Sympathetic Innervation of Human Alpha Cells

Abstract

Autonomic input is critical for the regulation of glucagon secretion to maintain blood glucose levels. Glucagon secretion is abnormal in patients with type 1 diabetes (T1D) and we hypothesize this results from dysfunctional sympathetic innervation of alpha-cells and may be present in at-risk autoantibody positive (AAb+) individuals. To characterize alpha-cell sympathetic innervation, pancreas tail samples were used from age-matched organ donors in three groups: controls (n=6), AAb+ (4), and T1D (4). Fresh frozen 40μm sections or 1-2 cm optically cleared samples were stained with markers for neuroendocrine cells (GCG, INS), total (NCAM)/ sympathetic nerves (tyrosine hydroxylase (TH)), and vascular smooth muscle cells (SMA) followed by confocal and Lightsheet imaging. To determine whether sympathetic fibers were decreased in AAb+ and/or T1D donors, TH+ fiber numbers were counted within islets and the percentage (%) of TH+ fibers/NCAM+ fibers determined. The TH+ fibers/islet were not significantly different between controls (1.5 ± 0.36, n=6), AAb+ (1.2 ± 0.27 (4)), and T1D (1.8 ± 0.57 (4)) donors (p = 0.07, ANOVA) with similar results for TH+ %. TH+ fibers were observed at alpha-cells in all donors. TH+ nerve densities were also similar between islets and exocrine regions between groups. Microarray analysis of single islet transcriptomes obtained using laser microdissection (n=120 islets, 13 control, 12 T1D) showed decreased expression of VMAT2 in insulin+ islets from T1D donors compared to control islets (p=0.047) while insulin- islets in T1D donors had significantly reduced VMAT2 levels compared to control islets (p<0.0001). These data indicate a potential direct mechanism by which the sympathetic nervous system may regulate alpha-cell glucagon secretion with dysregulation of a vesicular monoamine transporter that accumulates monoamines in synaptic vesicles in islets without beta-cells in T1D donors. Sympathetic innervation of alpha-cells could play a direct role in exacerbation of dysregulated glucagon secretion in T1D. Disclosure E.A. Butterworth: None. L.H. Nasif: None. K. Nasif: None. K.N. Carty: None. C.E. Mathews: None. M.A. Atkinson: Other Relationship; Self; Patent Issued. I.C. Gerling: None. M. Campbell-Thompson: None.

A 3D atlas of the dynamic and regional variation of pancreatic innervation in diabetes

“…In keeping with previous studies examining TH + fibers in human pancreatic samples ( 3 , 11 ), innervation density is similar in human exocrine and endocrine pancreas. We found that NF200 + innervation is present in human islets, in keeping with previous studies demonstrating TH + fibers in islets ( 3 , 11 ), but at a lower density than mouse islets. A smaller proportion of human β cells contact NF200 + fibers compared to mouse islets.…”

“…In our studies, the proportion of innervated islets and the finding that innervated islets were larger than noninnervated were similar in both humans and mice. Initial 2D imaging reported reduced islet innervation in human samples, but recent data from optically cleared human samples using markers for sympathetic nerves suggest that human islets, like mouse islets, have a dense neural network (9,11). In keeping with previous studies examining TH + fibers in human pancreatic samples (3,11), innervation density is similar in human exocrine and endocrine pancreas.…”

“…Initial 2D imaging reported reduced islet innervation in human samples, but recent data from optically cleared human samples using markers for sympathetic nerves suggest that human islets, like mouse islets, have a dense neural network (9,11). In keeping with previous studies examining TH + fibers in human pancreatic samples (3,11), innervation density is similar in human exocrine and endocrine pancreas. We found that NF200 + innervation is present in human islets, in keeping with previous studies demonstrating TH + fibers in islets (3,11), but at a lower density than mouse islets.…”

“…Whole-organ clearing and imaging are especially suited for the mapping of filamentous structures, particularly to delineating innervation across large distances that can be difficult to achieve using traditional serial sections and 2D imaging. Tissue clearing has been used previously in thick pancreatic sections (350 to 1000 m) (9,10,12,(42)(43)(44) and small pieces of pancreatic tissue (11) and then imaged with optometry or high-magnification confocal microscopy for detailed analysis [see (45)(46)(47) for review]. This has provided important information about islet characteristics and structural relationships over a close range.…”

“…In addition, thin filamentous structures, such as nerves, are difficult to quantify and trace over large volumes using this approach. Recent studies have applied confocal imaging of small pieces and thick sections of cleared pancreatic tissue to examine endocrine innervation (9)(10)(11)(12)(13)(14). These have revealed dense nerve processes within both mouse and human islets.…”