Decreased Plasma Lipoprotein Lipase in Hypoadiponectinemia: An association independent of systemic inflammation and insulin resistance

We investigated two different cohorts of patients. 1) A total of 206 nondiabetic men were recruited from the University Hospital Heidelberg, Department of Medicine. All individuals in this group had diagnosed or suspected coronary artery disease and underwent elective coronary angiography. The overt diagnosis of diabetes, according to the American Diabetes Association criteria (fasting plasma glucose ≥126 mg/dl) (13), was an exclusion criterion. 2) A total of 110 patients (82 male and 28 female subjects) with known type 2 diabetes were recruited from the Diabetes Outpatient Clinic of the University Hospital Heidelberg.

Contraindications for heparin, treatment with subcutaneous or intravenous heparin in the previous 72 h, severe kidney or liver disease, treatment with drugs known to affect LPL activity (such as fibrates) or adiponectin plasma levels (such as peroxisome proliferator–activated receptor γ agonists), and a fasting triglyceride level >11.4 mmol/l (1,000 mg/dl), suggesting secondary lipid disorders, were exclusion criteria in both nondiabetic and type 2 diabetic subjects. The study was approved by the Internal Ethics Committee of Heidelberg University, and each patient gave informed consent.

Total cholesterol, HDL cholesterol, and triglyceride concentrations were determined enzymatically with a Synchron LX-20 (Beckman Coulter, Munich, Germany). LDL and VLDL were separated by ultracentrifugation in a Beckman LM-8 ultracentrifuge in 100-μl volumes with a VT-51.2 rotor (Beckman Coulter). The atherogenic index was calculated by the following formula: (total cholesterol − HDL cholesterol)/HDL cholesterol.

After an overnight fast, venous blood samples were drawn into EDTA tubes before and 10 min after intravenous injection of 60 IU heparin (Braun Melsungen, Melsungen, Germany) per kilogram of body weight. The samples were immediately chilled to 4°C, centrifuged, and stored at −80°C until assayed. Postheparin LPL activity was determined with a triolein/phosphatidylcholine emulsion as described previously (14). Regular serum was used as the source for the LPL activator apolipoprotein C-II. Selective measurement of LPL was based on the inactivation of LPL by 1.0 mol/l NaCl. The samples were quantitated in duplicate, and postheparin plasma from pooled normal control subjects was used to correct for interassay variation. The intra-assay coefficient was 7.8% and the interassay coefficient 11.4%. LPL concentration in postheparin plasma was quantitated by enzyme-linked immunosorbent assay (ELISA) (Immundiagnostik, Bensheim, Germany) using monoclonal antibodies against the COOH-terminal domain of LPL for capture and quantitation.

In the morning after an overnight fast, venous blood was sampled for the measurement of plasma concentrations of adiponectin, glucose, insulin, and C-reactive protein (CRP). Adiponectin samples were quantitated in duplicate by ELISA (B-Bridge International, San Jose, CA), and plasma from four normal control subjects was used for interassay variation. Both the intra- and interassay coefficients of variation were <5.0%. Plasma glucose was measured by a glucose-oxidase method. Serum insulin immunoreactivity was determined from frozen serum by ELISA (CIS bio international, Gif-Sur-Yvette, France). Plasma concentrations of CRP in a highly sensitive assay (high-sensitivity CRP [hs-CRP]) were determined by ELISA (Dade Behring, Cupertino, CA).

The degree of insulin resistance was estimated by homeostatis model assessment (HOMA) according to the method described by Matthews et al. (15). In particular, an insulin resistance score (HOMA of insulin resistance [HOMA-IR]) was computed with the following formula: fasting serum glucose (mmol/l) × fasting serum insulin (μU/ml)/22.5.

Statistical analyses were performed with SPSS for Windows (version 11.0; SPSS, Chicago, IL). Spearman correlation coefficients were used to describe the association between adiponectin and other continuous variables of interest. Linear regression models were used to control for potentially confounding variables. The models, fitted for LPL activity as a dependent variable, included age, BMI, plasma triglycerides, HDL cholesterol, HOMA-IR, hs-CRP, and adiponectin plasma concentration, as well as independent variables, to demonstrate the relative contribution of each of these variables to the outcome variable. Variables that were not normally distributed, such as plasma adiponectin, triglycerides, and hs-CRP, were log transformed to better approximate normal distributions. Results are expressed as means ± SE. A P value <0.05 was considered statistically significant.

