PCSK7: A novel regulator of apolipoprotein B and a potential target against non-alcoholic fatty liver disease.

BACKGROUND: Epidemiological evidence links the proprotein convertase subtilisin/kexin 7 (PCSK7) to triglyceride (TG) metabolism. We associated the known PCSK7 gain-of-function non-coding SNP rs236918 with higher levels of plasma apolipoprotein B (apoB) and the loss-of-function coding variant p.Pro777Leu (SNP rs201598301) with lower apoB and TG. Herein, we aimed to unravel the in vivo role of liver PCSK7. METHODS: We biochemically defined the functional role of PCSK7 in lipid metabolism using hepatic cell lines and Pcsk7-/- mice. Our findings were validated following subcutaneous administration of hepatocyte-targeted N-acetylgalactosamine (GalNAc)-antisense oligonucleotides (ASOs) against Pcsk7. RESULTS: Independent of its proteolytic activity, membrane-bound PCSK7 binds apoB100 in the endoplasmic reticulum and enhances its secretion. Mechanistically, the loss of PCSK7/Pcsk7 leads to apoB100 degradation, triggering an unfolded protein response, autophagy, and ß-oxidation, eventually reducing lipid accumulation in hepatocytes. Non-alcoholic fatty liver disease (NAFLD) was induced by a 12-week high fat/fructose/cholesterol diet in wild type (WT) and Pcsk7-/- mice that were then allowed to recover on a 4-week control diet. Pcsk7-/- mice recovered more effectively than WT mice from all NAFLD-related liver phenotypes. Finally, subcutaneous administration of GalNAc-ASOs targeting hepatic Pcsk7 to WT mice validated the above results. CONCLUSIONS: Our data reveal hepatic PCSK7 as one of the major regulators of apoB, and its absence reduces apoB secretion from hepatocytes favoring its ubiquitination and degradation by the proteasome. This results in a cascade of events, eventually reducing hepatic lipid accumulation, thus supporting the notion of silencing PCSK7 mRNA in hepatocytes for targeting NAFLD.

Native low-density lipoproteins are priming signals of the NLRP3 inflammasome/interleukin-1ß pathway in human adipose tissue and macrophages.

Elevated plasma numbers of atherogenic apoB-lipoproteins (apoB), mostly as low-density lipoproteins (LDL), predict diabetes risk by unclear mechanisms. Upregulation of the NLRP3 inflammasome/interleukin-1 beta (IL-1ß) system in white adipose tissue (WAT) is implicated in type 2 diabetes (T2D); however, metabolic signals that stimulate it remain unexplored. We hypothesized that (1) subjects with high-apoB have higher WAT IL-1ß-secretion than subjects with low-apoB, (2) WAT IL-1ß-secretion is associated with T2D risk factors, and (3) LDL prime and/or activate the WAT NLRP3 inflammasome. Forty non-diabetic subjects were assessed for T2D risk factors related to systemic and WAT glucose and fat metabolism. Regulation of the NLRP3 inflammasome was explored using LDL without/with the inflammasome's priming and activation controls (LPS and ATP). LDL induced IL1B-expression and IL-1ß-secretion in the presence of ATP in WAT and macrophages. Subjects with high-apoB had higher WAT IL-1ß-secretion independently of covariates. The direction of association of LDL-induced WAT IL-1ß-secretion to T2D risk factors was consistently pathological in high-apoB subjects only. Adjustment for IL-1ß-secretion eliminated the association of plasma apoB with T2D risk factors. In conclusion, subjects with high-apoB have higher WAT IL-1ß-secretion that may explain their risk for T2D and may be related to LDL-induced priming of the NLRP3 inflammasome.ClinicalTrials.gov (NCT04496154): Omega-3 to Reduce Diabetes Risk in Subjects With High Number of Particles That Carry "Bad Cholesterol" in the Blood-Full Text View-ClinicalTrials.gov.

IL-6 Trans-Signaling Is Increased in Diabetes, Impacted by Glucolipotoxicity, and Associated With Liver Stiffness and Fibrosis in Fatty Liver Disease.

