Publications by authors named "Craig S Nunemaker"

58 Publications

Natural Compound α-PGG and Its Synthetic Derivative 6Cl-TGQ Alter Insulin Secretion: Evidence for Diminishing Glucose Uptake as a Mechanism.

Authors:
Xiaozhuo Chen Nigel A Daniels David Cottrill Yanyang Cao Xuan Wang Yunsheng Li Pratik Shriwas Yanrong Qian Michael W Archer Nicholas B Whitticar Ishrat Jahan Craig S Nunemaker Aili Guo

Diabetes Metab Syndr Obes 2021 24;14:759-772. Epub 2021 Feb 24.

Department of Internal Medicine, Division of Endocrinology, Diabetes and Metabolism, University of California at Davis (UC Davis) School of Medicine, UC Davis Health Science, Sacramento, CA, 95817, USA.

Purpose: Previously we showed that natural compound α-penta-galloyl-glucose (α-PGG) and its synthetic derivative 6-chloro-6-deoxy-1,2,3,4-tetra-O-galloyl-α-D-glucopyranose (6Cl-TGQ) act to improve insulin signaling in adipocytes by increasing glucose transport. In this study, we investigated the mechanism of actions of α-PGG and 6Cl-TGQ on insulin secretion.

Methods: Mouse islets and/or INS-1832/13 beta-cells were used to test the effects of our compounds on glucose-stimulated insulin secretion (GSIS), intracellular calcium [Ca] using fura-2AM, glucose transport activity via a radioactive glucose uptake assay, intracellular ATP/ADP, and extracellular acidification (ECAR) and mitochondrial oxygen consumption rates (OCAR) using Seahorse metabolic analysis.

Results: Both compounds reduced GSIS in beta-cells without negatively affecting cell viability. The compounds primarily diminished glucose uptake into islets and beta-cells. Despite insulin-like effects in the peripheral tissues, these compounds do not act through the insulin receptor in islets. Further interrogation of the stimulus-secretion pathway showed that all the key metabolic factors involved in GSIS including ECAR, OCAR, ATP/ADP ratios, and [Ca] of INS-1832/13 cells were diminished after the compound treatment.

Conclusion: The compounds suppress glucose uptake of the beta-cells, which consequently slows down the rates of glycolysis and ATP synthesis, leading to decrease in [Ca] and GSIS. The difference between adipocytes and beta-cells in effects on glucose uptake is of great interest. Further structural and functional modifications could produce new compounds with optimized therapeutic potentials for different target cells. The higher potency of synthetic 6Cl-TGQ in enhancing insulin signaling in adipocytes but lower potency in reducing glucose uptake in beta-cells compared to α-PGG suggests the feasibility of such an approach.
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http://dx.doi.org/10.2147/DMSO.S284295DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7917315PMC
February 2021

A Practical Guide to Rodent Islet Isolation and Assessment Revisited.

Authors:
Kathryn L Corbin Hannah L West Samantha Brodsky Nicholas B Whitticar William J Koch Craig S Nunemaker

Biol Proced Online 2021 Mar 1;23(1). Epub 2021 Mar 1.

Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, USA.

Insufficient insulin secretion is a key component of both type 1 and type 2 diabetes. Since insulin is released by the islets of Langerhans, obtaining viable and functional islets is critical for research and transplantation. The effective and efficient isolation of these small islands of endocrine cells from the sea of exocrine tissue that is the rest of the pancreas is not necessarily simple or quick. Choosing and administering the digestive enzyme, separation of the islets from acinar tissue, and culture of islets are all things that must be considered. The purpose of this review is to provide a history of the development of islet isolation procedures and to serve as a practical guide to rodent islet research for newcomers to islet biology. We discuss key elements of mouse islet isolation including choosing collagenase, the digestion process, purification of islets using a density gradient, and islet culture conditions. In addition, this paper reviews techniques for assessing islet viability and function such as visual assessment, glucose-stimulated insulin secretion and intracellular calcium measurements. A detailed protocol is provided that describes a common method our laboratory uses to obtain viable and functional mouse islets for in vitro study. This review thus provides a strong foundation for successful procurement and purification of high-quality mouse islets for research purposes.
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http://dx.doi.org/10.1186/s12575-021-00143-xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919091PMC
March 2021

Postnatal maturation of calcium signaling in islets of Langerhans from neonatal mice.

Authors:
Hannah L West Kathryn L Corbin Cathleen V D'Angelo Lauren M Donovan Ishrat Jahan Guoqiang Gu Craig S Nunemaker

Cell Calcium 2021 03 28;94:102339. Epub 2020 Dec 28.

Dept. of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, USA; Diabetes Institute, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, USA. Electronic address:

Pancreatic islet cells develop mature physiological responses to glucose and other fuels postnatally. In this study, we used fluorescence imaging techniques to measure changes in intracellular calcium ([Ca]) to compare islets isolated from mice on postnatal days 0, 4, and 12 with islets from adult CD-1 mice. In addition, we used publicly available RNA-sequencing data to compare expression levels of key genes in β-cell physiology with [Ca] data across these ages. We show that islets isolated from mice on postnatal day 0 displayed elevated [Ca] in basal glucose (≤4 mM) but lower [Ca] responses to stimulation by 12-20 mM glucose compared to adult. Neonatal islets displayed more adult-like [Ca] in basal glucose by day 4 but continued to show lower [Ca] responses to 16 and 20 mM glucose stimulation up to at least day 12. A right shift in glucose sensing (EC) correlated with lower fragment-per-kilobase-of-transcript-per-million-reads-mapped (FPKM) of Slc2a2 (glut2) and Actn3 and increased FPKM for Galk1 and Nupr1. Differences in [Ca] responses to additional stimuli were also observed. Calcium levels in the endoplasmic reticulum were elevated on day 0 but became adult-like by day 4, which corresponded with reduced expression in Atp2a2 (SERCA2) and novel K+-channel Ktd17, increased expression of Pml, Wfs1, Thada, and Herpud1, and basal [Ca] maturing to adult levels. Ion-channel activity also matured rapidly, but RNA sequencing data mining did not yield strong leads. In conclusion, the maturation of islet [Ca] signaling is complex and multifaceted; several possible gene targets were identified that may participate in this process.
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http://dx.doi.org/10.1016/j.ceca.2020.102339DOI Listing
March 2021

A Putative Prohibitin-Calcium Nexus in -Cell Mitochondria and Diabetes.

Authors:
Gaurav Verma Aparna Dixit Craig S Nunemaker

J Diabetes Res 2020 8;2020:7814628. Epub 2020 Oct 8.

HCOM-Biomedical Sciences, Ohio University, Athens Camp, US-45701 Ohio, USA.

