A structured weight loss program increases gut microbiota phylogenetic diversity and reduces levels of Collinsella in obese type 2 diabetics: A pilot study

The global obesity epidemic constitutes a major cause of morbidity and mortality challenging public health care systems worldwide. Thus, a better understanding of its pathophysiology and the development of novel therapeutic options are urgently needed. Recently, alterations of the intestinal microbiome in the obese have been discussed as a promoting factor in the pathophysiology of obesity and as a contributing factor to related diseases such as type 2 diabetes and metabolic syndrome. The present pilot study investigated the effect of a structured weight loss program on fecal microbiota in obese type 2 diabetics. Twelve study subjects received a low-calorie formula diet for six weeks, followed by a nine week food reintroduction and stabilization period. Fecal microbiota were determined by 16S rRNA gene sequencing of stool samples at baseline, after six weeks and at the end of the study after fifteen weeks. All study subjects lost weight continuously throughout the program. Changes in fecal microbiota were most pronounced after six weeks of low-calorie formula diet, but reverted partially until the end of the study. However, the gut microbiota phylogenetic diversity increased persistently. The abundance of Collinsella, which has previously been associated with atherosclerosis, decreased significantly during the weight loss program. This study underlines the impact of dietary changes on the intestinal microbiome and further demonstrates the beneficial effects of weight loss on gut microbiota. Trial registration: ClinicalTrials.gov NCT02970838.


Aims
In a dual center and prospective intervention study at the Department of Medicine A,

University Medicine Greifswald and the Department of Medicine B, University Medicine
Münster, the influence of a multimodal intervention therapy based on a standardized weight-loss program of parameters of metabolism in obese diabetics will be investigated.

Primary Endpoint
We estimated that the standardized weight-loss program effects a positive reduction of the HbA1c-value about -15 % next to a successful weight loss. Therefore, HbA1c-value is the primary endpoint.

Recruitment of the study population
After checking for in-and exclusion criteria through the study team, patients will be included in the trial. Every study patient will get an identification code, which will be saved at the University Medicine Greifswald. Blood samples and questionnaires will be collected.
In addition, for every patient a registration sheet will be created with the following data: identification code, written informed consent, master data, diagnosis, and therapy.

Exclusion criteria
• Intake of incretin analogs ≤ 3 month before study inclusion • Renal insufficiency or liver insufficiency (KDOQI > 3; Child Pugh > A) • Heart failure (NYHA > 3) • Diagnosed eating disorder • Mental retardation • Immobility • Age < 18 years • Pregnancy or breastfeeding • Alcohol and drug abuse • Exclusion from MRI-measurement: pacemaker, claustrophobia, metal-containing elements The retrospective data for HbA1c-value and weight will be used as controls. 9

Obesity and Diabetes mellitus Type 2
Obesity is a pathological increase in body fat mass (BMI > 30 kg/m²) associated with health risks [1]. According to the "Nationale Verzehrsstudie 2" Germany (Second National  [4]. Due to these numbers as well as an increasing incidence and prevalence, obesity and the secondary diseases must be regarded as one of the biggest health problems in Germany. There is a marked increased risk of morbidity and mortality, which results in the indication of lifelong patient care [5]. Large prospective studies have shown that an increasing BMI is associated with a variety of secondary diseases as well as an increasing reduction in life expectancy [1] [6]. The average life expectancy for obese persons at the age of 40 years without further risk factors (i.e. smoking) is reduced by seven years [7]. Not every obese person inevitably develops secondary diseases, but the risk increases with the extent and duration of obesity [1]. The major comorbidities and complications of obesity are: arterial hypertension, cardiovascular disease, type 2 diabetes mellitus, cancer and the development of fatty liver disease [8].
Obese persons have an up to 5-fold increased risk for developing diabetes mellitus [9][10]. The term diabetes mellitus encompasses a heterogeneous group of glucose metabolism disorders that are characterized by a pathological control of glucose metabolism with consecutive blood glucose elevation. This is caused by a lack of insulin or insufficient insulin action (insulin resistance). 80% of all diabetics have type 2 diabetes.
The main causes are insulin resistance with a relatively insulin deficiency leading to a predominantly secretory defect with insulin resistance [11].
Insulin is a peptide hormone and is secreted in the pancreatic Langerhans' β cells. At the same time, the increased glucose uptake leads to a decrease in the blood glucose concentration [12].
In obese persons often glucose intolerance occurs. That means, the glucose utilization in the muscle is reduced (peripheral insulin resistance) and an increased gluconeogenesis takes place in the liver (hepatic insulin resistance). In the early stages, an increased release of insulin with concomitant hyperinsulinemia compensated the insulin resistance.
Over time, there is a risk of a decrease in the secretory function of the pancreatic β cells [13]. Inadequate treatment of diabetes may lead to late complications including cardiovascular disease, retinopathy, and diabetic nephropathy, diabetic neuropathy, amputations, and autonomic nervous system disorders with paresis in the gastrointestinal tract [14].
Professional societies initially recommend lifestyle interventions for obesity before medical or surgical therapies are considered [8] [9]. Programs with comprehensive care by physicians, behavioral therapist, nutritionists and sport therapist, often in combination with formula diet as part of a structured weight-loss program, showed sufficient results in terms of sustainable weight loss [15][16] [17]. Such intensively managed lifestyle interventions can also be successful in patients with diabetes mellitus type 2 and can reduce diabetesassociated mortality [15] [18]. However, so far many research group investigated lifestyle interventions more for prevention than as therapy option [19][20] [21]. Therefore, the OPTIFAST®II-Kurzprogram (short program) for obese patients with diabetes mellitus type 2 will be investigated at the Department of Internal Medicine A, University Medicine Greifswald and the Department of Medicine B, University Medicine Münster. Therefore, overweight and obese patients with diabetes mellitus type 2 get the chance to lose weight and to improve their metabolism. Additional survey will observe the insulin sensitivity and the liver and pancreas fat content during weight loss.

