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Results: 8

1.
FIGURE 7.

FIGURE 7. From: Targeting Acyl-CoA:Diacylglycerol Acyltransferase 1 (DGAT1) with Small Molecule Inhibitors for the Treatment of Metabolic Diseases.

T863 enhanced insulin-stimulated glucose uptake in differentiated 3T3-L1 adipocytes. Differentiation of 3T3-L1 cells to adipocytes, compound treatment, and insulin-stimulated glucose uptake test are described under “Experimental Procedures.” Data represent mean ± S.E. (error bars) obtained from three independent experiments, each performed in triplicate. *, p < 0.05 versus vehicle.

Jingsong Cao, et al. J Biol Chem. 2011 December 2;286(48):41838-41851.
2.
FIGURE 8.

FIGURE 8. From: Targeting Acyl-CoA:Diacylglycerol Acyltransferase 1 (DGAT1) with Small Molecule Inhibitors for the Treatment of Metabolic Diseases.

Proposed mechanisms of action upon pharmacological inhibition of DGAT1 in the context of treatment of metabolic disorders. In small intestine, inhibition of DGAT1 resulted in dynamic changes in triglyceride and cholesterol absorption, which might in turn lead to changes in gut microenvironments and incretin release. Inhibition of hepatic DGAT1 improved steatosis caused by an excess of exogenous fatty acids (i.e. under high fat feeding) and up-regulated the expression level of IRS2, an important protein mediating insulin signaling. In adipose tissues, DGAT1 inhibition led to diminished fat biosynthesis and stimulated insulin-induced glucose uptake. The combined effects from these actions are believed to contribute to the beneficial outcomes of DGAT1 inhibition in vivo, including reduced body weight, improved insulin resistance, and hyperlipidemia, and alleviated hepatic steatosis.

Jingsong Cao, et al. J Biol Chem. 2011 December 2;286(48):41838-41851.
3.
FIGURE 5.

FIGURE 5. From: Targeting Acyl-CoA:Diacylglycerol Acyltransferase 1 (DGAT1) with Small Molecule Inhibitors for the Treatment of Metabolic Diseases.

Chronic treatment of DIO mice with T863 caused weight loss and improved insulin sensitivity. A, body weight of chow-fed or DIO mice treated with vehicle or T863. B, cumulative food intake in DIO mice treated with vehicle or T863. C and D, body composition of DIO mice treated with vehicle or T863. The fat mass (C) and lean mass (D) were analyzed by echo MRI on the 13th day of the treatment. E and F, fed state blood glucose (E) and plasma insulin (F) in DIO mice treated with vehicle or T863. G and H, blood glucose excursion curve (G) and serum insulin levels (H) in an OGTT test performed in DIO mice on the 15th day of the treatment. Mice were treated with vehicle or 30 mg/kg T863 as described under “Experimental Procedures.” Data are expressed as mean ± S.E. (error bars) (n = 7). *, p < 0.05 versus vehicle (Veh).

Jingsong Cao, et al. J Biol Chem. 2011 December 2;286(48):41838-41851.
4.
FIGURE 2.

FIGURE 2. From: Targeting Acyl-CoA:Diacylglycerol Acyltransferase 1 (DGAT1) with Small Molecule Inhibitors for the Treatment of Metabolic Diseases.

