An initial and fundamental thank you must go to the designers and administrators of Thanks to Dale Greiner (University of Massachusetts Medical School), Lenny Shultz (The Jackson Laboratory), Pedro Herrera (University of Geneva), Roland Stein (VUMC), William Russell (VUMC), and Larry Scheving (VUMC).
IN VIVO METABOLIC STRESS IMPAIRS ISLET TRANSCRIPTION
HUMAN ISLET PREPARATIONS DISTRIBUTED FOR RESEARCH
Univariate analysis of donor and islet variables 104 Stimulated insulin secretion in vitro does not correlate with function in vivo. Patterning of insulin secretion as assessed by perifusion 109 Gene expression differences between Group 1 and Group 5 111 islets.
LIST OF TABLES
LIST OF ABBREVIATIONS
BACKGROUND AND SIGNIFICANCE
The Pancreas Tissue compartments
Similar to the islet vasculature, nerve fibers and neurons are specifically denser around and in the islets. Expression of MafA, MafB, Nkx6.1 and Pdx1 contributes to mature β-cell function in the postnatal period.
Species Differences in Islet Physiology
When directly compared, human islets secrete more basal insulin, but the increase in secretion after high-glucose stimulation is smaller in human than in mouse (Figure 10E-F), and insulin content experiences a smaller increase in islets. human than mouse. after 24 hours high glucose treatment.46. Differences in glucose-responsive gene expression and glucose-stimulated insulin secretion in human and mouse islets.
Insulin
However, when insulin binds to the α-subunits, this repression is released and the two β-subunits transphosphorylate tyrosine residues (Figure 13).57-60. In skeletal muscle, the primary effect of insulin signaling is to promote the cellular uptake of glucose from the bloodstream.
Diabetes Mellitus
T2DM is associated with resistance to insulin signaling in the liver, skeletal muscle and adipose tissue, also called peripheral insulin resistance. The progression from normal glucose metabolism to insulin resistance against T2DM depends on a shift in the curve relating β-cell function to insulin sensitivity (Figure 15B).
Transplantation of Human Islets Clinical transplantation
As clinical transplantation has grown in popularity, the increase in human islet use for basic research has been dramatic. Transplantation of human islets, mostly in immunodeficient mouse models, has become a valuable and accepted way to study human islet biology.
Glucotoxicity and Lipotoxicity
Oxidative stress has been implicated in many events in the pathogenesis and progression of β-cell dysfunction and apoptosis,120 but the effects on transcription factor function have been of particular interest. Schematic depiction of the exchange of electrons in the mitochondrial membrane along the electron transport chain of proteins.
Aims of Dissertation
The contents of the insulin granule include the monomers of islet amyloid polypeptide (IAPP), a peptide involved in the progressive loss of β-cell function. the ER and Golgi, abnormal presence of IAPP/oligomer in the cytosol, interference with mitochondrial membrane integrity, fusion and/or rupture of secretory granules, and requirement of autophagy for oligomer degradation, rather than proteasomal degradation.
MATERIALS AND METHODS
Mouse Models
Original B6 mice; CBA Tg (Ins2-HBEGF) 6832) Ugfm/Sz were made by injecting the RIP-DTR construct into B6 eggs; CBA. These NSG-RIP-DTR mice express the human diphtheria toxin receptor (DTR) driven by the rat insulin promoter (RIP).
Islet isolation
EGFRfl/fl mus149 on a mixed background were kindly provided by William Russell and Larry Scheving at Vanderbilt University Medical Center. The C57Bl/6 EGFRfl/fl mouse was then crossed with the Tg(Ins2-Cre)1Herr,150 here called the "InsCre" mouse, also on the C57Bl/6 background, yielding InsCreposEGFRfl/+ animals, which were then crossed with EGFRfl/fl- mice to yield a population of InsCreposEGFRfl/fl pups.
Human islet acquisition
Islet perifusion Experimental protocol
The insulin content of each fraction was measured by radioimmunoassay and the insulin content values were normalized to 100 IEQ.146.
Islet transplantation
Islets were loaded onto Omnifit chromatography columns (Sigma, St. Louis, MO) with filter media (25um filter size) and immersed in a 37oC water bath. After the rat was fully anesthetized, an incision was made through the skin and muscle and the kidney was exposed.
Human Islet Assessment
Donor attributes,” characteristics of the human pancreas donor reported by the Organ Procurement Organization (OPO) to the islet isolation center, and “Islet attributes.” In vitro response” was defined at baseline (the insulin concentration of the last fraction collected before introduction of 16 .7 mM glucose), Peak1Max (highest point of Peak in response to 16.7 mM glucose), Peak2Max (highest point of Peak in response to 16.7 mM glucose + IBMX), Fold 1 (Peak1Max/Baseline), Fold 2 (Peak2Max/Baseline) and Peak Difference (Peak2Max-Peak1Max).A Peak is defined as having a collected fraction with an insulin concentration greater than 1.5 times that of the Baseline value.
Insulin content of pancreas and islet grafts
Protocols for viability and purity quantification are available on the IIDP website at http://iidp.coh.org/investigator_sops.aspx. Tissue human insulin or total insulin (mouse and human) was measured using species-specific radioimmunoassays from Millipore (Billerica, MA, catalog #RI-14K or #RI-13K, respectively), either in the laboratory or through the Vanderbilt University Hormone Assay and Analytical Services Core.
Genotyping
Glucose Tolerance Tests and Blood Glucose Measurements
Blood glucose values were measured from the tail vein notch at the zero-minute and fifteen-minute time points using an Accucheck Aviva glucometer and compatible strips (Roche, Indianapolis, IN).
