Abetalipoproteinemia is a human genetic disease that is characterized by a defect in the assembly or secretion of plasma very low density lipoproteins and chylomicrons. The microsomal triglyceride transfer protein (MTP), which is located in the lumen of microsomes isolated from the liver and intestine, has been proposed to function in lipoprotein assembly. MTP activity and the 88-kilodalton component of MTP were present in intestinal biopsy samples from eight control individuals but were absent in four abetalipoproteinemic subjects. This finding suggests that a defect in MTP is the basis for abetalipoproteinemia and that MTP is indeed required for lipoprotein assembly.

The C-aryl glucoside 6 (dapagliflozin) was identified as a potent and selective hSGLT2 inhibitor which reduced blood glucose levels in a dose-dependent manner by as much as 55% in hyperglycemic streptozotocin (STZ) rats. These findings, combined with a favorable ADME profile, have prompted clinical evaluation of dapagliflozin for the treatment of type 2 diabetes.

OBJECTIVE-The inhibition of gut and renal sodium-glucose cotransporters (SGLTs) has been proposed as a novel therapeutic approach to the treatment of diabetes. We have identified dapagliflozin as a potent and selective inhibitor of the renal sodium-glucose cotransporter SGLT2 in vitro and characterized its in vitro and in vivo pharmacology.RESEARCH DESIGN AND METHODS-Cell-based assays measuring glucose analog uptake were used to assess dapagliflozin's ability to inhibit sodium-dependent and facilitative glucose transport activity. Acute and multi-dose studies in normal and diabetic rats were performed to assess the ability of dapagliflozin to improve fed and fasting plasma glucose levels. A hyperinsulinemic-euglycemic clamp study was performed to assess the ability of dapagliflozin to improve glucose utilization after multi-dose treatment.RESULTS-Dapagliflozin potently and selectively inhibited human SGLT2 versus human SGLT1, the major cotransporter of glucose in the gut, and did not significantly inhibit facilitative glucose transport in human adipocytes. In vivo, dapagliflozin acutely induced renal glucose excretion in normal and diabetic rats, improved glucose tolerance in normal rats, and reduced hyperglycemia in Zucker diabetic fatty (ZDF) rats after single oral doses ranging from 0.1 to 1.0 mg/kg. Once-daily dapagliflozin treatment over 2 weeks significantly lowered fasting and fed glucose levels at doses ranging from 0.1 to 1.0 mg/kg and resulted in a significant increase in glucose utilization rate accompanied by a significant reduction in glucose production.CONCLUSIONS-These data suggest that dapagliflozin has the potential to be an efficacious treatment for type 2 diabetes.

The microsomal triglyceride transfer protein (MTP) is a dimeric lipid transfer protein consisting of protein disulfide isomerase and a unique 97-kDa subunit. In vitro, MTP accelerates the transport of triglyceride, cholesteryl ester, and phospholipid between membranes. It was recently demonstrated that abetalipoproteinemia, a hereditary disease characterized as an inability to produce chylomicrons and very low-density lipoproteins in the intestine and liver, respectively, results from mutations in the gene encoding the 97-kDa subunit of the microsomal triglyceride transfer protein. Downstream effects resulting from this defect include malnutrition, very low plasma cholesterol and triglyceride levels, altered lipid and protein compositions of membranes and lipoprotein particles, and vitamin deficiencies. Unless treated, abetalipoproteinemic subjects develop gastrointestinal, neurological, ophthalmological, and hematological abnormalities.

