Effect of Linagliptin + Metformin on Glucose Metabolism and Pancreatic Beta Cell Function in Patients With Prediabetes Who do Not Achieve Normoglycemia After 12 Months of Treatment With Metformin Alone
Phase: PHASE4
Conditions: Prediabetic State, Impaired Glucose Tolerance, Insulin Resistance
Sponsor: Universidad de Guanajuato

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Effect of Linagliptin + metformin on glucose metabolism and pancreatic β-cell 
function in patients with prediabetes who do not achieve normoglycemia after 12 
months of treatment with metformin alone 
 
RESCATHEME Project (Rescue After Therapy with Metformin for Diabetes Prevention) 
 
 
 
 
 Research Protocol 
 
 
Rodolfo Guardado-Mendoza MD PhD 
 Principal Investigator 
University of Guanajuato 
Hospital Regional de Alta Especialidad del Bajío, León, Guanajuato, México 
 
 
 
 
 
Collaborators 
Alberto Aguilar-García MD 
Edgar Durán-Pérez MD 
Mildred Alvarez-Canales MS 
Sara Stephania Salazar López MD 
Diana Farfán MD 
 
University of Guanajuato and Hospital Regional de Alta Especialidad del Bajío 
 
 
 
 
 
 
Last update: January 30, 2018 
 

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Introduction 
Type 2 diabetes (T2D) is a worldwide health problem, reaching estimated prevalence around 
18 % in countries like Mexico, and an unstoppable increase in the number of new cases, 
having a big economic impact due mainly to micro and macrovascular complications, and 
being the first cause of mortality in Mexico (1, 2) 
  
Physiopathology of hyperglycemia is complex and multifactorial in T2D. It is known that 
insulin resistance (IR) is one of the first observed abnormalities, leading to a decrease in the 
glucose uptake by muscle, increase in hepatic glucose output and lypolisis; different factors 
have been involved in IR (3-5).  IR stimulates insulin secretion from the pancreatic β cells, 
causing hyperinsulinemia to compensate IR and maintain normal glucose levels; however, if 
IR persist, β cell can be damaged by different factors; incretin defect, oxidative stress, and 
amyloid deposition (3, 6-8), causing a reduction on β-cell function and mass up to 50-80 % (3, 
4, 6, 9-12). Together with pancreatic β-cell dysfunction, α-cell dysfunction and 
hyperglucagonemia have been also involved in the development of hyperglycemia (6, 13-16); 
glucagon is one of the main regulators of hepatic glucose production, and together with the 
reduction on incretin hormones (GLP-1, GIP) and amyloid deposition are physiopathological 
defects that contribute to pancreatic islet dysfunction and hyperglycemia (17-21). 
 
Prediabetes is a condition with intermediate hyperglycemia, and based on the American 
Diabetes Association criteria(22) can be defined as: i) impaired fasting glucose (IFG): FPG 
≥5.5mmol/L [≥100mg/dl]; ii) impaired glucose tolerance (IGT): 2 h plasma glucose 
≥7.8mmol/L [140 - 199mg/dl]; and iii) IFG + IGT: FPG ≥5.5mmol/L [≥100mg/dl] and 2 h 
plasma glucose ≥7.8mmol/L [140 - 199mg/dl]. Most of the physiopathological abnormalities 
observed in T2D, have been already reported in prediabetes with a different severity (23), IR 
(24), pancreatic β-cell dysfunction (10, 11, 25, 26), pancreatic α-cells dysfunction and relative 
hyperglucagonemia (13, 15, 25), incretin defect, although it has been questioned whether 
incretin defect is a cause(27) or consequence of hyperglycemia in T2D (28). It has been 
previously shown that clinical and physiopathological characteristics are different between 
these three categories of prediabetes. IFG mainly presents hepatic insulin resistance and mild 

3 
 
impairment on firs phase insulin secretion (β-cell dysfunction), IGT is more related to muscle 
insulin resistance and first and second phase insulin secretion (β-cell dysfunction), while the 
combination of IFG+IGT present marked pancreatic β-cell dysfunction and whole body IR, 
with the greatest risk to develop T2D. 
 
