Mark C. Zielinski

Regional Differences in Islet Distribution in the Human Pancreas - Preferential Beta-Cell Loss in the Head Region in Patients with Type 2 Diabetes

While regional heterogeneity in islet distribution has been well studied in rodents, less is known about human pancreatic histology. To fill gaps in our understanding, regional differences in the adult human pancreas were quantitatively analyzed including the pathogenesis of type 2 diabetes (T2D). Cadaveric pancreas specimens were collected from the head, body and tail regions of each donor, including subjects with no history of diabetes or pancreatic diseases (n = 23) as well as patients with T2D (n = 12). The study further included individuals from whom islets were isolated (n = 7) to study islet yield and function in a clinical setting of islet transplantation. The whole pancreatic sections were examined using an innovative large-scale image capture and unbiased detailed quantitative analyses of the characteristics of islets from each individual (architecture, size, shape and distribution). Islet distribution/density is similar between the head and body regions, but is >2-fold higher in the tail region. In contrast to rodents, islet cellular composition and architecture were similar throughout the pancreas and there was no difference in glucose-stimulated insulin secretion in islets isolated from different regions of the pancreas. Further studies revealed preferential loss of large islets in the head region in patients with T2D. The present study has demonstrated distinct characteristics of the human pancreas, which should provide a baseline for the future studies integrating existing research in the field and helping to advance bi-directional research between humans and preclinical models.

Natural scenes in tactile texture

Sensory systems are designed to extract behaviorally relevant information from the environment. In seeking to understand a sensory system, it is important to understand the environment within which it operates. In the present study, we seek to characterize the natural scenes of tactile texture perception. During tactile exploration complex high-frequency vibrations are elicited in the fingertip skin, and these vibrations are thought to carry information about the surface texture of manipulated objects. How these texture-elicited vibrations depend on surface microgeometry and on the biomechanical properties of the fingertip skin itself remains to be elucidated. Here we record skin vibrations, using a laser-Doppler vibrometer, as various textured surfaces are scanned across the finger. We find that the frequency composition of elicited vibrations is texture specific and highly repeatable. In fact, textures can be classified with high accuracy on the basis of the vibrations they elicit in the skin. As might be expected, some aspects of surface microgeometry are directly reflected in the skin vibrations. However, texture vibrations are also determined in part by fingerprint geometry. This mechanism enhances textural features that are too small to be resolved spatially, given the limited spatial resolution of the neural signal. We conclude that it is impossible to understand the neural basis of texture perception without first characterizing the skin vibrations that drive neural responses, given the complex dependence of skin vibrations on both surface microgeometry and fingertip biomechanics.

Quantification of islet size and architecture

Human islets exhibit distinct islet architecture particularly in large islets that comprise of a relatively abundant fraction of α-cells intermingled with β-cells, whereas mouse islets show largely similar architecture of a β-cell core with α-cells in the periphery. In humans, islet architecture is islet-size dependent. Changes in endocrine cell mass preferentially occurred in large islets as demonstrated in our recent study on pathological changes of the pancreas in patients with type 2 diabetes.1 The size dependency of human islets in morphological changes prompted us to develop a method to capture the representative islet distribution in the whole pancreas section combined with a semi-automated analysis to quantify changes in islet architecture. The computer-assisted quantification allows detailed examination of endocrine cell composition in individual islets and minimizes sampling bias. The standard immunohistochemistry based method is widely applicable to various specimens, which is particularly useful for large animal studies but is also applied to a large-scale analysis of the whole organ section from mice. In this article, we describe the method of image capture, parameters measured, data analysis and interpretation of the data.

