Molecular Pathways Related to Short-term Fasting Response

Fasting Clinical Trial

Official title:

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

Clinical Trial Details — Status: Completed

Administrative data

• NCT number: NCT04259879
• Other study ID #: IMD PI0025
• Status: Completed
• Phase: N/A
• Start date: April 7, 2016
• Est. completion date: June 15, 2016

Study information

• Verified date: January 2020
• Source: IMDEA Food
• Contact: n/a
• Is FDA regulated: No
• Study type: Interventional

Clinical Trial Summary

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

Description:

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).

Recruitment information / eligibility

• Status: Completed
• Enrollment: 20
• Est. completion date: June 15, 2016
• Est. primary completion date: May 18, 2016
• Accepts healthy volunteers: Accepts Healthy Volunteers
• Gender: All
• Age group: 18 Years to 50 Years

Eligibility

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

Related Conditions & MeSH terms

• Fasting

Intervention

Other:
• Fasting
Food intake restriction

Locations

Country	Name	City	State
Spain	IMDEA Food	Madrid

Sponsors (2)

Lead Sponsor	Collaborator
IMDEA Food	Centro Nacional de Investigaciones Oncologicas CARLOS III

Country where clinical trial is conducted

Spain, 

References & Publications (22)

Antoni R, Johnston KL, Collins AL, Robertson MD. Effects of intermittent fasting on glucose and lipid metabolism. Proc Nutr Soc. 2017 Aug;76(3):361-368. doi: 10.1017/S0029665116002986. Epub 2017 Jan 16. Review. — View Citation

Arnason TG, Bowen MW, Mansell KD. Effects of intermittent fasting on health markers in those with type 2 diabetes: A pilot study. World J Diabetes. 2017 Apr 15;8(4):154-164. doi: 10.4239/wjd.v8.i4.154. — View Citation

Bouwens M, Afman LA, Müller M. Fasting induces changes in peripheral blood mononuclear cell gene expression profiles related to increases in fatty acid beta-oxidation: functional role of peroxisome proliferator activated receptor alpha in human peripheral blood mononuclear cells. Am J Clin Nutr. 2007 Nov;86(5):1515-23. — View Citation

Cahill GJ Jr, Owen OE, Morgan AP. The consumption of fuels during prolonged starvation. Adv Enzyme Regul. 1968;6:143-50. — View Citation

Di Biase S, Lee C, Brandhorst S, Manes B, Buono R, Cheng CW, Cacciottolo M, Martin-Montalvo A, de Cabo R, Wei M, Morgan TE, Longo VD. Fasting-Mimicking Diet Reduces HO-1 to Promote T Cell-Mediated Tumor Cytotoxicity. Cancer Cell. 2016 Jul 11;30(1):136-146. doi: 10.1016/j.ccell.2016.06.005. — View Citation

Gotthardt JD, Verpeut JL, Yeomans BL, Yang JA, Yasrebi A, Roepke TA, Bello NT. Intermittent Fasting Promotes Fat Loss With Lean Mass Retention, Increased Hypothalamic Norepinephrine Content, and Increased Neuropeptide Y Gene Expression in Diet-Induced Obese Male Mice. Endocrinology. 2016 Feb;157(2):679-91. doi: 10.1210/en.2015-1622. Epub 2015 Dec 14. — View Citation

Halberg N, Henriksen M, Söderhamn N, Stallknecht B, Ploug T, Schjerling P, Dela F. Effect of intermittent fasting and refeeding on insulin action in healthy men. J Appl Physiol (1985). 2005 Dec;99(6):2128-36. Epub 2005 Jul 28. — View Citation

Lee C, Raffaghello L, Brandhorst S, Safdie FM, Bianchi G, Martin-Montalvo A, Pistoia V, Wei M, Hwang S, Merlino A, Emionite L, de Cabo R, Longo VD. Fasting cycles retard growth of tumors and sensitize a range of cancer cell types to chemotherapy. Sci Transl Med. 2012 Mar 7;4(124):124ra27. doi: 10.1126/scitranslmed.3003293. Epub 2012 Feb 8. — View Citation

Lopez-Guadamillas E, Fernandez-Marcos PJ, Pantoja C, Muñoz-Martin M, Martínez D, Gómez-López G, Campos-Olivas R, Valverde AM, Serrano M. p21(Cip1) plays a critical role in the physiological adaptation to fasting through activation of PPARa. Sci Rep. 2016 Oct 10;6:34542. doi: 10.1038/srep34542. — View Citation

Mattson MP, Longo VD, Harvie M. Impact of intermittent fasting on health and disease processes. Ageing Res Rev. 2017 Oct;39:46-58. doi: 10.1016/j.arr.2016.10.005. Epub 2016 Oct 31. Review. — View Citation