Prevalence of prediabetes varies between 9.7 to 56.8 %; in Mexico it has been reported to be 
around 30 % (29). Eventually, most of the patients with prediabetes (70-80 %) will develop 
T2D during their life, and of note, the have a higher risk and may already present micro and 
macrovascular complications (30).   
Pancreatic β-cell function has been reported as one of the main factors involved in the 
progression from prediabetes to T2D in observational studies as well as in clinical trials (31-
33) 
 
Different pharmacologic and non-pharmacologic strategies have been used to reduces the 
progression risk from prediabetes to diabetes and prevent T2D. Lifestyle modification 
programs, based on physical activity and nutrition, have proved to be a useful strategy, 
reducing the risk up to 58 % in patients with IGT, in China and Finland (34) (35). The 
Diabetes Prevention Program Study (DPP) was designed to compare the risk reduction 
between an intensive lifestyle program vs placebo and metformin (36); DPP by the  first time 
evaluated the effect of metformin, an insulin sensitizer that mainly reduces hepatic glucose 
production(37); this study showed also a risk reduction of 58 % in the lifestyle group, and only 
31 % in the metformin group, in comparison to the placebo group; however, the main 
limitation of the lifestyle programs it´s the adherence to the program, and that besides of that, 
there is a progression from prediabetes to T2D and eventually most of the patients will 
develop T2D in the long  term follow-up, since in  the DPP Outcome Study(38) the 
cumulative T2D incidence at 15 years of follow-up was 55, 56 and 62 % in the lifestyle, 
metformin and placebo group, respectively.  
Other kind of drugs have been also used to prevent T2D; rosiglitazone in the DREAM study 
showed a 62 % risk reduction in patients with IGT to develop T2D (39), in the ACT-NOW 
Study, Pioglitazone showed a 72 % risk reduction in patients with IGT, but was associated 
with significant weight gain and edema, perhaps due to the used dose(40). 

4 
 
 
Few studies have been performed to evaluate the effect of incretin drugs to prevent T2D. If 
there is a reduction in the incretin effect in prediabetes, there is two ways of improving it, with 
GLP-1 receptor agonist or GLP-1 analogues, or with  the use of a DPP-IV inhibidor, that 
eventually will increase  endogenous GLP-1 levels two or three-fold. Most of the previous 
studies have been done with GLP-1 RA or agonist. Exenatide, a GLP-1 receptor agonist, after 
24-weeks, in patients with obesity and prediabetes, together with a lifestyle program showed 
reductions in calorie intake and glucose normalization, but without a difference in fasting, 2h 
glucose and T2D incidence, but with a mean body weight reduction of around 4.0 kg(41). 
Liraglutide 1.8 mg/d, also in patients with prediabetes or early T2D (<12 months), was 
compared to a lifestyle modification program in a small sample of patients (n=40), achieving 
in both groups a weight reduction of around 7.0 kg, and a slightly better improvement in 2h 
glucose and β-cell function in the liraglutide group(42); also in patients with prediabetes, 
liraglutide showed to improve glucose values during OGTT and β-cell function during 14 
weeks of treatment(43). At doses of 3.0 mg/d, liraglutide together with diet and exercise has 
shown a reduction in the incidence of T2D (HR 0.21 95% CI 0.13-0.34, p <0.0001), and 
improvement in glucose during OGTT in patients with overweight-obesity and prediabetes 
during a 3 year study (44). 
Endogenous GLP-1 is inactivated in 1-4 minutes by the DPP-IV, and with DPP-IV inhibitors 
the incretin effect gets improved; the use of DPP-IV inhibitors in prediabetes has been less 
explored, since there are few studies with short duration and small sample size; in an 8-week 
study, sitagliptin showed no effect in patients with IFG(45), but in patients with IGT it was 
reported a reduction in glucose levels as well as an improvement in insulin and glucagon 
levels during OGTT(46). In a 12-week study in patients with overweight and prediabetes, 
sitagliptin improved also glucose profile and β-cell function during OGTT (47).  Saxagliptin in 
a 24-week study (n = 5-7 per group), showed similar changes than metformin in fasting 
glucose and HbA1c and a slightly better improvement in 2 h glucose levels in patients with 
IFG or IGT(48). Linagliptin has been used only in 12-week study (n=8 per group) in 
comparison to metformin in overweight-obese patients with IGT, finding mainly a reduction 
in 2h glucose with linagliptin (49). 