The Effect of Surface Wave Propagation on Neural Responses to Vibration in Primate Glabrous Skin

Because tactile perception relies on the response of large populations of receptors distributed across the skin, we seek to characterize how a mechanical deformation of the skin at one location affects the skin at another. To this end, we introduce a novel non-contact method to characterize the surface waves produced in the skin under a variety of stimulation conditions. Specifically, we deliver vibrations to the fingertip using a vibratory actuator and measure, using a laser Doppler vibrometer, the surface waves at different distances from the locus of stimulation. First, we show that a vibration applied to the fingertip travels at least the length of the finger and that the rate at which it decays is dependent on stimulus frequency. Furthermore, the resonant frequency of the skin matches the frequency at which a subpopulation of afferents, namely Pacinian afferents, is most sensitive. We show that this skin resonance can lead to a two-fold increase in the strength of the response of a simulated afferent population. Second, the rate at which vibrations propagate across the skin is dependent on the stimulus frequency and plateaus at 7 m/s. The resulting delay in neural activation across locations does not substantially blur the temporal patterning in simulated populations of afferents for frequencies less than 200 Hz, which has important implications about how vibratory frequency is encoded in the responses of somatosensory neurons. Third, we show that, despite the dependence of decay rate and propagation speed on frequency, the waveform of a complex vibration is well preserved as it travels across the skin. Our results suggest, then, that the propagation of surface waves promotes the encoding of spectrally complex vibrations as the entire neural population is exposed to essentially the same stimulus. We also discuss the implications of our results for biomechanical models of the skin.

Evidence of non-pancreatic beta cell-dependent roles of Tcf7l2 in the regulation of glucose metabolism in mice

Non-coding variation within TCF7L2 remains the strongest genetic determinant of type 2 diabetes risk in humans. A considerable effort has been placed in understanding the functional roles of TCF7L2 in pancreatic beta cells, despite evidence of TCF7L2 expression in various peripheral tissues important in glucose homeostasis. Here, we use a humanized mouse model overexpressing Tcf7l2, resulting in glucose intolerance, to infer the contribution of Tcf7l2 overexpression in beta cells and in other tissues to the metabolic phenotypes displayed by these mice. Restoring Tcf7l2 expression specifically in beta cells to endogenous levels, in face of its overexpression elsewhere, results in impaired insulin secretion, reduced beta cell number and islet area, corroborating data obtained in humans showing similar phenotypes as a result of manipulations leading to Tcf7l2 loss of function. Interestingly, the persistent overexpression of Tcf7l2 in non-pancreatic tissues results in a significant worsening in glucose tolerance in vivo, indicating that Tcf7l2 overexpression in beta cells does not account for the glucose intolerance in the Tcf7l2 overexpression mouse model. Collectively, these data posit that Tcf7l2 plays key roles in glucose metabolism through actions beyond pancreatic beta cells, and further points to functionally opposing cell-type specific effects for Tcf7l2 on the maintenance of balanced glucose metabolism, thereby urging a careful examination of its role in non-pancreatic tissues as well as its composite metabolic effects across distinct tissues. Uncovering these roles may lead to new therapeutic targets for type 2 diabetes.

Case Report: Preservation of Reduced Numbers of Insulin-Positive Cells in Sulfonylurea-Unresponsive KCNJ11-related Diabetes

Context The most common genetic cause of permanent neonatal diabetes mellitus is activating mutations in KCNJ11, which can usually be treated using oral sulfonylureas (SUs) instead of insulin injections, although some mutations are SU unresponsive. In this work, we provide a report of the pancreatic islet endocrine cell composition and area in a patient with an SU-unresponsive KCNJ11 mutation (p.G334D), in comparison with age-matched controls. Case Description Pancreatic autopsy tissue sections from a 2-year-old female child diagnosed with KCNJ11-related diabetes at 4 days of age and 13 age-matched controls were stained with insulin, glucagon, somatostatin, pancreatic polypeptide, and Ki67 antibodies to determine islet endocrine cell composition and area. β-cell ultrastructure was assessed by electron microscopic (EM) analysis. The patients pancreas (sampling from head to tail) revealed insulin-positive cells in all regions. The pancreatic β-cell (insulin) area was significantly reduced compared with controls: 0.50% ± 0.04% versus 1.67% ± 0.20%, respectively (P < 0.00001). There were no significant differences in α-cell (glucagon) or δ-cell (somatostatin) area. EM analysis revealed secretory granules with a dense core typical of mature β-cells as well as granules with a lighter core characteristic of immature granules. Conclusions Our results suggest that mechanisms exist that allow preservation of β-cells in the absence of insulin secretion. It remains to be determined to what extent this reduction in β-cells may be reversible.