The role of mitochondria in apoptosis is well known; however, the mechanisms linking mitochondria to the proapoptotic effects of proinflammatory cytokines, hyperglycemia, and glucolipotoxicity are not completely understood. Complex Ca signaling has emerged as a critical contributor to these proapoptotic effects and has gained significant attention in regulating the signaling processes of mitochondria. In pancreatic -cells, Ca plays an active role in -cell function and survival. Prohibitin (PHB), a mitochondrial chaperone, is actively involved in maintaining the architecture of mitochondria. However, its possible interaction with Ca-activated signaling pathways has not been explored. The present review aims to examine potential crosstalk between Ca signaling and PHB function in pancreatic -cells. Moreover, this review will focus on the effects of cytokines and glucolipotoxicity on Ca signaling and its possible interaction with PHB. Improved understanding of this important mitochondrial protein may aid in the design of more targeted drugs to identify specific pathways involved with stress-induced dysfunction in the -cell.
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http://dx.doi.org/10.1155/2020/7814628DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7737164PMC
October 2020

Isolation and Assessment of Pancreatic Islets Versus Dispersed Beta Cells: A Straightforward Approach to Examine Cell-Cell Communication.

Authors:
Rachel T Scarl William J Koch Kathryn L Corbin Craig S Nunemaker

Methods Mol Biol 2020 Dec 15. Epub 2020 Dec 15.

Department of Biomedical Sciences Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, USA.

Islets of Langerhans, found in the pancreas, are microorgans essential for glucose homeostasis within the body. Many cells are found with an islet, such as beta cells (~70%), alpha cells (~20%), delta cells (~5%), F cells (~4%), and epsilon cells (1%), each with its own unique function. To better understand the roles of these cells and how cell communication alters their function, several techniques have been established such as islet isolation and beta cell dispersion. Here we describe how to isolate primary rodent islets, disperse pancreatic islets, measure intracellular calcium, and use immunofluorescent staining to distinguish beta cells and alpha cells.
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http://dx.doi.org/10.1007/7651_2020_338DOI Listing
December 2020

Pyruvate Kinase Controls Signal Strength in the Insulin Secretory Pathway.

Authors:
Sophie L Lewandowski Rebecca L Cardone Hannah R Foster Thuong Ho Evgeniy Potapenko Chetan Poudel Halena R VanDeusen Sophia M Sdao Tiago C Alves Xiaojian Zhao Megan E Capozzi Arnaldo H de Souza Ishrat Jahan Craig J Thomas Craig S Nunemaker Dawn Belt Davis Jonathan E Campbell Richard G Kibbey Matthew J Merrins

Cell Metab 2020 Nov;32(5):736-750.e5

Department of Medicine, Division of Endocrinology, Diabetes, and Metabolism, University of Wisconsin-Madison, Madison, WI 53705, USA; William S. Middleton Memorial Veterans Hospital, Madison, WI 53705, USA. Electronic address:

Pancreatic β cells couple nutrient metabolism with appropriate insulin secretion. Here, we show that pyruvate kinase (PK), which converts ADP and phosphoenolpyruvate (PEP) into ATP and pyruvate, underlies β cell sensing of both glycolytic and mitochondrial fuels. Plasma membrane-localized PK is sufficient to close K channels and initiate calcium influx. Small-molecule PK activators increase the frequency of ATP/ADP and calcium oscillations and potently amplify insulin secretion. PK restricts respiration by cyclically depriving mitochondria of ADP, which accelerates PEP cycling until membrane depolarization restores ADP and oxidative phosphorylation. Our findings support a compartmentalized model of β cell metabolism in which PK locally generates the ATP/ADP required for insulin secretion. Oscillatory PK activity allows mitochondria to perform synthetic and oxidative functions without any net impact on glucose oxidation. These findings suggest a potential therapeutic route for diabetes based on PK activation that would not be predicted by the current consensus single-state model of β cell function.
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http://dx.doi.org/10.1016/j.cmet.2020.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7685238PMC
November 2020

Reducing Glucokinase Activity to Enhance Insulin Secretion: A Counterintuitive Theory to Preserve Cellular Function and Glucose Homeostasis.

Authors:
Nicholas B Whitticar Craig S Nunemaker

Front Endocrinol (Lausanne) 2020 9;11:378. Epub 2020 Jun 9.

Diabetes Institute, Heritage College of Osteopathic Medicine, Ohio University, Athens OH, United States.

Pancreatic beta-cells are the only cells in the body that can synthesize and secrete insulin. Through the process of glucose-stimulated insulin secretion, beta-cells release insulin into circulation, stimulating GLUT4-dependent glucose uptake into peripheral tissue. Insulin is normally secreted in pulses that promote signaling at the liver. Long before type 2 diabetes is diagnosed, beta-cells become oversensitive to glucose, causing impaired pulsatility and overstimulation in fasting levels of glucose. The resulting hypersecretion of insulin can cause poor insulin signaling and clearance at the liver, leading to hyperinsulinemia and insulin resistance. Continued overactivity can eventually lead to beta-cell exhaustion and failure at which point type 2 diabetes begins. To prevent or reverse the negative effects of overstimulation, beta-cell activity can be reduced. Clinical studies have revealed the potential of beta-cell rest to reverse new cases of diabetes, but treatments lack durable benefits. In this perspective, we propose an intervention that reduces overactive glucokinase activity in the beta-cell. Glucokinase is known as the glucose sensor of the beta-cell due to its high control over insulin secretion. Therefore, glycolytic overactivity may be responsible for hyperinsulinemia early in the disease and can be reduced to restore normal stimulus-secretion coupling. We have previously reported that reducing glucokinase activity in prediabetic mouse islets can restore pulsatility and enhance insulin secretion. Building on this counterintuitive finding, we review the importance of pulsatile insulin secretion and highlight how normalizing glucose sensing in the beta cell during prediabetic hyperinsulinemia may restore pulsatility and improve glucose homeostasis.
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http://dx.doi.org/10.3389/fendo.2020.00378DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7296051PMC
June 2020

Chronic stimulation induces adaptive potassium channel activity that restores calcium oscillations in pancreatic islets in vitro.

Authors:
Nathan C Law Isabella Marinelli Richard Bertram Kathryn L Corbin Cara Schildmeyer Craig S Nunemaker

Am J Physiol Endocrinol Metab 2020 04 18;318(4):E554-E563. Epub 2020 Feb 18.

Department of Biomedical Sciences Heritage College of Osteopathic Medicine, Ohio University, Athens, Ohio.

Insulin pulsatility is important to hepatic response in regulating blood glucose. Growing evidence suggests that insulin-secreting pancreatic β-cells can adapt to chronic disruptions of pulsatility to rescue this physiologically important behavior. We determined the time scale for adaptation and examined potential ion channels underlying it. We induced the adaptation both by chronic application of the ATP-sensitive K [K(ATP)] channel blocker tolbutamide and by application of the depolarizing agent potassium chloride (KCl). Acute application of tolbutamide without pretreatment results in elevated Ca as measured by fura-2AM and the loss of endogenous pulsatility. We show that after chronic exposure to tolbutamide (12-24 h), Ca oscillations occur with subsequent acute tolbutamide application. The same experiment was conducted with potassium chloride (KCl) to directly depolarize the β-cells. Once again, following chronic exposure to the cell stimulator, the islets produced Ca oscillations when subsequently exposed to tolbutamide. These experiments suggest that it is the chronic stimulation, and not tolbutamide desensitization, that is responsible for the adaptation that rescues oscillatory β-cell activity. This compensatory response also causes islet glucose sensitivity to shift rightward following chronic tolbutamide treatment. Mathematical modeling shows that a small increase in the number of K(ATP) channels in the membrane is one adaptation mechanism that is compatible with the data. To examine other compensatory mechanisms, pharmacological studies provide support that Kir2.1 and TEA-sensitive channels play some role. Overall, this investigation demonstrates β-cell adaptability to overstimulation, which is likely an important mechanism for maintaining glucose homeostasis in the face of chronic stimulation.
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http://dx.doi.org/10.1152/ajpendo.00482.2019DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7191410PMC
April 2020

Intact pancreatic islets and dispersed beta-cells both generate intracellular calcium oscillations but differ in their responsiveness to glucose.

