Lipoprotein Lipase Activity Does Not Differ in the Serum Environment of Vegans and Omnivores

Although vegan diets have been reported to be associated with a reduced risk of cardiovascular disease, it was not known whether this might be partly due to vegan diets’ effects on plasma triglyceride metabolism. This study aimed to investigate if there are differences in the activity of lipoprotein lipase (LPL), an enzyme that functions at the vascular endothelium and is responsible for triglyceride breakdown, in sera obtained from vegans and omnivores. LPL activity was assessed using isothermal titration calorimetry, which allows measurements in undiluted serum samples, mimicking physiological conditions. Fasted sera from 31 healthy participants (12F 2M vegans, 11F 6M omnivores) were analyzed. The results indicated no significant differences in average LPL activity between the vegan and omnivore groups. Interestingly, despite similar triglyceride levels, there were considerable variations in LPL activity and total very-low-density lipoprotein triglyceride breakdowns between individuals within both groups. Biomarker analysis showed that vegans had lower total cholesterol and LDL-C levels compared to omnivores. These findings suggest that the lipid-related benefits of a vegan diet, in terms of atherogenic risk, may primarily stem from cholesterol reduction rather than affecting serum as a medium for LPL-mediated triglyceride breakdown. In healthy individuals, lipid-related changes in serum composition in response to a vegan diet are likely overshadowed by genetic or other lifestyle factors

Chiral hemicucurbit[8]uril as an anion receptor: selectivity to size, shape and charge distribution

A novel eight-membered macrocycle of the hemicucurbit[n]uril family, chiral (all-R)-cyclohexanohemicucurbit[8]uril (cycHC[8]) binds anions in a purely protic solvent with remarkable selectivity. The cycHC[8] portals open and close to fully encapsulate anions in a 1 : 1 ratio, resembling a molecular Pac-Man™. Comprehensive gas, solution and solid phase studies prove that the binding is governed by the size, shape and charge distribution of the bound anion. Gas phase studies show an order of SbF6− ≈ PF6− > ReO4− > ClO4− > SCN− > BF4− > HSO4− > CF3SO3− for anion complexation strength. An extensive crystallographic study reveals the preferred orientations of the anions within the octahedral cavity of cycHC[8] and highlights the importance of the size- and shape-matching between the anion and the receptor cavity. The solution studies show the strongest binding of the ideally fitting SbF6− anion, with an association constant of 2.5 × 105 M−1 in pure methanol. The symmetric, receptor cavity-matching charge distribution of the anions results in drastically stronger binding than in the case of anions with asymmetric charge distribution. Isothermal titration calorimetry (ITC) reveals the complexation to be exothermic and enthalpy-driven. The DFT calculations and VT-NMR studies confirmed that the complexation proceeds through a pre-complex formation while the exchange of methanol solvent with the anion is the rate-limiting step. The octameric cycHC[8] offers a unique example of template-controlled design of an electroneutral host for binding large anions in a competitive polar solvent.peerReviewe

Calculated diffusion coefficients of main LPL component from RICS measurements.

Calculated diffusion coefficients of main LPL component from RICS measurements.</p

Catalytic activity of LPL measured with DGGR after incubation in various conditions.

(A) 200 nM LPL was incubated in buffer A (▲), which contained 10 IU/ml heparin (◆), 50 mg/ml BSA (■) or both (●). Alternatively, LPL was incubated in LFHP (▼). LPL activity is expressed relative to the initial activity of the experiment that contained both heparin and BSA. Results at 75 minutes were compared by two-tailed Student’s t-test. *P(B) 200 nM LPL was incubated in macromolecularly crowded buffer A. LPL activity is expressed as relative fluorescence units per second (RFU/s). PEG 6 nor dextran 40 could stabilize LPL like BSA, indicating that macromolecular crowding alone is not sufficient for LPL stabilization. (C) 200 nM LPL was incubated in buffer A with various proteins. Casein and β-lactoglobulin were as efficient as BSA in stabilizing LPL, however lysozyme failed to exert any effect. This suggests that LPL stabilization by proteins depends on their specific characteristics such as charge or hydrophobicity.</p

Catalytic activity of LPL on triglyceride-rich lipoproteins in the presence of fatty acid acceptors and macromolecular crowders.