The clinical characteristics of the subjects studied are summarized in Table 1. Table 2 shows the association between adiponectin and selected variables for both nondiabetic and diabetic subjects. LPL activity and concentration were highly correlated with each other (P < 0.001) in both cohorts, as expected (16). Therefore, we mainly report the findings with LPL activity. In both nondiabetic and type 2 diabetic subjects, adiponectin levels were strongly correlated with postheparin plasma LPL activity (Fig. 1 and Table 2). As shown in Table 2, there was a significant negative correlation between plasma adiponectin levels and the concentration of plasma hs-CRP in both groups. HOMA-IR was negatively associated with plasma adiponectin in the nondiabetic group. Spearman correlation coefficients of plasma adiponectin with BMI and lipid parameters (HDL cholesterol and plasma triglycerides) were similar in the diabetic and nondiabetic subjects. We also found a significant relationship between postheparin LPL activity and HOMA-IR in both cohorts (data not shown). Patients with significant renal insufficiency have been shown to have elevated adiponectin plasma levels (17). In our patient cohort, however, very few subjects had increased plasma creatinine values. Adiponectin was not correlated with creatinine in either of our cohorts.

In multivariate analyses including markers of systemic inflammation (hs-CRP) and insulin resistance (HOMA-IR), the strongest predictive variable for LPL activity in the nondiabetic and diabetic groups was adiponectin in log₁₀ (r = 0.521 and r = 0.357, respectively) (Table 3, model 1). When HDL cholesterol and plasma triglycerides (Table 3, model 2) and finally age and BMI (Table 3, model 3) were included in the model, adiponectin remained the strongest predictive factor for LPL activity in both groups. Regression analyses revealed that plasma adiponectin levels account for 23% of the variation in LPL activity in nondiabetic subjects and 26% of the variation in LPL activity in type 2 diabetic patients.

Our data in two independent cohorts of patients demonstrate a significant relationship between the plasma levels of adiponectin and postheparin plasma LPL activity. This is of special importance because investigations on the regulation of plasma LPL activity in larger patient collectives are limited because of the complex procedure of the quantification of postheparin plasma LPL activity.

Similar findings of dramatically raised LPL activity associated with increased plasma adiponectin levels have recently been shown in an animal model (18). Our data also confirm a significant negative association between the plasma adiponectin level and the concentration of hs-CRP that has been previously reported (19). Thus, a proinflammatory state with a decreased plasma concentration of adiponectin may result in reduced LPL expression.

In vitro, insulin is a strong determinant of LPL activity (1). In clinical studies, however, conflicting data about the association of plasma insulin levels with LPL activity have been published (20,21). Therefore, it has been suggested that the HOMA index, integrating plasma insulin levels with glycemia, may be the more reliable parameter (4). Our results show a negative correlation between postheparin LPL activity and HOMA-IR in both cohorts studied, which is consistent with previous reports (4). Plasma adiponectin has been shown to be inversely correlated with insulin resistance (9). This capability of adiponectin to increase insulin sensitivity may therefore point to an indirect effect of adiponectin on plasma LPL activity. Our findings, however, show that the relationship between adiponectin and postheparin LPL activity and concentration is independent of both adiponectin effects on whole-body insulin sensitivity and systemic inflammation.

Multivariate regression analyses, including HOMA-IR as a marker of insulin resistance and hs-CRP as a marker of systemic inflammation, revealed that plasma adiponectin was the strongest independent factor influencing plasma LPL activity in both groups studied and accounted for 23 and 26% of the observed variances in posthaparin LPL activity, respectively.

In a previous study, adiponectin was associated with an antiatherogenic lipid profile (12), but the mechanism by which adiponectin influences lipid metabolism has not been elucidated. We now show for the first time a significant relationship between plasma adiponectin and lipid metabolism that is independent of inflammation and insulin resistance. We speculate that adiponectin, in addition to its anti-inflammatory properties, may directly stimulate the expression of LPL. Thus, increased LPL activity stimulated by increased adiponectin levels might result in elevated plasma HDL cholesterol and antiatherogenic changes in lipid profile because a positive association between LPL activity and HDL levels is known (22).

In summary, we demonstrate for the first time that plasma levels of adiponectin are significantly correlated with LPL activity in two independent cohorts, nondiabetic individuals and patients with type 2 diabetes. This relationship is independent of systemic inflammatory activity and unaffected by the link of adiponectin with insulin resistance.

This work was supported in part by a research grant from Pfizer (ATV-D-01-006G). J.G.S. has received support from the National Institutes of Health (Grant DK07296-23) since November 2002.