Materials and Methods
The definition of prediabetes and other metabolic parameters
Diabetes was defined by any of the following criteria: (1) FPG of ≥ 126 mg/dL; (2)
PPG-2h of ≥ 200mg/dL; (3) A1C of ≥ 6.5% (10, 23). Patients who had an A1C ≤5.6%
before operation were evaluated if they had new-onset prediabetes during their follow-up period (Figure 1B). Development of new-onset prediabetes was defined as having two or more components of the following criteria in a single test and/or meeting one or more components of the following criteria in two or more consecutive tests: (1) FPG of 100-125 mg/dL; (2) PPG-2h of 140-199 mg/dL; (3) A1C of 5.7-6.4% (10). For the purpose of
subgroup analysis in the IAT group, the presence of prediabetes before operation was defined by any of the above criteria. A1C was measured using a high performance liquid
chromatography method certified by the national glycohemoglobin standardization program.
Serum C-peptide (Immunotech, Marseille, France) and insulin (Medgenix, Niveles, Belgium) levels were measured in duplicate by immunoradiometric assay.
Insulin resistance levels were determined by the QUICKI (1/ [log (fasting insulin in µIU/ml) + log (fasting plasma glucose in mg/dl)]) and the HOMA-IR (fasting insulin [in picomoles per liter] × fasting glucose [in millimoles per liter]/135) (21). Three indices were calculated for insulin secretion: INSindex (∆insulin30min [in picomoles per liter]/∆glucose30min
[in millimoles per liter] during OGTT) (22); homeostasis model assessment for β-cell function (HOMA-BC = fasting insulin [in picomoles per liter] × 120/fasting glucose [in millimoles per liter]− 3.5])(21); and SUITO index (fasting C-peptide [in nanograms per milliliter] × 1500/[fasting glucose in mg/dl − 63]) (23).
Islet isolation and transplantation
The methods for islet isolation and transplantation have previously been described (8, 9, 17). Distal partial pancreatectomy was performed for pancreatic masses that required pathologic diagnosis. After a distal partial pancreatectomy, the pancreatic segment containing the tumor was immediately sent to the pathologist (a co-worker in the islet transplantation team) for a histopathologic decision on its candidacy for islet transplantation. The remainder of the pancreas was immediately placed in cold University of Wisconsin solution and transported in an icebox to the islet isolation laboratory. Pancreas samples were kept until an unequivocal diagnosis of their benign nature was made from frozen sections. The weight of the remainder of the pancreas was recorded before islet isolation. The pancreas was then isolated by organ distention using intraductal collagenase (Boehringer Mannheim, Mannheim, Germany) and digested using a modified Ricordi method. In all the 20 IATs, islet purification was performed using a COBE 2991 processor (COBE, Lakewood, CA) using a large-scale continuous density gradient prepared from Ficoll diatrizoate media. Islet purity was determined subjectively by visual assessment using two 100-µL sampling strips. Islet yields were expressed as islet equivalents (IEQ) (of islets 150 µm in diameter). Islets were purified with a continuous density gradient using Ficoll (Biocoll, Biochrom AG Seromed, Berlin, Germany) and University of Wisconsin solutions and a COBE 2991 cell processor (COBE BCT Inc., Lakewood, CO80215, USA). Final pellet volume ranged from 0.2 ml to 2.0 ml.
After islet isolation, purified islets were incubated for at least 24 to 48 hours at 37°C in culture medium. The incubation time was dependent on the definite pathologic decision.
Total transplanted islet mass was expressed as islet equivalent volume (IEQ) before culture.
To avoid islet loss by further manipulation, islets were not additionally counted after the culture. After confirmation of sterility, preparations were infused using a syringe by means of
percutaneous transhepatic portal vein catheterization using an 11-gauge silicon catheter (Vygon, Ecouen, France). Immediately before infusion, patients were administered 5,000 IU of intravenous heparin. Throughout the infusion, the portal venous and brachial arterial pressures were monitored. Pre- and post-islet infusion pressures were compared so that the volume effects of the islet infusion could be taken into consideration. All patients were placed under close care after pancreatectomy and underwent intensive glucose monitoring for at least seven days, with a target blood glucose level of approximately 100 mg/dL using continuous regular insulin infusion and intermittent insulin injections (NovoLet; Novo Nordisk, Bagsvaerd, Denmark).
Table S1. Comparison of islet isolation parameters of patients with and without prediabetes development within two years after surgery
No prediabetes
development within two years after IAT
(n = 5)
Prediabetes development within two years after IAT
(n = 10)
P-value
Digestion time (min) 25 (9-27) 26.5 (15-31) 0.438
Pancreas weight 20 (16-45) 35 (30-55) 0.060
Islet yield before purification (IEQ)
293111
(218260-330000)
352222
(156000-491000)
0.240
Islet yield before purification per pancreas weight (IEQ/g)
15789 (6514-16500)
9580 (5200-10919)
0.364
Total islet yield after purification (IEQ)
174945
(144000-297333)
165000
(135900-301056)
0.898
Islet yield after purification per pancreas weight(IEQ/g)
7579 (5184-14267)
4883 (3654-6608)
0.012
Recovery rate (%) 71.0 (48.0-100.0) 50.9 (33.6-93.9) 0.240 IAT, islet auto-transplantation; IEQ, islet equivalent
Table S2. Comparison of preoperative metabolic parameters in patients with and without diabetes development within one year after surgery.
