**3.2. Microsome triglyceride transfer protein inhibitor**

MTP is localized in the endoplasmic reticulum in hepatocytes and enterocytes, and MTP leads the transfer of triglyceride (TG) and cholesteryl ester between membranes [86]. The protein participates in the assembly of TG-rich lipoproteins, such as chylomicron particles in the small intestine and very low-density lipoprotein (VLDL) particles in the liver, thereby also participating in the mobilization and secretion of TG-rich lipoproteins from enterocytes and hepatocytes [87]. Since enteric MTP has been shown to play a critical role in the absorption of fat or cholesterol, the inhibition of MTP in small intestine is expected to induce the potential of weight loss as an antiobesity drug.

Since the *in vivo* effects of MTP inhibitors were reported, it has been pointed out that inhibition of hepatic MTP could lead to the potent blockade of VLDL release, resulting in reduced plasma lipids but inducing fatty liver and hepatic dysfunction [88]. In fact, while the potential benefits of MTP inhibition, such as lowering chylomicron-TG and VLDL-TG levels, are demonstrated in animal experiments and in clinical studies, several major toxicity issues affect the clinical development of MTP inhibitors [89]. In clinical studies of BAY 13-9952 and BMS-201038, for example, hepatotoxicity indicated by the elevation of transaminase level halted their developments. Therefore, the compounds designed to show a high selectively inhibition for intestine-MTP have been developed and lipid-absorption inhibitors are expected to show pharmacological effects, including weight loss, without any hepatotoxicity.

Mera et al. designed the compound, JTT-130, that would be rapidly metabolized during the absorption process to avoid inhibition of hepatic MTP after oral administration [90, 91]. JTT-130 was designed to be rapidly hydrolyzed to its inactive metabolite (M1) by cleavage of ester group in the structures. The IC50 values of JTT-130 on MTP inhibitory activities were 0.83 nM for TG transfer and 0.74 nM for cholesteryl ester (CE) transfer, respectively. No inhibitory effect of M1 on MTP was observed at concentrations of M1 increasing up to 30,000 nM. Antiobesity effects were investigated in a DIO model, Sprague-Dawley rat fed a 35% fat diet [15]. JTT-130 treatment decreased body weights with suppression of food intake (**Figure 2A** and **B**). Interestingly, the pharmacological effects were not observed in rats fed with the 3.1% fat diet (**Figure 2C** and **D**), and JTT-130 showed antiobesity effects in a dietary fat-dependent manner. The elevation of plasma levels of gut hormones, such as glucagon-like peptide-1 (GLP-1) and peptide YY (PYY), was observed in DIO rats, and the elevation of gut peptides may be related with body weight loss with JTT-130 treatment. The antiobesity effect of JTT-130 was also investigated using a genetic model, ZDF rat [92]. Male ZDF rats at 7 weeks of age were fed a regular diet with JTT-130 as a food admixture for 6 weeks. JTT-130 treatment decreased the food intake in the ZDF rats throughout the treatment period, resulting in reduction in the body weight in the first 4 weeks of the treatment period. However, the body weights of the JTT-130 treated ZDF rats were comparable to those of the control ZDF rats after 5 weeks of treatment. The body weight change is considered to be induced by the improvement of metabolic abnormalities in whole body with JTT-551 treatment.

In the JTT-551 100 m/kg group, the cumulative calorie intake tended to decrease from 2 weeks after treatment and significantly decreased from 6 weeks after treatment. Body weight in JTT-551 treatment tended to decrease dose-dependently and the decreases in the JTT-551 100 mg/kg group were significant from 5 to 6 weeks after treatment. PTP1B inhibitor is a unique target that shows not only an improvement of glucose metabolism but also an antiobesity effect

MTP is localized in the endoplasmic reticulum in hepatocytes and enterocytes, and MTP leads the transfer of triglyceride (TG) and cholesteryl ester between membranes [86]. The protein participates in the assembly of TG-rich lipoproteins, such as chylomicron particles in the small intestine and very low-density lipoprotein (VLDL) particles in the liver, thereby also participating in the mobilization and secretion of TG-rich lipoproteins from enterocytes and hepatocytes [87]. Since enteric MTP has been shown to play a critical role in the absorption of fat or cholesterol, the inhibition of MTP in small intestine is expected to induce the potential of

