Published: 07 November 2024
Valérie Lamantia, Simon Bissonnette, Myriam Beaudry, Yannick Cyr, Christine Des Rosiers, Alexis Baass & May Faraj

 

Abstract

Elevated numbers of atherogenic lipoproteins (apoB) predict the incidence of type 2 diabetes (T2D). We reported that this may be mediated via the activation of the NLRP3 inflammasome, as low-density lipoproteins (LDL) induce interleukin-1 beta (IL-1β) secretion from human white adipose tissue (WAT) and macrophages. However, mitigating nutritional approaches remained unknown. We tested whether omega-3 eicosapentaenoic and docosahexaenoic acids (EPA and DHA) treat LDL-induced upregulation of WAT IL-1β-secretion and its relation to T2D risk factors. Twelve-week intervention with EPA and DHA (2.7 g/day, Webber Naturals) abolished baseline group-differences in WAT IL-1β-secretion between subjects with high-apoB (N = 17) and low-apoB (N = 16) separated around median plasma apoB.

Post-intervention LDL failed to trigger IL-1β-secretion and inhibited it in lipopolysaccharide-stimulated WAT. Omega-3 supplementation also improved β-cell function and postprandial fat metabolism in association with higher blood EPA and mostly DHA. It also blunted the association of WAT NLRP3 and IL1B expression and IL-1β-secretion with multiple cardiometabolic risk factors including adiposity. Ex vivo, EPA and DHA inhibited WAT IL-1β-secretion in a dose-dependent manner. In conclusion, EPA and DHA treat LDL-induced upregulation of WAT NLRP3 inflammasome/IL-1β pathway and related T2D risk factors. This may aid in the prevention of T2D and related morbidities in subjects with high-apoB.

 

Introduction

In 2021, 529 million people or 6.1% of the world’s population were living with diabetes, 96% of which were type 2 diabetes (T2D)1. T2D increases the risk for cardiovascular disease (CVD) and stroke by 2–4 fold2 and remains the leading cause of death and disability worldwide1. However, T2D is largely preventable3. Uncovering new mechanisms fueling T2D and their treatment are vital for the prevention of T2D and CVD and the extension of a healthy life span in humans.

The bidirectional communication between the immune and metabolic pathways is widely appreciated as a regulator of homeostasis as well as disease resulting from dysregulated inflammation4,5. The NLRP3 inflammasome is a key innate sensor of metabolic stress that is implicated in the pathology of CVD and more recently T2D (NLRP3 for Nucleotide-binding domain and Leucine-rich repeat Receptor, containing a Pyrin domain 3)6,7,8. Activation of the NLRP3 inflammasome leads to the secretion of interleukin-1 beta (IL-1β), which is reported to inhibit insulin signaling in various cell types including adipocytes and β-cells6,7,8.

For IL-1β to be secreted, two signals are needed. The first is a priming signal that occurs via the activation of the nuclear factor-κB pathway, downstream of membrane receptors such as toll-like receptors, scavenger receptors and cytokine receptors including IL-1 receptor6,7,8. This leads to the transcriptional upregulation of NLRP3 and IL1B or post-translational modification of NLRP3 independent of its transcription9,10. Priming signals in macrophages include microbial lipopolysaccharide (LPS)7,11, palmitate12, and oxidized LDL13. The second signal is an activation signal that promotes the assembly of the inflammasome subunits, activation of caspase-1, cleavage of pro-IL-1β, and secretion of IL-1β. Activation signals in LPS-primed β-cells and macrophages include islet amyloid polypeptide oligomers8, ceramide14, adenosine triphosphate (ATP)15 and palmitate11.

While the upregulation of the NLRP3 inflammasome in white adipose tissue (WAT) is believed to promote metabolic stress and T2D, endogenous signals that stimulate the NLRP3 inflammasome in human WAT triggering IL-1β-secretion were unclear. Recently, we reported that native low-density lipoproteins (LDL), the most common form of atherogenic lipoproteins, are priming signals of the NLRP3 inflammasome that lead to IL-1β-secretion from human WAT and monocyte-derived macrophages16. Subjects with high plasma numbers of apoB-lipoproteins (high-apoB) had higher WAT IL-1β-secretion than subjects with low-apoB16. Importantly, WAT IL-1β-secretion induced by LDL (without/with ATP) was associated with risk factors for T2D mostly in subjects with high-apoB not low-apoB16. However, nutritional approaches that mitigate LDL-induced upregulation of human WAT NLRP3 inflammasome/ IL-1β pathway remained to be established.