Authors:
Rachel T Scarl Kathryn L Corbin Nicholas W Vann Hallie M Smith Leslie S Satin Arthur Sherman Craig S Nunemaker

Cell Calcium 2019 11 16;83:102081. Epub 2019 Sep 16.

Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, United States; Diabetes Institute, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, United States. Electronic address:

Pancreatic islets produce pulses of insulin and other hormones that maintain normal glucose homeostasis. These micro-organs possess exquisite glucose-sensing capabilities, allowing for precise changes in pulsatile insulin secretion in response to small changes in glucose. When communication among these cells is disrupted, precision glucose sensing falters. We measured intracellular calcium patterns in 6-mM-steps between 0 and 16 mM glucose, and also more finely in 2-mM-steps from 8 to 12 mM glucose, to compare glucose sensing systematically among intact islets and dispersed islet cells derived from the same mouse pancreas in vitro. The calcium activity of intact islets was uniformly low (quiescent) below 4 mM glucose and active above 8 mM glucose, whereas dispersed beta-cells displayed a broader activation range (2-to-10 mM). Intact islets exhibited calcium oscillations with 2-to-5-min periods, yet beta-cells exhibited longer 7-10 min periods. In every case, intact islets showed changes in activity with each 6-mM-glucose step, whereas dispersed islet cells displayed a continuum of calcium responses ranging from islet-like patterns to stable oscillations unaffected by changes in glucose concentration. These differences were also observed for 2-mM-glucose steps. Despite the diversity of dispersed beta-cell responses to glucose, the sum of all activity produced a glucose dose-response curve that was surprisingly similar to the curve for intact islets, arguing against the importance of "hub cells" for function. Beta-cells thus retain many of the features of islets, but some are more islet-like than others. Determining the molecular underpinnings of these variations could be valuable for future studies of stem-cell-derived beta-cell therapies.
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http://dx.doi.org/10.1016/j.ceca.2019.102081DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6983470PMC
November 2019

Comparing methods to normalize insulin secretion shows the process may not be needed.

Authors:
Kira G Slepchenko Kathryn L Corbin Craig S Nunemaker

J Endocrinol 2019 Mar 1. Epub 2019 Mar 1.

C Nunemaker, Biomedical Sciences, Ohio University, Athens, United States.

Glucose-stimulated insulin secretion (GSIS) is a well-accepted method to investigate the physiological and pathophysiological function of islets. However, there is little consensus about which method is best for normalizing and presenting GSIS data. In this study, we evaluated the sufficiency of islet area, total protein, total DNA, and total insulin content as parameters to normalize GSIS data. First, we tested if there is a linear correlation between each parameter and the number of islets (10, 20, 30, and 40 islets). Islet area, total protein, and insulin content produced excellent linear correlations with islet number (R2 >0.9 for each) from the same islet material. Insulin secretion in 11mM glucose also correlated reasonably well for islet area (R2=0.69), protein (R2=0.49), and insulin content (R2=0.58). DNA content was difficult to reliably measure and was excluded from additional comparisons. We next measured GSIS for 18 replicates of 20 islets each, measuring 3mM and 11mM glucose to calculate the stimulation index and to compare each normalization parameter. Using these similar islet masses for each replicate, none of the parameters produced linear correlations with GSIS (R2<0.05), suggesting that inherent differences in GSIS dominate small differences in islet mass. We conclude that when comparing GSIS for islets of reasonably similar size (<50% variance), normalization does not improve the representation of GSIS data. Normalization may be beneficial when substantial differences in islet mass are involved. In such situations, we suggest that using islet cross-sectional area is superior to other commonly used techniques for normalizing GSIS data.
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http://dx.doi.org/10.1530/JOE-18-0542DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6983291PMC
March 2019

Reducing Glucokinase Activity Restores Endogenous Pulsatility and Enhances Insulin Secretion in Islets From db/db Mice.

Authors:
Ishrat Jahan Kathryn L Corbin Avery M Bogart Nicholas B Whitticar Christopher D Waters Cara Schildmeyer Nicholas W Vann Hannah L West Nathan C Law Jeffrey S Wiseman Craig S Nunemaker

Endocrinology 2018 11;159(11):3747-3760

Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, Ohio.

An early sign of islet failure in type 2 diabetes (T2D) is the loss of normal patterns of pulsatile insulin release. Disruptions in pulsatility are associated with a left shift in glucose sensing that can cause excessive insulin release in low glucose (relative hyperinsulinemia, a hallmark of early T2D) and β-cell exhaustion, leading to inadequate insulin release during hyperglycemia. Our hypothesis was that reducing excessive glucokinase activity in diabetic islets would improve their function. Isolated mouse islets were exposed to glucose and varying concentrations of the glucokinase inhibitor d-mannoheptulose (MH) to examine changes in intracellular calcium ([Ca2+]i) and insulin secretion. Acutely exposing islets from control CD-1 mice to MH in high glucose (20 mM) dose dependently reduced the size of [Ca2+]i oscillations detected by fura-2 acetoxymethyl. Glucokinase activation in low glucose (3 mM) had the opposite effect. We then treated islets from male and female db/db mice (age, 4 to 8 weeks) and heterozygous controls overnight with 0 to 10 mM MH to determine that 1 mM MH produced optimal oscillations. We then used 1 mM MH overnight to measure [Ca2+]i and insulin simultaneously in db/db islets. MH restored oscillations and increased insulin secretion. Insulin secretion rates correlated with MH-induced increases in amplitude of [Ca2+]i oscillations (R2 = 0.57, P < 0.01, n = 10) but not with mean [Ca2+]i levels in islets (R2 = 0.05, not significant). Our findings show that correcting glucose sensing can restore proper pulsatility to diabetic islets and improved pulsatility correlates with enhanced insulin secretion.
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http://dx.doi.org/10.1210/en.2018-00589DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6202857PMC
November 2018

Metformin Inhibits Mouse Islet Insulin Secretion and Alters Intracellular Calcium in a Concentration-Dependent and Duration-Dependent Manner near the Circulating Range.

Authors:
Lindor Gelin Jiewen Li Kathryn L Corbin Ishrat Jahan Craig S Nunemaker

J Diabetes Res 2018 18;2018:9163052. Epub 2018 Mar 18.

Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, USA.

