Islet Cell and ST2 Axis Dysregulation in Post-Transplant Diabetes Mellitus
Conditions: Diabetes Mellitus, Cancer
Sponsor: Vanderbilt-Ingram Cancer Center

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Islet Cell and ST2 Axis Dysregulation in Post-Transplant Diabetes Mellitus 
 
Principal Investigators: 
 
Brian Engelhardt, M.D., M.S.C.I. 
 
Co-Investigators / Key Study Personnel: 
 
Dae Kwang Jung, B.S. 
 
Division of Hematology-Oncology 
Department of Medicine 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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Table of Contents 
 
 
1.0 Rationale and Specific Aims 
       
 3 
 
 
 
 
 
 
 
 
2.0 Introduction and Background        
 4 
 
3.0 Preliminary Data  
 
 
 
 
 
 
 
  6 
 
 
 
 
 
 
 
  
4.0 Inclusion/Exclusion Criteria 
       
 8 
5.0 Enrollment 
         
 9 
6.0 Study Procedures 
       10 
7.0 Risks and Toxicity Management 
 
 
 
 
 
14 
 
8.0 Reporting of Adverse Events or Unanticipated Problems  
 
15 
  
          
9.0 Study Withdrawal/Discontinuation  
     1
5 
 
 
                    
                      
10.0 Statistical Considerations 
      16 
 
 
 
 
 
 
          
11.0 Privacy/Confidentiality Issues 
     1
8 
 
 
 
 
 
 
          
12.0 Followup and Record Retention 
     18 
 
 
 
 
 
 
          
13.0 References 
        19 
 
 
14.0 Appendix 
 
 
A. Study Table 
 
 
 
 
 
 
 
22 
 
 
 
 
 
 
 
 
          
 
 
 
 
 
 
 
 
          
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

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1.0 Rationale and Specific Aims: 
 
The development of type 2 diabetes mellitus is a significant complication for long-term 
cancer survivors including patients treated with allogeneic hematopoietic stem cell 
transplantation (HCT).  New onset, post-transplant diabetes mellitus (PTDM) occurs in 
up to 60% of HCT recipients and negatively impacts transplant survival (1). Currently 
8,000 HCT’s are performed each year in the United States primarily for patients with 
high risk hematological disorders.  These individuals comprise a well defined group that 
is at significant risk for developing new metabolic abnormalities including PTDM.  
 
We have found that PTDM resembles type 2 diabetes mellitus. Normally, type 2 diabetes 
mellitus results from a combination of insulin resistance and decreased insulin secretion. 
The transition from compensated insulin resistance to frank diabetes occurs as a result 
of β-cell failure mediated by metabolic overload and inflammation. In animal models, loss 
of IL-33 signaling through its receptor serum STimulation-2 (ST2) decreases 
immunosuppressive ST2+ regulatory T cells (Tregs) in visceral adipose tissue, increases 
Th1 inflammation, worsens insulin resistance, and promotes β-cell stress and diabetes 
conversion. We found that new-onset PTDM is characterized by: 1) elevated fasting C-
peptide levels pre-HCT; 2) impaired islet response to hyperglycemia and GLP-1 after 
HCT with decreased β-cell insulin secretion and blunted α-cell suppression; and 3) 
increased post-transplant serum levels of soluble ST2 (sST2), a decoy receptor which 
sequesters IL-33. We hypothesize that in PTDM, initial β-cell compensation progresses 
to exhaustion during the course of HCT, which coincides with increased tissue demand 
for insulin due to changes in IL-33 signaling, inflammation, and/or hyperglucagonemia.  
We propose to test this hypothesis with the following aims in a prospective, 
observational study in HCT recipients. 
 
Specific Aim 1. To determine if changes in islet cell physiology are detectable 
before or after matched related donor (MRD) HCT in patients developing new-
onset PTDM. 
 
Subaim 1A. To determine if a compensatory increase in glucose stimulated insulin 
secretion (GSIS) by β-cells precedes PTDM development in patients without 
diabetes undergoing MRD HCT. Based on our C-peptide and oral glucose tolerance 
test (OGTT) data, we hypothesize that PTDM-prone individuals maintain euglycemia 
pre-transplant via enhanced β-cell secretory capacity followed by progressive β-cell 
dysfunction and relative insulin insufficiency after transplant. In a prospective MRD HCT 
cohort without diabetes, β-cell function and secretory capacity will be directly measured 
with hyperglycemic clamps before and 90 days after transplant. 
 
Subaim 1B. To determine if excess glucagon secretion and impaired α-cell 
response to glucose or GLP-1 contributes to the hyperglycemia of PTDM. We will 
test the hypothesis that PTDM patients have increased glucagon levels and that glucose 
and GLP-1 induced α-cell suppression are incomplete. To assess α-cell dysregulation, 
glucose-induced glucagon suppression will be measured during a hyperglycemic clamp 
and during 2 OGTTs either with or without GLP-1 infusion, before and 90 days after 
HCT. 
 
Specific Aim 2. To determine if the IL-33/ST2 axis promotes immune/islet cell 
dysregulation during PTDM.  We hypothesize that PTDM is characterized by 
decreased protective IL-33/ST2 signaling in adipose tissue at rest (i.e. pre-transplant) 

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followed by increased systemic, inflammatory signaling during stress (i.e. post-
transplant), which leads to β-cell compensation and exhaustion, respectively. To 
investigate the ST2 pathway, we will measure adipose and plasma levels of IL-33 and 
sST2 and quantify ST2 expression on circulating Tregs and Th1 cells before and after 
HCT from our MRD cohort. IL-33 effects on T cells and on human islet function will be 
assessed in vitro.  
 
2.0 Introduction and Background 
 
Role of HCT 
 
Over 30,000 allogeneic HCTs are performed yearly, with the primary indication being 
hematological malignancy (2). Although malignancy relapse is the most important cause 
for death after transplant, HCT recipients who are cured of cancer continue to be at 
increased risk of developing significant secondary medical conditions that can impact 
transplant survival negatively (3, 4). Of particular interest is PTDM, due to its high 
prevalence after transplant and its detrimental effects on cardiovascular health (5, 6). 
 