Merl V, Peters A, Oltmanns KM, Kern W, Born J, Fehm HL, Schultes B. Serum adiponectin concentrations during a 72-hour fast in over- and normal-weight humans. Int J Obes (Lond). 2005 Aug;29(8):998-1001. — View Citation

Nuttall FQ, Almokayyad RM, Gannon MC. Comparison of a carbohydrate-free diet vs. fasting on plasma glucose, insulin and glucagon in type 2 diabetes. Metabolism. 2015 Feb;64(2):253-62. doi: 10.1016/j.metabol.2014.10.004. Epub 2014 Oct 8. — View Citation

Nuttall FQ, Almokayyad RM, Gannon MC. The ghrelin and leptin responses to short-term starvation vs a carbohydrate-free diet in men with type 2 diabetes; a controlled, cross-over design study. Nutr Metab (Lond). 2016 Jul 22;13:47. doi: 10.1186/s12986-016-0106-x. eCollection 2016. — View Citation

Pietrocola F, Pol J, Vacchelli E, Rao S, Enot DP, Baracco EE, Levesque S, Castoldi F, Jacquelot N, Yamazaki T, Senovilla L, Marino G, Aranda F, Durand S, Sica V, Chery A, Lachkar S, Sigl V, Bloy N, Buque A, Falzoni S, Ryffel B, Apetoh L, Di Virgilio F, Madeo F, Maiuri MC, Zitvogel L, Levine B, Penninger JM, Kroemer G. Caloric Restriction Mimetics Enhance Anticancer Immunosurveillance. Cancer Cell. 2016 Jul 11;30(1):147-160. doi: 10.1016/j.ccell.2016.05.016. — View Citation

Prokesch A, Graef FA, Madl T, Kahlhofer J, Heidenreich S, Schumann A, Moyschewitz E, Pristoynik P, Blaschitz A, Knauer M, Muenzner M, Bogner-Strauss JG, Dohr G, Schulz TJ, Schupp M. Liver p53 is stabilized upon starvation and required for amino acid catabolism and gluconeogenesis. FASEB J. 2017 Feb;31(2):732-742. doi: 10.1096/fj.201600845R. Epub 2016 Nov 3. — View Citation

Raffaghello L, Safdie F, Bianchi G, Dorff T, Fontana L, Longo VD. Fasting and differential chemotherapy protection in patients. Cell Cycle. 2010 Nov 15;9(22):4474-6. Epub 2010 Nov 15. — View Citation

Ruderman NB. Muscle amino acid metabolism and gluconeogenesis. Annu Rev Med. 1975;26:245-58. Review. — View Citation

Safdie FM, Dorff T, Quinn D, Fontana L, Wei M, Lee C, Cohen P, Longo VD. Fasting and cancer treatment in humans: A case series report. Aging (Albany NY). 2009 Dec 31;1(12):988-1007. — View Citation

Tinkum KL, Stemler KM, White LS, Loza AJ, Jeter-Jones S, Michalski BM, Kuzmicki C, Pless R, Stappenbeck TS, Piwnica-Worms D, Piwnica-Worms H. Fasting protects mice from lethal DNA damage by promoting small intestinal epithelial stem cell survival. Proc Natl Acad Sci U S A. 2015 Dec 22;112(51):E7148-54. doi: 10.1073/pnas.1509249112. Epub 2015 Dec 7. — View Citation

Tinkum KL, White LS, Marpegan L, Herzog E, Piwnica-Worms D, Piwnica-Worms H. Forkhead box O1 (FOXO1) protein, but not p53, contributes to robust induction of p21 expression in fasted mice. J Biol Chem. 2013 Sep 27;288(39):27999-8008. doi: 10.1074/jbc.M113.494328. Epub 2013 Aug 5. — View Citation

Varady KA, Bhutani S, Church EC, Klempel MC. Short-term modified alternate-day fasting: a novel dietary strategy for weight loss and cardioprotection in obese adults. Am J Clin Nutr. 2009 Nov;90(5):1138-43. doi: 10.3945/ajcn.2009.28380. Epub 2009 Sep 30. — View Citation

Vasconcelos AR, Yshii LM, Viel TA, Buck HS, Mattson MP, Scavone C, Kawamoto EM. Intermittent fasting attenuates lipopolysaccharide-induced neuroinflammation and memory impairment. J Neuroinflammation. 2014 May 6;11:85. doi: 10.1186/1742-2094-11-85. — View Citation

* Note: There are 22 references in all — Click here to view all references

Outcome

Type	Measure	Description	Time frame	Safety issue
Primary	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 ?Ct method (?Ct = [Ct of gene of interest - Ct of housekeeping]). The housekeeping genes used for input normalization were ß-actin (ACTB) and ribosomal protein lateral stalk subunit P0 (RPLP0).	Baseline, 24 hours and 48 hours later
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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
Secondary	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