, Biophysical characterization of DGAT1 inhibitors. A, structure of T863. B and C, Lineweaver-Burk plots for oleoyl-CoA or 1,2-DOG in the presence of T863 at various concentrations. The data were obtained from the CPM fluorescent assay performed in the 384-well format as described under “Experimental Procedures.” The pattern of fitting curves suggests that T863 is competitive against oleoyl-CoA (B) and uncompetitive against 1,2-DOG (C). D, T863 competes with the binding of acyl-CoA to hDGAT1 microsomes in a dose-dependent manner. Increasing concentrations of T863 were incubated with 50 μm [14C]oleoyl-CoA and DGAT1 microsomes containing 0.5 μg of total protein in 100 μl of reaction mixture. Filtration was performed as described under “Experimental Procedures.” E, the radiolabeled DGAT1 inhibitor T863 shows specific binding to hDGAT1 microsomes. 3H-Labeled T863 at various concentrations was incubated with wild-type or DGAT1-overexpressing Sf-9 cell microsomes for 90 min, followed by filtration to remove unbound radioligand. Retained radioligand was detected with a liquid scintillation counter. F, DGAT1 inhibitors T863 and compound 4a compete with the radiolabeled [3H]T863 for binding to hDGAT1. Increasing concentrations of compound T863 or 4a were incubated with DGAT1-microsomes and [3H]T863 for 90 min before filtration. See “Experimental Procedures” for a detailed description of the radioligand filtration assay. Data represent mean ± S.E. (error bars) obtained from three independent experiments, each performed in triplicate.

Jingsong Cao, et al. J Biol Chem. 2011 December 2;286(48):41838-41851.
5.
FIGURE 6.

FIGURE 6. From: Targeting Acyl-CoA:Diacylglycerol Acyltransferase 1 (DGAT1) with Small Molecule Inhibitors for the Treatment of Metabolic Diseases.

Chronic treatment of DIO mice with T863 improved hepatic steatosis, induced liver IRS-2 expression, and caused a modest increase in insulin-stimulated hepatic Akt phosphorylation. A–C, liver weight (A), representative size and morphology (B, top), Oil Red O-stained sections (B, bottom), and liver triglyceride content (C) in normal chow-fed or DIO (High-fat) mice treated with vehicle or T863 for 15 days. Data are expressed as mean ± S.E. (n = 7). *, p < 0.05 versus vehicle. D and E, Western blot analysis of hepatic IRS-2, IRS-1, and total Akt (D) and phosphorylated Akt protein at residue Ser-473 and total Akt (E) in DIO mice treated with vehicle or T863 for 2 weeks. Liver homogenates containing 50 μg of total proteins were subjected to Western analysis probed by antibodies as indicated. In E, mice were fasted overnight and given an intraperitoneal injection of insulin (20 units/kg) or saline at 15 min prior to liver sample collection. Phosphorylation status is also presented as the ratio between phosphorylated and total Akt protein. Data are mean ± S.E. (error bars) (n = 4).

Jingsong Cao, et al. J Biol Chem. 2011 December 2;286(48):41838-41851.
6.
FIGURE 1.

FIGURE 1. From: Targeting Acyl-CoA:Diacylglycerol Acyltransferase 1 (DGAT1) with Small Molecule Inhibitors for the Treatment of Metabolic Diseases.

Development of a fluorescence-based assay for high throughput screening of DGAT1 inhibitors. A, schematic of the fluorescent CPM assay for DGAT1. The assay is designed to detect CoASH released from acyl-CoA in a DGAT1-mediated reaction with a thio-reactive probe, CPM. The yielding product CoA-CPM emits strong fluorescence at 460 nm. B, Triton X-100 increases the assay window for the DGAT1 CPM assay. C, Triton X-100 markedly increases TAG formation, as assessed by the TLC-based DGAT1 assay. D, time and protein dependence of DGAT1 activity and a standard curve of CoASH in the CPM assay. The assay was performed in a 96-well plate as described under “Experimental Procedures.” E, substrate kinetics of DGAT1 activity in the CPM assay. Assays were conducted in a 96-well plate by using 0.5 μg of microsomes with the indicated concentrations of oleoyl-CoA or 1,2-DOG in the presence of 200 μm 1,2-DOG or 100 μm oleoyl-CoA, respectively. F, a reference DGAT1 inhibitor, T863, shows comparable IC50 values in the CPM and the TLC assay. The percentage inhibition is calculated by the formula, % inhibition = (RUhigh − RUcpd)/(RUhigh − RUlow) × 100, where RUhigh, RUcpd, and RUlow are readings (fluorescence or radioactivity) obtained from reaction without the addition of T863, with T863 at different concentration, or with T863 at 10 μm, respectively. Data shown in B, D, and F represent mean ± S.E. (error bars) obtained from three independent experiments; data in C and E are representative of three independent experiments with similar results.