Metformin is considered the first-line treatment for type 2 diabetes. While metformin primarily increases insulin sensitivity, evidence also suggests that metformin affects the activity of insulin-secreting pancreatic islets. This study was designed to systematically examine the direct effects of metformin by measuring insulin secretion and the kinetics of the calcium response to glucose stimulation in isolated mouse islets using varying concentrations (20 M, 200 M, and 1 mM) and durations (~1, 2, and 3 days) of metformin exposure. We observed both concentration- and duration-dependent inhibitory effects of metformin. Concentrations as little as 20 M (nearing circulating therapeutic levels) were sufficient to reduce insulin secretion following 3-day treatment. Concentrations of 200 M and 1 mM produced more pronounced effects more rapidly. With 1 mM metformin, islets showed severe impairments in calcium handling, inhibition of insulin secretion, and increased cell death. No stimulatory effects of metformin were observed for any experimental endpoint. We conclude that the direct effects of metformin on islets are inhibitory at near-physiological concentrations. Beneficial effects of metformin observed on islets under various stressors may occur by "resting" fatigued cellular processes. However, metformin may have unintended consequences on normally functioning islets within the circulating range that require further evaluation.
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http://dx.doi.org/10.1155/2018/9163052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5971297PMC
October 2018

Can NMDA Receptors Get β-Cells Toxically Excited?

Authors:
Craig S Nunemaker Chien Li

Endocrinology 2017 11;158(11):3709-3710

Obesity Research, Novo Nordisk Research Center, Seattle, Washington 98109.

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http://dx.doi.org/10.1210/en.2017-00832DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5695841PMC
November 2017

STEAP4: its emerging role in metabolism and homeostasis of cellular iron and copper.

Authors:
Rachel T Scarl C Martin Lawrence Hannah M Gordon Craig S Nunemaker

J Endocrinol 2017 Sep 2;234(3):R123-R134. Epub 2017 Jun 2.

Diabetes InstituteHeritage College of Osteopathic Medicine, Ohio University, Athens, Ohio, USA

Preserving energy homeostasis in the presence of stressors such as proinflammatory cytokines and nutrient overload is crucial to maintaining normal cellular function. Six transmembrane epithelial antigen of the prostate 4 (STEAP4), a metalloreductase involved in iron and copper homeostasis, is thought to play a potentially important role in the cellular response to inflammatory stress. Genome-wide association studies have linked various mutations in with the development of metabolic disorders such as obesity, metabolic syndrome and type 2 diabetes. Several studies have shown that expression of is modulated by inflammatory cytokines, hormones and other indicators of cellular stress and that STEAP4 may protect cells from damage, helping to maintain normal metabolic function. STEAP4 appears to be particularly relevant in metabolically oriented cells, such as adipocytes, hepatocytes and pancreatic islet cells. These cells struggle to maintain their function in iron or copper overloaded states, presumably due to increased oxidative stress, suggesting STEAP4's role in metal homeostasis is critical to the maintenance of cellular homeostasis in general, and in preventing the onset of metabolic disease. In this review, we explore genetic associations of with metabolic disorders, and we examine STEAP4 tissue expression, subcellular localization, regulation, structure and function as it relates to metabolic diseases. We then examine how STEAP4's role as a regulator of cellular iron and copper may relate to type 2 diabetes.
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http://dx.doi.org/10.1530/JOE-16-0594DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166870PMC
September 2017

STEAP4 expression in human islets is associated with differences in body mass index, sex, HbA1c, and inflammation.

Authors:
Hannah M Gordon Neil Majithia Patrick E MacDonald Jocelyn E Manning Fox Poonam R Sharma Frances L Byrne Kyle L Hoehn Carmella Evans-Molina Linda Langman Kenneth L Brayman Craig S Nunemaker

Endocrine 2017 Jun 12;56(3):528-537. Epub 2017 Apr 12.

Department of Biomedical Sciences, Ohio University, Athens, OH, USA.

Objective: STEAP4 (six-transmembrane epithelial antigen of the prostate 4) is a metalloreductase that has been shown previously to protect cells from inflammatory damage. Genetic variants in STEAP4 have been associated with numerous metabolic disorders related to obesity, including putative defects in the acute insulin response to glucose in type 2 diabetes.

Purpose: We examined whether obesity and/or type 2 diabetes altered STEAP4 expression in human pancreatic islets.

Methods: Human islets were isolated from deceased donors at two medical centers and processed for quantitative polymerase chain reaction. Organ donors were selected by status as non-diabetic or having type 2 diabetes. Site 1 (Edmonton): N = 13 type 2 diabetes donors (7M, 6F), N = 20 non-diabetic donors (7M, 13F). Site 2 (Virginia): N = 6 type 2 diabetes donors (6F), N = 6 non-diabetic donors (3M, 3F).

Results: STEAP4 showed reduced islet expression with increasing body mass index among all donors (P < 0.10) and non-diabetic donors (P < 0.05) from Site 1; STEAP4 showed reduced islet expression among type 2 diabetes donors with increasing hemoglobin A1c. Islet STEAP4 expression was also marginally higher in female donors (P < 0.10). Among type 2 diabetes donors from Site 2, islet insulin expression was reduced, STEAP4 expression was increased, and white blood cell counts were increased compared to non-diabetic donors. Islets from non-diabetic donors that were exposed overnight to 5 ng/ml IL-1β displayed increased STEAP4 expression, consistent with STEAP4 upregulation by inflammatory signaling.

Conclusions: These findings suggest that increased STEAP4 mRNA expression is associated with inflammatory stimuli, whereas lower STEAP4 expression is associated with obesity in human islets. Given its putative protective role, downregulation of STEAP4 by chronic obesity suggests a mechanism for reduced islet protection against cellular damage.
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http://dx.doi.org/10.1007/s12020-017-1297-2DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6166871PMC
June 2017

Zinc Transport Gets Its Zing Back: Double-Knockout of ZnT7 and ZnT8 Reveals the Importance of Zinc Transporters to Insulin Secretion.

Authors:
Craig S Nunemaker Richard K P Benninger

Endocrinology 2016 12;157(12):4542-4544

Diabetes Institute and Department of Biomedical Sciences (C.S.N.), Heritage College of Osteopathic Medicine, Ohio University, Athens, Ohio 45701; and Barbara Davis Center for Diabetes and Department of Bioengineering (R.K.P.B.), University of Colorado Anschutz Medical Campus, Aurora, Colorado 80045.

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http://dx.doi.org/10.1210/en.2016-1797DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5133344PMC
December 2016

An Automated Perifusion System for Modifying Cell Culture Conditions over Time.

Authors:
Nicholas B Whitticar Elisha W Strahler Parthiban Rajan Savas Kaya Craig S Nunemaker

Biol Proced Online 2016 21;18:19. Epub 2016 Nov 21.

Diabetes Institute, Ohio University, Athens, OH USA.

Background: Cells are continuously exposed to changes in their environment. Endocrine systems, in particular, communicate by rhythms and feedback loops. In this study, we developed an automated system to produce such conditions for cultured cells in a precisely timed manner. We utilized a programmable pair of syringe pumps for inflow and a peristaltic pump for outflow to create rhythmic pulses at 5-min intervals in solutions that mimic the endogenous patterns of insulin produced by pancreatic islets as a test case.