Measurements were performed using ITC. LPL activity is expressed as heat rate (μJ/s). The substrate mixture contained CM/VLDL (adjusted to 0.5 mM triglycerides), 50 mg/ml BSA (0.75 mM) or 10 mg/ml β-cyclodextrin (8.81 mM) (CYC) and either 10% PEG 6 or 10% dextran 40 (DEX).</p

Interaction of LPL with BSA as studied using ITC, stabilization of LPL, and SPR.

(A) An example ITC thermogram for the titration of BSA into a solution with bLPL, obtained after subtraction of the BSA dilution effect. The concentration of bLPL in the cell was 1.695 μM. (B) Fitted isotherm for the binding between BSA and bLPL from the ITC experiment on panel A. (C) Enzymatic stability of LPL in the presence of BSA as determined with DGGR. 200 nM bLPL or hLPL was incubated for 60 minutes in buffer A at various BSA concentrations. The values are calculated relative to the initial activity of LPL in the same conditions. BSA stabilized both bLPL and hLPL in a concentration-dependent manner. (D) SPR sensograms showing binding of BSA to biotinylated bLPL that was attached to pre-immobilized neutravidin. (E) SPR sensorgrams showing BSA binding to bLPL that was attached to pre-immobilized GPIHBP1. In D and E, BSA concentrations are shown on sensograms. Non-specific binding sensorgrams of BSA to streptavidin and GPIHBP1, respectively, have been subtracted. (F) Plateau values of sensorgrams plotted against BSA concentration. ●—Binding of BSA with 0.1 IU/ml heparin to GPIHBP1-bound bLPL. ▲—Binding of BSA to biotinylated bLPL. ◆—Binding of BSA to 5D2-bound bLPL. Dashed line—1:1 ratio of immobilized bLPL to bound BSA in the experiment with GPIHBP1-bound bLPL. The results indicate that BSA can bind to neutravidin-bound LPL or GPIHBP1-bound LPL but not 5D2-bound LPL.</p

Addition of heparin to LPL incubation with albumin.

190 nM LPL with 10 nM LPL-ATTO610 was incubated at room temperature in 20 mM HEPES, 150 mM NaCl, pH 7.4 buffer with 50 mg/ml BSA to mimic conditions used for FCS measurements. After 15 minutes, final concentration of 10 IU/ml heparin or water was added to the mixture. LPL activity expressed as heat rate was measured with ITC at 25°C in 1.31 mM triglycerides human plasma after a 5 nM LPL injection. Data is presented as mean of two independent measurements. (TIF)</p

Incubation of heparin treated LPL with triton X-100 abolishes formation of LPL helices.

When 200 nM of LPL is mixed with 10 IU/ml heparin, the formation of LPL helices was observed (Fig 6D). When 200 nM LPL with 10 IU/ml heparin was treated with increasing concentrations of the detergent triton X-100, the LPL helices were progressively dissolved. (A)—At 0.005% triton X-100 concentration the beginning of LPL helix dissolution can be observed, with the helices pulling apart (upper panel) or dissolving from one end of the helix (lower panel). (B)—At 0.05% triton X-100 LPL helices are no longer observed, although some clumps of LPL are visible. (C)—at 0.5% triton X-100 the LPL particles are disperse and do not appear aggregated. Scale bars are 100 nm. (TIF)</p

Effect of LPL incubation concentration on its activity as measured with ITC.

LPL activity, expressed as heat rate (μJ/s), after incubation of LPL at various concentrations with 50 mg/ml BSA with (▲) or without (●) 10 IU/ml heparin in buffer A. The changes in residual LPL activity suggest that LPL oligomerization triggered by BSA is dependent on LPL concentration. This dependence disappears with the combined use of heparin and BSA which blocks the formation of LPL oligomers.</p

LPL activity measured in various substrate systems.

LPL activity was measured in substrate systems with increasing complexity: soluble fluorescent substrate DGGR (TIF)</p