Marine-source omega-3 eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were reported to inhibit the priming and activation of the NLRP3 inflammasome in macrophages in vitro17,18. Moreover, a meta-analysis in healthy adults reported that supplementation with 1.2–5.2 g/day of EPA and DHA for 6–24 weeks (EPA:DHA ratio of ~ 1.5:1 to 2.4:1) reduces IL-1β-secretion from cultured LPS-primed human monocytes19. Thus, we tested the hypotheses that EPA and DHA treat LDL-induced upregulation of the WAT NLRP3 inflammasome/ IL-1β pathway and its relation to T2D risk factors in humans.

 

Methodology

Study objectives, population and design

This work represents the post-intervention outcomes of a clinical trial with 12-week supplementation with EPA and DHA that was conducted at the Montréal Clinical Research Institute (IRCM). The central hypothesis of the 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, which can be treated by EPA and DHA. Subject recruitment was completed between 2013 and 2019. Nine-hundred and thirty subjects were screened, of whom 40 subjects were included (N = 27 females and N = 13 males)16. Baseline data reporting the effects of LDL on the WAT NLRP3 inflammasome/ IL-1β pathway in vivo and ex vivo were recently published16.

The hypotheses tested post-intervention were that supplementation with EPA and DHA 1) induces a greater reduction in WAT IL-1β-secretion in subjects with high-apoB than low-apoB eliminating baseline group-differences (primary hypothesis), 2) induces a greater reduction in risk factors for T2D in subjects with high-apoB than low-apoB (secondary hypothesis), and 3) reduces the baseline associations of WAT IL-1β-secretion with risk factors for T2D (secondary hypothesis). Moreover, we tested whether EPA and DHA inhibit LDL-induced priming and/or activation of WAT NLRP3 inflammasome ex vivo (secondary hypothesis). The sample size of N = 20/group was powered to test baseline and post-intervention group-differences in the primary outcome (WAT IL-1β-secretion)16.

Inclusion and exclusion criteria were reported16. Briefly, the trial included 45–74-year-old non-smoker males and postmenopausal females with body mass index greater than 20 kg/m2, sedentary lifestyle and low-moderate alcohol consumption. Exclusion criteria were chronic disease (e.g. CVD, diabetes, inflammatory), medications affecting metabolism, and allergy to seafood or fish. The trial was registered at ClinicalTrial.org (identifier: NCT04496154) on 03/08/2020. Sample analysis was blinded using subjects’ identification number.

Subjects were placed on a 4-week weight-stabilization period after which baseline outcome measures were conducted16. Three-day food reports were completed by the subjects and verified by the study dietitian. Assessment of the risk factors for T2D were conducted on 2 separate days, 1–4 weeks apart. Baseline measures were repeated following the intervention and post-intervention food records were completed in the last week of the intervention.

Intervention with EPA and DHA

Subjects followed a 12-week supplementation with 2.7 g/day EPA and DHA (3 softgels of Webbers Naturals Triple Strength Omega-3). Each softgel contains 1425 mg fish oil concentrate from anchovy, sardines and/or mackerel providing 600 mg EPA and 300 mg DHA in ethyl ester form. This supplementation received the Internationally Verified Omega-3 certification. This certification program ensures that the marine oil meets 90–165% of the label claim for EPA and DHA, has an oxidation limit (Totox value) ≤ 25 mEq/kg, and ≤ accepted limits for environmental toxins, heavy metals and microbiological contamination20. Subjects received their supply of omega-3 and had their body weight recorded monthly at IRCM. They were advised to consume the softgels with food and to preserve the opened bottle in the fridge to reduce light exposure and oxidation. They were also instructed to maintain their habitual dietary and physical activity during the intervention.