5 
 
Linagliptin is a potent and selective DPP-IV inhibitor with a long half-life to  be used once 
daily; it is the only DPP-IV inhibitor cleared mainly by the liver (˃80 %) and by consequence 
it can be used without dose adjustment in patients with high risk or any kind of renal 
dysfunction (50); besides this, linagliptin has shown in recent clinical trials a safe profile 
regarding renal and cardiovascular function (51), and has proven efficacy in T2D treatment as 
a monotherapy or combined therapy (52) 
 
In human islets from non-diabetic and diabetic donors, linagliptine has shown to  better 
improve insulin secretion, β-cell function and α-cell function in non-diabetic islets, suggesting 
that this kind of drugs could have a better effect at very early stages of the disease, like 
prediabetes (53).  
Few studies have tried to evaluate integral and realistic interventions to prevent T2D, focusing 
these therapies to the group of patients with prediabetes at the highest risk to develop T2D, 
combining oral and practical medications with a realistic and real life feasible lifestyle 
program, and moreover, no study has deeply evaluated the physipathological effects of such 
therapies. 
The goal of this work was to evaluate the effect of linagliptin + metformin to improve glucose 
metabolism and pancreatic β-cell function in patients with prediabetes who do not achieve 
normoglycemia after 12 months of treatment with metformin alone. 
 
Methods 
 
Study design and participants 
The RESCATHEME project (Rescue After Therapy with Metformin for Diabetes Prevention) 
is a single center double-blind randomized clinical trial performed in patients with IGT.  
Patients will be selected from a metabolic cohort study performed at the Metabolic Research 
Laboratory in the University of Guanajuato, México, as part of the University Cohort Project 
CARE-In-DEEP Study (Cardiometabolic Risk Evaluation and Interdisciplinary Diabetes 
Education and Early Prevention). For this particular study, patients will be screened with 
anthropometrical, physical activity, nutritional, biochemical and metabolic evaluation, 
including oral glucose tolerance test. Patients are eligible for enrollment in the study according 

6 
 
to the following criteria: i) persistence of impaired glucose tolerance (2 h glucose 140-
199mg/dl [7.8 to 11.0 mmol per liter]) during a single oral glucose tolerance test after 12 
months of treatment with 1700mg/day of metformin, and ii) age between 18 – 65 years; 
exclusion criteria: i) treatment with drugs affecting glucose levels during the previous 3 
months, ii) previous pathological conditions affecting glucose metabolism or body weight 
(thyroid disease, Cushing´s syndrome, Acromegaly); iii) excesive alcohol intake (acute or 
chronic), iv) fasting plasma triglyceride >400mg/dl, v) pregnancy, vi) Systolic blood pressure 
>180mmHg or diastolic blood pressure >105mmHg (subjects could be re-screened after 
hypertension treatment).  Written informed consent will be obtained from all participants and 
the study was approved by the Ethical Council at the Hospital Regional de Alta Especialidad 
del Bajío (CEI-35-16). 
 
Procedures 
Anthropometrical and body composition measurements. Weight will be measured while 
participants are barefoot and wearing minimal clothing. Height is obtained while the 
participants are standing barefoot with their shoulders in a normal position. BMI (kg/m2) is 
obtained from standardized measurements of weight and height and is computed as a ratio of 
weight (kg):height squared (m2). Waist circumference will be measured at the high point of 
the iliac crest at the end of normal expiration to the nearest 0.1 cm. Body composition will be 
assessed with electrical bioimpedance through a Tanita Scale SC-240. All measurements will 
be performed by personal trained to use standardized procedures and reproducibility is 
evaluated, resulting in concordance coefficients between 0.88 and 0.94. Weight will be 
recorded at least every two months, while electrical bioimpedance at basal and at EOS.    
 