Since the *in vivo* effects of MTP inhibitors were reported, it has been pointed out that inhibition of hepatic MTP could lead to the potent blockade of VLDL release, resulting in reduced plasma lipids but inducing fatty liver and hepatic dysfunction [88]. In fact, while the potential benefits of MTP inhibition, such as lowering chylomicron-TG and VLDL-TG levels, are demonstrated in animal experiments and in clinical studies, several major toxicity issues affect the clinical development of MTP inhibitors [89]. In clinical studies of BAY 13-9952 and BMS-201038, for example, hepatotoxicity indicated by the elevation of transaminase level halted their developments. Therefore, the compounds designed to show a high selectively inhibition for intestine-MTP have been developed and lipid-absorption inhibitors are expected to show

Mera et al. designed the compound, JTT-130, that would be rapidly metabolized during the absorption process to avoid inhibition of hepatic MTP after oral administration [90, 91]. JTT-130 was designed to be rapidly hydrolyzed to its inactive metabolite (M1) by cleavage of ester group in the structures. The IC50 values of JTT-130 on MTP inhibitory activities were 0.83 nM for TG transfer and 0.74 nM for cholesteryl ester (CE) transfer, respectively. No inhibitory effect of M1 on MTP was observed at concentrations of M1 increasing up to 30,000 nM. Antiobesity effects were investigated in a DIO model, Sprague-Dawley rat fed a 35% fat diet [15]. JTT-130 treatment decreased body weights with suppression of food intake (**Figure 2A** and **B**). Interestingly, the pharmacological effects were not observed in rats fed with the 3.1% fat diet (**Figure 2C** and **D**), and JTT-130 showed antiobesity effects in a dietary fat-dependent manner. The elevation of plasma levels of gut hormones, such as glucagon-like peptide-1 (GLP-1) and peptide YY (PYY), was observed in DIO rats, and the elevation of gut peptides may be related with body weight loss with JTT-130 treatment. The antiobesity effect of JTT-130 was also investigated using a genetic model, ZDF rat [92]. Male ZDF rats at 7 weeks of age were fed a regular diet with JTT-130 as a food admixture for 6 weeks. JTT-130 treatment decreased the food intake in the ZDF rats throughout the treatment period, resulting in reduction in the body

pharmacological effects, including weight loss, without any hepatotoxicity.

possibly by enhancement of leptin signaling.

58 Adiposity - Omics and Molecular Understanding

weight loss as an antiobesity drug.

**3.2. Microsome triglyceride transfer protein inhibitor**

**Figure 2.** Effects of microsomal triglyceride transfer protein inhibitor, JTT-130 on body weights and food intake in Sprague-Dawley rats on a 35% fat diet (A, B) and a 3.1% fat diet (C, D). Rats in the JTT-130 treatment groups were fed with the drug as a 0.029% food admixture (approximately 10 mg/kg/day), beginning at 10 weeks of age [15]. Data represent mean ± standard deviation (*n* = 6). \**p* < 0.05, \*\**p* < 0.01: significantly different from control group.

Furthermore, JTT-130 treatment has been reported as ameliorating impaired glucose and lipid metabolism in ZDF rats [92], and attenuates dyslipidemia in hyprlipidemic hamsters and rabbits [93]. It is expected that intestine-specific MTP inhibitors will be useful in treatment of diabetes and atherosclerosis as well as obesity.

#### **3.3. Acyl-CoA: diacylglycerol acyltransferase 1 inhibitor**

DGAT1 is an enzyme that catalyzes the final step of TG synthesis, i.e., synthesis of TG from diacylglycerol and fatty acyl-CoA. DGAT1 is expressed in various organs, and is especially highly expressed in the small intestine, fat tissue, and testes [94]. The enzyme is involved in TG absorption from the small intestine and fat accumulation in adipose tissues [95]. Indeed, DGAT1-knockout (−/−) mice show resistance to the antiobesity effects of a HF diet; wherein body weight gain is suppressed, fat weight and TG contents decrease, and energy consumption in the liver and skeletal muscles accelerates, as well as observing improvements in insulin and leptin resistance, in comparison with wild-type mice [96]. Since the inhibition of DGAT1 is expected to result in two kinds of pharmacological effects: (1) inhibition of fat absorption in the small intestine and (2) inhibition of fat synthesis in adipose tissues, DGAT1 inhibitors are likely to become a good therapeutic option for obesity.

