Molecular Pathways Related to Short-term Fasting Response

Study Overview
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Brief Summary
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This study will evaluate the effect of short-term fasting (36 hours) in gene expression in blood cells in healthy volunteers.

Detailed Description
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Fasting is a nutritional intervention consisting on the restriction of nutrient intake during a relatively long period of time. It elicits a profound metabolic reprogramming aimed at shifting nutrient supply from external food intake to internal stored nutrients. Periodic activation of this complex response, termed periodic or intermittent fasting (IF), elicits numerous protective effects against aging, metabolic alterations, neurological disorders and cardiovascular health. Short-term fasting is protective in different stress scenarios, including ischemia reperfusion, bouts of inflammation and chemotherapy-induced toxicity, and improves the anti-tumor efficacy of chemotherapy. Although the basic physiology of fasting is well known, the molecular mechanisms underlying its beneficial effects are not yet completely understood. In mammals, the response to short-term fasting (from 12 to 48 hours) in terms of nutrient mobilization through the bloodstream has been extensively studied. Fasting follows sequential phases, during which nutrients are released from different storing depots. First, glucose is released from glycogen stores in the liver and muscle. Upon depletion of glycogen, two fasting mechanisms are activated: fatty acids are exported from the adipose tissue into the bloodstream in the form of free fatty acids (FFAs), reaching the liver where they are used to produce ketone bodies, a process termed ketogenesis. Also, gluconeogenesis is activated in the liver, generating glucose mainly from glycerol (released during lipolysis) and amino acids, that originate mainly from muscle breakdown. All these physiological responses are tightly regulated by hormonal and molecular mechanisms. At the hormonal level, fasting induces a decrease in blood insulin, leptin and ghrelin, and an increase in glucagon levels, while blood adiponectin remains unchanged. Also, several signal transduction pathways are affected by fasting. PPARalpha, a nuclear receptor of fatty acids, becomes activated by the fasting-mediated increase in blood Free fatty Acids (FFAs) and triggers the expression of many target genes in several tissues, including blood cells. It has been shown that the Cyclin Dependent Kinase (CDK) inhibitor p21 is highly upregulated during short-term fasting in many mouse tissues. Moreover, it is known that p21-null mice are unable to endure normal periods of fasting and that p21 is required for the full activation of PPARa target genes both in vivo and in isolated hepatocytes. In the current study, the investigators wanted to study for the first time molecular mechanisms of fasting that still remained unexplored, specially the expression induction of p21 and PPARalpha signalling pathway. For this, the investigators analyzed blood samples from healthy volunteers subjected to 36 hours of fasting, to explore gene expression in Peripheral Blood Mononuclear Cells (PBMCs).

Official Title
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Evaluation of p21 Induction and Molecular Pathways Related to Short-term Fasting Response

Conditions
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Fasting

Intervention / Treatment
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* Other: Fasting


Participation Criteria
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Eligibility Criteria
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Inclusion Criteria: Men and women between 18 - 50 years old. BMI >20<30 Adequate education level and comprehension of the clinical study Willingness to participate in the study as a volunteer and to provide written consent Exclusion Criteria: BMI <20 (thinness) BMI >30 (obesity) Abnormal low glucose levels after fasting Having donated blood less than 8 weeks before starting the study Subjects who report special discomfort after previous periods of short fasting Diagnosis of type 2 Diabetes mellitus (insulin-dependent) Dyslipidemia under pharmacological treatment High blood pressure under pharmacological treatment Dementia, neurological disease or reduction of cognitive function Severe illness (hepatic disease, renal disease, etc Taking medications that could affect the lipid and glycemic profiles (statins, fibrate, diuretics, corticoids, anti-inflammatory, hypoglycemic or insulin) 30 days before the beginning of the study. Taking medications or substances for weight loss management (15 days before the beginning of the study) Pregnancy or lactation

Ages Eligible for Study
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Minimum Age: 18 Years
Maximum Age: 50 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: Basic Science
Intervention Model: Single Group Assignment
Interventional Model Description: This study was an Interventional study. There were three evaluations: the basal one was an initial evaluation after overnight fasting, the second evaluation 24 hours later (36 hours fasting) and the third one 24 hours post-refeeding.
Masking: None (Open Label)


Arms and Interventions

| Participant Group/Arm | Intervention/Treatment |
| --- | --- |
| Experimental: Fasting<br>The participants will follow a short-term fasting period for 36 hours | Other: Fasting<br>* Food intake restriction<br>|

What is the study measuring?
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Primary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Changes in gene expression in PBMCs after fasting | Expression analysis of p21, Pyruvate Dehydrogenase Kinase 4 (PDK4), Carnitine palmitoyltransferase 1 (CPT1), Adipophilin (ADFP) and Solute carrier family 25, member 50 (SLC25A50) were performed in a HT-7900 Fast Real time polymerase chain reaction (PCR). Quantifications were made applying the ΔCt method (ΔCt = [Ct of gene of interest - Ct of housekeeping]). The housekeeping genes used for input normalization were β-actin (ACTB) and ribosomal protein lateral stalk subunit P0 (RPLP0). | Baseline, 24 hours and 48 hours later | 

Secondary Outcome Measures

| Outcome Measure | Measure Description | Time Frame |
| --- | --- | --- |
| Changes in Insulin levels in response to fasting | Insulin levels (International Units per milliliter) were measured with a kit from Abbott Laboratories, by luminescent immunoassay using the Architect instrument from Abbott Laboratories. | Baseline, 24 hours and 48 hours later | 
| Changes in Free Fatty Acids levels in response to fasting | Free fatty acids levels (moles per milliliter) were evaluated with a kit from Abbott Laboratories, by enzymatic spectrophotometric assays using an Architect instrument from Abbott Laboratories. | Baseline, 24 hours and 48 hours later | 
| Changes ketone bodies in response to fasting | Ketone bodies concentration (moles per milliliter) will be measured with a kit from Sigma-Aldrich, by an enzymatic spectrophotometric assay using an microplate reader from Thermo Fisher. | Baseline, 24 hours and 48 hours later | 
| Changes in leptin levels in response to fasting | Leptin levels (nanograms per milliliter) were measured with a kit from Mercodia by a non-competitive automatic ELISA immunoanalysis | Baseline, 24 hours and 48 hours later | 
| Changes in lipid profile in response to fasting | To evaluate lipid improvements the following measurements were considered: Triacylglycerol, Total Cholesterol, low Density Lipoprotein and High-Density Lipoprotein measured by routine laboratory (CQS, Madrid, Spain) methods. | Baseline, 24 hours and 48 hours later | 
| Subjective evaluation of tolerance to fasting | To evaluate the tolerance to fasting, participants will fill in a fasting tolerance test based on the symptoms they feel, this will result in a final score of tolerance to fasting. | 36 hours of fasting | 

 Terms related to the study
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Keywords Provided by Centre Hospitalier Valida
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Short-term Fasting, molecular mechanisms, p21, Peripheral blood mononuclear cells (PBMCs), PPARalpha