Nutritional and Physical Activity Evaluation. A semi-quantitative food frequency 
questionnaire (FFQ) previously validated (54), will be applied to evaluate dietary intake. This 
questionnaire included data regarding the consumption of 116 food items. For each food, a 
commonly used portion size (e.g. 1 slice of bread or 1 cup of coffee) is specified on the FFQ 
and participants reported their frequency of consumption of each specific food over the 
previous year. Participants chose from 10 possible responses, ranging from “never” to “6 or 
more times per day.” For our analysis, the reported frequency for each food item is converted 

7 
 
into a daily intake. Total energy intake is computed by summing the energy intakes from all 
foods. The PA level of participants is assessed using a self-administered questionnaire that is 
verified when the patient assist for the metabolic evaluation. The questionnaire has a validated 
Spanish translation which has been adapted for use in the Mexican population (55). The 
questionnaire is self-administered and estimates the minutes devoted to the practice of 
different recreational physical activities during a typical week in the last year (including 
walking, running, cycling, aerobics, dancing, and swimming as well as playing football, 
volleyball, basketball, tennis, fronton, baseball, softball, and squash, among other activities). 
Each item includes time intervals that allow participants to detail the exact number of minutes 
or hours they dedicate to each form of recreational PA, as well as the intensity of each PA 
(light, moderate, vigorous). The total duration of each recreational PA is expressed in minutes 
per day. We calculated the number of hours per week devoted to each activity, which were 
then multiplied by the intensity of each activity, defined as multiples of the metabolic 
equivalent (MET) of sitting quietly. We used the Compendium of Physical Activities to assign 
METs to each activity (56). We then add the average weekly energy expenditure attributable 
to each activity to derive the total MET-hours per week. Energy intake and physical activity 
will be evaluated at basal and at the end of the study. 
 
Metabolic evaluation.  
Oral glucose tolerance test (OGTT):  All subjects will be admitted to the Metabolic Research 
Laboratory of the Department of Medicine and Nutrition, Division of Health Sciences at the 
University of Guanajuato the day of the study between 7 and 8 AM, and a catheter will be 
placed into an antecubital vein for all blood withdrawal.  Subjects will not be allowed to eat or 
drink anything after 10 PM on the night before, until the study is completed.  After the 
intravenous catheter is placed and the first blood sample is drawn, the patients ingest 75 grams 
of glucose.  Serum samples for glucose and insulin measurement will be drawn at -15, and 0 
minutes and every 30 minutes thereafter for two hours. Prediabetes is diagnosed based on the 
OGTT; and only patients with IGT (2 h glucose 140-199 mg/dl), and ideally together, 
although not exclusively, with IFG (FG 100-125mg/dl) will be included in this study. Patients 
with newly diagnosed T2D will be excluded from the study. 
 

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Measurements. Glucose will be measured with an Analox glucoanalyzer GM9 (Analox 
Instruments) and by colorimetric glucose oxidase (Vitros 5600; Ortho Clinical Diagnostics). 
Lipid levels are measured by dry chemistry with colorimetric method (Vitros 5600; Ortho 
Clinical Diagnostics). Insulin will be measured by chemiluminiscent immunometric assay 
(IMMULITE 2000 Immunoassay system, Siemens), and Glucagon by Quantikine ELISA 
Immunoassay (R&D Systems Inc, Minneapolis, USA) with an intra-assay CV of 2.7 – 3.6 %. 
HbA1c will be determined using high-performance liquid chromatography with a DS-5 
Analyzer (Drew Scientific, Inc. Miami, FL, USA).  
 
Calculations. The incremental and the AUC for glucose and insulin during the OGTT are 
calculated according to the trapezoidal rule. Insulin secretion is calculated dividing the 
AUCinsulin0_120 by the AUCglucose0_120 during the OGTT. The insulin secretion/insulin 
resistance (IS/IR) index (disposition index = DI) during OGTT is calculated as 
(AUCinsulin0_120 / AUCglucose0_120)*Matsuda index (57). Insulin sensitivity during OGTT is 
calculated from the Matsuda index (58), and at fasting with the homeostasis model assessment 
(HOMA-IR). From the hyperglycemic clamp the incremental and AUC for glucose, insulin 
and C-peptide are also calculated according to the trapezoidal rule. Different measurements of 
pancreatic β-cell function will be derived from the hyperglycemic clamp: DI will be obtained 
as a product of insulin sensitivity (Matsuda index) and the incremental AUC for insulin and C-
peptide from 0 to 15 and from 0 to 120 minutes. 
 