Many people living with diabetes also have nonalcoholic fatty liver disease (NAFLD). Interleukin-6 (IL-6) is involved in both diseases, interacting with both membrane-bound (classical) and circulating (trans-signaling) soluble receptors. We investigated whether secretion of IL-6 trans-signaling coreceptors are altered in NAFLD by diabetes and whether this might associate with the severity of fatty liver disease. Secretion patterns were investigated with use of human hepatocyte, stellate, and monocyte cell lines. Associations with liver pathology were investigated in two patient cohorts: 1) biopsy-confirmed steatohepatitis and 2) class 3 obesity. We found that exposure of stellate cells to high glucose and palmitate increased IL-6 and soluble gp130 (sgp130) secretion. In line with this, plasma sgp130 in both patient cohorts positively correlated with HbA1c, and subjects with diabetes had higher circulating levels of IL-6 and trans-signaling coreceptors. Plasma sgp130 strongly correlated with liver stiffness and was significantly increased in subjects with F4 fibrosis stage. Monocyte activation was associated with reduced sIL-6R secretion. These data suggest that hyperglycemia and hyperlipidemia can directly impact IL-6 trans-signaling and that this may be linked to enhanced severity of NAFLD in patients with concomitant diabetes. ARTICLE HIGHLIGHTS: IL-6 and its circulating coreceptor sgp130 are increased in people with fatty liver disease and steatohepatitis. High glucose and lipids stimulated IL-6 and sgp130 secretion from hepatic stellate cells. sgp130 levels correlated with HbA1c, and diabetes concurrent with steatohepatitis further increased circulating levels of all IL-6 trans-signaling mediators. Circulating sgp130 positively correlated with liver stiffness and hepatic fibrosis. Metabolic stress to liver associated with fatty liver disease might shift the balance of IL-6 classical versus trans-signaling, promoting liver fibrosis that is accelerated by diabetes.

Sex-Specific Models to Predict Insulin Secretion and Sensitivity in Subjects with Overweight and Obesity.

Sex-specific differences exist in insulin secretion (ISec) and sensitivity (IS) in humans. However, current fasting indices used to estimate them, such as HOMA and QUICKI, are not sex-specific. We aimed to develop sex-specific models to improve the prediction of ISec and IS by fasting measures in adults with overweight/obesity. A post hoc analysis was conducted on baseline data of two clinical trials completed between 2010 and 2020 (37 men and 61 postmenopausal women, 45-73 years, BMI > 25 kg/m2, without chronic disease). Glucose-induced insulin or C-peptide secretions and IS were measured using gold-standard Botnia-clamps, which is a 1 h intravenous glucose tolerance test followed by a 3 h hyperinsulinemic-euglycemic clamp. Stepwise regression analysis using anthropometric and fasting plasma glucose, insulin, and lipoprotein-related measures was used to predict ISec and IS. First-phase, second-phase and total glucose-induced ISec were predicted by a combination of fasting plasma insulin and apoB without or with plasma glucose, triglyceride, and waist circumference in women (R2 = 0.58-0.69), and by plasma insulin and glucose without or with BMI and cholesterol in men (R2 = 0.41-0.83). Plasma C-peptide, alone in men or followed by glucose in women, predicted C-peptide secretion. IS was predicted by plasma insulin and waist circumference, followed by HDL-C in women (R2 = 0.57) or by glucose in men (R2 = 0.67). The sex-specific models agreed with the Botnia-clamp measurements of ISec and IS more than with HOMA or QUICKI. Sex-specific models incorporating anthropometric and lipoprotein-related parameters allowed better prediction of ISec and IS in subjects with overweight or obesity than current indices that rely on glucose and insulin alone.

Lower plasma PCSK9 in normocholesterolemic subjects is associated with upregulated adipose tissue surface-expression of LDLR and CD36 and NLRP3 inflammasome.

BACKGROUND: LDL-cholesterol lowering variants that upregulate receptor uptake of LDL, such as in PCSK9 and HMGCR, are associated with diabetes via unclear mechanisms. Activation of the NLRP3 inflammasome/interleukin-1 beta (IL-1ß) pathway promotes white adipose tissue (WAT) dysfunction and type 2 diabetes (T2D) and is regulated by LDL receptors (LDLR and CD36). We hypothesized that: (a) normocholesterolemic subjects with lower plasma PCSK9, identifying those with higher WAT surface-expression of LDLR and CD36, have higher activation of WAT NLRP3 inflammasome and T2D risk factors, and; (b) LDL upregulate adipocyte NLRP3 inflammasome and inhibit adipocyte function. METHODOLOGY: Post hoc analysis was conducted in 27 overweight/ obese subjects with normal plasma LDL-C and measures of disposition index (DI during Botnia clamps) and postprandial fat metabolism. WAT was assessed for surface-expression of LDLR and CD36 (immunohistochemistry), protein expression (immunoblot), IL-1ß secretion (AlphaLISA), and function (3 H-triolein storage). RESULTS: Compared to subjects with higher than median plasma PCSK9, subjects with lower PCSK9 had higher WAT surface-expression of LDLR (+81%) and CD36 (+36%), WAT IL-1ß secretion (+284%), plasma IL-1 receptor-antagonist (+85%), and postprandial hypertriglyceridemia, and lower WAT pro-IL-1ß protein (-66%), WAT function (-62%), and DI (-28%), without group-differences in body composition, energy intake or expenditure. Adjusting for WAT LDLR or CD36 eliminated group-differences in WAT function, DI, and postprandial hypertriglyceridemia. Native LDL inhibited Simpson-Golabi Behmel-syndrome (SGBS) adipocyte differentiation and function and increased inflammation. CONCLUSION: Normocholesterolemic subjects with lower plasma PCSK9 and higher WAT surface-expression of LDLR and CD36 have higher WAT NLRP3 inflammasome activation and T2D risk factors. This may be due to LDL-induced inhibition of adipocyte function.