Impaired glucose homeostasis in HCT recipients (PTDM) 
 
HCT recipients are over 3 times more likely to report a diagnosis of diabetes than their 
sibling counterparts (7). Our prospective data indicates that early onset PTDM occurs in 
up to 60% of individuals with no history of diabetes, and it can persist long-term in about 
10-30% of these patients with a negative impact on survival (1, 6-11).  Risk factors for 
diabetes in HCT recipients have been described including: older age, nonwhite ethnicity, 
steroid exposure, and total body irradiation, however the actual mechanisms causing 
PTDM are unknown (1, 6, 7, 10-13). We have found that new-onset PTDM is 
characterized by: 1) elevated fasting C-peptide levels pre-HCT; 2) impaired islet 
response to hyperglycemia and GLP-1 after HCT; and 3) increased post-transplant 
serum levels of soluble serum STimulation-2 (sST2), a decoy receptor which sequesters 
IL-33 (1, 14). We hypothesize that in PTDM, initial β-cell compensation progresses to 
exhaustion during the course of HCT, which coincides with increased tissue demand for 
insulin due to changes in IL-33 signaling, inflammation, and/or hyperglucagonemia. 
 
Type 2 diabetes mellitus results from a combination of impaired tissue response to 
insulin action and decreased insulin secretion. Due to compensatory changes in β-cell 
function, frank hyperglycemia occurs when pancreatic β-cells can no longer meet the 
insulin demands needed for glycemic control, an event termed β-cell failure. Distinct to 
transplantation is the use of diabetogenic immunosuppressive medications. There are at 
least 3 lines of evidence indicating that PTDM is not just an iatrogenic complication of 
immunosuppressive drugs. 1) Despite near universal treatment of HCT recipients with 
calcineurin inhibitors, not all patients develop PTDM. 2) The diagnosis of PTDM 
generally precedes corticosteroid treatment (1, 8, 14). 3)  Rates of diabetes among HCT 
survivors off of immunosuppression are still higher compared to their sibling controls. 
Therefore the assumption that PTDM is merely a manifestation of diabetogenic 
medications is flawed (7). Since islet cells are major regulators of glucose homeostasis, 
their physiology during PTDM needs to be investigated with validated techniques 
including the oral glucose tolerance test (OGTT) and hyperglycemic clamp. 
 
 
 

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IL-33/ST2 axis and immune/metabolic dysregulation 
 
Type 2 diabetes mellitus is an inflammatory disease characterized by the accumulation 
of IFN-γ secreting T cells (Th1 cells) and depletion of immunosuppressive Foxp3+ 
regulatory T cells (Tregs) in adipose tissue (15-18). The balance between Th1 cells and 
Tregs is a critical determinant of insulin sensitivity/demand which in turn influences β-cell 
function and stress. IL-33 is a dual cytokine with both inflammatory and regulatory 
functions. Normally, adipose tissue expresses IL-33, however cytokine levels are lower 
during obesity and insulin resistance (19). The IL-33 receptor, serum STimulation-2 
(ST2) can exist as a membrane bound protein on immune cells including Tregs but not 
usually Th1 cells, or as a soluble form (sST2) where it functions as a decoy receptor 
sequestering excess IL-33 (19, 20). In pre-clinical obesity models, the administration of 
IL-33 increases ST2+Tregs in adipose tissue and improves glucose tolerance compared 
to PBS-injected control animals (21, 22). Resolution of visceral adipose tissue 
inflammation by ST2+Tregs decreases insulin requirements and improves fasting blood 
glucose levels (17, 18). Similarly, our collaborators have shown in a murine model of 
GVHD, that blockade of sST2 with a neutralizing antibody increased IL-33, expanded 
ST2+Tregs, decreased inflammation, and improved transplant survival (20). However IL-
33 functions as a double-edged sword.  In the setting of HCT where multiple noxious 
stimuli are present, Th1 cells which usually do not express ST2, can acquire the receptor 
and become responsive to IL-33 released from damaged tissue resulting in lethal 
alloreactivity (23-25). Theoretically, this ST2+Th1 response could exacerbate 
inflammation leading to impaired insulin action, increased insulin demand, and increased 
β-cell work after HCT. IL-33 also directly induces β-cell endoplasmic reticulum stress 
which accompanies the insulin secretory defect of type 2 diabetes (26). Thus, the IL-
33/ST2 axis could affect PTDM conversion through several different mechanisms 
depending on the differential expression of ST2 by Tregs or Th1 cells,or whether IL-33 
exerts its action predominantly in the periphery by determining insulin needs or by 
affecting β-cell function directly. Although IL-33’s influence on glucose homeostasis 
during periods of quiescence have been described, IL-33’s metabolic affects following 
transplant of an allogeneic immune system and in the presence of ST2+Th1 cells during 
HCT is unknown. 
Rationale and clinical application   
 
HCT recipients are at significant risk for developing new-onset PTDM, which in turn 
confers inferior survival. The initiating events and mechanisms that culminate in PTDM 
development remain understudied, and formal recommendations for screening and 
treatment are lacking. In this proposal, we will use OGTT and hyperglycemic clamps to 
characterize the onset and role of pancreatic β- and α-cell dysfunction in metabolic 
complications after HCT. These results will be adapted into future screening tools for 
PTDM and translated into therapeutic clinical trials targeting either insulin insufficiency or 
glucagon excess. By analyzing IL-33 signaling and ST2 expression on Tregs and Th1 
cells sequentially before and after transplant, we will 1) investigate the relationship 
between islet function and ST2 immune regulation during HCT, 2) establish an immune 
timeline for the development of PTDM, and 3) develop a biological rationale for why 
treatment/ prevention of metabolic complications could alter ST2 signaling and improve 
HCT outcomes. We propose that understanding the immunology and the metabolic 
abnormalities generating PTDM will promote rapid improvements in the care of HCT 
recipients. 
 

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3.0 Preliminary Data 
 
Incidence and risk factors for early PTDM 
 
We prospectively analyzed PTDM incidence (defined as a weekly fasting glucose ≥ 126 
mg/dL or random glucose ≥ 200 mg/dL) during the first 100 days of HCT in 84 patients 
without diabetes (1). New-onset PTDM was diagnosed in 50 (60%) patients at a median 
of 23 days after HCT and prior to the start of steroids in the majority. PTDM was 
associated with inferior survival when analyzed in a time-dependent manner [hazard 
ratio (HR), 3.27; 95% confidence interval (CI), 1.3-8.2; P=0.01]. In multivariate analysis, 
elevated, pre-transplant fasting C-peptide level independently predicted both new-onset 
PTDM [odds ratio (OR), 5.9; 95% Cl, 1.77-20.22; P=0.004] and increased mortality (HR, 
1.05; 95%CI, 1.01-1.09; P=0.013) (1). This prospective study indicates that 
compensatory β-cell changes, as evidenced by increased fasting C-peptide level, could 
be present prior to HCT (and before exposure to immunosuppression) and that changes 
in glucose metabolism could lead to inferior HCT outcomes. 
 