Randomization and masking. Patients will be randomly assigned in a 1:1 ratio to receive 
linagliptin/metformin 2.5/850mg every 12 h + lifestyle modification program, or metformin 
850mg every 12 h + lifestyle modification program during 6 months. Randomization will be 
performed using an electronic random numbers table by a Nutritionist not involved in the 
study. Participants and investigators involved in the patients follow-up and outcome 
measurements will be masked to treatment allocation during the entire study.  
 
Interventions. i) Linagliptin + metformin + lifestyle: Patients allocated to this group will start 
linagliptin/metformin pills of 2.5/850mg once daily during the first month, and after that the 
dose will be increased to 2.5/850mg twice daily from the second month until the end of the 

9 
 
study (24 months). ii) Metformin + lifestyle: Patients on this group will continue metformin 
pills of 850mg once daily during the first month, and increased to 850mg twice daily thereafter 
until the end of the study (6 months). Both groups will receive the same lifestyle 
implementation program based on a prescribed diet every two months seeking to reduce their 
body weight at least by 5-7 %, adjusting their energy requirements based on their weight every 
two months, and composed from 55-60 % of carbohydrates, 25-30 % fat, and 10-15 % 
proteins. If the patient is sedentary, he or she will be advised to start with 45 min/week of mild 
to moderate exercise with a specific activity according to patients preference; patients will be 
counseled to increase the duration and frequency or intensity of exercise every two weeks until 
reaching 150min/week of moderate activity or 75 min/week of intense activity. If the patient 
was already physically active, it will be recommended to continue like this and vary its 
exercise routines.  
 
Follow-up visits and outcome. Patients will have a follow-up visit every month. Every 
appointment is about 30-45 minutes; medications tolerance and side effects will be recorded in 
every patients´ visit. Nutritional and physical activity assessment according to the patient´s 
weight will be performed by a Nutritionist every month.  Monthly adherence to medications 
will be evaluated by pill counting; nutritional adherence and energy intake will be evaluated at 
6 months by a food frequency questionnaire, and physical activity at 0 and 6 months. At basal 
and at 6 months patients will have an OGTT. Primary objective is to evaluate at 6 months 
glucose profile during OGTT, insulin secretion and pancreatic β-cell function by the OGTT 
DI, and regression to normoglycemia; T2D will be diagnosed and confirmed by at least two 
consecutive measurements of the same criteria: glycated haemoglobin ≥ 6.5 %, fasting glucose 
≥ 126mg/dl, or 2 h glucose ≥ 200 mg/dl.  
 
Statistical analysis 
The sample size calculation was based on the fact that we expected an improvement of at least 
60 % on the pancreatic β-cell function and glucose profile during the OGTT in patients with 
the combined treatment and at least an improvement of 20% on the metformin group, based on 
previous reports about glucose levels and pancreatic β-cell function in prediabetic patients 
before and after treatment (40, 59); in order to be able to detect a minimum difference of 40 % 

10 
 
between groups in the quantitative measurement of pancreatic β-cell function, and assuming 
an alpha error of 5% and a beta error of 20%, we require approximately 14 patients per group.   
The primary analysis is the comparison of the change in glucose profile during OGTT as well 
as measurements of pancreatic β-cell function during OGTT. Intergroup comparisons between 
absolute values and delta values will be performed with a t test for independent groups; intra-
group comparisons will be performed using a paired t-test. Non-numerical variables will be 
compared between groups with the chi squared test.  
Statistical analyses and graphics will be performed using SPSS Version 21.0 (SPSS Inc) and 
GraphPad Prism 5.0. Statistical significance is considered when p value is less than 0.05. 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
 
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