White Adipose Tissue Surface Expression of LDLR and CD36 is Associated with Risk Factors for Type 2 Diabetes in Adults with Obesity.

OBJECTIVE: Human conditions with upregulated receptor uptake of low-density lipoproteins (LDL) are associated with diabetes risk, the reasons for which remain unexplored. LDL induce metabolic dysfunction in murine adipocytes. Thus, it was hypothesized that white adipose tissue (WAT) surface expression of LDL receptor (LDLR) and/or CD36 is associated with WAT and systemic metabolic dysfunction. Whether WAT LDLR and CD36 expression is predicted by plasma lipoprotein-related parameters was also explored. METHODS: This was a cross-sectional analysis of 31 nondiabetic adults (BMI > 25 kg/m2 ) assessed for WAT surface expression of LDLR and CD36 (immunohistochemistry), WAT function, WAT and systemic inflammation, postprandial fat metabolism, and insulin resistance (IR; hyperinsulinemic-euglycemic clamp). RESULTS: Fasting WAT surface expression of LDLR and CD36 was negatively associated with WAT function (3 H-triglyceride storage, r = -0.45 and -0.66, respectively) and positively associated with plasma IL-1 receptor antagonist (r = 0.64 and 0.43, respectively). Their expression was suppressed 4 hours postprandially, and reduced LDLR was further associated with IR (M/Iclamp , r = 0.61 women, r = 0.80 men). Plasma apolipoprotein B (apoB)-to-PCSK9 ratio predicted WAT surface expression of LDLR and CD36, WAT dysfunction, WAT NLRP3 inflammasome priming and disrupted cholesterol-sensing genes, and systemic IR independent of sex and body composition. CONCLUSIONS: Higher fasting and lower postprandial WAT surface expression of LDLR and CD36 is associated with WAT dysfunction, systemic inflammation, and IR in adults with overweight/obesity, anomalies that are predicted by higher plasma apoB-to-PCSK9 ratio.

LDL, LDL receptors, and PCSK9 as modulators of the risk for type 2 diabetes: a focus on white adipose tissue.

Type 2 diabetes (T2D) and cardiovascular disease (CVD) share many risk factors such as obesity, unhealthy lifestyle, and metabolic syndrome, whose accumulation over years leads to disease onset. However, while lowering plasma low-density lipoprotein cholesterol (LDLC) is cardio-protective, novel evidence have recognised a role for common LDLC-lowering variants ( e.g. in HMGCR, PCSK9, and LDLR) and widely used hypocholesterolemic drugs that mimic the effects of some of these variants (statins) in higher risk for T2D. As these conditions decrease plasma LDLC by increasing tissue-uptake of LDL, a role for LDL receptor (LDLR) pathway was proposed. While underlying mechanisms remain to be fully elucidated, work from our lab reported that native LDL directly provoke the dysfunction of human white adipose tissue (WAT) and the activation of WAT NLRP3 (Nucleotide-binding domain and Leucine-rich repeat Receptor, containing a Pyrin domain 3) inflammasome, which play a major role in the etiology of T2D. However, while elevated plasma numbers of apolipoprotein B (apoB)-containing lipoproteins (measured as apoB, mostly as LDL) is associated with WAT dysfunction and related risk factors for T2D in our cohort, this relation was strengthened in regression analysis by lower plasma proprotein convertase subtilisin/kexin type 9 (PCSK9). This supports a central role for upregulated pathway of LDLR and/or other receptors regulated by PCSK9 such as cluster of differentiation 36 (CD36) in LDL-induced anomalies. Targeting receptor-mediated uptake of LDL into WAT may reduce WAT inflammation, WAT dysfunction, and related risk for T2D without increasing the risk for CVD.

The deubiquitinating enzyme USP19 modulates adipogenesis and potentiates high-fat-diet-induced obesity and glucose intolerance in mice.