Implications for β-cell failure and α-cell dysregulation during PTDM development 
 
Glucose homeostasis is maintained by the coordinated secretion of insulin by pancreatic 
β-cells and glucagon by α-cells (27, 28). In addition to blood glucose, this process is 
regulated by the gut-derived incretin hormones glucagon-like peptide-1 (GLP-1) and 
glucose-dependent insulinotropic polypetide (GIP) which are secreted postprandially, 
promoting insulin release and glucagon suppression (29). In a prospective observational 
cohort study funded by the NHLBI a standard OGTT was performed on HCT recipients 
without established diabetes before and 90 days after transplant (Figure 1). Although 
insulin levels appear similar before and after transplant in the 2 groups, only in patients 
maintaining post-transplant euglycemia are β-cells able to prevent significant glucose 
excursions during the challenge (bottom panel B). Compared to pre-transplant values, 
blood sugars were elevated at all time points after HCT with no corresponding increase 
in insulin secretion by the PTDM group (top panel A). These data are most consistent 
with the development of a new β-cell defect after HCT. 
 
Glucagon is secreted in response to hypoglycemia where it promotes hepatic glycogen 
breakdown and gluconeogenesis. Our preliminary data suggests that PTDM patients 
have elevated fasting and post-prandial glucagon levels when compared to their pre-
transplant values and when compared to individuals maintaining euglycemia after HCT. 
This inappropriate glucagon elevation in PTDM patients exists despite factors known to 
inhibit α-cells including elevations in blood glucose, insulin, and GLP-1 (Figure 1). Taken 
together these data indicate a role for hyperglucagonemia and impaired α-cell regulation 
in PTDM 

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Figure 1. OGTT suggests β-cell failure and impaired glucagon suppression during 
PTDM after HCT. Representative data from 8 HCT recipients without a pre-
transplant diagnosis of diabetes is shown. Blood glucose, insulin, C-peptide, 
glucagon, and GLP-1 were measured every 30 minutes for 2 hours during a 
standard OGTT performed before and 90 days after HCT. Median values are 
depicted and data is stratified for the development of new-onset PTDM (A) or 
maintenance of euglycemia (B).  
 
GLP-1 is an important regulator of glucagon suppression. As expected, after an oral 
glucose load, GLP-1 is secreted with a corresponding increase in insulin and decline in 
glucagon (Figure 1). We hypothesize that the exaggerated GLP-1 secretion in PTDM 
patients after transplant represents an incomplete compensatory mechanism meant to 
restore homeostasis which will be explored further in Aim 1B with possible therapeutic 
ramifications.  
 
PTDM is Th-1 skewed and characterized by altered ST2 signaling  
Elevated fasting C-peptide levels pre-transplant and PTDM development after HCT both 
predict inferior survival (1, 8). Our collaborators have demonstrated that pre-transplant 
elevations in the decoy receptor sST2 are seen with obesity and that high sST2 after 
HCT is a validated biomarker for non-relapse mortality due to immune dysregulation (30-
32). Published literature indicates that low IL-33 levels are involved in insulin resistance 
in non-transplant animal models of diabetes (21, 22). Therefore we sought to examine 
sST2 as a predictor of PTDM independent of GVHD.  In a group of HCT recipients 
without preexisting diabetes, serum sST2 was elevated at neutrophil engraftment in 
patients who were prospectively diagnosed with new-onset PTDM after HCT (Figure 2). 
This increase in engraftment sST2 was confirmed in a separate cohort of HCT recipients 
who were retrospectively diagnosed with PTDM (14). We also confirmed that the 
majority of PTDM diagnoses occurred before steroids exposure (67% in cohort 1 and 
93% in cohort 2). Since PTDM generally occurs before steroids then steroids should not 
influence the development of PTDM for the majority. After adjustment for myeloablative 
chemotherapy and grade 3-4 acute GVHD (factors known to influence sST2 and 
survival), high engraftment sST2 (≥75th percentile of combined cohorts 1 and 2) 
predicted increased PTDM (HR, 2.25; 95%CI, 1.07-4.75; P= 0.03) and non-relapse 
mortality risk (HR, 8.54; 95%CI, 1.62-45.15; P = 0.01) (14). Thus, the data suggests that 
dysregulation of the IL-33/ST2 axis may influence metabolic complications after HCT 
which in turn presages clinical GVHD, steroid exposure, and mortality. 
 

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Figure 2. Increased sST2 after HCT 
is a validated PTDM predictor. 
Serum sST2 was measured by 
ELISA at engraftment in 36 
euglycemic HCT recipients followed 
prospectively for PTDM (cohort 1). 
Results were confirmed in a second 
cohort of 26 patients without pre-
existing diabetes retrospectively 
analyzed for PTDM (cohort 2). 
 
Type 2 diabetes mellitus is characterized by systemic inflammation (15-18). Using 
established cytometric assays, we found that by day+10 after HCT inflammatory IFN-γ+ 
Th1 cells were increased in patients prospectively followed for PTDM (Figure 3). This 
inflammatory response in PTDM could be driven by IL-33/ST2 disequilibrium and 
upregulation of ST2 by Th1 cells following HCT (Figure 4). The net 
effect of this IL-33/ST2 signaling after HCT could be Th1 
inflammation which overwhelms immune regulation and causes β-
cell stress 
 
Figure 3. PTDM is characterized by an inflammatory / Th-1 
skewed environment.  A cohort of 18 HCT recipients were 
prospectively followed for PTDM. The frequency of circulating 
Th1 cells were determined by flow cytometry. 
Figure 4. ST2 is expressed by a significant 
population of Th1 cells but not Th17 cells. HCT 
recipient PBMCs were stimulated for 5 hours 
with PMA/ionomycin/monensin. Cells were 
surface-stained, fixed and permeabilized and 
then intracellularly stained with an assay 
containing: CD14, CD3, CD4, ST2, IL-17, IFN-γ, 
and amine viability dye. Viable CD3+CD4+ T 
cells were identified followed by gating on ST2 
vs. IFN-γ or IL-17 to determine the frequency of 
ST2+Th1 (A) or ST2+Th17 cells (B), respectively. 
 
In summary, our data suggests that immune dysregulation and changes in IL-33/ST2 
signaling contributes to the pathogenesis of PTDM which begins before transplant with 
compensatory β-cell changes followed by islet dysfunction manifesting as insulin 
insufficiency and hyperglucagonemia after HCT. 
 
