Plasma Trimethylamine N-oxide Elevation Induced by L-carnitine Supplementation and Insulin Resistance

Study Overview
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Brief Summary
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The primary aims of the current study: using L-carnitine supplementation modulate the level of plasma trimethylamine N-oxide to assess its effect on circulating cytokines related to diabetes and metabolic syndrome; using simulated night-shift work intervention as a stress factor, explore the effect of circulating metabolites on insulin sensitivity The secondary aim is to evaluate the effect of carnitine supplementation on gut microbiome composition.

Detailed Description
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Subjects will be randomly divided in two groups: supplemented by L-carnitine 2g/day for 12 weeks, and placebo group receiving leucine in identical gelatine capsules. Subjects who successfully complete the protocol will be divided into four subgroups - two with a changed circadian cycle and two controls (supplemented and placebo). Subgroups with a changed daily cycle will be able to sleep between 8:00 and 17:00 for four consecutive days, while during the night they will stay active in the laboratory. The oral glucose tolerance test (OGTT) will be performed before and after the circadian cycle modification. Glucose levels will be monitored by FreeStyle Libre Sensor. Moreover, the activity of the participants will be monitored by wearable activity trackers. Before supplementation, as well as before circadian cycle modification and after finishing the whole experimental procedure, fasting blood samples will be collected for determination of plasma trimethylamine N-oxide (TMAO), trimethylamine (TMA), carnitine (free and acyl derivatives), protein markers of diabetes and inflammation. Moreover, the stool samples will be collected before and after 12 weeks of supplementation, to determine the composition of the gut microbiome. In addition diet of participants will be monitored.

Official Title
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The Role of Gut Microbiota Metabolite, Trimethylamine N-oxide, in the Insulin Resistance Development

Conditions
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Insulin Resistance

Intervention / Treatment
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* Dietary Supplement: L-carnitine
* Dietary Supplement: L-leucine
* Behavioral: Change of the circadian cycle


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: healthy volunteers, must be able to swallow tablets Exclusion Criteria: smokers, cardiovascular disease liver disease kidney disease gastrointestinal disorders (including stomach ulcers and erosions) diabetes

Ages Eligible for Study
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Minimum Age: 20 Years
Maximum Age: 30 Years

Sexes Eligible for Study
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All

Accepts Healthy Volunteers
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Accepts Healthy Volunteers

Study Plan
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How is the study designed?
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Design Details

Primary Purpose: Prevention
Allocation: Randomized
Intervention Model: Parallel Assignment
Masking: Double


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: carnitine<br>2 grams of L-carnitine per day for 12 weeks | Dietary Supplement: L-carnitine<br>* L-carnitine-L-tartrate<br>Behavioral: Change of the circadian cycle<br>* Reducing sleep at night for four consecutive days (enabling sleep between 8:00 and 17:00)<br>|
| Placebo Comparator: leucine<br>2 grams of L-leucine per day for 12 weeks | Dietary Supplement: L-leucine<br>* L-leucine<br>Behavioral: Change of the circadian cycle<br>* Reducing sleep at night for four consecutive days (enabling sleep between 8:00 and 17:00)<br>|
| Experimental: modified circadian cycle<br>no sleep at night for four consecutive days (enabling sleep between 8:00 and 17:00). | Dietary Supplement: L-carnitine<br>* L-carnitine-L-tartrate<br>Dietary Supplement: L-leucine<br>* L-leucine<br>|
| Active Comparator: normal circadian cycle<br>sleep at night for four consecutive days | Dietary Supplement: L-carnitine<br>* L-carnitine-L-tartrate<br>Dietary Supplement: L-leucine<br>* L-leucine<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Circulating microbiome metabolites | Determination of plasma trimethylamine and trimethylamine N-oxide using ultra performance liquid chromatography - tandem mass spectrometer (UPLC-MS/MS) method | 13 weeks | 
| Circulating carnitine metabolites | Determination of plasma free, total, and acyl-L-carnitines using ultra performance liquid chromatography - tandem mass spectrometer (UPLC-MS/MS) method | 13 weeks | 
| Circulating diabetes biomarkers | insulin, C-peptide, ghrelin, leptin, resistin using enzyme-linked immunosorbent assay | 13 weeks | 
| Circulating inflammatory biomarkers | tumor necrosis factor, C-reactive protein using enzyme-linked immunosorbent assay | 13 weeks | 
| Oral Glucose Tolerance Test | The subjects will consume 75 g of glucose solution drink within a 5-minute time frame. The glucose level will be continuously monitored with the FreeStyle Libre Sensor. | 1 week | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| gut microbiome composition | 16 S rRNA sequencing will be used to analyze the gut microbiome in the stool samples | 13 weeks |