AIMS/HYPOTHESIS: Elucidating the molecular mechanisms of fat accumulation and its metabolic consequences is crucial to understanding and treating obesity, an epidemic disease. We have previously observed that Usp19 deubiquitinating enzyme-null mice (Usp19-/-) have significantly lower fat mass than wild-type (WT) mice. Thus, this study aimed to provide further understanding of the role of ubiquitin-specific peptidase 19 (USP19) in fat development, obesity and diabetes. METHODS: In this study, the metabolic phenotypes of WT and Usp19-/- mice were compared. The stromal vascular fractions (SVFs) of inguinal fat pads from WT and Usp19-/- mice were isolated and cells were differentiated into adipocytes in culture to assess their adipogenic capacity. Mice were fed a high-fat diet (HFD) for 18 weeks. Body composition, glucose metabolism and metabolic variables were assessed. In addition, following insulin injection, signalling activity was analysed in the muscle, liver and adipose tissue. Finally, the correlation between the expression of Usp19 mRNA and adipocyte function genes in human adipose tissue was analysed. RESULT: Upon adipogenic differentiation, SVF cells from Usp19-/- failed to accumulate lipid and upregulate adipogenic genes, unlike cells from WT mice. Usp19-/- mice were also found to have smaller fat pads throughout the lifespan and a higher percentage of lean mass, compared with WT mice. When fed an HFD, Usp19-/- mice were more glucose tolerant, pyruvate tolerant and insulin sensitive than WT mice. Moreover, HFD-fed Usp19-/- mice had enhanced insulin signalling in the muscle and the liver, but not in adipose tissue. Finally, USP19 mRNA expression in human adipose tissue was positively correlated with the expression of important adipocyte genes in abdominal fat depots, but not subcutaneous fat depots. CONCLUSIONS/INTERPRETATION: USP19 is an important regulator of fat development. Its inactivation in mice exerts effects on multiple tissues, which may protect against the negative metabolic effects of high-fat feeding. These findings suggest that inhibition of USP19 could have therapeutic potential to protect from the deleterious consequences of obesity and diabetes.

The Association of Polyunsaturated Fatty Acid δ-5-Desaturase Activity with Risk Factors for Type 2 Diabetes Is Dependent on Plasma ApoB-Lipoproteins in Overweight and Obese Adults.

Background: δ-5 and δ-6 desaturases (D5D and D6D) catalyze the endogenous conversion of n-3 (ω-3) and n-6 (ω-6) polyunsaturated fatty acids (PUFAs). Their activities are negatively and positively associated with type 2 diabetes (T2D), respectively, by unclear mechanisms. Elevated plasma apoB-lipoproteins (measured as plasma apoB), which can be reduced by n-3 PUFA intake, promote T2D risk factors. Objective: The aim of this study was to test the hypothesis that the association of D5D and D6D activities with T2D risk factors is dependent on plasma apoB. Methods: This is a pooled analysis of 2 populations recruited for 2 different metabolic studies. It is a post hoc analysis of baseline data of these subjects [n = 98; 60% women (postmenopausal); mean ± SD body mass index (in kg/m2): 32.8 ± 4.7; mean ± SD age: 57.6 ± 6.3 y]. Glucose-induced insulin secretion (GIIS) and insulin sensitivity (IS) were measured using Botnia clamps. Plasma clearance of a high-fat meal (600 kcal/m2, 66% fat) and white adipose tissue (WAT) function (storage of 3H-triolein-labeled substrate) were assessed in a subpopulation (n = 47). Desaturase activities were estimated from plasma phospholipid fatty acids. Associations were examined using Pearson and partial correlations. Results: While both desaturase activities were positively associated with percentage of eicosapentaenoic acid, only D5D was negatively associated with plasma apoB (r = -0.30, P = 0.003). Association of D5D activity with second-phase GIIS (r = -0.23, P = 0.029), IS (r = 0.33, P = 0.015, in women) and 6-h area-under-the-curve (AUC6h) of plasma chylomicrons (apoB48, r = -0.47, P = 0.020, in women) was independent of age and adiposity, but was eliminated after adjustment for plasma apoB. D6D activity was associated in the opposite direction with GIIS (r = 0.24, P = 0.049), IS (r = -0.36, P = 0.004) and AUC6h chylomicrons (r = 0.52, P = 0.004), independent of plasma apoB. Both desaturases were associated with plasma interleukin-1-receptor antagonist (D5D: r = -0.45, P < 0.001 in women; D6D: r = -0.33, P = 0.007) and WAT function (trend for D5D: r = 0.30, P = 0.05; D6D: r = 0.39, P = 0.027) independent of any adjustment. Conclusions: Association of D5D activity with IS, lower GIIS, and plasma chylomicron clearance is dependent on plasma apoB in overweight and obese adults.

High plasma apolipoprotein B identifies obese subjects who best ameliorate white adipose tissue dysfunction and glucose-induced hyperinsulinemia after a hypocaloric diet.

