Omega-3 to Reduce Diabetes Risk in Subjects With High Number of Particles That Carry "Bad Cholesterol" in the Blood

• ClinicalTrials.gov Identifier: NCT04496154
• Recruitment Status: Completed
• First Posted: August 3, 2020
• Last Update Posted: August 5, 2020
• Sponsor: May Faraj, PDt, PhD
• Collaborator: Canadian Institutes of Health Research (CIHR)
• Information provided by (Responsible Party): May Faraj, PDt, PhD, Institut de Recherches Cliniques de Montreal

Study Description

Brief Summary:

In this project, investigators explored the role of the particles that carry "bad cholesterol" in the blood (termed LDL) that are known to promote heart disease, in the promotion of type 2 diabetes (T2D) in humans. In specific, they investigated how these particles may induce the activation of an immune pathway in human fat tissue leading to multiple anomalies that favors T2D. They also explored whether omega-3 fatty acids, which are the type of fat found in fish oils can counterbalance the negative effects of LDL in fat tissue, thus providing a natural way to help reduce the risk for T2D in subjects with elevated blood LDL.

To do so, 41 subjects who were free of disease or medication affecting metabolism were enrolled at the Montreal Clinical Research Institute between 2013 and 2019 and were placed on an intervention with omega-3 fatty acids supplementation for 12 weeks (2.7 g/day, Triple Strength Omega-3 from Webbers Naturals). Investigators examined the effects of LDL and omega-3 on risk factors for T2D before and after the intervention in the whole body and specifically in fat tissue biopsies taken from the hip region. Eighty percent of the subjects who were enrolled into the study completed the intervention.

Condition or disease	Intervention/treatment	Phase
Type 2 Diabetes; Insulin Sensitivity/Resistance; Inflammatory Response; Fatty Acids, Omega-3	Dietary Supplement: Omega-3 fatty acids (2.7 g/d, EPA:DHA, 2:1)	Not Applicable

Detailed Description:

Diabetes-attributed deaths, mostly type 2 diabetes (T2D), total more than 40,000 per year, out of which 80% are secondary to cardiovascular disease and stroke. Research from the investigators' lab and others suggest that elevated atherogenic apoB-lipoproteins may not be a mere consequence of T2D but also a cause. They reported that high number of apoB-lipoproteins (apoB) may directly induce subcutaneous white adipose tissue dysfunction and related risk factors for T2D in humans; however underlying mechanisms were yet unclear.

Strong evidence implicates a specific innate immunity complex, the NLRP3 inflammasome (NLRP3 for Nucleotide-binding domain and Leucine-rich repeat Receptor, containing a Pyrin domain 3) in white adipose tissue dysfunction and associated metabolic anomalies in mice and humans. Preliminary evidence from the investigators lab and their collaborator (Dr Maya Saleh, at McGill University) indicated that native apoB-lipoproteins activate the NLRP3 inflammasome in murine bone marrow derived macrophages. On the other hand, omega-3 fatty acids were reported to inhibit the NLRP3 inflammasome in immune cells.

Thus the hypotheses that were examined in this study were that:

Primary hypothesis:

1. Subjects with high plasma apoB have higher NLRP3 inflammasome activity in white adipose tissue indicated by higher interleukin 1 beta (IL-1β) secretion.

   Higher white adipose tissue IL-1β secretion was expected to be associated with risk factors for T2D documented in subjects with high plasma apoB, namely white adipose tissue dysfunction, systemic inflammation, postprandial hypertriglyceridemia, insulin resistance and hyperinsulinemia

   Secondary hypotheses:

2. Twelve-week supplementation with omega-3 fatty acids (2.7 g/day, eicosapentaenoic acid (EPA) and docosahexaenoic (DHA), 2:1) induces a greater reduction in white adipose tissue NLRP3 inflammasome activity and related risk factors for T2D in subjects with high plasma apoB.
3. Subject native LDL (representing > 90% of apoB-lipoproteins) directly prime and/or activate the NLRP3 inflammasome in subject own white adipose tissue ex vivo, while EPA/DHA inhibit it.

Forty-one subjects (34% men) were enrolled in the study, of whom 33 subjects completed the 12-week omega-3 intervention (drop out/exclusion rate = 20%). Baseline and post-intervention risk factors for T2D measured in these subjects were; white adipose tissue and systemic NLRP3 inflammasome activity, dietary fat clearance (after a high fat meal), adipose tissue function (ex vivo after a subcutaneous adipose tissue needle biopsy), and insulin secretion and sensitivity (by gold-standard Botnia clamp technique).

