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.

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 suggests that elevated atherogenic apoB-lipoproteins, mostly low-density lipoproteins (LDL) may not be a mere consequence of T2D but also a cause. They reported that high numbers of apoB-lipoproteins (apoB) induce subcutaneous white adipose tissue (WAT) dysfunction and predict several risk factors for T2D in humans. However, mechanisms underlying LDL-induced abnormalities and nutritional approaches to target them remain unexplored.

Strong evidence implicates a specific innate immunity system, the NLRP3 inflammasome/ interleukin 1 beta (IL-1β) pathway in WAT dysfunction and associated T2D risk factors in mice and humans (NLRP3 for Nucleotide-binding domain and Leucine-rich repeat Receptor, containing a Pyrin domain 3). Preliminary evidence from the investigator's lab and their collaborator (Dr Maya Saleh, at McGill University) indicated that native apoB-lipoproteins activate the NLRP3 inflammasome leading to IL-1β secretion in murine bone marrow derived macrophages. On the other hand, fish-oil derived omega-3 fatty acids, eicosapentaenoic and docosahexaenoic acids (EPA and DHA), were reported to inhibit the NLRP3 inflammasome/ IL-1β pathway in immune cells.

Thus, the central hypothesis of this trial was that apoB-lipoproteins act as metabolic danger-associated molecular patterns that activate the NLRP3 inflammasome in WAT leading to WAT dysfunction and associated risks for T2D in humans. This can be treated by EPA and DHA supplementation.

The specific hypotheses examined in 2 parts of this trial, at baseline and post-intervention, were:

Part A: At baseline (mechanisms):

Primary hypothesis:

1. Compared to subjects with low plasma apoB, subjects with high plasma apoB have higher WAT NLRP3 inflammasome activity indicated by higher WAT IL-1β secretion.

   Secondary hypotheses:

2. WAT IL-1β secretion is associated with risk factors for T2D (WAT dysfunction, systemic inflammation, postprandial hypertriglyceridemia, insulin resistance and hyperinsulinemia).

3. Ex vivo, subjects' native LDL prime and/or activate the NLRP3 inflammasome in subjects' own WAT.

Part B: Post-intervention (treatment of the baseline mechanisms):

Primary hypothesis:

1. Compared to subjects with low plasma apoB, twelve-week supplementation with EPA and DHA induces a greater reduction in WAT IL-1β secretion in subjects with high plasma apoB eliminating baseline group-differences.

   Secondary hypotheses:

2. Compared to subjects with low plasma apoB, twelve-week supplementation with EPA and DHA induces a greater reduction in risk factors for T2D in subjects with high plasma apoB.

3. Twelve-week supplementation with EPA and DHA reduces the baseline associations of WAT IL-1β secretion with risk factors for T2D

4. Ex vivo, EPA and DHA inhibit LDL-induced priming and/or activation of subjects' WAT NLRP3 inflammasome.

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%). For statistical analysis, subjects were stratified into 2 groups based on baseline median plasma apoB per sex. The 2 groups with high plasma apoB and low plasma apoB were characterized and compared for the primary and secondary outcomes at baseline and following the omega-3 intervention.

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

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.