Background: To optimize the prevention of type 2 diabetes (T2D), high-risk obese subjects with the best metabolic recovery after a hypocaloric diet should be targeted. Apolipoprotein B lipoproteins (apoB lipoproteins) induce white adipose tissue (WAT) dysfunction, which in turn promotes postprandial hypertriglyceridemia, insulin resistance (IR), and hyperinsulinemia. Objective: The aim of this study was to explore whether high plasma apoB, or number of plasma apoB lipoproteins, identifies subjects who best ameliorate WAT dysfunction and related risk factors after a hypocaloric diet. Design: Fifty-nine men and postmenopausal women [mean ± SD age: 58 ± 6 y; body mass index (kg/m2): 32.6 ± 4.6] completed a prospective study with a 6-mo hypocaloric diet (-500 kcal/d). Glucose-induced insulin secretion (GIIS) and insulin sensitivity (IS) were measured by 1-h intravenous glucose-tolerance test (IVGTT) followed by a 3-h hyperinsulinemic-euglycemic clamp, respectively. Ex vivo gynoid WAT function (i.e., hydrolysis and storage of 3H-triolein-labeled triglyceride-rich lipoproteins) and 6-h postprandial plasma clearance of a 13C-triolein-labeled high-fat meal were measured in a subsample (n = 25). Results: Postintervention first-phase GIISIVGTT and total C-peptide secretion decreased in both sexes, whereas second-phase and total GIISIVGTT and clamp IS were ameliorated in men (P < 0.05). Baseline plasma apoB was associated with a postintervention increase in WAT function (r = 0.61) and IS (glucose infusion rate divided by steady state insulin (M/Iclamp) r = 0.30) and a decrease in first-phase, second-phase, and total GIISIVGTT (r = -0.30 to -0.35) without sex differences. The association with postintervention amelioration in WAT function and GIISIVGTT was independent of plasma cholesterol (total, LDL, and HDL), sex, and changes in body composition. Subjects with high baseline plasma apoB (1.2 ± 0.2 g/L) showed a significant increase in WAT function (+105%; P = 0.012) and a decrease in total GIISIVGTT (-34%; P ≤ 0.001), whereas sex-matched subjects with low plasma apoB (0.7 ± 0.1 g/L) did not, despite equivalent changes in body composition and energy intake and expenditure. Conclusions: High plasma apoB identifies obese subjects who best ameliorate WAT dysfunction and glucose-induced hyperinsulinemia, independent of changes in adiposity after consumption of a hypocaloric diet. We propose that subjects with high plasma apoB represent an optimal target group for the primary prevention of T2D by hypocaloric diets. This trial was registered at BioMed Central as ISRCTN14476404.

ApoB-lipoproteins and dysfunctional white adipose tissue: Relation to risk factors for type 2 diabetes in humans.

BACKGROUND: Elevated plasma apoB is an independent predictor of T2D; however, underlying mechanisms remain unclear. Chronic reduction in white adipose tissue (WAT) function promotes T2D. We reported that differentiation of preadipocytes and acute incubation of human WAT with LDL induce their dysfunction (decreased hydrolysis and storage of triglyceride-rich lipoproteins [TRL]). OBJECTIVE: To examine the hypothesis that the association of plasma apoB with T2D risk factors, hypertriglyceridemia (hyperTG), insulin resistance (IR), and hyperinsulinemia, was dependent on WAT dysfunction. METHODS: Thirty normoglycemic subjects were enrolled (≥27 kg/m2, 45-74 years). Fasting gynoid WAT biopsy was obtained followed by the ingestion of a 13C-triolein-labeled-high-fat meal. WAT function was measured ex vivo as the hydrolysis and storage of 3H-triolein-labeled-TRL as 3H-lipids over 4 hours. Insulin sensitivity and secretion were measured by Botnia clamps. RESULTS: WAT function correlated with higher insulin sensitivity (M/Iclamp r = 0.60) and faster plasma clearance of chylomicrons in women (iAUC6hrs apoB48, r = -0.60). Plasma apoB correlated with WAT dysfunction (r = -0.52), postprandial hyperTG (iAUC6hrs-TG, r = 0.51, 13C-TG, r = 0.48), IR (M/Iclamp r = -0.38) and hyperinsulinemia (second phase-glucose-induced-insulin-secretion, r = 0.41). Co-incubation of subjects' WAT with their LDL increased medium accumulation of 3H-TRL and 3H-NEFA with no sex differences. Adjusting for WAT function eliminated the association of plasma apoB with IR independent of sex and body fat or adipocyte diameter. Its association with other risk factors was unaffected. CONCLUSIONS: Association of plasma apoB with IR in obese subjects is dependent on gynoid WAT dysfunction. We propose that targeting hyperapoB, without increasing their uptake into nonhepatic peripheral tissues, ameliorates WAT function and risk for T2D.

Association between osteocalcin gamma-carboxylation and insulin resistance in overweight and obese postmenopausal women.

AIM: In mice, osteocalcin (OCN) acts as a bone-derived hormone promoting insulin sensitivity and glucose tolerance. In that species, OCN endocrine action is inhibited when its first glutamic acid residue (Glu13) is γ-carboxylated (Gla). The importance of this posttranslational modification for OCN function in human is still unclear. Our objectives were to identify an assay to assess γ-carboxylation of human OCN on its first Glu residue (Glu17) and to test its association with insulin resistance and inflammation profile in overweight women. METHODS: Several ELISAs were tested to determine their specificity toward various forms of human OCN. Associations between OCN γ-carboxylation and determinants of glucose tolerance, insulin sensitivity, liver function and subclinical inflammation were then investigated in 129 non-diabetic overweight and obese postmenopausal women. RESULTS: We identified assays allowing the measurement of total OCN (tOCN) and the ratio of Gla17/tOCN. Circulating Gla17/tOCN levels correlated negatively with insulin sensitivity assessed by hyperinsulinemic-euglyceamic clamp (P=0.02) or insulin sensitivity index derived from oral glucose tolerance test (P=0.00003), and positively with insulin resistance assessed by HOMA-IR (P=0.0005) and with markers of subclinical inflammation and liver enzymes, including C-reactive protein (CRP; P=0.007) and aspartate aminotransferase (AST; P=0.009). CONCLUSIONS: γ-carboxylation of OCN on Glu17 residue correlates with insulin resistance and subclinical inflammation, suggesting that γ-carboxylation of OCN negatively regulates its endocrine action in humans.