Results: This perifusion system was first tested by measuring trypan blue absorbance, which was intermittently added and washed out at 3:3 and 2:3 min (in:out). Absorbance corresponded with patterns of trypan blue delivery. We then created patterns of forced oscillations in islets by intermittently switching between solutions containing 28 millimolar (mM) glucose (producing high levels of intracellular calcium ([Ca]) and insulin secretion) and 28 mM glucose + calcium-channel blocker nifedipine (producing low levels of [Ca] and insulin secretion). Forced perifusion effects were monitored by fura-2 AM fluorescence measurements of [Ca]. Islets showed uniform oscillations in [Ca] at time intervals consistent with the perifusion pattern, mimicking endogenous pulsatility.

Conclusions: This study highlights a valuable method to modify the environment of the cell culture over a period of hours to days.
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http://dx.doi.org/10.1186/s12575-016-0049-7DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5117600PMC
November 2016

Considerations for Defining Cytokine Dose, Duration, and Milieu That Are Appropriate for Modeling Chronic Low-Grade Inflammation in Type 2 Diabetes.

Authors:
Craig S Nunemaker

J Diabetes Res 2016 23;2016:2846570. Epub 2016 Oct 23.

Diabetes Institute, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, USA; Department of Biomedical Sciences, Heritage College of Osteopathic Medicine, Ohio University, Athens, OH, USA.

Proinflammatory cytokines have been implicated in the pathophysiology of both type 1 diabetes (T1D) and type 2 diabetes (T2D). T1D is an autoimmune disease involving the adaptive immune system responding to pancreatic beta-cells as antigen-presenting cells. This attracts immune cells that surround pancreatic islets (insulitis) and secrete cytokines, such as IL-1beta, IFN-gamma, and TNF-alpha, in close proximity to pancreatic beta-cells. In contrast, there is little evidence for such a focused autoimmune response in T2D. Instead, the innate immune system, which responds to cellular damage and pathogens, appears to play a key role. There are three major sources of proinflammatory cytokines that may impact islet/beta-cell function in T2D: (1) from islet cells, (2) from increased numbers of intraislet macrophages/immune cells, and (3) from increased circulating levels of proinflammatory cytokines due to obesity, presumably coming from inflamed adipose tissue. These differences between T1D and T2D are reflected by significant differences in the cytokine concentration, duration, and milieu. This review focuses on chronic versus acute cytokine action, cytokine concentrations, and cytokine milieu from the perspective of the pancreatic islet in T2D. We conclude that new cytokine models may be needed to reflect the pathophysiology of T2D more effectively than what are currently employed.
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http://dx.doi.org/10.1155/2016/2846570DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5097812PMC
June 2017

Islet Hypersensitivity to Glucose Is Associated With Disrupted Oscillations and Increased Impact of Proinflammatory Cytokines in Islets From Diabetes-Prone Male Mice.

Authors:
Kathryn L Corbin Christopher D Waters Brett K Shaffer Gretchen M Verrilli Craig S Nunemaker

Endocrinology 2016 05 4;157(5):1826-38. Epub 2016 Mar 4.

Department of Biomedical Sciences (K.L.C., C.S.N.), Heritage College of Osteopathic Medicine, and Diabetes Institute (K.L.C., C.S.N.), Ohio University, Athens, Ohio 45701; and Departments of Medicine (C.D.W., B.K.S., G.M.V.) and Biomedical Engineering (C.D.W.), University of Virginia, Charlottesville, Virginia 22908.

Pulsatile insulin release is the primary means of blood glucose regulation. The loss of pulsatility is thought to be an early marker and possible factor in developing type 2 diabetes. Another early adaptation in islet function to compensate for obesity is increased glucose sensitivity (left shift) associated with increased basal insulin release. We provide evidence that oscillatory disruptions may be linked with overcompensation (glucose hypersensitivity) in islets from diabetes-prone mice. We isolated islets from male 4- to 5-week-old (prediabetic) and 10- to 12-week-old (diabetic) leptin-receptor-deficient (db/db) mice and age-matched heterozygous controls. After an overnight incubation in media with 11 mM glucose, we measured islet intracellular calcium in 5, 8, 11, or 15 mM glucose. Islets from heterozygous 10- to 12-week-old mice were quiescent in 5 mM glucose and displayed oscillations with increasing amplitude and/or duration in 8, 11, and 15 mM glucose, respectively. Islets from diabetic 10- to 12-week-old mice, in contrast, showed robust oscillations in 5 mM glucose that declined with increasing glucose. Similar trends were observed at 4-5-weeks of age. A progressive left shift in maximal insulin release was also observed in islets as db/db mice aged. Reducing glucokinase activity with 1 mM D-mannoheptulose restored oscillations in 11 mM glucose. Finally, overnight low-dose cytokine exposure negatively impacted oscillations preferentially in high glucose in diabetic islets compared with heterozygous controls. Our findings suggest the following: 1) islets from frankly diabetic mice can produce oscillations, 2) elevated sensitivity to glucose prevents diabetic mouse islets from producing oscillations in normal postprandial (11-15 mM glucose) conditions, and 3) hypersensitivity to glucose may magnify stress effects from inflammation or other sources.
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http://dx.doi.org/10.1210/en.2015-1879DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4870867PMC
May 2016

An Islet-Targeted Genome-Wide Association Scan Identifies Novel Genes Implicated in Cytokine-Mediated Islet Stress in Type 2 Diabetes.

Authors:
Poonam R Sharma Aaron J Mackey Eden A Dejene James W Ramadan Carl D Langefeld Nicholette D Palmer Kent D Taylor Lynne E Wagenknecht Richard M Watanabe Stephen S Rich Craig S Nunemaker

Endocrinology 2015 Sep 27;156(9):3147-56. Epub 2015 May 27.

Department of Medicine (P.R.S., E.A.D., J.W.R., C.S.N.), Center for Public Health Genomics (A.J.M., S.S.R.), and Department of Chemistry (E.A.D.), University of Virginia, Charlottesville, Virginia 22904; Department of Biochemistry (N.D.P.), Center for Genomics and Personalized Medicine Research (N.D.P.), Center for Diabetes Research (N.D.P.), Center for Public Health Genomics (C.D.L., N.D.P., L.E.W.), Department of Biostatistical Sciences (C.D.L.), and Division of Public Health Sciences (L.E.W.), Wake Forest School of Medicine, Winston-Salem, North Carolina 27157; Department of Physiology and Biophysics (R.M.W.), Department of Preventive Medicine, and USC Diabetes and Obesity Research Institute (R.M.W.), Keck School of Medicine of University of Southern California, Los Angeles, California 90033; and Institute for Translational Genomics and Population Sciences (K.D.T.) and Department of Pediatrics (K.D.T.), Los Angeles Biomedical Research Institute at Harbor-UCLA Medical Center, Torrance, California 90502.