Variable Non-DM (n=15) DM (n=5) P-value
Age (years) 45 (23-62) 57 (34-68) 0.088
Sex (women/men) 15/0 2/3 0.053
Location of mass (head:body:tail) 0:12:3 0:5:0 0.339 Extent of resection (50%:50-
60%) 10:5 4:1 1.000
No. of patients with prediabetesa 5 4
Fasting plasma glucose (mg/dl) 85 (67-102) 96 (76-119) 0.025
PPG-2h (mg/dl) 121.5 (61-185) 132 (83-189) 0.032
Hemoglobin A1C 5.2 (4.2-5.8) 5.4 (4.8-5.6) 0.350
Fasting insulin (µIU/ml) 6.8 (2.9-18.5) 10.2 (8.0-12.8) 0.049 Fasting C-peptide (ng/ml) 1.69 (0.80-3.70) 1.87 (1.25-3.46) 0.920 Stimulated C-peptide (ng/ml) 8.62 (2.03-13.42) 7.44 (5.39-13.3) 0.910 QUICKI index 0.158(0.135-0.181) 0.143 (0.137-0.156) 0.050
HOMA-IR 1.63 (0.72-4.49) 3.04 (1.74-4.20) 0.050
INS index 60.3 (3.3-383.7) 60.4 (2.5-371.4) 0.716
HOMA-BC 7537.8
(1734.2-19698.5)
3835.2
(2760.7-9285.8) 0.275 SUITO index 158.4(57.1-398.4) 95.1(34.1-152.3) 0.162
DM, diabetes mellitus; PPG-2h, post-challenge plasma glucose at two hours; QUICKI, quantitative insulin sensitivity check index; HOMA-IR, homeostasis model assessment for insulin resistance; INS index, insulinogenic index; HOMA-BC, homeostasis model assessment for β-cell function; SUITO, secretory unit of islet transplant objects index.
a Having increased risk for diabetes development according to current criteria by the American Diabetes Association (10)
Figure S1.
Receiver-operator characteristic analysis curve for potential predictors of diabetes development within 12 months after islet autotransplantation. The area under the curve for total islet mass before purification (IEQ, preCOBE), islet mass per pancreas weight before purification (IEQ/g, preCOBE), total islet mass after purification (IEQ, postCOBE), and islet mass per pancreas weight after purification (IEQ/g, postCOBE) was 0.507 (95% confidence interval (CI) [0.208-0.805], p = 0.965), 0.600 (95% CI [0.331-0.869], p = 0.513), 0.793 (95%
CI [0.583-1.000], p = 0.055), and 0.933 (95% CI [0.820-1.000], p = 0.005), respectively.
Figure S2
Comparison of glycometabolic parameters between the patients who did not undergo IAT and patients who underwent IAT with islet yield <5154IEQ/g and >5154IEQ/g before and after distal pancreatectomy. (A) Fasting plasma glucose levels. (B) Post-challenge plasma glucose levels. (C) Hemoglobin A1C. * p < 0.05 by Mann-Whitney U test. Bars and error bars represent medians and ranges, respectively.
0 50 100 150
A
control <5154IEQ/g >5154IEQ/g
FPG Baseline
2 year post-op
mg/dL
0 100 200 300
B
control <5154IEQ/g >5154IEQ/g
PPG-2h
Baseline 2 year post-op
*
mg/dL
0 2 4 6 8
C
control <5154IEQ/g >5154IEQ/g
HbA1c
Baseline 2 year post-op
%
Figure S3
A.
B.
Preoperative glycometabolic parameters of patients with prediabetes and high levels of insulin resistance. (A) Fasting plasma glucose (FPG), post-challenge plasma glucose at 2 hours (PPG-2h), and hemoglobin A1C (A1C) of patients who had prediabetes at preoperative stage according to islet isolation yield per resected pancreas mass (>5154IEQ / g and <5154
control <5154IEQ/g >5154IEQ/g 0
50 100 150
FPG(mg/dL)
control <5154IEQ/g >5154IEQ/g 0
50 100 150
200 PPG-2h(mg/dL)
control <5154IEQ/g >5154IEQ/g 4.0
4.5 5.0 5.5 6.0
6.5 A1C(%)
HOMA-IR
control <5154IEQ/g >5154IEQ/g 0
2 4 6 8
IEQ / g). Control represents the patients who did not undergo IAT. (B) Preoperative HOMA- IR of patients who had HOMA-IR of >2.0 at preoperative stage according to islet isolation yield per resected pancreas mass (>5154IEQ / g and <5154 IEQ / g). Control represents the patients who did not undergo IAT.