Enrichment of Triglyceride-Rich Lipoproteins with Apolipoprotein C-I Is Positively Associated with Their Delayed Plasma Clearance Independently of Other Transferable Apolipoproteins in Postmenopausal Overweight and Obese Women.

Background: The role of plasma apolipoprotein (apo) C-I in cardiometabolic risk in humans is unclear. However, in vitro studies showed a dual role for apoC-I, both protective and harmful, depending on the carrier lipoprotein.Objective: We tested the hypothesis that triglyceride (TG)-rich lipoprotein (TRL) apoC-I, not total or HDL apoC-I, is associated with delayed postprandial plasma clearance of TRLs, independently of apoC-II, apoC-III, and apoE.Methods: This cross-sectional study examines the plasma clearance of a 13C-triolein-labeled high-fat meal (68% fat energy) in 20 postmenopausal overweight and obese women [body mass index (in kg/m2) ≥27; aged 45-74 y] as the increment change in area under the 6-h postprandial curves (iAUC6h) of TRL parameters. Lipoproteins were fractionated by fast-protein LC. Transferable apolipoproteins were measured by ELISA. TRL enrichment with apolipoproteins was calculated by dividing their TRL concentrations by TRL apoB. The effects of human apoC-I and apoC-III on the hydrolysis and storage of 3H-triolein-labeled TRLs were tested in 3T3-L1 adipocytes.Results: TRL apoC-I was positively associated with plasma apo B-48 and total and non-HDL TGs, cholesterol, and apoB (r = 0.52-0.97) and negatively with HDL cholesterol (r = -0.52) and LDL diameter (r = -0.91) (P < 0.05). Total and HDL apoC-I were correlated only with total (r = 0.62) and HDL (r = 0.75) cholesterol. Women with high fasting TRL enrichment with apoC-I (99-365 µmol apoC-I/µmol apoB), but not apoC-II, apoC-III, or apoE, had higher iAUC6h for TGs (+195%), 13C-TGs (+319%), and apo B-48 (+186%) than those with low enrichment (14-97 µmol apoC-I/µmol apoB). The 4-h postprandial increase in TRL apoC-I was associated with a 4-h increase in TRL TGs and iAUC6h for TGs, 13C-TGs, and apo B-48 (r = 0.74-0.86, P < 0.001), independently of 4-h changes in TRL apoB, apoC-II, apoC-III, or apoE. ApoC-I and apoC-III inhibited 3H-TRL clearance by adipocytes by >75% (P < 0.001).Conclusions: TRL enrichment with apoC-I is positively associated with postprandial hypertriglyceridemia and remnant accumulation in postmenopausal overweight and obese women, independently of apoC-II, apoC-III, or apoE, which may be due to inhibiting TRL clearance by adipocytes. Reducing TRL apoC-I may ameliorate delayed postprandial plasma clearance of TRLs and associated risks in humans.

Nutritional management of hyperapoB.

Plasma apoB is a more accurate marker of the risk of CVD and type 2 diabetes (T2D) than LDL-cholesterol; however, nutritional reviews targeting apoB are scarce. Here we reviewed eighty-seven nutritional studies and present conclusions in order of strength of evidence. Plasma apoB was reduced in all studies that induced weight loss of 6-12 % using hypoenergetic diets (seven studies; 5440-7110 kJ/d; 1300-1700 kcal/d; 34-50 % carbohydrates; 27-39 % fat; 18-24 % protein). When macronutrients were compared in isoenergetic diets (eleven studies including eight randomised controlled trials (RCT); n 1189), the diets that reduced plasma apoB were composed of 26-51 % carbohydrates, 26-46 % fat, 11-32 % protein, 10-27 % MUFA, 5-14 % PUFA and 7-13 % SFA. Replacement of carbohydrate by MUFA, not SFA, decreased plasma apoB. Moreover, dietary enriching with n-3 fatty acids (FA) (from fish: 1·1-1·7 g/d or supplementation: 3·2-3·4 g/d EPA/DHA or 4 g/d EPA), psyllium (about 8-20 g/d), phytosterols (about 2-4 g/d) or nuts (30-75 g/d) also decreased plasma apoB, mostly in hyperlipidaemic subjects. While high intake of trans-FA (4·3-9·1 %) increased plasma apoB, it is unlikely that these amounts represent usual consumption. Inconsistent data existed on the effect of soya proteins (25-30 g/d), while the positive association of alcohol consumption with low plasma apoB was reported in cross-sectional studies only. Five isoenergetic studies using Mediterranean diets (including two RCT; 823 subjects) reported a decrease of plasma apoB, while weaker evidence existed for Dietary Approaches to Stop Hypertension (DASH), vegetarian, Nordic and Palaeolithic diets. We recommend using a Mediterranean dietary pattern, which also encompasses the dietary components reported to reduce plasma apoB, to target hyperapoB and reduce the risks of CVD and T2D.