Improved coating of pancreatic islets with regulatory T cells to create local immunosuppression by using the biotin-polyethylene glycol-succinimidyl valeric acid ester molecule

BACKGROUND We showed that T regulatory (Treg) cells can be attached to the surface of pancreatic islets providing local immunoprotection. Further optimization of the method can improve coating efficiency, which may prolong graft survival. In this study, we compared the effectiveness of two different molecules used for binding of the Tregs to the surface of pancreatic islets. Our aim was to increase the number of Treg cells attached to islets without compromising islets viability and function. METHODS The cell surface of human Treg cells and pancreatic islets was modified using biotin-polyethylene glycol-N-hydroxylsuccinimide (biotin-PEG-NHS) or biotin-PEG-succinimidyl valeric acid ester (biotin-PEG-SVA). Then, islets were incubated with streptavidin as islet/Treg cells binding molecule. Treg cells were stained with CellTracker CM-DiL dye and visualized using a Laser Scanning Confocal Microscope. The number of Treg cells attached per islets surface area was analyzed by Imaris software. The effect of coating on islet functionality was determined using the glucose-stimulated insulin response (GSIR) assay. RESULTS The coating procedure with biotin-PEG-SVA allowed for attaching 40% more Treg cells per 1 μm(2) of islet surface. Although viability was comparable, function of the islets after coating using the biotin-PEG-SVA molecule was better preserved than with NHS molecule. GSIR was 62% higher for islets coated with biotin-PEG-SVA compared to biotin-PEG-NHS. CONCLUSION Coating of islets with Treg cells using biotin-PEG-SVA improves effectiveness with better preservation of the islet function. Improvement of the method of coating pancreatic islets with Treg cells could further facilitate the effectiveness of this novel immunoprotective approach and translation into clinical settings.

Stereological analyses of the whole human pancreas

The large size of human tissues requires a practical stereological approach to perform a comprehensive analysis of the whole organ. We have developed a method to quantitatively analyze the whole human pancreas, as one of the challenging organs to study, in which endocrine cells form various sizes of islets that are scattered unevenly throughout the exocrine pancreas. Furthermore, the human pancreas possesses intrinsic characteristics of intra-individual variability, i.e. regional differences in endocrine cell/islet distribution, and marked inter-individual heterogeneity regardless of age, sex and disease conditions including obesity and diabetes. The method is built based on large-scale image capture, computer-assisted unbiased image analysis and quantification, and further mathematical analyses, using widely-used software such as Fiji/ImageJ and MATLAB. The present study includes detailed protocols of every procedure as well as all the custom-written computer scripts, which can be modified according to specific experimental plans and specimens of interest.

Distinct function of the head region of human pancreas in the pathogenesis of diabetes

The large size of the human pancreas challenges unbiased quantitative analyses that require a practical stereological approach. While many histological studies of the pancreas in the past lacked regional information, we have shown marked heterogeneity within an individual, where islet distribution/density is relatively low in the head and gradually increases through the body toward the tail region by > 2-fold.1, 2 Studies focusing on the tail region may be prone to overestimation of β-cell/islet mass when normalizing measured values per person by using pancreas weight or volume. In this article, beyond technical issues, we discuss the pathophysiological importance of studying the head region of the human pancreas regarding its unique characteristics in early development, and the anatomical disposition that may lead to a preferential loss of β-cells in patients with type 2 diabetes and the development of pancreatic cancer.