Return of First-phase Insulin Secretion in Type 2 Diabetes is Associated With Depletion of Pancreas Lipid
Phase: NA
Conditions: Type 2 Diabetes Mellitus
Sponsor: University of Alabama at Birmingham

Return of First-phase Insulin Secretion in Type 2 Diabetes is 
Associated With Depletion of Pancreas Lipid 
Study Protocol & Statistical Analysis Plan 
NCT03430310 
Date of Last Protocol Approval: 12/13/2023 
Barbara Gower, PhD, Principal Investigator 
University of Alabama at Birmingham 
Birmingham, AL 35294 

Methods 
Participants and Study Design 
Participants were AA and EA adults, determined by self-reported race, with T2D and 
were recruited based on the following: Inclusion criteria were diagnosis within the past 10 years; 
treated with diet, metformin, SGLT2-inhibitors, DPP-IV inhibitors, or GLP-1 RAs; adult men 
and women aged 35-65 yr; HbA1c <8.0; and with a BMI of 25-50 kg/m2. Exclusion criteria were 
a change in body weight of >5 kg in the past 6 months; use of glucocorticoids; and tobacco or 
recreational drug use. Medication was discontinued 1 week prior to baseline testing for 
metformin and SGLT2-inhibitors, and 4 weeks prior to testing for DPP-IV-inhibitors and GLP-1 
receptor agonists. Fasting glucose was monitored throughout the study, and any patient who 
demonstrated fasting glucose >200 mg/dL on 3 consecutive days was returned to medication. 
Routine physical activity was recorded by questionnaire at screening, and all participants were 
asked to maintain their usual level of physical activity throughout the study. 
 
The study design was a 12-week randomized clinical trial with two arms: carbohydrate 
restricted (CR) and higher carbohydrate (HC).  Both diets were developed by a Registered 
Dietitian who met with the study participants weekly. Participants were given detailed 
instructions on daily meal plans. Groceries were delivered to each participant weekly by a local 
delivery service. Participants prepared their own meals and snacks, at an energy level calculated 
to be weight-maintaining, based on the instructions provided by the study dietitian. Participants 
reported their body weight to the dietitian weekly, and their diet prescription was altered if their 
weight changed by more than 1 kg. The CR diet was composed of ~9% energy from 
carbohydrate, 26% energy from protein, and 65% energy from fat, whereas the HC diet was 

composed of ~55% energy from carbohydrate, 25% energy from protein, and 20% energy from 
fat. The UAB Institutional Review Board approved the study. 
 
Procedures 
At baseline and after 12 weeks of diet therapy, participants underwent a standard, 75 g 
oral glucose tolerance test (OGTT) and a hyperglycemic clamp. The OGTT was 3 hours in 
length, with blood sampled at -30, -15, -5, 10, 20, 30, 60, 90, 120, 150 and 180 min relative to 
the start of glucose ingestion for measurement of glucose, insulin, and C-peptide. 
The clamp protocol involved three stages: a 30-min stage to evaluate first-phase beta-cell 
response, a 2-hour phase to evaluate insulin sensitivity, and a final, 30-min phase to evaluate 
maximal beta-cell response to arginine. The first phase was conducted by administering a bolus 
of glucose over 2 min at time zero followed by an intravenous administration of glucose (as D20) 
to achieve a blood glucose concentration 50 mg/dL above that morning’s fasted glucose 
concentration (G + 50). Formulas were used based on the RISE protocol (18) to determine both 
the bolus amount [weight (kg) x 50 mg/dL x 1.1 / 180] and to estimate the glucose infusion rate 
[weight (kg) x 5 x 60 / 180]; this rate was adjusted as necessary to achieve G + 50. Blood was 
sampled at -20 and -5 min prior to the start of glucose administration; then at 2, 4, 6, 8, 10, 15, 
20, 25, and 30 min after the start of glucose administration. Glucose was measured at bedside, 
and sera were stored for later assessment of glucose, insulin, and C-peptide. After the 30-min 
first-phase stage, blood glucose was brought to 200 mg/dL and maintained there for the next 2 
hours to determine insulin sensitivity. During this time, blood was sampled every 5 minutes. 
After the 150-min blood sample was collected, a second bolus of D20 was administered over 2 
min [weight (kg) x 100 mg/dL x 1.1 / 180]. Blood glucose was brought to 300 mg/dL and 