Genome-wide association studies in human type 2 diabetes (T2D) have renewed interest in the pancreatic islet as a contributor to T2D risk. Chronic low-grade inflammation resulting from obesity is a risk factor for T2D and a possible trigger of β-cell failure. In this study, microarray data were collected from mouse islets after overnight treatment with cytokines at concentrations consistent with the chronic low-grade inflammation in T2D. Genes with a cytokine-induced change of >2-fold were then examined for associations between single nucleotide polymorphisms and the acute insulin response to glucose (AIRg) using data from the Genetics Underlying Diabetes in Hispanics (GUARDIAN) Consortium. Significant evidence of association was found between AIRg and single nucleotide polymorphisms in Arap3 (5q31.3), F13a1 (6p25.3), Klhl6 (3q27.1), Nid1 (1q42.3), Pamr1 (11p13), Ripk2 (8q21.3), and Steap4 (7q21.12). To assess the potential relevance to islet function, mouse islets were exposed to conditions modeling low-grade inflammation, mitochondrial stress, endoplasmic reticulum (ER) stress, glucotoxicity, and lipotoxicity. RT-PCR revealed that one or more forms of stress significantly altered expression levels of all genes except Arap3. Thapsigargin-induced ER stress up-regulated both Pamr1 and Klhl6. Three genes confirmed microarray predictions of significant cytokine sensitivity: F13a1 was down-regulated 3.3-fold by cytokines, Ripk2 was up-regulated 1.5- to 3-fold by all stressors, and Steap4 was profoundly cytokine sensitive (167-fold up-regulation). Three genes were thus closely associated with low-grade inflammation in murine islets and also with a marker for islet function (AIRg) in a diabetes-prone human population. This islet-targeted genome-wide association scan identified several previously unrecognized candidate genes related to islet dysfunction during the development of T2D.
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http://dx.doi.org/10.1210/en.2015-1203DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4541617PMC
September 2015

Stress-induced dissociations between intracellular calcium signaling and insulin secretion in pancreatic islets.

Authors:
Farhan M Qureshi Eden A Dejene Kathryn L Corbin Craig S Nunemaker

Cell Calcium 2015 May 23;57(5-6):366-375. Epub 2015 Mar 23.

Department of Medicine, University of Virginia, Charlottesville, VA.

In healthy pancreatic islets, glucose-stimulated changes in intracellular calcium ([Ca(2+)]i) provide a reasonable reflection of the patterns and relative amounts of insulin secretion. We report that [Ca(2+)]i in islets under stress, however, dissociates with insulin release in different ways for different stressors. Islets were exposed for 48h to a variety of stressors: cytokines (low-grade inflammation), 28mM glucose (28G, glucotoxicity), free fatty acids (FFAs, lipotoxicity), thapsigargin (ER stress), or rotenone (mitochondrial stress). We then measured [Ca(2+)]i and insulin release in parallel studies. Islets exposed to all stressors except rotenone displayed significantly elevated [Ca(2+)]i in low glucose, however, increased insulin secretion was only observed for 28G due to increased nifedipine-sensitive calcium-channel flux. Following 3-11mM glucose stimulation, all stressors substantially reduced the peak glucose-stimulated [Ca(2+)]i response (first phase). Thapsigargin and cytokines also substantially impacted aspects of calcium influx and ER calcium handling. Stressors did not significantly impact insulin secretion in 11mM glucose for any stressor, although FFAs showed a borderline reduction, which contributed to a significant decrease in the stimulation index (11:3mM glucose) observed for FFAs and also for 28G. We also clamped [Ca(2+)]i using 30mM KCl+250μM diazoxide to test the amplifying pathway. Only rotenone-treated islets showed a robust increase in 3-11mM glucose-stimulated insulin secretion under clamped conditions, suggesting that low-level mitochondrial stress might activate the metabolic amplifying pathway. We conclude that different stressors dissociate [Ca(2+)]i from insulin secretion differently: ER stressors (thapsigargin, cytokines) primarily affect [Ca(2+)]i but not conventional insulin secretion and 'metabolic' stressors (FFAs, 28G, rotenone) impacted insulin secretion.
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http://dx.doi.org/10.1016/j.ceca.2015.03.002DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4417449PMC
May 2015

Increased serum CXCL1 and CXCL5 are linked to obesity, hyperglycemia, and impaired islet function.

Authors:
Craig S Nunemaker H Grace Chung Gretchen M Verrilli Kathryn L Corbin Aditi Upadhye Poonam R Sharma

J Endocrinol 2014 Aug 13;222(2):267-76. Epub 2014 Jun 13.

Division of Endocrinology and MetabolismDepartment of MedicineDepartment of Biomedical EngineeringDepartment of BiologyUniversity of Virginia, PO Box 801413, Charlottesville, Virginia 22908, USA.

Proinflammatory cytokines are thought to play a significant role in the pathogenesis of type 2 diabetes (T2D) and are elevated in the circulation even before the onset of the disease. However, the full complement of cytokines involved in the development of T2D is not known. In this study, 32 serum cytokines were measured from diabetes-prone BKS.Cg-m+/+Lepr(db)/J (db/db) mice and heterozygous age-matched control mice at 5 weeks (non-diabetic/non-obese), 6-7 weeks (transitional-to-diabetes), or 11 weeks (hyperglycemic/obese) and then correlated with body weight, blood glucose, and fat content. Among these 32 cytokines, C-X-C motif ligand 1 (CXCL1) showed the greatest increase (+78%) in serum levels between db/db mice that were hyperglycemic (blood glucose: 519±23 mg/dl, n=6) and those that were non-hyperglycemic (193±13 mg/dl, n=8). Similarly, increased CXCL1 (+68%) and CXCL5 (+40%) were associated with increased obesity in db/db mice; note that these effects could not be entirely separated from age. We then examined whether islets could be a source of these chemokines. Exposure to cytokines mimicking low-grade systemic inflammation (10 pg/ml IL1β+20 pg/ml IL6) for 48 h upregulated islet CXCL1 expression by 53±3-fold and CXCL5 expression by 83±10-fold (n=4, P<0.001). Finally, overnight treatment with the combination of CXCL1 and CXCL5 at serum levels was sufficient to produce a significant decrease in the peak calcium response to glucose stimulation, suggesting reduced islet function. Our findings demonstrated that CXCL1 and CXCL5 i) are increased in the circulation with the onset of T2D, ii) are produced by islets under stress, and iii) synergistically affect islet function, suggesting that these chemokines participate in the pathogenesis of T2D.
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http://dx.doi.org/10.1530/JOE-14-0126DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4135511PMC
August 2014

Episodic hormone secretion: a comparison of the basis of pulsatile secretion of insulin and GnRH.

Authors:
Craig S Nunemaker Leslie S Satin

Endocrine 2014 Sep 8;47(1):49-63. Epub 2014 Mar 8.

Division of Endocrinology and Metabolism, Department of Medicine, University of Virginia, P.O. Box 801413, Charlottesville, VA, 22901, USA,

Rhythms govern many endocrine functions. Examples of such rhythmic systems include the insulin-secreting pancreatic beta-cell, which regulates blood glucose, and the gonadotropin-releasing hormone (GnRH) neuron, which governs reproductive function. Although serving very different functions within the body, these cell types share many important features. Both GnRH neurons and beta-cells, for instance, are hypothesized to generate at least two rhythms endogenously: (1) a burst firing electrical rhythm and (2) a slower rhythm involving metabolic or other intracellular processes. This review discusses the importance of hormone rhythms to both physiology and disease and compares and contrasts the rhythms generated by each system.
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http://dx.doi.org/10.1007/s12020-014-0212-3DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC4382805PMC
September 2014

Slow oscillations of KATP conductance in mouse pancreatic islets provide support for electrical bursting driven by metabolic oscillations.