WAT apoC-I secretion: role in delayed chylomicron clearance in vivo and ex vivo in WAT in obese subjects.

Reduced white adipose tissue (WAT) LPL activity delays plasma clearance of TG-rich lipoproteins (TRLs). We reported the secretion of apoC-I, an LPL inhibitor, from WAT ex vivo in women. Therefore we hypothesized that WAT-secreted apoC-I associates with reduced WAT LPL activity and TRL clearance. WAT apoC-I secretion averaged 86.9 ± 31.4 pmol/g/4 h and 74.1 ± 36.6 pmol/g/4 h in 28 women and 11 men with BMI ≥27 kg/m(2), respectively, with no sex differences. Following the ingestion of a (13)C-triolein-labeled high-fat meal, subjects with high WAT apoC-I secretion (above median) had delayed postprandial plasma clearance of dietary TRLs, assessed from plasma (13)C-triolein-labeled TGs and apoB48. They also had reduced hydrolysis and storage of synthetic (3)H-triolein-labeled ((3)H)-TRLs in WAT ex vivo (i.e., in situ LPL activity). Adjusting for WAT in situ LPL activity eliminated group differences in chylomicron clearance; while adjusting for plasma apoC-I, (3)H-NEFA uptake by WAT, or body composition did not. apoC-I inhibited in situ LPL activity in adipocytes in both a concentration- and time-dependent manner. There was no change in postprandial WAT apoC-I secretion. WAT apoC-I secretion may inhibit WAT LPL activity and promote delayed chylomicron clearance in overweight and obese subjects. We propose that reducing WAT apoC-I secretion ameliorates postprandial TRL clearance in humans.

The apoB-to-PCSK9 ratio: A new index for metabolic risk in humans.

BACKGROUND: Proprotein convertase subtilisin/kexin type 9 (PCSK9) shuttles low-density lipoprotein (LDL) receptors for degradation, thus upregulates LDL plasma clearance. Although PCSK9 loss of function is cardioprotective, its role in metabolic risks remains unknown. Increased apoB-lipoproteins uptake into nonhepatic tissues such as white adipose tissue (WAT) induces their dysfunction, which may be favored by lower plasma PCSK9. We hypothesized that lower plasma PCSK9 relative to apoB, or higher apoB-to-PCSK9 ratio, is a better predictor of metabolic disturbances than PCSK9 alone in humans. METHODS: Thirty-three men and 48 postmenopausal women (>27 kg/m(2), aged 45-74 years, normoglycemic) underwent in-depth assessment of glucose and fat metabolism using high-fat meals, WAT biopsies, intravenous glucose-tolerance tests, and hyperinsulinemia clamps. RESULTS: Plasma apoB correlated positively with fasting and postprandial triglycerides and chylomicron clearance (R = 0.44-0.66) and glucose-stimulated insulin secretion (R = 0.24) and negatively with insulin sensitivity (R = -0.28) and gynoid WAT in situ lipoprotein lipase activity (ie, ex vivo WAT function, R(2) = 0.34). Neither PCSK9 nor LDL cholesterol associated with these risks. In regression analysis that adjusted for body mass index, lower plasma PCSK9 strengthened the association of apoB to WAT dysfunction and insulin resistance. Moreover, plasma apoB-to-PCSK9 ratio correlated positively with all these metabolic risks and further associated positively with android-to-gynoid fat ratio (R = 0.41) and negatively with gynoid fat mass (R = -0.23, all P ≤ .05). No significant sex differences existed in these associations. CONCLUSIONS: Lower plasma PCSK9 relative to apoB associates with metabolic risks and WAT dysfunction in normoglycemic obese subjects. We hypothesize that the plasma apoB-to-PCSK9 ratio provides a better clinical index than PCSK9 alone for monitoring early metabolic disturbances that may be promoted by reduction in plasma PCSK9.

Changes in total and central fat mass after a hypocaloric diet associate with changes of apoC-I in postmenopausal obese women.