4.0 Inclusion/Exclusion Criteria 
 
Inclusion Criteria for Patients: 
 
- Age ≥ 18 years 
- Patients undergoing MRD allogeneic HCT 
 
 
Exclusion Criteria for Patients: 

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- Patients who have not received an allogeneic HCT 
- Recent or current history of diabetes mellitus, defined as: 1) diabetes therapy within 6 
months of enrollment, or 2) fasting blood glucose at “pre-admit” (screening) visit ≥ 
126mg/dL 
- Pregnancy or breastfeeding 
- Unrelated donor, umbilical cord blood, mismatched, or haploidentical transplants 
- Patients receiving T cell depletion or thymoglobulin as part of their transplant 
- Patients on established, chronic corticosteroid therapy (> 10 mg /day of prednisone or 
prednisone equivalent) prior to transplant. Established, chronic corticosteroid therapy is 
defined as daily dosing of > 10 mg / day of prednisone or prednisone equivalent for at 
least 2 weeks prior to the start of conditioning/chemotherapy or plans to continue pre-
transplant corticosteroids (> 10 mg /day of prednisone or prednisone equivalent) 
indefinitely after transplantation.  
- Inability to give informed consent 
- Any condition which, in the opinion of the investigator, might interfere with study 
objective 
- Any reason which, in the opinion of the investigator, adds additional risk to the patient 
 
Additional exclusion criteria (Aim 1 only): 
-Diagnosis of diabetes by standard oral glucose tolerance testing prior to transplant (2-
hour plasma glucose value ≥ 200 mg/dL). 
 
Inclusion Criteria for Donors: 
 
- Age ≥ 18 years 
- Donors undergoing stem cell collection for match related allogeneic stem cell transplant 
 
 
Exclusion Criteria for Donors: 
 
- Individuals not donating stem cells 
- Pregnancy or breastfeeding 
- Inability to give informed consent 
- Any condition which, in the opinion of the investigator, might interfere with study 
objective 
 
 
5.0 Enrollment 
 
Potential study candidates will be recruited from the Hematology-Oncology Clinics at the 
Vanderbilt Ingram Cancer Center (VICC) by a member of the research team prior to their 
allogeneic stem cell transplant. Potential participants including patients and donors will 
be identified at the time of acceptance for transplant by their treating physicians. Patients 
and donors who may qualify for participation will be provided a copy of the IRB-approved 
Informed Consent document to read. Subjects who express interest will then meet with a 
member of our research team during their regularly scheduled clinic visit.  Interested 
subjects will be screened for inclusion and exclusion criteria.  Standard of care 
laboratories (i.e. pregnancy test if indicated and fasting blood glucose) will be reviewed.  

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Those subjects who sign consent and who fulfill all inclusion and exclusion criteria will 
then be enrolled in the study.   
 
 
 
6.0 Study Procedures 
 
Sources of materials- Donors 
 
Donors who have given informed consent will undergo standard of care evaluations or 
study visits at either the Outpatient Transplant Unit or the inpatient Myelosuppression 
Unit at Vanderbilt University Medical Center. Study blood will be obtained once prior to 
beginning the stem cell mobilization procedure. A single blood draw of 50-60 ml will be 
collected once. Venous blood will be obtained via peripheral venipuncture and preferably 
will be collected during routine laboratory analysis.  Samples will be coded with a unique 
alphanumeric identifier. Clinical information and research data will be obtained by donor 
interview, medical chart review, and from laboratory testing. Information will be stored in 
a password-protected electronic database. After the blood draw no further testing will be 
performed on the donor. 
 
A single sample of 50-60 mL of blood will be collected from the donor at one time point 
during this study. 
 
Sources of materials- Patients 
 
Study participants who have given informed consent will undergo standard of care 
evaluations or study visits at either the Outpatient Transplant Unit, the inpatient 
Myelosuppression Unit, or the Clinical Research Center (CRC) at Vanderbilt University 
Medical Center. Two oral glucose tolerance tests and 1 hyperglycemic clamp will be 
performed on separate days prior to transplant and then each procedure will be repeated 
once between day+80 to day+100 (+/- 10 days) after transplant (Figure 5).  In addition to 
the 6 research visits, study blood will be obtained at the following timepoints: pre-
transplant (prior to beginning chemotherapy), Day+10, Day+20, Day+30, and Day+90 
(all +/- 7 days) (the day of stem cell infusion is considered Day 0).  A total of 550 mL of 
blood will be collected from the patient over approximately 110 days (over 3 to 4 
months).  Blood will be drawn for research purposes at the same time as routine blood 
sampling when feasible. Venous blood will be obtained by accessing the central venous 
catheter using sterile technique or via peripheral venipuncture.  Samples will be coded 
with a unique alphanumeric identifier at the time of collection so that patient identity will 
be available only to study investigators and research staff. Clinical information, 
outcomes, and research data will be obtained by patient interview, medical chart review, 
and from laboratory testing. Information will be stored in a password-protected electronic 
database.   
 
 
 
 
 
 
 

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Figure 5. Study schema and timeline 
 
 
OGTT 
Two OGTTs (without and with GLP-1 infusion) will be performed prior to HCT and 
after an 8 hour overnight fast (water and medications allowed) in patients with a 
screening blood glucose < 126 mg/dL. Plasma samples will be analyzed for glucose, 
insulin, glucagon, C-peptide, GLP-1 every 30 minutes from Time -30 minutes to Time 
120 minutes (6 time points) as per physician discretion. Remaining aliquots will be 
stored. At time 0, participants will ingest 75 g of water-free glucose dissolved in 300 mL 
of water. Pre-HCT patients will be categorized per the 2-hour OGTT plasma glucose 
value as normal (<140 mg/dL), impaired glucose tolerance (140-199mg/dL), or diabetic 
(≥ 200 mg/dL) (33, 34).  Study patients diagnosed with diabetes by standard OGTT prior 
to HCT will not be monitored for de novo PTDM. Study patients diagnosed with diabetes 
by standard OGTT prior to HCT will not undergo further testing as outlined in aim 1 after 
the diagnosis is made, however the immune / metabolic studies as outlined for aim 2 will 
be performed in these individuals. 
 
The second OGTT procedure will be repeated on a different day with exogenous GLP-1 
infusion using similar methods as described above. At Time -30 minutes, a 2.0 
pmol/kg/min infusion of GLP-1 will be given IV and continued for the remainder of the 
procedure (150 minutes). This concentration of GLP-1 is well tolerated (35). GLP-1 (7-
36) amide acetate will be purchased from Clinalfa, Bachem (Weil am Rhein, Germany). 
Vanderbilt Investigational Drug Service (IDS) has experience preparing this medication 
for use in clinical investigation (36). Biologically active GLP-1 is rapidly metabolized by 
the proteolytic enzyme DPP-4 with a half-life < 2 minutes in the circulation. Thus, the 
physiologic effects of GLP-1 should be negligible shortly after the infusion is stopped at 
time = 120 minutes (29). 
 