maintained there with a variable infusion of D20. Blood was sampled every 5 minutes to minute 
180. After the 180-min blood collection, 5 g arginine (10% in saline) was infused over 5 min. 
Blood was sampled every 2 min from 182 to 190 min to capture maximal C-peptide secretion.  
Serum samples were stored at -70oC until analyzed. Glucose was measured on a SIRRUS 
chemistry analyzer (Stanbio Laboratory, Boerne, TX) using glucose oxidase reagent; the 
minimum detectable value was 2 mg/dl; inter-assay CV was 4.48%; and intra-assay CV was 
1.28%. Insulin was assayed using immunofluorescence technology (TOSOH AIA900, TOSOH 
Bioscience, South San Francisco, CA); assay sensitivity was 0.5 uU/ml; inter-assay CV was 
3.95% and intra-assay CV was 1.49%. C-peptide was analyzed on the TOSOH; assay sensitivity 
was 0.04 ng/ml; inter-assay CV was 6.81% and intra-assay CV was 1.67%. 
For this study, the lipid content of pancreas and liver was be assessed by MRI using the 
3-point Dixon approach. This method provides high-resolution contiguous images of the 
abdomen in breathhold durations and its intrinsic 3D imaging capability allows whole organs and 
spatial variations in ectopic fat across any region of the imaged anatomy to be evaluated. The 3-
point M Dixon fat fraction (FF) is computed from the separated water and fat signals on a voxel-
by-voxel basis. The accuracy of the fat fraction metric from this multi-echo fat quantification 
method has been validated in several pancreatic/hepatic fat quantification 
studies using the following equation: Fat fraction3-point M Dixon = Signal Intensity 
(SI)FAT/(SIWATER+SIFAT) x 100%. 
We will attempt to image both the head and tail of the pancreas, however all regions of 
the pancreas show similar associations with beta-cell function. Visceral fat volume also was 
assessed in a 10 cm region of interest. Trans-axial abdominal images was obtained using 3D 
volumetric T1-weighted magnetization prepared rapid acquisition gradient echo (MPRAGE). 

The echo time, repetition time, and pulse flip angles will be selected to optimize the signal-
intensity contrast between adipose and non-adipose tissue. Image acquisition was done at the 
Civitan International Neuroimaging Laboratory under the oversight of Dr. Bolding using a state-
of-the-art Prisma 3.0-Tesla whole-body MRI scanner. Image analysis was done by Dr. Goss 
using Slice-O-Matic software for visceral fat volume, and Osirix MD on an iMAC computer for 
assessing the signal intensity of a region of interest on the pancreas and liver to determine the fat 
fraction. By quantifying visceral adipose, and pancreatic and hepatic lipid, we will be able to 
examine whether these depots change in tandem during the intervention, and which depot is most 
closely associated with improvements in insulin sensitivity and beta-cell function. 
Body composition: Body composition was assessed using dual-energy X-ray 
absorptiometry (DXA) in the Metabolism Core Laboratory of UAB’s Nutrition Obesity Research 
Center (NORC) with a Lunar iDXA instrument (GE Healthcare, Madison, WI) with the enCORE 
2011 v15 (sp2) software package. Total body fat mass and lean body mass (LBM) in kg, as well 
as total body %fat, was recorded. 
Resting energy expenditure (REE) and substrate utilization (respiratory quotient, RQ): 
REE and RQ was determined in the NORC Core Laboratory by indirect calorimetry (TrueOne 
2400, Parvo Medics, Sandy, UT). Participants were tested in the fasted (10h) condition following 
a 30-min supine rest. 
Calculations 
OGTT: Fasting values were determined from the average of the -30, -15, and -5 minute 
time points. The first-phase C-peptide index (CPI) was calculated as the difference in C-peptide 
from 0 to 30 minutes divided by the difference in serum glucose from 0 to 30 minutes ([C-
peptide30min - C-peptide0min] / [glucose30min – glucose0min]). The Matsuda Insulin Sensitivity Index 

(ISI) was used as a measure of whole-body insulin sensitivity due to its ability to capture both 
hepatic and skeletal muscle-derived insulin sensitivity. The Disposition Index (DI), an index of 
beta-cell function, was calculated as the product of C-peptide secretion and insulin sensitivity, as 
reported: DI = CPI x ISI.   
Hyperglycemic clamp: The RISE protocol was used to calculate first-phase C-peptide 
response as the mean incremental response above baseline over minutes 2-10, and the maximal 
C-peptide response as the mean incremental response during minutes 184-190 above the mean at 
min 170-180. Insulin sensitivity was calculated as described for the hyperglycemic clamp. 
 