The OPTIFAST®-Program
In a one-week preparatory phase, a comprehensive health analysis will be conducted to

Measurement of clinical relevant parameters
The measurements of the following parameters are standard routine in the hospital:

Anthropometric data
During the intervention period, patients will be weighted weekly. Body mass index is calculated as body weight divided by height squared [kg/m²]. In addition, waist and hip circumference will be measured midway between the superior iliac spine and the lower rib margin without exerting pressure on the body surface.

Bioelectric impedance analysis
Bioelectric impedance analysis is used for determination of body composition of patients in week 1, 7 and 15. It is a widely used and non-invasive method for body composition and nutritional status based on electrical conductivity of the different body tissues.
Technically two electrodes are placed on the patient`s body surface and a weak electric field is be generated that is not felt by the patient. There are two types of electric resistances that are determined; one is water resistance (R out of which body fluid content, lean body mass and fat will be determined. Cellular resistance (Xc) is the second parameter and gives information on organ and muscular mass of the body. Final body composition is calculated by Data Input® [22].

OptiDiet
To compare food intake of the patients before and after the intervention, patients will complete a nutritional protocol with all food and beverages they consumed in seven days.
To calculate the average intake, the software OptiDiet® will be used.

HOMA-Index
The homeostasis-model-assessment-(HOMA)-index will be used for calculating insulin

Magnetic resonance imaging
Magnetic resonance imaging (MRT 1.5 Tesla Magnetom Avanto, Siemens HealthCare, Erlangen) is a valid tool for fat quantification (liver fat, visceral fat, pancreas fat). There is a standardized protocol including allocation for the right position, adjustment and calibration.
One measurement will last 10 minutes. The MRI is a diagnostic assessment tool without using ionizing ration (X ray). Instead, airwaves and magnetic fields were used. No contrast media is given to the patient. All contraindication will be checked before the first measurement using an additional clinical accepted information sheet.

Quality of Life
The questionnaire SF-12 will be used for the measurement of quality of life. The SF-12 is the short form of the measurement tool SF-36 Health Survey. Both, the general health status and the impairment in every-day work will be assessed. The results are presented as scale values between 0 and 100. A lower score equals a lower quality of life.

Sleep quality
The sleep quality before and after the intervention will be assessed using the Pittsburgh sleep quality index (PSQI). The questionnaire conducted retrospectively for the last four Many research groups investigated the diagnostic validity including sensitivity and specificity. The sensitivity was over 80% (80-100%), the specificity (only analyzed in three trials) showed equally high values (83-87%) [24].

Initiation of the study
After approval of the study by the local ethics committee recruitment could be started. In addition, this study will be registered at the database ClinicalTrials.gov (NCT). Figure 1 shows the study procedure. Before the weight-loss program started, patients will be properly informed and after written informed consent, the baseline measurements will be collected. The baseline measurements will include: MRI-and BIA-measurements, blood samples, anthropometric data, 7-day food-diary, questionnaire on quality of life and sleep quality. These measurements will be repeated after 15 weeks weight-loss program.

Study procedure
The weight-loss program will last 15 weeks and consisted of the OPTIFAST®-Kurzprogram (short program) and additional measurements. Weight, waist and hip circumference will be measured weekly. An additional MRI-and BIA-measurement will be conducted in week 7. Two additional blood samples will be conducted for patients` safety.
The necessary medical care will be ensured for the whole study period.
For assessing long-term effects, two follow-up measurements are planned after three, six and nine month. In case of weight gain, patients will have the possibility to receive a food replacement therapy.

Retrospective data: weight and HbA1c
Prospective data measurements:

Study termination
The study will be terminated in presence of the following factors: • Patient`s wish: the patient can withdraw from the study at any time he/she wants.
• If the therapy is disadvantageous for the patient. Any deviation or early termination of the study has to be recorded by a member of the study group. If possible, examinations that were originally scheduled at the end of the study should be brought forward. The patient has the right to withdraw from the study at any time. Any adverse event will have to be recorded and the patient observed until clarification of the adverse event.

Financial support
Nestlé Health Science Germany support the study by granting study participants a 15 % discount for the formula diet.

Statistical considerations
The retrospective data of HbA1c-values before the intervention will be used as controls for the paired sample. Die changes before intervention will be compared with changes during the intervention Therefore, Wilcoxon signed-rank test will be used. In case of normal distribution, the t-test can be used also. Based on recent literature, sample size calculation resulted in n = 35 with a power of 80% and n = 57 with a power of 95% ( Figure   2). Statistical analysis will be done using STATA 11 software.

Ethical and data safety issues
Data will be saved electronically in a pseudonymized way using an identification code. All data will be stored on one computer belonging to the Department of Medicine A, University Medicine Greifswald. Only members of the study group are authorized to have access to pseudonymized data.