Jingsong Cao, et al. J Biol Chem. 2011 December 2;286(48):41838-41851.
7.
FIGURE 4.

FIGURE 4. From: Targeting Acyl-CoA:Diacylglycerol Acyltransferase 1 (DGAT1) with Small Molecule Inhibitors for the Treatment of Metabolic Diseases.

Effects of T863 on intestinal fat absorption. A, oral administration of T863 significantly decreased serum TAG level in an acute lipid challenge test. In this experiment, C57/BL6 mice were first treated with an oral dose of compounds or vehicle. One hour later, a bolus of corn oil was administered via gavage, followed by measurement of serum triglyceride at 1 h after the lipid challenge. B, time course study of fat absorption in mice treated with vehicle or T863. Compound administration and lipid challenge followed the same procedure as described in A. Blood samples were withdrawn at 0, 0.5, 1, 4, 8, and 24 h after lipid challenge for measurement of serum triglyceride level. C, DGAT1 inhibition delayed the intestinal absorption of fat. Shown are sections of jejunum stained with Oil Red O. Bar, 100 μm. D, impaired formation of triglyceride in intestinal segment from compound-treated mice. C57/BL6 mice were first treated with an oral dose of compounds or vehicle. One hour later, the small intestine segments were collected and incubated with 14C-labeled oleic acid for 0.5 h. Total lipids were extracted and subjected for TLC analysis to measure TAG formation. Quantitative data are mean ± S.E. (error bars) (n = 5). *, p < 0.05 versus vehicle (Veh).

Jingsong Cao, et al. J Biol Chem. 2011 December 2;286(48):41838-41851.
8.
FIGURE 3.

FIGURE 3. From: Targeting Acyl-CoA:Diacylglycerol Acyltransferase 1 (DGAT1) with Small Molecule Inhibitors for the Treatment of Metabolic Diseases.

T863 inhibited DGAT activity in primary tissues and TAG formation in a cell-based model. A, inhibition of in vitro enzymatic activity by T863. The acyltransferase activity was measured by TLC-based assays in which membrane fractions prepared from Sf-9 cells overexpressing hDGAT1, mouse DGAT1, human MGAT3, human MGAT2, or human DGAT2, mouse small intestine (Sm. intest.), or white adipose tissue (WAT) were used as enzymatic sources. See “Experimental Procedures” for a detailed description of the TLC-based acyltransferase activity assays. Images shown on the left are the radiolabeled TAG or DAG (human MGAT2 only) bands captured from the TLC plate. The radioactivity of TAG or DAG bands was quantitated and used to calculate the percentage inhibition of enzymatic activity by T863 (with vehicle-treated sample as a control). Data shown on the right represent mean ± S.E. obtained from three independent experiments. The values of IC50 calculated for each enzymatic source were also listed. Veh, vehicle (1% DMSO). B, inhibition of TAG formation by T863 in a cell-based assay. HEK293 cells overexpressing hDGAT1 were incubated with 10 μm [14C]oleic acid (50 Ci/mmol) in the absence (Veh) or presence of T863 at various concentrations for 4 h. Cellular lipids were extracted and analyzed by TLC as described under “Experimental Procedures.” The top panel is a representative TLC image showing formation of radiolabeled TAG. The quantitative values of TAG bands were used to calculate the percentage inhibition of DGAT1 activity in the presence of T863 and plotted against the concentrations of T863 (bottom); data shown here represent mean ± S.E. (error bars) obtained from three independent experiments. C, increased cellular phospholipid (PL) formation upon DGAT1 inhibition. Metabolic labeling experiments were performed as described above. Cellular lipids were extracted and analyzed by TLC to monitor formation of TAG (top) or phospholipid (bottom).

Jingsong Cao, et al. J Biol Chem. 2011 December 2;286(48):41838-41851.

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