Dapagliflozin is a potent and selective sodium glucose cotransporter‐2 (SGLT2) inhibitor which promotes urinary glucose excretion and induces weight loss. Since metabolic compensation can offset a negative energy balance, we explored the potential for a compensatory physiological response to the weight loss induced by dapagliflozin. Dapagliflozin was administered (0.5–5 mpk; p.o.) to diet‐induced obese (DIO) rats with or without ad libitum access to food for 38 days. Along with inducing urinary glucose excretion, chronic administration of dapagliflozin dose‐dependently increased food and water intake relative to vehicle‐treated controls. Despite this, it reduced body weight by 4% (relative to controls) at the highest dose. The degree of weight loss was increased by an additional 9% if hyperphagia was prevented by restricting food intake to that of vehicle controls. Neither oxygen consumption (vO2) or the respiratory exchange ratio (RER) were altered by dapagliflozin treatment alone. Animals treated with dapagliflozin and pair‐fed to vehicle controls (5 mpk PF‐V) showed a reduction in RER and an elevation in nonfasting β‐hydroxybutyrate (BHBA) relative to ad libitum‐fed 5 mpk counterparts. Fasting BHBA was elevated in the 1 mpk, 5 mpk, and 5 mpk PF‐V groups. Serum glucose was reduced in the fasted, but not the unfasted state. Insulin was reduced in the non‐fasted state. These data suggest that in rodents, the persistent urinary glucose excretion induced by dapagliflozin was accompanied by compensatory hyperphagia, which attenuated the weight loss induced by SGLT2 inhibition. Therefore, it is possible that dapagliflozin‐induced weight loss could be enhanced with dietary intervention.

In the current paper, we have conducted a detailed characterization of this reaction in test tube, intact cell culture, and animal models. Enzymatically, we found that triacylglycerol (TAG) synthesis from MAG by DGAT1 does not behave according to classic Michaelis-Menten kinetics. At low concentrations of 2-MAG (<50 M), the major acylation product by DGAT1 was TAG; however, increased concentrations of 2-MAG (50 -200 M) resulted in decreased TAG formation. This unique product/substrate relationship is similar to MGAT3 but distinct from DGAT2 and MGAT2. We have also found that XP620 is an inhibitor that selectively inhibits the acylation of MAG by DGAT1 (IC 50 of human DGAT1: 16.6 ؎ 4.0 nM (MAG as substrate) and 1499 ؎ 318 nM (DAG as substrate); IC 50 values of human DGAT2, MGAT2, and MGAT3 are >30,000 nM). Using this pharmacological tool, we have shown that ϳ76 and ϳ89% of the in vitro TAG synthesis initiated from MAG is mediated by DGAT1 in Caco-2 cell and rat intestinal mucosal membranes, respectively. When applied to intact cultured cells, XP620 substantially decreased but did not abolish apoB secretion in differentiated Caco-2 cells. It also decreased TAG and DAG syntheses in primary enterocytes. Last, when delivered orally to rats, XP620 decreased absorption of orally administered lipids by ϳ50%. Based on these data, we conclude that the acylation of acylglycerols by DGAT1 is important for dietary fat absorption in the intestine.Dietary fat absorption in mammals is critical for growth and development. In the small intestine, dietary triacylglycerol (TAG) 2 is first hydrolyzed by pancreatic lipases into free fatty acid and 2-monoacylglycerol (MAG) that are readily taken up by the enterocytes. Upon appearance in the enterocytes, 2-MAG is first acylated by acyl coenzyme A:monoacylglycerol (MGAT) to form diacylglycerol (DAG); DAG is further acylated by acyl coenzyme A:diacylglycerol acyltransferase (DGAT) to re-synthesize TAG. TAG molecules are then packaged with other lipids, such as cholesteryl ester, retinyl ester, and phospholipids to form chylomicron lipoprotein particles, which are ultimately secreted into the lymph to serve as an energy source for the whole body (1-3).Recently, multiple membrane-bound acyltransferases have been identified, which are implicated in the sequential MAG and DAG acylation reactions in the gut. Chronologically, DGAT1 was the first enzyme identified (4). It belongs to the acyl coenzyme A:cholesterol acyltransferase gene family (5), which possesses up to nine transmembrane domains (6). A detailed analysis of DGAT1 knock-out mice, which have been shown to be resistant to a high fat diet-induced obesity (7), revealed that DGAT1 deletion caused a substantial decrease, but not a total ablation, of chylomicron formation following an acute challenge of orally administered fat (8). Subsequently, DGAT2 and its homologues were identified as a seven-member gene family (9). Although DGAT1 and DGAT2 are capable of catalyzing the same DGAT enzyme reaction, they bear little sequence resemblan...

