimal. We hypothesized that high glucose or free fatty acids are responsible for the dysfunctional up‐regulation of β‐cell EP3 in vivo. Finally, we demonstrate the relevance of targeting this pathway in vivo, as mice lacking the EP3 effector, Gαz, specifically in the β‐cell are protected from the β‐cell dysfunction induced by inflammation.

Type 1 diabetes (T1D) occurs when β‐cell death causes insufficient β‐cell mass to maintain normoglycemia. Although select T1D patients are candidates for transplantation, no pharmaceutical cure for T1D exists. Such cures would augment the residual β‐cell mass had by most, if not all, T1D patients. We showed the α subunit of the heterotrimeric Gz protein, Gαz, inhibits production of cAMP, a second messenger proposed to potentiate β‐cell function and mass. In an obesity‐linked T2D model, we demonstrated islets from Gαz‐null mice have constitutively increased β‐cell cAMP production, insulin secretion, and replicative capacity. We hypothesized Gαz‐null mice subjected to chemical induction of T1D would have improved β‐cell replication, survival, and, ultimately, mass, especially when treated with drugs known to stimulate cAMP production. To test our hypothesis, we induced diabetes in mice with streptozotocin. The Gαz‐null mutation partially protected against hyperglycemia, as did treatment with the known cAMP potentiator, exendin‐4. Combining the Gαz‐null mutation with exendin‐4 treatment was completely protective. These pancreas sections had both increased β‐cell replication and decreased apoptosis. In vitro assays with novel Gαz variants are pinpointing critical downstream signaling pathways. Overall, our results support targeting Gαz signaling to preserve and regenerate functional β‐cell mass.Grant Funding Source: Supported by the Juvenile Diabetes Research Foundation