Insulin tolerance tests
Glucose-arginine stimulation
All in vivo insulin secretion data used in the retrospective analyzes of human islet preparations (Chapter IV) reflect human islets transplanted into normoglycemic mice on a regular diet, and the data were normalized to the number of transplanted islet equivalents.
Serum lipid quantification
Percent fat and lean mass
Compound preparation and delivery Diphtheria toxin
Recombinant murine EGF (PeproTech, Rocky Hill, NJ, #315-09) was reconstituted from lyophilized powder in MilliQ water to a concentration of 1.0 µg/µL. Using the molecular weight of EGF, 50 nM concentrations were calculated and used in static islet culture media. Reconstituted EGF was stored at 4oC for up to one week or at -20oC for longer periods.
Islet static culture with EGF
Tissue Collection, Fixation, and Preparation
Immunohistochemistry
Before adding secondary antibodies, slices were washed three times for 10 min each in 0.1% Triton X-100 to remove unbound primary antibodies. Sections were incubated in secondary antibodies at room temperature for one hour before three 15-min washes in 0.1% Triton X-100, followed by three 5-min washes in neat 1X PBS.
Imaging
Electron microscopy
Dehydrated samples were then incubated in 100% ethanol and propylene oxide, then in two washes of pure propylene oxide. Embedded tissue sections of 500 nm thickness were stained with 1% toluidine blue and imaged on a light microscope to locate the location of islets. Thin sections (60-80nm) were cut, collected on copper mesh grids and stained with 2% uranyl acetate and lead citrate.
Morphometric analysis
Detection of apoptosis, superoxide, and amyloid
Quantitative RT-PCR
Secreted insulin was analyzed from culture medium and was normalized to the insulin content after cell lysis (cell lysis buffer: 1M Tris, Triton x-100, glycerol, 5M NaCl, 0.2M EGTA, protease inhibitor tablet).
Statistical analysis General statistics
For univariate analyses, statistical significance (calculation of a P value) was assessed by a Student's t test of the regression coefficient. Eight nodes were chosen to optimally capture the characteristic features of the island response curve while preserving degrees of freedom. The spline analyzes were performed to assess the effects of categorical variables, allowing different insulin secretion response curves to be fitted within different categories of the variable.
IN VIVO METABOLIC STRESS IMPAIRS ISLET TRANSCRIPTION FACTOR EXPRESSION AND INSULIN SECRETION IN HUMAN ISLETS
Introduction
However, alternative approaches involving studies of glucose and/or lipid excess in islet cell lines and islets in culture, whether murine or human culture, do not mimic islets in vivo, as cultured islets lack vascularization and innervation, however, islet culture itself leads to changes in islet function and gene expression. In addition, such in vitro studies are challenged by the selection of individual lipid species, lipid concentrations, and/or glucose concentrations. Because of these experimental limitations and species differences, the mechanisms by which excess glucose and/or lipids specifically impair human islet function in vivo are not fully understood.
Results
HFD induces insulin resistance in NSG mice. or 45% HFD impaired GTT in the NSG mice. Chronic hyperglycemia or chronic insulin resistance reduces antioxidant enzyme expression and increases superoxide levels in human islet grafts. The unfolded protein response is not upregulated in response to chronic hyperglycemia or chronic insulin resistance.
Discussion
Insulin resistance and hyperglycemia potently stimulated mouse β cell proliferation, but not human β cell proliferation. Moreover, HFD increased both peri-islet amyloid and intracellular lipid deposits in human islets. These included lack of metabolic stress-induced β-cell proliferation (Figure 32C and J), decreased insulin gene expression and unchanged insulin content (Figure 30B and C), appearance of intracellular lipid droplets (Figure 32C, F, G and J). , lack of UPR induction (Figure 34I), levels of the reactive oxygen species superoxide (Figure 34F), and changes in transcription factor expression (Figure 37D) in the human islets. Using three models of metabolic stress on human islets in vivo, this work demonstrates that hyperglycemia and insulin resistance impair stimulated insulin secretion in human islets in vivo, and this is at least partially due to reduced expression of NKX6.1 and/or MAFB.
HUMAN ISLET PREPARATIONS DISTRIBUTED FOR RESEARCH EXHIBIT A VARIETY OF INSULIN SECRETORY PROFILES
In vitro stimulated insulin secretion does not correlate with in vivo function of reactive islet preparations. Our linear regression and spline modeling results show that in vitro insulin secretion from human islet preparations has improved over the years studied. The majority of islet preparations from each center and year (except center 15 and year 2004) have a reactive profile (Group 1).
INVESTIGATING THE ROLE OF EGFR SIGNALING IN ADULT β CELL PHYSIOLOGY
Of the four, the EGF receptor (also known as ErbB1 or EGFR) seems particularly involved. The identity of the signaling pathway initiated by EGFR signaling is determined by specific phosphotyrosines. Given the ubiquitous importance of EGFR signaling in many organ systems, the pancreatic and islet phenotypes of the global knockout.
CONCLUSION
Summary of findings
Our extensive analyzes of human islet preparations for research have provided a new and informative classification system for in vitro insulin secretion profiles. These analyzes showed that overall the performance of human islet preparations improved over time, suggesting better isolation and handling techniques. To better inform decisions prior to human islet transplantation studies and to address general aspects of human islet biology, we were interested in whether in vitro function correlates with in vivo function.
Significance and future directions Glucotoxicity and lipotoxicity in human islets