First testing day for body composition, glucose-induced insulin secretion (GIIS) and insulin sensitivity (IS)

Body composition was measured by dual energy X-ray absorptiometry (GE Healthcare, Little Chalfont, UK), after which GIIS and IS were measured by gold-standard Botnia clamps as standard16,21,22,23,24,25,26,27,28. Briefly, GIIS and C-peptide secretion were measured during a 1-h intravenous glucose tolerance test (IVGTT). The first- and second-phase insulin and C-peptide secretions were calculated as the area under the curve (AUC) of their plasma levels during the first 10 min and the following 50 min of the IVGTT, respectively. The IVGTT was immediately followed by a 3-h hyperinsulinemic euglycemic clamp, during which IS was measured as the glucose infusion rate divided by steady state plasma insulin (M/Iclamp). The disposition index (DI) was calculated as the 1st phase or total C-peptide secretionIVGTT multiplied by IS (M/Iclamp). Fasting blood samples collected from this day were used to isolate native LDL and measure circulating fatty acid (FA) profile.

Second testing day for basal metabolic rate and postprandial plasma fat clearance and for the collection of WAT biopsies

Basal metabolic rate was measured after 12-h fast by indirect calorimetry (Vmax Encore; Carefusion). Subjects then consumed a standardized high-fat meal (600 kcal/m2, 68% fat, 36% saturated fat, 18% carbohydrate) 16,21,23,24,25,26,28. Postprandial plasma clearance rates of fat and chylomicrons were calculated as the AUC of plasma triglycerides (TG) and apoB48 over 6 h, respectively. Fasting WAT biopsies were collected from the right hip by needle-liposuction under local anesthesia (Xylocaine 20 mg/mL, AstraZeneca) using a syringe. Biopsies were conducted between 7:30 and 9:30 am over a period of 2–2.5 min per biopsy. WAT biopsies were immediately washed with antibiotic/antifungal-supplemented Hanks’ Balanced Salt solution at 37 °C and processed as follows when sufficient yield was available 16,21,23,24,25,26,28: a portion was snap-frozen in liquid nitrogen for the measurement of mRNA and protein expressions, and another was used fresh within 45–60 min after the WAT biopsy to assess IL-1β-secretion ex vivo.

Plasma and WAT-secreted parameters

Plasma lipids, apoB and apoA1 were measured by an automated analyzer (Cobas Integra 400; Roche Diagnostics), plasma apoB48 and proprotein convertase subtilisin/kexin type 9 (PCSK9) by enzyme-linked immunosorbent assay kits (BioVendor and CircuLex MBL International, respectively), plasma glucose by an automated analyzer (YSI 2300 STAT Plus) and serum insulin and C-peptide by radioimmunoassay kits (Millipore Corporation). IL-1β accumulation in WAT culture medium was measured by alpha-LISA® kits (Perkin Elmer, Canada)16.

Plasma and red blood cell (RBC) FA profile

To assess the compliance to the intervention and to have an objective measure of EPA and DHA bioavailability, the concentrations of FA in the phospholipid (PL) layer of plasma (i.e. lipoproteins) and RBC (i.e. long-term bioavailability) were measured by gas chromatography–mass spectrometry as published16,27. Briefly, PL were separated on an aminopropyl column, and the FA were converted to methyl esters for analysis. The FA concentrations were calculated using external and internal isotope-labeled standards and expressed in molar concentrations and percent (%) of total FA. The post-intervention % shift in omega-3 FA in RBC was calculated by subtracting the baseline from the post-intervention % omega-3 FA in RBC.

Regulation of WAT NLRP3 inflammasome/IL-1β pathway

WAT samples were incubated for 4 h with the following inflammasome priming conditions16: medium alone or supplemented with native LDL or LPS. Medium was removed and WAT samples were washed and re-incubated for 3 h with the following activating conditions: medium alone or supplemented with native LDL or ATP. Medium accumulation of IL-1β was then quantified (termed IL-1β-secretion). The medium used was Dulbecco’s-Modified Eagle Medium containing 5% fetal bovine serum (Gibco/Thermo Fisher).

Native LDL was isolated from fasting blood and used within 1–4 weeks on subject’s own WAT16. LDL of 1.2 g/L apoB was used as it corresponds to the 75th percentile in Canadians29, was previously used to induce human WAT dysfunction23,28, and represents average levels of our cohorts with high-apoB24,28,30,31. LPS was used as the positive control for the inflammasome priming (Sigma-Aldrich L4591, 0.3 μg/ml) and ATP as that for activation (Sigma-Aldrich A2383, 3 mmol/L) as determined in baseline pilot kinetic studies16. WAT experiments used 5–10 mg WAT/well and 2–4 wells/condition16.

