Publications by authors named "Nicholas B Whitticar"

5 Publications

  • Page 1 of 1

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

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.

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

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

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

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

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

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

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