Statistics: 
a. Analyses. The primary analysis for each aim will be a mixed model, using all available data 
and utilizing the estimated correlation between measurement times. The mixed model is less 
biased towards statistically significant results in the context of missing data, and/or when a 
moderate to high correlation exists between outcome measures. For Aim 1, the primary outcome 
will be pancreas lipid. Statistical models will include time, diet, and the time-by-diet interaction. 
Other potential covariates (e.g., gender, race, total body fat, adherence to diet) will be explored 
in preliminary analyses. Similar analyses will be conducted for hepatic lipid and visceral adipose 
tissue. For Aim 2, the primary outcome will be beta-cell function, as reflected in first phase C-
peptide secretion from the hyperglycemic clamp. Statistical models will include time, diet, and 
the time-by-diet interaction. Other potential covariates (e.g., gender, race, total body fat, 
adherence to diet, insulin sensitivity) will be explored in preliminary analyses. Similar analyses 
will be conducted for PhiD from the MMTT, and insulin sensitivity from the hyperglycemic 
clamp. Food records will be coded to assess adherence as a dichotomous variable, and adherence 

will be defined as +/- 10 percentage points of the targeted carbohydrate intake. For Aim 3, the 
primary variable of interest is the race-by-diet interaction term, which will be added to the 
models from Aims 1 and 2. If either of these interaction terms is significant, the data will be 
analyzed separately within each race group. Influential data points and outliers will be examined 
in a sensitivity analyses. 
Correlative analyses will be conducted between changes (week 12 minus baseline) in 
measures of glucose metabolism (e.g., glucose AUC from liquid meal test, HbA1c), markers of 
inflammation, visceral fat, and hepatic/pancreatic lipid with Pearson’s correlations coefficients 
(adjusting for relevant confounding variables). The correlation between change in pancreas lipid 
and change in first-phase C-peptide secretion will be examined, adjusting for insulin sensitivity. 
For all analyses, variables known to deviate from a normal distribution will be log transformed. 
All statistical tests will be two-sided and will be performed using an alpha level of 0.05. 
Statistical analyses will be performed using SAS Version 9.4®. Baseline descriptive 
characteristics will summarized as means (SD) or proportions to include demographic, clinical, 
and other measures. 
 
b. Power calculation. Based on our published data, a sample size of 15 in each group will have 
80% power to detect a difference in means of -89.700 (the difference between a Group 1 mean, 
μ1, of 6.400 and a Group 2 mean, μ2, of 96.100) in first-phase response at week 12 assuming 
that the common standard deviation is 84.639 using a two group t-test with a 0.050 two-sided 
significance level. Because we believe that the association between pancreas lipid and beta-cell 
function will be stronger in African-Americans, we have elected to target a final sample size of 

15 participants per diet-race group (total of 60 participants with complete data). Using the Fisher 
r-to-z transformation, a value of z can be calculated that can be applied to assess the 
significance of the difference between two correlation coefficients, ra and rb, found in two 
independent samples. Given 30 subjects in each race group, we will be able to detect a difference 
of 0.3 with a one-sided alpha of 0.05. We will recruit 80 participants (20 per diet-race group) to 
account for missing data, potential drop-outs, or for participants who have to withdraw from the 
study due to problems with glycemic control.  
 
c. Randomization 
The study statistician (Dr. Desmond) will prepare a closed envelope randomization for the two 
treatment arms stratified by race group using Proc Plan in SAS Ver. 9.4®. Patients will receive 
their group assignment following baseline testing. 
 
Patient management following the study: 
Given that patients were removed from medication prior to study initiation, and were 
carefully monitored for glycemic control during the study, it is important to ensure that they 
make a smooth transition to a free-living situation. Following final testing, all study participants 
will be given access to continued diet/nutrition support, provided by the study team, to assure a 
smooth transition to a free-living diet. Patients will be able to choose their preferred diet (they 
may choose to stay on their assigned intervention diet, or to transition to a different diet). 
Medical management, including of medication type and dosage, will be available during this 
transition. All patients will demonstrate stable glycemic control on a given diet and medication 
regimen before exiting the study. 

Supplementary Table 1.  Insulin sensitivity from the hyperglycemic clamp and OGTT 
(Matsuda Index) by race and diet. 
Insulin sensitivity 
measure 
Baseline Mean 
(SD) 
Week 12 Mean 
(SD) 
Change Mean 
(SEM) 
P for change 
Hyperglycemic clamp 
EA HC 
9.13 (7.42) 
15.87 (16.65) 
5.31 (4.17) 
0.24 
EA CR 
6.33 (5.74) 
9.03 (6.06) 
2.3 (2.74) 
0.42 
AA HC 
6.65 (4.49) 
8.12 (4.77) 
1.28 (0.72) 
0.09 
AA CR 
10.24 (8.99) 
12.16 (19.96) 
4.07 (3.42) 
0.26 
Matsuda Index (OGTT) 
EA HC 
2.46 (1.91) 
3.36 (2.27) 
0.89 (0.49) 
0.1 
EA CR 
2.08 (1.02) 
2.74 (1.35) 
0.66 (1) 
0.083 
AA HC 
2.18 (1.16) 
5.99 (2.98) 
0.66 (0.32) 
0.06 
AA CR 
3.11 (3.99) 
4.39 (2.94) 
1.28 (0.75) 
0.11 
Abbreviations: EA: European American, AA: African American, CR: Carbohydrate restricted diet, HC: 
Higher carbohydrate diet.