BACKGROUND: We previously reported the secretion of apolipoprotein apoC-I, apoC-II, apoC-III, and apoE from adipose tissue in postmenopausal obese women, suggesting their potential regulation by energy balance in humans. OBJECTIVE: We examined the changes of these apolipoproteins, in relation to changes in cardiometabolic risks, following a hypocaloric diet in overweight/obese women. METHODS/RESULTS: A total of 137 postmenopausal overweight/obese women who were free of chronic disease were examined at baseline, 56 women of whom were reevaluated following a 6-month hypocaloric diet. At baseline, there was no association between the plasma transferable apolipoproteins with any index of adiposity, insulin sensitivity, lipids, or inflammation, except for apoE with peripheral fat mass (r = 0.18, P < .05), and apoC-II and apoC-III with cholesterol (r = 0.23 and r = 0.20 respectively, P < .05). The hypocaloric diet reduced adiposity, insulin resistance, and inflammatory markers but had no significant effects on plasma transferable apolipoproteins or lipids, whose average concentrations were within normal range at baseline. The changes in total and central, but not peripheral, fat mass associated with changes of apoC-I only (r = 0.28 and r = 0.43; respectively, P < .05). Post-weight-loss apoC-I increased in some women (52%) yet it decreased in others, however there were no differences in cardiometabolic risk factors between the 2 groups. CONCLUSIONS: Plasma apoC-I, apoC-II, apoC-III, and apoE are not associated with adiposity, insulin sensitivity, or inflammation in obese but healthy postmenopausal women. Post-weight-loss changes of total and central fat mass associate with changes of apoC-I.

The effect of insulin on circulating PCSK9 in postmenopausal obese women.

BACKGROUND: PCSK9 (proprotein convertase subtilisin/kexin type 9) promotes the degradation of the LDLR (LDL receptor) in hepatocytes, leading to an increase in plasma LDL-C (LDL cholesterol). Previous animal studies have shown that insulin stimulates PCSK9 transcription and observational human studies showed a positive correlation between plasma PCSK9 concentration and fasting insulinemia. OBJECTIVE: The purpose of this study was to investigate the effects of chronic and acute hyperinsulinemia on PCSK9 in a large cohort of human subjects as well as at a cellular level. METHODS: The in vivo effect of hyperinsulinemia on plasma PCSK9 concentration was studied using euglycemic-hyperinsulinemic clamps in 82 non-diabetic post-menopausal obese patients. We studied the in vitro effects of insulin stimulation on PCSK9 mRNA as well as on protein expression and secretion in HepG2 and Huh7 cells. RESULTS: Analysis of the pre and post-clamp data revealed a 15.4% (p<0.001) lowering of plasma PCSK9 concentration after acute insulin induction. In vitro studies post-insulin stimulation showed that mRNA levels of PCSK9 reduced by 25% in HepG2 cells (p<0.027) and by 59% in Huh7 cells (p<0.01). Intracellular concentration of PCSK9 were 10% lower in HepG2 cells (p<0.05) and 35% lower in Huh7 cells (p<0.05). CONCLUSIONS: Our results show an inhibitory effect of acute hyperinsulinemia on PCSK9 in humans both in vitro and in vivo. This data may assist in evaluating PCSK9 levels in individuals on insulin therapy.

Caspases and inflammasomes in metabolic inflammation.

Inflammation is an important contributor to the development of metabolic disease. Recent work has strongly implicated the inflammasome and caspase-1 as having a pivotal role in the regulation of metabolism, obesity, insulin resistance and cardiovascular disease. Through multiple murine and human studies we now know that the inflammasome can be activated by metabolic triggers in vivo. Clinical studies also reveal the inflammasome to be a potential candidate for therapeutic intervention and provide a clear incentive for future work on this inflammatory pathway.

Circulating endocannabinoids in insulin sensitive vs. insulin resistant obese postmenopausal women. A MONET group study.

OBJECTIVE: To measure the circulating levels of endocannabinoids and related molecules at fasting, after acute hyperinsulinemia and after weight loss in insulin sensitive vs. insulin resistant obese postmenopausal women. DESIGN AND METHODS: The sample consisted of 30 obese postmenopausal women (age: 58.9 ± 5.2 yrs; BMI: 32.9 ± 3.6 kg/m(2) ). Subjects underwent a 3-hour hyperinsulinaemic-euglycaemic clamp (HEC) (glucose disposal rate (M-value): 10.7 ± 3.3 mg min(-1) kg(-1) FFM) and 6-month weight loss intervention. Participants were classified as insulin sensitive obese (ISO) or insulin resistant obese (IRO) based on a predefined cutoff. Plasma levels of the endocannabinoids, anandamide (AEA), 2-arachidonoylglycerol (2-AG), and of the AEA-related compounds, palmitoylethanolamide (PEA) and oleoylethanolamide (OEA), were measured by liquid chromatography-mass spectrometry. RESULTS: IRO presented higher levels of 2-AG (P < 0.05) independently of the HEC and weight loss, whereas the HEC had an independent inhibitory effect on AEA, PEA, and OEA levels (P < 0.05) in both groups. Furthermore, there was an independent stimulatory effect of weight loss only on PEA levels in both groups (P < 0.05). CONCLUSIONS: This study is the first to show that higher circulating levels of the endocannabinoid 2-AG are found in IRO compared to ISO postmenopausal women, and that weight loss is associated with an increase in PEA, a PPAR-α ligand.