Fructose and Glucose and TAS1R2 in Type 1 Diabetes (TAS1R2)

INTRODUCTION:

Fructose is transported via the portal vein to the liver, where is rapidly taken up by hepatocytes and phosphorylated to fructose-1-phosphate independent of insulin action by the enzyme fructokinase. Thus, under normal conditions, fructose is converted mainly to glucose, glycogen, and lactate, and to a small extent also to triglycerides. However a hypertriglyceridemia has been occur during high intakes of fructose.

Large amounts of fructose, unlike glucose, may result in increased lactate, pyruvate, and uric acid level in healthy subjects and patients with uncontrolled diabetes. However, uric acid also has pro-oxidative properties. Possibly, the uric acid neutralizes oxidative damage.

Sweetness is very attractive to patients with diabetes possibly due to genetic variants. The sweet taste receptor is a heterodimer of 2 protein subunits, T1R2 (taste receptor, type 1, member 2) and taste receptor, type 1, member 3, located on human chromosome 1. T1R2 is the component specific to sweet taste perception because taste receptor, type 1, member 3 is also involved in the detection of umami when it dimerizes with taste receptor, type 1, member 1.

At moment, none study assessed TAS1R variants in individuals with type 1 diabetes.

EXPERIMENTAL DESIGN:

This is a randomized, single-blind, two-way, crossover study conducted at the Research Centre (Clementino Fraga Filho University Hospital) including 30 adults with type 1 diabetes.

All volunteers will be instruct to complete the usual dietary record, register their capillary blood glucose and to abstain from vigorous physical activity, alcohol, high-fat foods and excess caffeine for 24 days before each study day.

One day before each study day, we will call to volunteer to ask about possible events that may influence the biochemical results (such as: infections, flu, fever). If we detect some, the test will be rescheduled.

Basal insulin doses will be retained, and no bolus insulin will be allowed in the morning of the day study. The study day will be performed if the blood glucose target level is between 70 and 200 mg/dL in the morning.

All volunteers will be assessed two-way crossover in a single blind, randomized fashion, with the two studies in the same subject, 1 week apart. The study was divided into two-days (the order of which was randomized): one-day with a solution containing glucose and the other with a solution containing glucose.

The subjects were admitted to the Research Centre (Nutritional Laboratory) at approximately 7-h on the day of study to test capillary blood glucose, collect arterial venous blood sample, assessed appetite sensations. After these measurements, they will rapidly drink the test solution.

Palatability ratings of the solution will assessed immediately after. Appetite sensations and will be tested using VAS scores immediately before and 70, 130, and 180 minutes after solution.

The second arterial venous blood sample will be collected 185 minutes after solution and test capillary blood glucose before and 30, 90, 120, and 180 minutes after solution.

We excluded volunteers who not complete both tests.

TEST SOLUTION Solution will be containing 75g of glucose or the same amount of fructose diluted in 200 mL of water.These amounts were calculated considering the usual oral glucose tolerance test. In addition, at moment, no studies indicate the ability capacity of fructose in patients with type 1 diabetes, however, it has been suggested that the absorption capacity of fructose in healthy adults varies from 30g to 40g.

USUAL DIETARY ASSESSMENT Usual dietary intake will be evaluated from 3-day diet records, and 24-hour recalls will be performed in the day study. All dietary records will be analyzed using a local nutritional software.

ANTHROPOMETRIC ASSESSMENT BMI will be calculated as body weight in kilograms divided by the square of height in meters.

Waist circumference will be determined as the average of two measurements calculated to the nearest 0.1cm midway between the lower rib margin and the ilial crest after a normal expiration.

Body composition will be measured by tetrapolar bioelectrical impedance(Biodynamic Model 450).

BIOCHEMICAL ASSESSMENT All arterial venous blood sample will be collected by trained healthcare professionals with disposable materials in the Research Centre (Nutritional Laboratory) and analyzed at Clinical Analysis Laboratory (located on Federal University).

Glycosylated hemoglobin, total cholesterol, HDL, creatinine, aspartate aminotransferase, alanine aminotransferase, and creatinine will be assessed to characterize the study population. Glucose, triglycerides, uric acid, pyruvic acid, lactate, and malonaldehyde will be measured before and 180 min after ingestion of solution

Glycosylated hemoglobin will be performed by high-performance liquid chromatography. Glucose, total cholesterol, HDL and triglycerides will be measured by enzymatic colorimetric method. LDL cholesterol will be calculated by Friedewald equation. Creatinine will be assessed by kinetic colorimetric method to calculates the creatinine clearance using the Cockcroft-Gault. Pyruvic acid, aspartate aminotransferase and alanine aminotransferase will be determined by ultraviolet kinetic method. Uric acid and lactate assessed by enzyme kinetic method. Malonaldehyde wil be detected by enzyme-linked immunosorbent assay.

GENOTYPING Deoxyribonucleic acid (DNA) will be isolated from whole blood using the DNA Multi-Sample Kit (Applied Biosystems®) and samples were stored at -20 degree Celsius until amplification assays in a Research Laboratory (Laboratory of Molecular Biology, Federal University).

The Ile191Val (rs35874116) polymorphism in TAS1R2 gene will be detected by using a TaqMan allelic discrimination assay (ABI number C_55646_20; Applied Biosystems, Foster City, CA) with real-time polymerase chain reaction on an ABI 7000 Sequence Detection System (Applied Biosystems). Conditions of the polymerase chain reaction is 95 degree Celsius for 10 min and 40 cycles of 95 degree Celsius for 15 s and 60 degree Celsius for 1 min. Genotyping will be verified by using positive control subjects in each 30-well plate as well as rerunning ≥ 5% of the samples, which were 100% concordant.

APPETITE AND PALATABILITY ASSESSMENT Visual analogue scales will be used to assess hunger, satiety, fullness, prospective food consumption, and palatability of the test solution, and appetite sensations will be evaluated palatability, taste, aftertaste, smell, and visual appeal of the solutions.

Both ratings consists of a visual analogue scale is a premeasured 100-mm scale that can be used to represent symptom intensity visually. Each scale is anchored at its ends by opposing feelings descriptors. Volunteers will be asked to describe their feelings by placing a vertical mark along the line that best represents the intensity of a given feelings about the solution. The distance of this mark from the appropriate anchor is measured and then translated into a score, ranging from 0 (none or best possible) to 10 (worst possible).

DIABETES TYPE 1 MANAGEMENT The investigators only included volunteers in a basal-bolus regimen with insulin infusion pump or insulin analogs (basal: detemir or glargine; bolus: lispro, glulisine or aspart) to avoid glucose oscillations during the experiment.

All volunteers will be instructed to not modify basal insulin doses and not take the morning bolus insulin. The subjects will be admitted in the Research Centre at approximately 7-h and will be test theirs capillary blood glucose, and if the blood glucose target level is between 70 and 200 mg/dL the experiment will be performed.

Before and during the experiment, we will test the capillary blood glucose levels 5-times using Accu-Check® Active, Roche, and will be canceled in glucose ≥350 mg/dL. In this case, we will take a bolus correction insulin dosage.

STATISTICAL Statistical analyzes will be performed with statistical software and significance level of 5%.

Quantitative variables will be described as the mean and standard deviation. Mann-Whitney test will be used for between-group comparison and Wilcoxon test to compare the effects of solution in each group. Spearman's correlation coefficient and regression will also be used.