A total of about 90 ml of blood will be drawn for the 2 OGTTs. The 2 OGTTs will be 
repeated after transplant between day +80 to day +100 +/- 10 days. 
 
 

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Adipose tissue microdialysis 
During the OGTT without GLP-1 infusion, a microdialysis catheter will be inserted into 
abdominal subcutaneous adipose tissue using local anesthesia and sterile technique.  At 
the start of the OGTT, the microdialysis pump containing T1 perfusion fluid (M Dialysis) 
will be infused at 1 μl/min. After 60 minutes the microdialysate will be discarded and a 
fresh microdialysate sample will be obtained over 90 minutes. Samples will be collected 
on ice and stored at -80° C until analysis.  
 
Hyperglycemic clamp 
 
After an overnight fast, pre-HCT patients will undergo a hyperglycemic clamp procedure, 
as described by DeFronzo et. al. and by our collaborators (37, 38). Arterialized blood will 
be collected by using a heating pad  after insertion of a venous catheter (39). Baseline 
values for glucose, insulin, C-peptide, and glucagon will be calculated by averaging the 
measurements obtained at -20, -10, and -1 minute before initiation of the glucose 
infusion. At time 0, a 200 mg/kg priming dose of 20% dextrose (Hospira) will be given for 
10 minutes. Thereafter, blood glucose will be measured approximately every 5 minutes 
and plasma glucose will be maintained at 200 mg/dL with a variable infusion of 20% 
dextrose solution for 150 minutes (37, 38). Insulin, C-peptide, and glucagon will be 
measured 3 times during the first 10 minutes, and then every 10 minutes from 90 to 120 
minutes. At time 120 minutes, 5 gm of the secretagogue L-arginine will be injected over 
1 minute while the patient is hyperglycemic (40). Insulin, C-peptide, and glucagon levels 
will be measured 3 times from time 120-130 minutes and then every 10 minutes from 
130 to 150 minutes. At 150 minutes the glucose infusion and the procedure will be 
stopped.  
The clamp, 2 OGTTs, and microdialysis procedure will be repeated after HCT between 
day+80 to +100 (+/- 10 days). Patients diagnosed with PTDM requiring treatment after 
HCT will be maintained on short-acting insulin which will be held the morning of the 
studies. Oral antidiabetes medications are not routinely used at our HCT center, 
however if prescribed, these agents will be washed out for 5 days prior to the procedure 
and the patient will be maintained on short-acting insulin as above. 
 
Calculations 
First-phase GSIS will be calculated in 2 ways: 1) the difference between the maximum 
plasma insulin level during the first 10 minutes and the average of the 3 baseline insulin 
values (ΔInsulin0-10) or 2) as the AUC for insulin above baseline value from time= 0 to 10 
minutes (AUCΔInsulin0-10). Second or late-phase GSIS will be calculated in the same 
manner but from time 90-120 minutes (37). These analyses will be repeated using C-
peptide and glucagon measurements. Maximum hormone secretion will be determined 
by the acute insulin, C-peptide, and glucagon response to L-arginine (AIRarg, ACRarg, and 
AGRarg, respectively). These values will be calculated as the mean hormone level for the 
first 10 minutes after arginine stimulation minus their average baseline levels (37). 
 
Immune / Metabolic Studies 
 

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In addition to the 6 study visits, this protocol will involve 5 separate blood draws for the 
patient [pre-transplant, Day +10, Day +20, Day +30, and Day +90 (all +/- 7 days)].  
During each blood draw, no more than 50 ml of blood will be drawn for serum and 
lymphocyte analysis. Aliquots will be analyzed using standard immunologic techniques 
and by flow cytometry as outlined in the preliminary data section. Blood will be drawn for 
research purposes at the same time as routine blood sampling. Venous blood will be 
obtained by accessing the central venous catheter using sterile technique or via 
peripheral venipuncture.  Samples will be coded with a unique alphanumeric identifier at 
the time of collection so that patient identity will be available only to study investigators 
and research staff.  Codes will be sequential numbers and letters.    
 
Blood samples and database will be maintained indefinitely by the study investigators or 
the laboratory of Crowe/Engelhardt and the research staff. A total of 250 ml of blood will 
be drawn on 5 occasions for lymphocyte and serum analysis. 
 
 
Static islet incubation 
 
Human islets will be obtained from the NIDDK supported, Integrated Islet Distribution 
Program (http://iidp.coh.org/). In this program, pancreatic islets are prepared from 
cadaver donors by islet isolation centers around the USA. This human tissue can be 
sent to more than 100 investigators without identifying personal health information. 
Investigators are unable to track any information related to the pancreas donor. The only 
information received is gender, age, BMI, and whether or not the donor had diabetes. 
Because of these factors, this research is generally considered non-human research. 
After receipt of the de-identified tissue, islet set viability and secretory function will be 
assessed during exposure to various cytokines including IL-33. Vanderbilt Islet 
Procurement and Analysis Core will perform islet experiments using standard 
techniques. 
 
Diagnosis of PTDM 
 
PTDM is defined by the occurrence of a fasting blood glucose level ≥126 mg/dL or a 
random blood glucose level ≥200 mg/dL at any point from day +1 (day after stem cell 
infusion) to day +100 after transplant.  Fasting blood glucose level will be assessed 
weekly during routine outpatient visits from day 1 to day 100 after transplant.  Patients 
will be evaluated following an 8 hour overnight fast (water and medications allowed) 
during standard of care visits.  Random blood glucose levels from day +1 to day +100 
after transplant will be assessed during standard of care visits or during hospitalization. 
For hospitalized patients and patients receiving total parenteral nutrition (TPN), all blood 
glucose determinants will be considered random blood glucose levels for purposes of 
analysis. 
 
Data Management and Safety 
 
All data will be collected prospectively until day 100 post-transplant.  Information will be 
stored in a password protected electronic database.  After day 100, the database will be 
updated when the patient is seen in the clinic for their annual visit (i.e. 1 year after 
transplant). In addition, the standard of care in the transplant program is to discuss 
complex inpatients and outpatients (within the first 100 days) on a weekly basis. Detailed 
minutes of these meetings are routinely maintained. Major events (e.g.: unexpected 

Version 1.1 dated December 1, 2017 
14
toxicity and all deaths) are routinely reviewed in a Quality Assurance Meeting and 
detailed minutes are kept. The above measures will ensure that study patients are 
monitored closely after transplant and that data will be accurate. 
 
A total of 550 mL of blood will be collected from the patient over approximately 110 days 
(over 3 to 4 months) for this study. 
 