Study Design

• Study Type: Interventional (Clinical Trial)
• Actual Enrollment: 41 participants
• Allocation: N/A
• Intervention Model: Single Group Assignment
• Masking: None (Open Label)
• Primary Purpose: Prevention
• Official Title: The Inflammasome and Dysfunctional Adipose Tissue: Why Should apoB-lipoproteins be Targeted in Humans
• Actual Study Start Date: September 5, 2013
• Actual Primary Completion Date: January 22, 2020
• Actual Study Completion Date: February 24, 2020

Arms and Interventions

Arm	Intervention/treatment
Experimental: Omega-3 fatty acids; 3 oral softgels (600 mg EPA and 300 mg DHA / softgel), Triple Strength Omega-3 from Webber Naturals	Dietary Supplement: Omega-3 fatty acids (2.7 g/d, EPA:DHA, 2:1); Triple Strength Omega-3 from Webber Naturals

Outcome Measures

Primary Outcome Measures :

1. Fasting white adipose tissue NLRP3 inflammasome activation [ Time Frame: Baseline ]
   White adipose tissue medium accumulation of interleukin 1 beta (IL-1β) ex vivo over 4 hours (pg/mg tissue by AlphaLISA)

Secondary Outcome Measures :

1. Fasting white adipose tissue NLRP3 inflammasome activation [ Time Frame: Change at 12 weeks from baseline ]
   White adipose tissue medium accumulation of interleukin 1 beta (IL-1β) ex vivo over 4 hours (pg/mg tissue by AlphaLISA)
2. White adipose tissue inflammation profile [ Time Frame: Baseline ]
   Fasting and 4 hour-postprandial change in NLRP3 inflammasome related inflammatory parameters; including protein and gene expression of IL1B and NLRP3 (by immunohistochemistry and RT-PCR), secretion of IL-1β (per mg tissue by AlphaLISA) and macrophage infiltration of white adipose tissue (by RT-PCR)
3. White adipose tissue inflammation profile [ Time Frame: Change at 12 weeks from baseline ]
   Fasting and 4 hour-postprandial change in NLRP3 inflammasome related inflammatory parameters; including protein and gene expression of IL1B and NLRP3 (by immunohistochemistry and RT-PCR), secretion of IL-1β (per mg tissue by AlphaLISA) and macrophage infiltration of white adipose tissue (by RT-PCR)
4. White adipose tissue function ex vivo [ Time Frame: Baseline ]
   Fasting in situ lipoprotein lipase activity (nmol 3H-triglyceride/mg tissue)
5. White adipose tissue function ex vivo [ Time Frame: Change at 12 weeks from baseline ]
   Fasting in situ lipoprotein lipase activity (nmol 3H-triglyceride/mg tissue)
6. Postprandial fat metabolism [ Time Frame: Baseline ]
   Area under the 6 hour time curve of plasma triglycerides (mmol/hour) after a high-fat meal (66% fat)
7. Postprandial fat metabolism [ Time Frame: Change at 12 weeks from baseline ]
   Area under the 6 hour time curve of plasma triglycerides (mmol/hour) after a high-fat meal (66% fat)
8. Systemic inflammation [ Time Frame: Baseline ]
   Fasting plasma inflammatory parameters including IL-1Ra and IL-1β (pg/mL by AlphaLISA)
9. Systemic inflammation [ Time Frame: Change at 12 weeks from baseline ]
   Fasting plasma inflammatory parameters including IL-1Ra and IL-1β (pg/mL by AlphaLISA)
10. Insulin sensitivity and secretion [ Time Frame: Baseline ]
    Glucose-induced insulin secretion (uU/mL/min) and insulin sensitivity (glucose infusion rate mg/kg/min) measured by Botnia clamp
11. Insulin sensitivity and secretion [ Time Frame: Change at 12 weeks from baseline ]
    Glucose-induced insulin secretion (uU/mL/min) and insulin sensitivity (glucose infusion rate mg/kg/min) measured by Botnia clamp
12. Fatty acid profile in the phospholipid fraction of plasma and red blood cells [ Time Frame: Baseline ]
    (As μmol/L by gas chromatography mass spectrometry)
13. Fatty acid profile in the phospholipid fraction of plasma and red blood cells [ Time Frame: Change at 12 weeks from baseline ]
    (As μmol/L by gas chromatography mass spectrometry)
14. Body composition [ Time Frame: Baseline ]
    Fat and lean body mass (as kg by dual energy x-ray absorptiometry)
15. Body composition [ Time Frame: Change at 12 weeks from baseline ]
    Fat and lean body mass (as kg by dual energy x-ray absorptiometry)
16. Energy expenditure [ Time Frame: Baseline ]
    Fasting and postprandial during the 6-hour high fat meal challenge (as kcal/hr using indirect calorimetry)
17. Energy expenditure [ Time Frame: Change at 12 weeks from baseline ]
    Fasting and postprandial during the 6-hour high fat meal challenge (as kcal/hr using indirect calorimetry)
18. Energy intake [ Time Frame: Baseline ]
    (Average of 3 day energy intake as kcal/day collected by 3-day dietary records)
19. Energy intake [ Time Frame: Change at 12 weeks from baseline ]
    (Average of 3 day energy intake as kcal/day collected by 3-day dietary records)