The microsomal triglyceride transfer protein (MTP), which catalyses the transport of triglyceride, cholesteryl ester and phospholipid between phospholipid surfaces, is a heterodimer composed of the multifunctional protein, protein disulphide isomerase, and a unique large subunit with an apparent M(r) of 88K (refs 1-3). It is isolated as a soluble protein from the lumen of the microsomal fraction of liver and intestine. The large subunit of MTP was not detectable in four unrelated subjects with abetalipoproteinaemia, a rare autosomal recessive disease characterized by a defect in the assembly or secretion of plasma lipoproteins that contain apolipoprotein B (ref. 6). We report here the isolation and sequencing of complementary DNA encoding the large subunit of MTP. A comparison of this sequence to corresponding genomic sequences from two abetalipoproteinaemic subjects revealed a homozygous frameshift mutation in one subject and a homozygous nonsense mutation in the other. The results indicate that a defect in the gene for the large subunit of MTP is the proximal cause of abetalipoproteinaemia in these two subjects, and that MTP is required for the secretion of plasma lipoproteins that contain apolipoprotein B.

Microsomal triglyceride transfer protein (MTP) is critical for the assembly and secretion of apolipoprotein B (apoB) lipoproteins. Its activity is classically measured by incubating purified MTP or cellular homogenates with donor vesicles containing radiolabeled lipids, precipitating the donor vesicles, and measuring the radioactivity transferred to acceptor vesicles. Here, we describe a simple, rapid, and sensitive fluorescence assay for MTP. In this assay, purified MTP or cellular homogenates are incubated with small unilamellar donor vesicles containing quenched fluorescent lipids (triacylglycerols, cholesteryl esters, and phospholipids) and different types of acceptor vesicles made up of phosphatidylcholine or phosphatidylcholine and triacylglycerols. Increases in fluorescence attributable to MTP-mediated lipid transfer are measured after 30 min. MTP's lipid transfer activity could be assayed using apoB lipoproteins but not with high density lipoproteins as acceptors. The assay was used to measure MTP activity in cell and tissue homogenates. Furthermore, the assay was useful in studying the inhibition of the cellular as well as purified MTP by its antagonists.This new method is amenable to automation and can be easily adopted for large-scale, high-throughput screening. -Athar, H., J. Iqbal, X-C. Jiang, and M. M. Hussain. A simple, rapid, and sensitive fluorescence assay for microsomal triglyceride transfer protein.

Patients with abetalipoproteinemia, a disease caused by defects in the microsomal triglyceride transfer protein (MTP), do not produce apolipoprotein B-containing lipoproteins. It was hypothesized that small molecule inhibitors of MTP would prevent the assembly and secretion of these atherogenic lipoproteins. To test this hypothesis, two compounds identified in a high-throughput screen for MTP inhibitors were used to direct the synthesis of a highly potent MTP inhibitor. This molecule (compound 9) inhibited the production of lipoprotein particles in rodent models and normalized plasma lipoprotein levels in Watanabe-heritable hyperlipidemic (WHHL) rabbits, which are a model for human homozygous familial hypercholesterolemia. These results suggest that compound 9, or derivatives thereof, has potential applications for the therapeutic lowering of atherogenic lipoprotein levels in humans.

To Recent studies showing that a defect in the MTP is the proximal cause of abetalipoproteinemia indicate that this protein is required for assembly and secretion of apoBcontaining lipoproteins (10, 11). Abetalipoproteinemic patients have only trace amounts of plasma apoB-containing lipoproteins, resulting in extremely low TG and cholesterol levels (12). The cause of this phenotype is a defect in the pathway responsible for assembly and secretion of apoBcontaining lipoproteins since the apoB gene (13, 14) and lipid synthesis (12) have been shown to be normal. Thus, it is clear that MTP is required for the efficient assembly and secretion of apoB-containing lipoprotein particles.Although studies of abetalipoproteinemic patients indicate that MTP is required for the production of plasma lipoproteins containing apoB, the role of MTP in this process remains unclear. Also, it is not known whether MTP is the only tissue-specific factor needed by hepatocytes and enterocytes to synthesize and secrete these particles. To address these issues, apoB-53 was expressed in either a nonlipoprotein-producing cell line (HeLa) or a derivative cell line stably expressing the large subunit ofMTP and MTP activity. These cells were evaluated for their ability to secrete apoBcontaining lipoproteins into the tissue culture medium. tTo whom reprint requests should be addressed. MATERIALS AND METHODS 7628The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.