7.0 Risks and Toxicity Management 
 
Risks 
 
1) Study visits at Vanderbilt  can be inconvenient 
2) Venipuncture, blood sampling, and catheter insertion can cause mild discomfort, 
bruising, and rarely infection 
3) Repeated blood sampling can worsen anemia, which is monitored frequently after 
transplant 
4) Symptoms of hyperglycemia including: thirst, frequent urination, and dry mouth may 
develop during the hyperglycemic clamp. Symptoms will be monitored closely and 
minimized by keeping blood glucose levels at 200 mg/dL by adjusting the glucose 
infusion. 
5) Arginine is a potent stimulator of insulin and glucagon secretion and will be used 
during the hyperglycemic clamp. Arginine is commonly employed as a diagnostic test for 
growth hormone deficiency. It may cause flushing, nausea, or vomiting in about 3% of 
subjects using doses 6 times the amount used in this study. Since arginine can cause 
vein irritation, it will be administered by a central venous catheter or large vein to 
minimize these symptoms. 
6) GLP-1 [Clinalfa, Bachem (Weil am Rhein, Germany)] will be infused intravenously 
during the OGTT.  GLP-1 can cause nausea, vomiting, headache, and hypoglycemia 
(usually in patients on combination therapy for diabetes). The dose of GLP-1 in this 
study is well tolerated and the hormone is rapidly metabolized with a half-life < 2 minutes 
in the circulation. Thus the effects of GLP-1 should be negligible shortly after stopping 
the infusion. Based on previous experience, GLP-1 should have no significant side 
effects at the dose given in this study. 
For toxicity attributed to the study, the NCI Common Terminology Criteria for Adverse 
Events (CTCAE) version 5.0 will be used to characterize the severity. 
 
Protection against risk 
1) To minimize patient discomfort and venipuncture, blood draws for research purposes 
will be coordinated with routine clinical blood sampling when possible and will use 
existing central venous catheters when available 
 
2) Nurse or physician will be present during study days 

Version 1.1 dated December 1, 2017 
15
 
3) Anemia is common after HCT and patients are intensively followed with complete 
blood counts.  Patients routinely receive blood transfusions for a hematocrit <24. Blood 
for research purposes will not be drawn if hematocrit is known to be <24 and the patient 
has not yet received transfusion. Once transfusion has been performed, study blood will 
be drawn. 
 
4) Patients will be monitored closely during study visits and glucose infusions will be 
adjusted to prevent symptomatic hyperglycemia during hyperglycemic clamps. 
5) A unique alphanumeric code will be assigned to each study participant and their study 
samples to protect their confidentiality.  Clinical information and laboratory data will be 
kept in a password protected database maintained by and accessible to the study 
investigators and research staff.  The key to decode patient identity will be kept in a 
password-protected database. Only the investigators and research staff will have access 
to the database. 
 
 
8.0 Reporting of Adverse Events or Unanticipated Problems 
 
Any untoward medical event will be classified as an adverse event (AE), regardless of its 
causal relationship with the study. An adverse event will be classified as serious (SAE) if 
it a) results in death, b) is life-threatening, c) requires inpatient hospitalization or 
prolongation of existing hospitalization, d) results in persistent or significant disability or 
incapacity, or e) is a congenital anomaly or birth defect.   
 
Patients undergoing HCT are at high risk for AE’s and often require multiple admissions to the 
hospital during the first 3-4 months after transplantation. For the purposes of this study, only the 
following SAE’s will be reported to the IRB: 
 
-Failure of neutrophil engraftment by day 35 after transplant.  Neutrophil engraftment is defined 
as the first of 3 consecutive days of absolute neutrophil count ≥ 0.5 x 109/L. 
 
-Any death not related to chemotherapy/conditioning/immunosuppression regimen, progression 
of malignancy, or GVHD (only until day +100 post transplant) 
 
-Any grade 4 or higher non-hematologic toxicity (not related to 
chemotherapy/conditioning/immunosuppression regimen, progression of malignancy, or GVHD) 
(Only until day +100 post transplant). 
 
Deaths not related to chemotherapy/conditioning regimen/immunosuppression regimen, 
progression of malignancy, or GVHD be reported to the IRB within 24 hours of the Investigators 
knowledge of the occurrence and all other toxicities will be reported within 7 days of the 
Investigator’s knowledge of occurrence. 
 
 
9.0 Study Withdrawal/Discontinuation  
 
Patients will be followed after transplantation.  Individuals may withdraw from the study 
at any time by notifying one of the study investigators.  Withdrawal from the study will be 
noted in the database.  Following withdrawal, no subsequent blood collections for study 

Version 1.1 dated December 1, 2017 
16
purposes will be obtained, and no further clinical information will be collected for this 
study. 
 
In addition, the investigator has the right to withdraw a patient from the trial for any of the 
following reasons: 
 
-protocol violation 
 
-non-compliance 
 
-any condition which, in the opinion of the investigator, might interfere with study 
objective 
 
-any reason which, in the opinion of the investigator, adds additional risk to the patient 
 
10.0 Statistical Considerations 
 
10.1 Power Analysis and Sample Size Calculation 
 
D.3.c. Sample size calculation for Aims 1 and 2. 
β-cell function, glucagon suppression, and IL-33 signaling are the primary outcomes for 
the aims. Sample size was calculated using a 2-group t-test with PS Software (version 
3.0, Dupont&Plummer, 2009) at a 0.05 two-sided significance level. We estimate a 50% 
incidence of PTDM by day+100 (1, 8, 14). The sample size provides at least 80% power 
to detect a biologically meaningful difference in mean between patients with and without 
PTDM for each aim. The same patient cohort will be monitored for all aims. We will 
enroll 34 patients to ensure at least 80% power for each aim. Assuming the dropout rate 
is about 10% (early death or pre-HCT diagnosis of diabetes by OGTT), an additional 6 
subjects will be recruited (41-43).  Forty subjects are feasible to accrue over 4 years, 
given that 151 patients underwent allogeneic HCT at Vanderbilt in 2015.  
Subaim 1A primary outcome: second-phase GSIS. A mean difference of 7.5 μU/mL 
represents a significant difference in second-phase insulin release between healthy 
controls and individuals at risk for type 2 diabetes (44-46). A sample size of 17 patients 
in each group (either with or without PTDM) has 80% power to detect at least a 7.5 
μU/mL difference  in insulin secretion between groups using a standard deviation of 7.5 
μU/mL. 
Subaim 1B primary outcome: glucagon secretion during OGTT. AUC was 
calculated for each patient to present as total glucagon secretion during OGTT. Our 
preliminary data observed 8.83± 0.73 pg/mL•min (mean ± SD) in log(AUC) scale for 
patients with PTDM (Figure 4). With a sample size of 9 each group, there is 80% power 
to detect a log(AUC) change of from 8.83 pg/mL•min to 7.83 pg/mL•min. 
Aim 2 primary outcome: IL-33 signaling Extrapolating our preliminary and pre-clinical 
data, a mean difference of 70 pg/mL is likely to represent a significant difference in IL-33 
signaling between groups (14, 20). With a sample size of 17 in each group, there is 80% 
power to detect a 70 pg/mL difference in IL-33 production between groups using a 
standard deviation of about 70 pg/ml.  
 