Other Outcome Measures:

1. Post-hoc analyses of white adipose tissue receptors for apoB-lipoproteins [ Time Frame: Baseline ]
   Fasting and 4 hour-postprandial change in white adipose tissue surface-expression LDLR and CD36 (% of control by immunohistochemistry in white adipose tissue slides)
2. Post-hoc analyses of white adipose tissue receptors for apoB-lipoproteins [ Time Frame: Change at 12 weeks from baseline ]
   Fasting and 4 hour-postprandial change in white adipose tissue surface-expression LDLR and CD36 (% of control by immunohistochemistry in white adipose tissue slides)
3. Post-hoc analyses of white adipose tissue genes related to lipid and lipoprotein metabolism [ Time Frame: Baseline ]
   Fasting and 4-hour postprandial change in gene expression including LDLR, CD36, SREBP1, SREBP2 and HMGCR (by RT-PCR)
4. Post-hoc analyses of white adipose tissue genes related to lipid and lipoprotein metabolism [ Time Frame: Change at 12 weeks from baseline ]
   Fasting and 4-hour postprandial change in gene expression including LDLR, CD36, SREBP1, SREBP2 and HMGCR (by RT-PCR)

Eligibility Criteria

• Ages Eligible for Study: 45 Years to 74 Years (Adult, Older Adult)
• Sexes Eligible for Study: All
• Accepts Healthy Volunteers: Yes

Criteria

Inclusion Criteria:

Men and post-menopausal women:

• Having a body mass index (BMI) > 20 kg/m2
• Aged between 45 and 74 years
• Having confirmed menopausal status (FSH ≥ 30 U/l)
• Non-smoker
• Sedentary (less than 2 hours of structured physical exercise (ex: sports club) per week)
• Low alcohol consumption: less than 2 alcoholic drinks/day

Exclusion Criteria:

• Subjects with elevated risk of cardiovascular disease (≥ 20% of calculated Framingham Risk Score) who require immediate medical intervention by lipid-lowering agents OR who cannot be placed on a 4 weeks wash-out period from their lipid-lowering medication at screening (i.e. upon admission to IRCM clinic).
• Subjects with systolic blood pressure > 160 mmHg or diastolic blood pressure > 100 mmHg
• Prior history of cardiovascular events (like stroke, transient ischemic attack, myocardial infarction, angina, heart failure...)
• Prior history of cancer within the last 3 years
• Thyroid disease - untreated
• Type 1 or 2 diabetes or fasting glucose > 7.0 mmol/L
• Claustrophobia
• Anemia - Hb < 120 g/L
• Creatinine > 100 μmol/L
• Hepatic dysfunction - AST/ALT > 3 times normal limit
• Blood coagulation problems (i.e. bleeding predisposition)
• Autoimmune diseases
• Chronic inflammatory diseases

• Concomitant medications

  • Hormone replacement therapy (except thyroid hormone at a stable dose)
  • Systemic corticosteroids
  • Anti-psychotic medications - psycho-active medication
  • Anticoagulant treatment (Aspirin, NSAIDs, warfarin, coumadin..)
  • Adrenergic agonist
  • Anti-hypertensive
  • Weight-loss
• Known substance abuse
• Allergy to seafood or fish
• Cancellation of the same scheduled testing visit, twice
• Lack of time to participate in the full length of the study (18 weeks)
• Have exceeded the annual total allowed radiation dose (like X-ray scans and/or tomography in the previous year or in the year to come) according to the physician's judgement.
• All other medical or psychological conditions deemed inappropriate according to the physician

