Identification of a botanical inhibitor of intestinal diacylglyceride acyltransferase 1 activity via in vitro screening and a parallel, randomized, blinded, placebo-controlled clinical trial

Abstract

Background: Diacylglyceride acyltransferase 1 (DGAT1) is the enzyme that adds the final fatty acid on to a diacylglyceride during triglyceride (TG) synthesis. DGAT1 plays a key role in the repackaging of dietary TG into circulating TG rich chylomicrons. A growing amount of research has indicated that an exaggerated postprandial circulating TG level is a risk indicator for cardiovascular and metabolic disorders. The aim of this research was to identify a botanical extract that inhibits intestinal DGAT1 activity and attenuates postprandial hypertriglyceridemia in overweight and obese humans.

Methods: Twenty individual phytochemicals and an internal proprietary botanical extract library were screened with a primary cell-free DGAT1 enzyme assay that contained dioleoyl glycerol and palmitoleoyl Coenzyme A as substrates plus human intestinal microsomes as the DGAT1 enzyme source. Botanical extracts with IC50 values < 100 μg/mL were evaluated in a cellular DGAT1 assay. The cellular DGAT1 assay comprised the analysis of (14)C labeled TG synthesis in cells incubated with (14)C-glycerol and 0.3 mM oleic acid. Lead botanical extracts were then evaluated in a parallel, double-blind, placebo-controlled clinical trial. Ninety healthy, overweight and obese participants were randomized to receive 2 g daily of placebo or individual botanical extracts (the investigational product) for seven days. Serum TG levels were measured before and after consuming a high fat meal (HFM) challenge (0.354 L drink/shake; 77 g fat, 25 g carbohydrate and 9 g protein) as a marker of intestinal DGAT1 enzyme activity.

Results: Phenolic acids (i.e., gallic acid) and polyphenols (i.e., cyanidin) abundantly found in nature appeared to inhibit DGAT1 enzyme activity in vitro. Four polyphenolic rich botanical extracts were identified from in vitro evaluation in both cell-free and cellular model systems: apple peel extract (APE), grape extract (GE), red raspberry leaf extract (RLE) and apricot/nectarine extract (ANE) (IC50 = 1.4, 5.6, and 10.4 and 3.4 μg/mL, respectively). In the seven day clinical trial, compared to placebo, only GE significantly reduced the baseline subtracted change in serum TG AUC following consumption of the HFM (AUC = 281 ± 37 vs. 181 ± 30 mg/dL*h, respectively; P = 0.021). Chromatographic characterization of the GE revealed a large number of closely eluting components containing proanthocyanidins, catechins, anthocyanins and their secondary metabolites that corresponded with the observed DGAT1 enzyme inhibition in the cell-free model.

Conclusion: These data suggest that a dietary GE has the potential to attenuate postprandial hypertriglyceridemia in part by the inhibition of intestinal DGAT1 enzyme activity without intolerable side effects.

Trial registration: This trial was registered with ClinicalTrials.gov NCT02333461.

Figures

Fig. 1

Role of DGAT1 in the assimilation of dietary TG into circulating TG rich chylomicrons (CM). Dietary TG are first broken down into monoacylglycerides (MG) and fatty acids (FA) by a host of pancreatic lipases. MG and FA are then absorbed into the small intestinal enterocytes and repackaged into diacylglyercides (DG) by monoacylglyceride acyltransferase transferase. DGAT1 then acylates the DG into TG (yellow circles), which are incorporated into CM and secreted into the lymphatic circulation then enter the blood circulation via the thoracic duct

Fig. 2

Clinical trial participant flow

Fig. 3

Percent DGAT1 enzyme inhibition and IC50 values for single phytochemicals in the cell-free assay. Twenty individual phytochemicals were screened through the cell-free DGAT1 enzyme assay. Six of the twenty phytochemicals were dose dependent inhibitors of DGAT1 enzyme activity (IC50 ranged from 0.667 to 8.60 μM, compared to A-922500 = 40nM) and all were phenolic acids or polyphenols. Results are the mean of duplicates

Fig. 4

Percent DGAT1 enzyme inhibition and IC50 values for the four lead botanical extracts. a APE, GE, RLE and ANE exhibited dose responsive inhibition of DGAT1 enzyme activity in the cell-free assay. The IC50 values ranged from 1.41 to 10.4 μg/mL and 17.1 ng/mL for A-922500. Results are the mean of triplicates. b The cellular DGAT1 assay was comprised of adding [14C]-glycerol to label newly synthesized TG and 0.3 mM oleic acid/BSA to stimulate DGAT1 activity. All botanical extracts and A-922500 inhibited oleic acid induced DGAT1 enzyme activity as measured by 14C label TG levels. GE was statistically more potent than APE, RLE and ANE, defined as greatest inhibition at the lowest dose (100 μg/mL) (Two-way ANOVA with Bonferroni's multiple comparisons test; P < 0.001). Result are the mean ± SEM (n = 3). Different letters, within each dose, indicate statistically significant

Fig. 5

Baseline (fasting levels) subtracted change in serum TG levels following a HFM challenge after seven days of placebo or investigational product. a Baseline subtracted serum TG response over a 6 h period. GE significantly reduced the serum TG levels at 2 h (p = 0.014) and 4 h (p = (0.029) after the HFM compared to placebo. b AUC for the baseline subtracted serum TG was also significantly reduced by seven days of dietary GE (p = 0.021). Fasting serum TG = 95.8 ± 8.8; 94.7 ± 6.8; 90.3 ± 6.2; 102.1 ± 13.8; 103.8 ± 9.8 mg/dL, for placebo, APE, GE, RLE and ANE, respectively. Results are mean ± SEM. Comparisons between placebo and investigational product test groups were made using unpaired t-test (*P < 0.05)

Fig. 6

Cell-free DGAT1 inhibition from chromatographic fractionation of GE with overlay of the corresponding LC-MS and LC-UV chromatograms. a The LC-MS chromatogram was generated using base peak index utility to differentiate major compounds. b The LC-UV chromatogram represents the sum of all wavelengths to better show the complex mixture and abundance of phytochemicals in the GE extract. DGAT1 enzyme inhibition (right side x-axis) correlates with the LC-UV pattern and abundance (left side x-axis), showing that multiple phytochemicals in the GE extract are responsible for the observed inhibition. Identities of numbered peaks are given in Table 5