Version 1.1 dated December 1, 2017 
17
10.2 Statistical Analysis Plan 
 
Primary analysis of Subaim 1A 
We will compare pre-transplant insulin secretion (first- and second-phase GSIS) among 
patients who do or do not go on to develop PTDM. Wilcoxon rank sum test will be 
applied to compare the population mean difference between these two groups. 
Multivariable logistic regression will evaluate whether GSIS is an independent predictor 
of PTDM after adjusting for the following covariates: gender, conditioning (ablative vs. 
reduced intensity), or acute GVHD requiring steroids. The estimated OR and 95% CI of 
the OR will be provided to measure the effect of the association. We will take a similar 
approach for the primary analysis of Aims 1B and 2 which will compare post-transplant 
total glucagon secretion during the OGTT (AUCGlucagon0-120) and serum IL-33 levels 
among patients with or without PTDM, respectively.  
Secondary analyses for Subaims 1A and 1B   
1) determining if glucagon secretion and OGTT results  pre-transplant predict PTDM 
development (herein OGTT results refers to 2 hour blood glucose and peak/nadir/total 
hormone levels during OGTT), 2) determining if GLP-1 infusion increases insulin 
secretion and glucagon suppression compared to OGTT values without GLP-1, 3) 
comparing GSIS, glucagon suppression, and OGTT results at day +90 among patients 
with or without established PTDM, and 4) comparing the change in GSIS, glucagon 
suppression, and OGTT results at baseline to those determined at day +90 after 
transplant in the entire cohort or among groups stratified based on PTDM, conditioning 
regimen (ablative vs. reduced intensity) or for the presence or absence of acute GVHD 
treated with steroids.  
Secondary analyses for Aim 2 
1) determining if PTDM is predicted by pre- or post-transplant levels of IL-33/sST2 from 
serum/tissue microdialysate, or by the frequency of circulating ST2+Th1 cells and ST2+ 
Tregs, 2) analyzing whether IL-33/sST2 levels and ST2+Th1 cells and ST2+Tregs 
correlate with insulin/glucagon secretion during OGTT and hyperglycemic clamps, 3) test 
whether in vitro exposure of HCT recipient CD4+ T cells to IL-33 promotes proliferation 
and inflammatory Th1 immunity, and 4) test if in vitro exposure of human islets to IL-33 
impairs insulin secretion and decreases islet cell viability in culture.  
General statistical plan 
Data will be presented using means and standard deviations for continuous variables, as 
well as percentage and frequency for categorical variables. Investigations for outliers 
and assumptions for statistical analysis, e.g., normality and homoscedasticity will be 
made for the parametric test such as t-test and mixed model. If necessary, data will be 
transformed utilizing appropriate transformations such as the log or square root. 
Comparison between two independent groups, e.g., with or without PTDM, will be 
carried out using either two-sample t-test or Wilcoxon rank sum test for continuous 
variables and Chi-square test or Fisher’s exact test for categorical variables. A 
Spearman correlation will be used to test the relationship between 2 continuous 
variables.  For matched analyses and for the comparison of the changes between 
baseline and day+90 after transplant in the entire cohort, the mean difference will be 
compared with paired Student’s t-test or Wilcoxon signed rank test for continuous 

Version 1.1 dated December 1, 2017 
18
variables and a McNemar’s test for categorical variables. To compare the differences 
between baseline (pre-transplant) and day +90 test results among various groups we will 
apply linear mixed model to continuous outcomes and generalized linear mixed model to 
binary/ordinal outcomes. Groups of interest will be created by stratifying patients based 
on PTDM, conditioning regimen (ablative vs. reduced intensity) or acute GVHD treated 
with steroids. All data will be stored in a computerized database and analyzed with R 
version 3.3.1 (R Foundation for Statistical Computing, Vienna, Austria).  P-values will be 
2-tailed and considered significant at P <0.05. 
 
11.0 Privacy/Confidentiality Issues 
 
A unique alphanumeric code will be assigned to each study participant to protect their 
confidentiality.  Clinical information and laboratory data will be kept in a password 
protected database maintained by and accessible to the study investigators and 
research staff.  The key to decode patient identity will be kept in a password protected 
database. Only the investigators and research staff will have access to the database.  All 
communications with non-investigators of this study and any publication of results will 
identify patients by code only to protect patient identity. 
 