Authors:
Jianhua Ren Arthur Sherman Richard Bertram Paulette B Goforth Craig S Nunemaker Christopher D Waters Leslie S Satin

Am J Physiol Endocrinol Metab 2013 Oct 6;305(7):E805-17. Epub 2013 Aug 6.

Department of Pharmacology and Brehm Diabetes Center, University of Michigan Medical School, Ann Arbor, Michigan;

We used the patch clamp technique in situ to test the hypothesis that slow oscillations in metabolism mediate slow electrical oscillations in mouse pancreatic islets by causing oscillations in KATP channel activity. Total conductance was measured over the course of slow bursting oscillations in surface β-cells of islets exposed to 11.1 mM glucose by either switching from current clamp to voltage clamp at different phases of the bursting cycle or by clamping the cells to -60 mV and running two-second voltage ramps from -120 to -50 mV every 20 s. The membrane conductance, calculated from the slopes of the ramp current-voltage curves, oscillated and was larger during the silent phase than during the active phase of the burst. The ramp conductance was sensitive to diazoxide, and the oscillatory component was reduced by sulfonylureas or by lowering extracellular glucose to 2.8 mM, suggesting that the oscillatory total conductance is due to oscillatory KATP channel conductance. We demonstrate that these results are consistent with the Dual Oscillator model, in which glycolytic oscillations drive slow electrical bursting, but not with other models in which metabolic oscillations are secondary to calcium oscillations. The simulations also confirm that oscillations in membrane conductance can be well estimated from measurements of slope conductance and distinguished from gap junction conductance. Furthermore, the oscillatory conductance was blocked by tolbutamide in isolated β-cells. The data, combined with insights from mathematical models, support a mechanism of slow (∼5 min) bursting driven by oscillations in metabolism, rather than by oscillations in the intracellular free calcium concentration.
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http://dx.doi.org/10.1152/ajpendo.00046.2013DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3798703PMC
October 2013

Circulating levels of IL-1B+IL-6 cause ER stress and dysfunction in islets from prediabetic male mice.

Authors:
Christina M O'Neill Christine Lu Kathryn L Corbin Poonam R Sharma Stacey B Dula Jeffrey D Carter James W Ramadan Wenjun Xin Jae K Lee Craig S Nunemaker

Endocrinology 2013 Sep 8;154(9):3077-88. Epub 2013 Jul 8.

University of Virginia, Department of Medicine, Charlottesville, Virginia 22908, USA.

Elevated levels of circulating proinflammatory cytokines are associated with obesity and increased risk of type 2 diabetes, but the mechanism is unknown. We tested whether proinflammatory cytokines IL-1B+IL-6 at low picogram per milliliter concentrations (consistent with serum levels) could directly trigger pancreatic islet dysfunction. Overnight exposure to IL-1B+IL-6 in islets isolated from normal mice and humans disrupted glucose-stimulated intracellular calcium responses; cytokine-induced effects were more severe among islets from prediabetic db/db mice that otherwise showed no signs of dysfunction. IL-1B+IL-6 exposure reduced endoplasmic reticulum (ER) calcium storage, activated ER stress responses (Nos2, Bip, Atf4, and Ddit3 [CHOP]), impaired glucose-stimulated insulin secretion, and increased cell death only in islets from prediabetic db/db mice. Furthermore, we found increased serum levels of IL-1B and IL-6 in diabetes-prone mice at an age before hyperglycemia was exhibited, suggesting that low-grade systemic inflammation develops early in the disease process. In addition, we implanted normal outbred and inbred mice with subcutaneous osmotic mini-pumps containing IL-1B+IL-6 to mimic the serum increases found in prediabetic db/db mice. Both IL-1B and IL-6 were elevated in serum from cytokine-pump mice, but glucose tolerance and blood glucose levels did not differ from controls. However, when compared with controls, isolated islets from cytokine-pump mice showed deficiencies in calcium handling and insulin secretion that were similar to observations with islets exposed to cytokines in vitro. These findings provide proof of principle that low-grade systemic inflammation is present early in the development of type 2 diabetes and can trigger ER stress-mediated islet dysfunction that can lead to islet failure.
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http://dx.doi.org/10.1210/en.2012-2138DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3749476PMC
September 2013

Vesicular nucleotide transporter-mediated ATP release regulates insulin secretion.

Authors:
Jessica C Geisler Kathryn L Corbin Qin Li Andrew P Feranchak Craig S Nunemaker Chien Li

Endocrinology 2013 Feb 19;154(2):675-84. Epub 2012 Dec 19.

Department of Pharmacology, University of Virginia Health System, Charlottesville, VA 22908, USA.

Extracellular ATP plays a critical role in regulating insulin secretion in pancreatic β cells. The ATP released from insulin secretory vesicles has been proposed to be a major source of extracellular ATP. Currently, the mechanism by which ATP accumulates into insulin secretory granules remains elusive. In this study, the authors identified the expression of a vesicular nucleotide transporter (VNUT) in mouse pancreas, isolated mouse islets, and MIN6 cells, a mouse β cell line. Immunohistochemistry and immunofluorescence revealed that VNUT colocalized extensively with insulin secretory granules. Functional studies showed that suppressing endogenous VNUT expression in β cells by small hairpin RNA knockdown greatly reduced basal- and glucose-induced ATP release. Importantly, knocking down VNUT expression by VNUT small hairpin RNA in MIN6 cells and isolated mouse islets dramatically suppressed basal insulin release and glucose-stimulated insulin secretion (GSIS). Moreover, acute pharmacologic blockade of VNUT with Evans blue, a VNUT antagonist, greatly attenuated GSIS in a dose-dependent manner. Exogenous ATP treatment effectively reversed the insulin secretion defect induced by both VNUT knockdown and functional inhibition, indicating that VNUT-mediated ATP release is essential for maintaining normal insulin secretion. In contrast to VNUT knockdown, overexpression of VNUT in β cells resulted in excessive ATP release and enhanced basal insulin secretion and GSIS. Elevated insulin secretion induced by VNUT overexpression was reversed by pharmacologic inhibition of P2X but not P2Y purinergic receptors. This study reveals VNUT is expressed in pancreatic β cells and plays an essential and novel role in regulating insulin secretion through vesicular ATP release and extracellular purinergic signaling.
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http://dx.doi.org/10.1210/en.2012-1818DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3548185PMC
February 2013

Manganese-enhanced magnetic resonance imaging detects declining pancreatic β-cell mass in a cyclophosphamide-accelerated mouse model of type 1 diabetes.

Authors:
Patrick F Antkowiak Brian K Stevens Craig S Nunemaker Marcia McDuffie Frederick H Epstein

Diabetes 2013 Jan 29;62(1):44-8. Epub 2012 Aug 29.

Department of Biomedical Engineering, University of Virginia, Charlottesville, Virginia, USA.