Contacts and Locations

Sponsors and Collaborators

May Faraj, PDt, PhD

Canadian Institutes of Health Research (CIHR)

Investigators

• Principal Investigator: May Faraj, PDt, PhD; Montreal Clinical Research Institute/ University of Montreal

More Information

Publications of Results:

Simon Bissonnette, Valerie Lamantia, Yannick Cyr, Viviane Provost, Marie Devaux, May Faraj. Native LDL are Priming Signals of White Adipose Tissue NLRP3 Inflammasome in Overweight and Obese Subjects. Atherosclerosis Supplements (Abstract). 2018; 32:123

Other Publications:

Lamantia V, Bissonnette S, Wassef H, Cyr Y, Baass A, Dufour R, Rabasa-Lhoret R, Faraj M. ApoB-lipoproteins and dysfunctional white adipose tissue: Relation to risk factors for type 2 diabetes in humans. J Clin Lipidol. 2017 Jan-Feb;11(1):34-45.e2. doi: 10.1016/j.jacl.2016.09.013. Epub 2016 Oct 3.

Bissonnette S, Saint-Pierre N, Lamantia V, Cyr Y, Wassef H, Faraj M. Plasma IL-1Ra: linking hyperapoB to risk factors for type 2 diabetes independent of obesity in humans. Nutr Diabetes. 2015 Sep 28;5(9):e180. doi: 10.1038/nutd.2015.30.

Lamantia V, Bissonnette S, Provost V, Devaux M, Cyr Y, Daneault C, Rosiers CD, Faraj M. The Association of Polyunsaturated Fatty Acid delta-5-Desaturase Activity with Risk Factors for Type 2 Diabetes Is Dependent on Plasma ApoB-Lipoproteins in Overweight and Obese Adults. J Nutr. 2019 Jan 1;149(1):57-67. doi: 10.1093/jn/nxy238.

Skeldon AM, Faraj M, Saleh M. Caspases and inflammasomes in metabolic inflammation. Immunol Cell Biol. 2014 Apr;92(4):304-13. doi: 10.1038/icb.2014.5. Epub 2014 Feb 11.

Lamantia V, Sniderman A, Faraj M. Nutritional management of hyperapoB. Nutr Res Rev. 2016 Dec;29(2):202-233. doi: 10.1017/S0954422416000147. Epub 2016 Nov 8.

Faraj M. LDL, LDL receptors, and PCSK9 as modulators of the risk for type 2 diabetes: a focus on white adipose tissue. J Biomed Res. 2020 Mar 12;34(4):251-259. doi: 10.7555/JBR.34.20190124.

• Responsible Party: May Faraj, PDt, PhD, Professor, Institut de Recherches Cliniques de Montreal
• ClinicalTrials.gov Identifier: NCT04496154
• Other Study ID Numbers: 2013-14 and 2014-756; NUT273741 ( Other Grant/Funding Number: Canadian Institutes of Health Research (CIHR) )
• First Posted: August 3, 2020
• Last Update Posted: August 5, 2020
• Last Verified: August 2020

Individual Participant Data (IPD) Sharing Statement:

• Plan to Share IPD: No
• Plan Description: Biological samples (plasma and adipose tissue) can be made available for analysis by other investigators. However data statistical analysis incorporating complete IPD must be conducted by the research team of Dr May Faraj as per subject consent form.

• Studies a U.S. FDA-regulated Drug Product: No
• Studies a U.S. FDA-regulated Device Product: No

Keywords provided by May Faraj, PDt, PhD, Institut de Recherches Cliniques de Montreal:

HyperapoB; ApoB-lipoproteins; White adipose tissue; NLRP3 inflammasome; Fat metabolism; Insulin sensitivity and secretion; Systemic inflammation; Eicosapentaenoic acid (EPA); Docosahexaenoic acid (DHA)

Additional relevant MeSH terms:

Insulin Resistance; Hypersensitivity; Immune System Diseases; Hyperinsulinism; Glucose Metabolism Disorders; Metabolic Diseases