 
12.0 Followup and Record Retention 
 
All research records including study results will be retained for at least 6 years after the 
study is finished. At that time, the research data that has not been put in a participant’s 
medical record and all s tudy samples will be k ept for an u nknown length of time. Any 
research data that has been put into a participant’s medical record w ill be k ept for an 
unknown length of time.    
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Version 1.1 dated December 1, 2017 
19
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7. 
Baker KS, Ness KK, Steinberger J, Carter A, Francisco L, Burns LJ, et al. 
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8. 
Engelhardt BG, Jagasia SM, Crowe JE, Jr., Griffith ML, Savani BN, Kassim AA, 
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Lee SJ, Seaborn T, Mao FJ, Massey SC, Luu NQ, Schubert MA, et al. Frequency 
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Majhail NS, Challa TR, Mulrooney DA, Baker KS, Burns LJ. Hypertension and 
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Taskinen M, Saarinen-Pihkala UM, Hovi L, Lipsanen-Nyman M. Impaired glucose 
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12. 
Oudin C, Simeoni MC, Sirvent N, Contet A, Begu-Le Coroller A, Bordigoni P, et 
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13. 
Griffith ML, Jagasia M, Jagasia SM. Diabetes mellitus after hematopoietic stem 
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15. 
Kahn SE, Hull RL, Utzschneider KM. Mechanisms linking obesity to insulin 
resistance and type 2 diabetes. Nature. 2006;444(7121):840-6. 
16. 
Hotamisligil GS. Inflammation and metabolic disorders. Nature. 
2006;444(7121):860-7. 
17. 
Winer S, Chan Y, Paltser G, Truong D, Tsui H, Bahrami J, et al. Normalization of 
obesity-associated insulin resistance through immunotherapy. Nat Med. 2009;15(8):921-
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18. 
Feuerer M, Herrero L, Cipolletta D, Naaz A, Wong J, Nayer A, et al. Lean, but not 
obese, fat is enriched for a unique population of regulatory T cells that affect metabolic 
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19. 
Molofsky AB, Savage AK, Locksley RM. Interleukin-33 in Tissue Homeostasis, 
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Zhang J, Ramadan AM, Griesenauer B, Li W, Turner MJ, Liu C, et al. ST2 
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21. 
Miller AM, Asquith DL, Hueber AJ, Anderson LA, Holmes WM, McKenzie AN, et 
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22. 
Vasanthakumar A, Moro K, Xin A, Liao Y, Gloury R, Kawamoto S, et al. The 
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Yang Q, Li G, Zhu Y, Liu L, Chen E, Turnquist H, et al. IL-33 synergizes with 
TCR and IL-12 signaling to promote the effector function of CD8+ T cells. European 
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24. 
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25. 
Reichenbach DK, Schwarze V, Matta BM, Tkachev V, Lieberknecht E, Liu Q, et 
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26. 
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27. 
Halban PA, Polonsky KS, Bowden DW, Hawkins MA, Ling C, Mather KJ, et al. 
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28. 
Quesada I, Tuduri E, Ripoll C, Nadal A. Physiology of the pancreatic alpha-cell 
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29. 
Baggio LL, Drucker DJ. Biology of incretins: GLP-1 and GIP. Gastroenterology. 
2007;132(6):2131-57. 
30. 
Vander Lugt MT, Braun TM, Hanash S, Ritz J, Ho VT, Antin JH, et al. ST2 as a 
marker for risk of therapy-resistant graft-versus-host disease and death. N Engl J Med. 
2013;369(6):529-39. 
31. 
Ponce DM, Hilden P, Mumaw C, Devlin SM, Lubin M, Giralt S, et al. High day 28 
ST2 levels predict for acute graft-versus-host disease and transplant-related mortality 
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32. 
Gleimer M, Li Y, Chang L, Paczesny S, Hanauer DA, Frame DG, et al. Baseline 
body mass index among children and adults undergoing allogeneic hematopoietic cell 

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transplantation: clinical characteristics and outcomes. Bone Marrow Transplant. 
2015;50(3):402-10. 
33. 
Inzucchi SE. Clinical practice. Diagnosis of diabetes. N Engl J Med.367(6):542-
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34. 
Diagnosis and classification of diabetes mellitus. Diabetes Care.33 Suppl 1:S62-
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35. 
Hare KJ, Knop FK, Asmar M, Madsbad S, Deacon CF, Holst JJ, et al. Preserved 
inhibitory potency of GLP-1 on glucagon secretion in type 2 diabetes mellitus. J Clin 
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36. 
Devin JK, Pretorius M, Nian H, Yu C, Billings FTt, Brown NJ. Dipeptidyl-
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37. 
Luther JM, Byrne LM, Yu C, Wang TJ, Brown NJ. Dietary sodium restriction 
decreases insulin secretion without affecting insulin sensitivity in humans. J Clin 
Endocrinol Metab. 2014;99(10):E1895-902. 
38. 
DeFronzo RA, Tobin JD, Andres R. Glucose clamp technique: a method for 
quantifying insulin secretion and resistance. Am J Physiol. 1979;237(3):E214-23. 
39. 
Abumrad NN, Rabin D, Diamond MP, Lacy WW. Use of a heated superficial 
hand vein as an alternative site for the measurement of amino acid concentrations and 
for the study of glucose and alanine kinetics in man. Metabolism. 1981;30(9):936-40. 
40. 
van Haeften TW, Voetberg GA, Gerich JE, van der Veen EA. Dose-response 
characteristics for arginine-stimulated insulin secretion in man and influence of 
hyperglycemia. J Clin Endocrinol Metab. 1989;69(5):1059-64. 
41. 
Engelhardt BG, Sengsayadeth SM, Jagasia M, Savani BN, Kassim AA, Lu P, et 
al. Tissue-specific regulatory T cells: biomarker for acute graft-vs-host disease and 
survival. Exp Hematol.40(12):974-82 e1. 
42. 
Engelhardt BG, Griffith ML, Crowe JE, Jr., Savani BN, Kassim AA, Yoder SM, et 
al. Interaction between Post-Transplant Diabetes Mellitus and Regulatory T Cell 
Phenotype. Blood. 2011;118(21):in press. 
43. 
Will new diagnostic criteria for diabetes mellitus change phenotype of patients 
with diabetes? Reanalysis of European epidemiological data. DECODE Study Group on 
behalf of the European Diabetes Epidemiology Study Group. Bmj. 1998;317(7155):371-
5. 
44. 
Polonsky KS, Given BD, Hirsch L, Shapiro ET, Tillil H, Beebe C, et al. 
Quantitative study of insulin secretion and clearance in normal and obese subjects. J 
Clin Invest. 1988;81(2):435-41. 
45. 
Karam JH, Grodsky GM, Forsham PH. Excessive insulin response to glucose in 
obese subjects as measured by immunochemical assay. Diabetes. 1963;12:197-204. 
46. 
Mitrakou A, Kelley D, Mokan M, Veneman T, Pangburn T, Reilly J, et al. Role of 
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Appendix A 
 
 
 
Study Table - Donor 
 
 
 
 
 
 
 
 
Baseline Pre-
Collection 
Study 
Blood 
collection 
 
X 
Informed 
Consent 
X 
 
Review of 
Eligibility 
X 
 
Data 
Collection 
X 
X 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 

Version 1.1 dated December 1, 2017 
23
Study Table - Patient 
 
 
 
 
 
 
 
 
 
 
Baseline Pre-
Transplant 
Day 10 
post- 
transplant 
Day 20 
post- 
transplant 
Day 30 
post- 
transplant 
Day 80-
100 post- 
transplant 
D365 
post- 
transplant 
D730 
post- 
transplant 
Study Blood 
collection 
X 
 
X 
X 
X 
X 
 
 
OGTT / 
Microdialysis - 
CRC visit 
 
X  
 
 
 
X  
 
 
OGTT / GLP-1 
Infusion -  
CRC visit 
 
X 
 
 
 
X 
 
 
Hyperglycemic 
Clamp -  
CRC visit 
 
X 
 
 
 
X 
 
 
Informed 
Consent 
X 
 
 
 
 
 
 
 
Review of 
Eligibility 
X 
 
 
 
 
 
 
 
Data 
Collection 
X 
X 
X 
X 
X 
X 
X 
X 
 
 
 
All dates for blood collection +/- 7 days, all dates for procedures / study visits +/- 10 days 
 
Oral glucose tolerance testing and the hyperglycemic clamps will be performed on 
separate days as scheduling allows