Currently, there is no ideal noninvasive method to quantify the progressive loss of pancreatic β-cell mass (BCM) that occurs in type 1 diabetes. Magnetic resonance imaging has detected gross differences in BCM between healthy and diabetic mice using the contrast agent manganese, which labels functional β-cells and increases the water proton relaxation rate (R1), but its ability to measure gradations in BCM during disease progression is unknown. Our objective was to test the hypothesis that measurements of the manganese-enhanced pancreatic R1 could detect decreasing BCM in a mouse model of type 1 diabetes. We used cyclophosphamide-accelerated BDC2.5 T-cell receptor transgenic nonobese diabetic mice, which experience development of type 1 diabetes during a 7-day time period after cyclophosphamide injection, whereas transgene-negative mice do not. We measured the manganese-enhanced pancreatic R1 before cyclophosphamide injection (day 0) and on days 3, 4, 5, and 7 afterward. Pancreatic R1 remained constant in transgene-negative mice and decreased stepwise day-to-day in transgene-positive mice, mirroring their loss of BCM, confirmed by pancreatic insulin measurements and histology. Changes in R1 in transgene-positive mice occurred before elevations in blood glucose, a clinical indicator of diabetes, suggesting potential for early noninvasive detection of changes in functional BCM.
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http://dx.doi.org/10.2337/db12-0153DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3526033PMC
January 2013

Salivary gland hypofunction induced by activation of innate immunity is dependent on type I interferon signaling.

Authors:
Seshagiri-Rao Nandula Paromita Dey Kathryn L Corbin Craig S Nunemaker Harini Bagavant Umesh S Deshmukh

J Oral Pathol Med 2013 Jan 5;42(1):66-72. Epub 2012 Jun 5.

Division of Nephrology, Center for Immunity Inflammation and Regenerative Medicine, University of Virginia, Charlottesville, VA 22908, USA.

Background: Activation of innate immunity through polyinosinic:polycytidylic acid [poly(I:C)] causes acute salivary gland hypofunction. As a major consequence of poly(I:C) treatment is type I interferon (IFN) production, this study was undertaken to investigate their role in salivary gland dysfunction.

Methods: Different strains of mice deficient in either interferon alpha receptor (IFNAR1(-/-)) or IL-6(-/-), or IL-10(-/-), or EBI3(-/-) were treated with poly(I:C). Salivary gland function was determined by measuring pilocarpine-induced saliva volume. Gene expression levels were measured by real-time PCR. Ca(2+) mobilization studies were performed using ex-vivo acinar cells.

Results: A single injection of poly(I:C) rapidly induced salivary gland hypofunction in wild-type B6 mice (41% drop in saliva volumes compared to PBS-treated mice). In contrast, the loss of function in poly(I:C)-treated IFNAR(-/-) mice was only 9.6%. Gene expression analysis showed reduced levels of Il-6, Il-10, and Il-27 in submandibular glands of poly(I:C)-treated IFNAR(-/-) mice. While salivary gland dysfunction in poly(I:C)-treated IL-10(-/-) and EBI3(-/-) mice was comparable to wild-type mice, the IL-6(-/-) mice were more resistant, with only a 21% drop in function. Pilocarpine-induced Ca(2+) flux was significantly suppressed in acinar cells obtained from poly(I:C)-treated wild-type mice.

Conclusions: Our data demonstrate that a combined action of type I IFNs and IL-6 contributes toward salivary gland hypofunction. This happens through interference with Ca(2+) mobilization within acinar cells. Thus, in acute viral infections and diseases like Sjögren's syndrome, elevated levels of type I IFNs and IL-6 can directly affect glandular function.
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http://dx.doi.org/10.1111/j.1600-0714.2012.01181.xDOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3443546PMC
January 2013

Apolipoprotein A-IV improves glucose homeostasis by enhancing insulin secretion.

Authors:
Fei Wang Alison B Kohan Tammy L Kindel Kathryn L Corbin Craig S Nunemaker Silvana Obici Stephen C Woods W Sean Davidson Patrick Tso

Proc Natl Acad Sci U S A 2012 Jun 22;109(24):9641-6. Epub 2012 May 22.

Departments of Pathology and Laboratory Medicine, University of Cincinnati, Cincinnati, OH 45237, USA.

Apolipoprotein A-IV (apoA-IV) is secreted by the small intestine in response to fat absorption. Here we demonstrate a potential role for apoA-IV in regulating glucose homeostasis. ApoA-IV-treated isolated pancreatic islets had enhanced insulin secretion under conditions of high glucose but not of low glucose, suggesting a direct effect of apoA-IV to enhance glucose-stimulated insulin release. This enhancement involves cAMP at a level distal to Ca(2+) influx into the β cells. Knockout of apoA-IV results in compromised insulin secretion and impaired glucose tolerance compared with WT mice. Challenging apoA-IV(-/-) mice with a high-fat diet led to fasting hyperglycemia and more severe glucose intolerance associated with defective insulin secretion than occurred in WT mice. Administration of exogenous apoA-IV to apoA-IV(-/-) mice improved glucose tolerance by enhancing insulin secretion in mice fed either chow or a high-fat diet. Finally, we demonstrate that exogenous apoA-IV injection decreases blood glucose levels and stimulates a transient increase in insulin secretion in KKAy diabetic mice. These results suggest that apoA-IV may provide a therapeutic target for the regulation of glucose-stimulated insulin secretion and treatment of diabetes.
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http://dx.doi.org/10.1073/pnas.1201433109DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3386109PMC
June 2012

Connexin-36 gap junctions regulate in vivo first- and second-phase insulin secretion dynamics and glucose tolerance in the conscious mouse.

Authors:
W Steven Head Meredith L Orseth Craig S Nunemaker Leslie S Satin David W Piston Richard K P Benninger

Diabetes 2012 Jul 17;61(7):1700-7. Epub 2012 Apr 17.

Department of Molecular Physiology and Biophysics, Vanderbilt University, Nashville, Tennessee, USA.

Insulin is secreted from the islets of Langerhans in coordinated pulses. These pulses are thought to lead to plasma insulin oscillations, which are putatively more effective in lowering blood glucose than continuous levels of insulin. Gap-junction coupling of β-cells by connexin-36 coordinates intracellular free calcium oscillations and pulsatile insulin release in isolated islets, however a role in vivo has not been shown. We test whether loss of gap-junction coupling disrupts plasma insulin oscillations and whether this impacts glucose tolerance. We characterized the connexin-36 knockout (Cx36(-/-)) mouse phenotype and performed hyperglycemic clamps with rapid sampling of insulin in Cx36(-/-) and control mice. Our results show that Cx36(-/-) mice are glucose intolerant, despite normal plasma insulin levels and insulin sensitivity. However, Cx36(-/-) mice exhibit reduced insulin pulse amplitudes and a reduction in first-phase insulin secretion. These changes are similarly found in isolated Cx36(-/-) islets. We conclude that Cx36 gap junctions regulate the in vivo dynamics of insulin secretion, which in turn is important for glucose homeostasis. Coordinated pulsatility of individual islets enhances the first-phase elevation and second-phase pulses of insulin. Because these dynamics are disrupted in the early stages of type 2 diabetes, dysregulation of gap-junction coupling could be an important factor in the development of this disease.
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http://dx.doi.org/10.2337/db11-1312DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC3379660PMC
July 2012