To assess the direct effect of EPA and DHA on WAT IL-1β-secretion ex vivo, EPA and DHA were purchased from Sigma Aldrich and used at a ratio of 2:1 as in the omega-3 softgels. They were co-incubated with LDL, LPS and/or ATP during the priming and activation periods. Their effects were compared to equal concentrations of palmitate and oleate. All FA were bound to albumin (0.105 mmol/L, US biological, low endotoxin), sterilized, sealed under nitrogen and stored at − 80 °C. Final concentrations of FA (50, 100, and 200 µmol/L) were measured by a commercial kit (Fujifilm Wako Pure Chemical Corporation)32.

WAT mRNA and protein expressions

mRNA expressions of genes related to inflammation and the NLRP3 inflammasome subunits (IL1B, NLRP3, CASP1,ADGRE1, MCP1, IL10) and WAT lipid metabolism and function (LDLR, CD36, HMGCR, SREBP1c, SREBP2, PPARG, ADIPOQ) were analyzed by real-time polymerase chain reaction using RotorGene Q (Qiagen) using HPRT as a reference gene16. WAT proteins were extracted in radioimmunoprecipitation assay buffer and pro-IL-1β was quantified by western blot using an internal control made with pooled WAT from 5 subjects. The list of primers and antibodies were previously reported25,26.

Statistical analysis

Data are presented as mean ± standard error of the mean. As WAT and/or LDL were insufficient to complete all experiments in some subjects, group-differences between subjects with low-apoB and high-apoB were analyzed by 2-way ANOVA for repeated measures based on a mixed-model with interaction (Figs.1, 2A–B, and 3). When the interaction was significant, inter or intra-subject differences were further analyzed by unpaired or paired t-test, respectively. The effects of FA on WAT IL-1β-secretion ex vivo were analyzed by 1-way ANOVA for repeated-measures based on a mixed-model (Fig. 8). ANOVA analyses were conducted with Geisser-Greenhouse correction and with controlling for false-discovery rate. Data with large intersubject-variability (i.e. raw data for IL-1β-secretion, gene expression, insulin and C-peptide secretionsIVGTT, and AUC6hrs of plasma TG) were LOG10 transformed before being used in analyses. Non-parametric Wilcoxon signed rank test was used when data could not be LOG10 transformed (i.e. % changes in Fig. 2C–H). Pearson correlation was used to examine the association between variables in the low-apoB and high-apoB groups separately and data were pooled when no group-differences in the regression lines existed (Figs. 4, 5, 6, and 7). Statistical analyses were performed using SPSS (V26) and GraphPad Prism (V 9.4) with significance set at p < 0.05.

 

Results

Forty subjects were included in this trial. Three subjects dropped out for lack of time/personal reasons, 2 for being unreachable, 1 for being diagnosed with cancer, and 1 for inability to swallow the omega-3 softgels. One female did not undergo the second testing day with WAT biopsies for lack of time and another was excluded by the investigators for oversensitivity to the insulin infusion during the clamp (Supplementary Fig. S1). Thus, this analysis was conducted on 33 subjects who completed the trial and were stratified based on baseline median fasting plasma apoB per sex.

Supplementation with EPA and DHA increased % EPA, docosapentaenoic acid (DPA, intermediate between EPA and DHA in the biosynthesis pathway33), DHA and total omega-3 FA in plasma and RBC confirming subject compliance (Fig. 1, Table 1). There was also a post-intervention decrease in % omega-6 FA and omega-6/omega-3 ratio. Similar changes in omega-3 and omega-6 FA were observed using their molar concentrations (Supplementary Fig. S2). Subjects with high-apoB had higher % EPA and DHA and lower omega-6/omega-3 ratio in RBC (Table 1). Plasma % EPA and/or DHA, % total omega-3, % total omega-6, and omega-6/omega-3 ratio were positively correlated with their counterparts in RBC (Supplementary Fig S3). There were no post-intervention changes in body composition or fasting metabolic parameters, including apoB, except for a decrease in systolic blood pressure and plasma FA and PCSK9 and an increase in plasma high-density lipoprotein cholesterol (HDL-C) and apoB/PCSK9 ratio (Table 1). Subjects with high-apoB had higher fasting plasma total cholesterol, LDL-C, TG and apoB/PCSK9 ratio and % fat intake and lower % carbohydrate intake. There were no post-intervention changes in dietary intake and expenditure in either group.