Tomimoto et al. reported antiobesity effects with JTT-553, which was discovered as a novel DGAT1 inhibitor [97]. A single administration of JTT-553 inhibited the increase of plasma TG levels after olive oil loading in Sprague-Dawley (SD) rats, suggesting that JTT-553 inhibited fat absorption in the small intestine. Furthermore, JTT-553 suppressed TG synthesis in adipose tissues [98]. The antiobesity effects of JTT-553 were investigated in DIO rats, SD rats fed a 35% fat diet, and a genetic model, the KKAy mouse. In DIO rats, body weight and visceral fat in the JTT-553 administration group decreased dose-dependently; however, the suppressive effects of JTT-553 on body weight were not observed with the 3.1% fat diet. Interestingly, the antifeeding effects of JTT-553 were observed in DIO rats, which was not observed in DGAT1 knockout (−/−) mice. A single administration of JTT-553 decreased food consumption depending on dietary fat content. The difference in appetite between DGAT1 inhibitor-treated and knockout mice remains unknown. In KKAy mice, JTT-553 decreased the food intake and body weight (**Figure 3**). Repeated administration of JTT-553 showed decreases of the liver and fat weights, and the liver TG content. The DGAT1 inhibitor was considered to suppress food consumption via the elevation of levels of gut hormones, such as GLP-1, in plasma [99]. Furthermore, JTT-553 was administrated to DIO mice and antiobesity effects and antidiabetic effects were investigated at the same time [98]. JTT-553 decreased body weight and food consumption, and treatment resulted in improvements in hyperinsulinemia and hyperlipidemia. In the glucose tolerance test, JTT-553 treatment resulted in ameliorations of insulin resistance. In addition, JTT-553 treatment resulted in significant reductions in fat mass, and increased glucose utilization of epididymal adipose tissues in the presence of insulin.

**Figure 3.** Effects of Acyl CoA: diacylglycerol acyltransferase1 inhibitor, JTT-553 on body weights gain in KKAy mice on a 35% fat diet [98]. JTT-553 was dosed as food admixture to KKAy mice for 5 weeks. Data represent mean ± standard deviation (*n* = 7–8). \**p* < 0.05, \*\**p* < 0.01: significantly different from 35% control group, #*p* < 0.05, ##*p* < 0.01: significantly different from 3.1% control group.

#### **3.4. Acyl-CoA: monoacylglycerol acyltransferase 2 inhibitor**

MGAT2 is an enzyme that catalyzes the esterification of monoacylglycerol (MG), i.e., synthesis of diglycerides from MG and fatty acyl-CoA [100, 101]. The genes encoding three MGATs, MGAT1, MGAT2, and MGAT3 have been identified [102–104]. MGAT1 is mainly expressed in the heart, lung, skeletal muscle, and pancreas, but not in the small intestine. Both MGAT2 and MGAT3 are mainly expressed in human small intestine, whereas only MGAT2 is expressed in mouse small intestine [102].

Tomimoto et al. reported antiobesity effects with JTT-553, which was discovered as a novel DGAT1 inhibitor [97]. A single administration of JTT-553 inhibited the increase of plasma TG levels after olive oil loading in Sprague-Dawley (SD) rats, suggesting that JTT-553 inhibited fat absorption in the small intestine. Furthermore, JTT-553 suppressed TG synthesis in adipose tissues [98]. The antiobesity effects of JTT-553 were investigated in DIO rats, SD rats fed a 35% fat diet, and a genetic model, the KKAy mouse. In DIO rats, body weight and visceral fat in the JTT-553 administration group decreased dose-dependently; however, the suppressive effects of JTT-553 on body weight were not observed with the 3.1% fat diet. Interestingly, the antifeeding effects of JTT-553 were observed in DIO rats, which was not observed in DGAT1 knockout (−/−) mice. A single administration of JTT-553 decreased food consumption depending on dietary fat content. The difference in appetite between DGAT1 inhibitor-treated and knockout mice remains unknown. In KKAy mice, JTT-553 decreased the food intake and body weight (**Figure 3**). Repeated administration of JTT-553 showed decreases of the liver and fat weights, and the liver TG content. The DGAT1 inhibitor was considered to suppress food consumption via the elevation of levels of gut hormones, such as GLP-1, in plasma [99]. Furthermore, JTT-553 was administrated to DIO mice and antiobesity effects and antidiabetic effects were investigated at the same time [98]. JTT-553 decreased body weight and food consumption, and treatment resulted in improvements in hyperinsulinemia and hyperlipidemia. In the glucose tolerance test, JTT-553 treatment resulted in ameliorations of insulin resistance. In addition, JTT-553 treatment resulted in significant reductions in fat mass, and

increased glucose utilization of epididymal adipose tissues in the presence of insulin.