The extent ofin vitro nonenzymatic glycosylation of purified rat brain tubulin was dependent on time and glucose concentration. Tubulin glycosylation profoundly inhibited GTPdependent tubulin polymerization. Electron microscopy and NaDodSO4/polyacrylamide gel electrophoresis showed that glycosylated tubulin forms high molecular weight amorphous aggregates that are not disrupted by detergents or reducing agents. The amount of covalently bound NaB3H4-reducible sugars in tubulin recovered from brain of streptozotocin-induced diabetic rats was dramatically increased as compared with tubulin recovered from normal rat brain. Moreover, tubulin recovered from diabetic rat brain exhibited less GTP-induced polymerization than tubulin from nondiabetic controls. The possible implications of these data for diabetic neuropathy are discussed.Diabetes mellitus is often accompanied by the gradual development of diverse and multifocal pathology (1). Insulin and antibiotics have contributed substantially to the management of carbohydrate abnormalities and infection (2). However, deterioration in the microcirculation of retina and kidney and diabetic neuropathy continue to present serious clinical problems (3). It is still unclear whether these complications are uniquely the result of carbohydrate disturbances due to intermittent insulin deficiency or also reflect more subtle genetic or metabolic factors that are, at best, indirectly related to insulin levels (4).Recent data have supported the hypothesis that increased nonenzymatic as well as enzymatic glycosylation ofcellular components, as a result of the elevated glucose concentrations of diabetes, may play a contributory role in diabetic microangiopathy (5), and diabetic cataractogenesis (6). Modification, by the nonenzymatic addition of glucose, has now been reported for a variety of serum, cellular, and interstitial proteins and phospholipids (7-10), suggesting the possibility that unanticipated additional functional changes may yet be observed to result from nonenzymatic (or enzymatic) glycosylation.We present here evidence for in vitro and in vivo glycosylation of brain tubulin as a consequence of exposure to elevated glucose concentrations. The in vitro nonenzymatic glycosylation oftubulin results in the formation ofamorphous aggregates oftubulin and tubulin-associated proteins that remain insoluble in 8 M urea or NaDodSO4. The in vivo formation of glycosylation-induced tubulin aggregates was found to compete with ordered tubulin polymerization in diabetic rats. METHODS AND MATERIALSTubulin Isolation and Measurement. Tubulin was purified from male weanling Sprague-Dawley rat brain according to the procedure of Berkowitz et al. (11). Rats were sacrificed by occipital trauma and cervical dislocation. Brains were removed and weighed and 0.75 ml of purification buffer (11) [50 mM 4-morpholineethanesulfonic acid (Sigma)/2 mM EGTA/1 mM MgSO4/4.0 M glycerol, pH 6.9] was added per gram of brain.Brains were homogenized in a loose-fitting motor-driven Teflon/glass homogeniz...

Microvessels isolated from rat epididymal fat exhibit differential vesicular ingestion rates for unmodified and nonenzymatically glycosylated rat albumin. While unmodified rat albumin is excluded from ingestion by endothelial micropinocytic vesicles, glycosylated albumin is avidly taken up by endocytosis. Interaction ofalbumin and glycosylated albumin with endothelium was studied with a double-label fluorescence assay of micropinocytosis. When glycosylated albumin was present at a concentration of 6% with respect to total albumin (the level found in "non diabetic" serum), only glycosylated albumin was ingested. At higher concentrations of glycosylated albumin (those found in diabetic serum), both albumin and glycosylated albumin are ingested. Glycosylation of endothelial membrane components results in stimulated ingestion of glycosylated albumin, persistent exclusion of unmodified albumin, and unaltered micropinocytic ingestion of native ferritin. These results indicate that nonenzymatic glycosylation of serum albumin may result in rapid vesicle-mediated extravasation of albumin. Chronic microvascular leakage of glycosylated albumin could contribute to the pathogenesis of diabetic microangiopathy.Molecular "languages" shape the complex interactions that support life processes. Unique symbols, fidelity of transcription, and rules of grammar are features of the genetic code (1). Specificity ofrecognition and informational content are also apparent in the interactions ofhormones with receptors (2), antigens with antibodies (3), and substrates with enzymes (4). In each ofthese examples, accurate recognition of a unique molecular conformation is crucial for the underlying process. Moreover, lapses in the fidelity of recognition can produce catastrophic results for the systems involved.Endothelial micropinocytosis provides a bidirectional large pore conduit for the transendothelial transport of macromolecules. Another form of molecular language is manifest in the interactions of such molecules with the caveolar (plasmalemmal vesicle) membrane components of endothelial cells. The use of this molecular language in the process ofrecognition-dependent endocytosis provides a discrimination function for transendothelial transport. There results a striking heterogeneity in the rates oftransendothelial vesicular transport ofa variety ofserum components (5-8).The reasons for these differences must be sought in specific interactions between each of the components and putative recognition sites in lumenal endothelial membranes. Such sites are situated within or adjacent to the stomata of caveolae and actively regulate the process of adsorptive endocytosis. Serum albumin appears virtually excluded from ingestion by micropinocytic vesicles (5). This exclusion cannot be attributed solely to the molecular dimensions or the net charge of albumin. Ferritin, which is larger and of similar net charge, is readily ingested by endothelial vesicles. The fine molecular details that govern these recognition processes, and thus modula...

Identification of MCHR1 antagonists with a preclinical safety profile to support clinical evaluation as antiobesity agents has been a challenge. Our finding that a basic moiety is not required for MCHR1 antagonists to achieve high affinity allowed us to explore structures less prone to off-target activities such as hERG inhibition. We report the SAR evolution of hydroxylated thienopyrimidinone ethers culminating in the identification of 27 (BMS-819881), which entered obesity clinical trials as the phosphate ester prodrug 35 (BMS-830216).