**Figure 3.** Effects of Acyl CoA: diacylglycerol acyltransferase1 inhibitor, JTT-553 on body weights gain in KKAy mice on a 35% fat diet [98]. JTT-553 was dosed as food admixture to KKAy mice for 5 weeks. Data represent mean ± standard deviation (*n* = 7–8). \**p* < 0.05, \*\**p* < 0.01: significantly different from 35% control group, #*p* < 0.05, ##*p* < 0.01: significantly

MGAT2 is an enzyme that catalyzes the esterification of monoacylglycerol (MG), i.e., synthesis of diglycerides from MG and fatty acyl-CoA [100, 101]. The genes encoding three MGATs,

**3.4. Acyl-CoA: monoacylglycerol acyltransferase 2 inhibitor**

different from 3.1% control group.

60 Adiposity - Omics and Molecular Understanding

MGAT2 is involved in the resynthesis of TG in the intestine, and plays an important role in the assembly and secretion of chylomicrons. In fact, MGAT2 KO mice demonstrate reduced fat uptake in the small intestine and delay in the absorption of fat into circulation [105]. In addition, the elevation of postprandial GLP-1 and not PYY levels are observed in MGAT2 KO mice fed a HF diet [106]. The chronic function of MGAT2 on metabolic disorders is investigated using MGAT2 KO mice. MGAT2 deficient mice are protected from HF diet-induced obesity and glucose intolerance [106]. Moreover, MGAT2 deficiency results in increased metabolic rates, decreased food consumption, and protection from obesity in genetically obese Agouti mice, suggesting that MGAT2 regulates energy balance [106, 107]. The intestinal function of MGAT2 and the effect of this function on obesity are also investigated using intestine-specific MGAT2 KO mice [108]. Intestinal-specific deletion of MGAT2 alters TG metabolism in the small intestine and delays fat absorption. These mice are protected from obesity and impair glucose metabolism when feed a HF diet. Thus, there is considerable interest that inhibition of MGAT2 is a feasible target for obesity and other metabolic disorders caused by excess dietary calories. Although, the physiological role of MGAT2 has been mainly investigated using genetically modified mice, the detailed pharmacological characteristics of MGAT2 inhibitors have not been reported.

Okuma et al. reported the pharmacological profile of JTP-103237, which was discovered as a novel MGAT2 inhibitor. A single administration of JTT-103237 reduced plasma TG after lipid loading. In addition, JTT-103237 increased MG and fatty acid content, which are MGAT2 substrates, in the small intestine. A single administration of JTT-103237 tended to elevate plasma levels of GLP-1 and PYY after olive oil loading, and the antifeeding effect of JTT-103237 was observed independent of dietary fat content. After repeated dosing, JTT-103237 reduced food consumption and body weight, and increased energy expenditure in DIO mice. Furthermore, JTT-103237 reduced hepatic steatosis in high sucrose and very low fat (HSVLF)-fed mice, through the suppression of TG synthesis related genes, such as sterol regulatory elementbinding protein (SREBP)-1c, fatty acid synthesis, and stearoyl-CoA desaturase (SCD)-1. The inhibition of hepatic MGAT2 activity is considered to directly reduce hepatic TG synthesis. 2- MG content in the small intestine is considered to increase by administration of MGAT2 inhibitor. The effects of 2-MG on food intake and diarrhea were evaluated and compared with the long-chain fatty acid (LCFA) in rats by intrajejunal infusion [109]. 2-MG did not induce diarrhea under the condition in which it comparably reduced food intake as compared with LCFA, suggesting that 2-MG stimulates satiety without inducing diarrhea, different from LCFA. From these findings, MGAT2 inhibition may prove to be a useful strategy target for treating obesity and related metabolic disorders.
