PMC:4195273 / 66666-77744 JSONTXT

{"project":"TEST0","denotations":[{"id":"25352770-183-191-357785","span":{"begin":272,"end":276},"obj":"[\"11564703\"]"},{"id":"25352770-206-214-357786","span":{"begin":295,"end":299},"obj":"[\"16099925\"]"},{"id":"25352770-231-239-357787","span":{"begin":320,"end":324},"obj":"[\"15724149\"]"},{"id":"25352770-233-241-357788","span":{"begin":347,"end":351},"obj":"[\"16131814\"]"},{"id":"25352770-226-234-357789","span":{"begin":368,"end":372},"obj":"[\"15927717\"]"},{"id":"25352770-235-243-357790","span":{"begin":390,"end":394},"obj":"[\"16762458\"]"},{"id":"25352770-231-239-357791","span":{"begin":412,"end":416},"obj":"[\"16621045\"]"},{"id":"25352770-124-132-357792","span":{"begin":2407,"end":2411},"obj":"[\"11564703\"]"},{"id":"25352770-147-155-357793","span":{"begin":2430,"end":2434},"obj":"[\"16099925\"]"},{"id":"25352770-168-176-357794","span":{"begin":2451,"end":2455},"obj":"[\"15927717\"]"},{"id":"25352770-190-198-357795","span":{"begin":2473,"end":2477},"obj":"[\"16762458\"]"},{"id":"25352770-234-242-357796","span":{"begin":2745,"end":2749},"obj":"[\"11564703\"]"},{"id":"25352770-225-233-357797","span":{"begin":2902,"end":2906},"obj":"[\"16762458\"]"},{"id":"25352770-122-130-357798","span":{"begin":3031,"end":3035},"obj":"[\"16099925\"]"},{"id":"25352770-143-151-357799","span":{"begin":3052,"end":3056},"obj":"[\"15927717\"]"},{"id":"25352770-154-162-357800","span":{"begin":3213,"end":3217},"obj":"[\"11564703\"]"},{"id":"25352770-235-243-357801","span":{"begin":3517,"end":3521},"obj":"[\"11564703\"]"},{"id":"25352770-226-234-357802","span":{"begin":3538,"end":3542},"obj":"[\"15927717\"]"},{"id":"25352770-232-240-357803","span":{"begin":3560,"end":3564},"obj":"[\"16762458\"]"},{"id":"25352770-232-240-357804","span":{"begin":4542,"end":4546},"obj":"[\"10854588\"]"},{"id":"25352770-233-241-357805","span":{"begin":4564,"end":4568},"obj":"[\"11223006\"]"},{"id":"25352770-235-243-357806","span":{"begin":4570,"end":4574},"obj":"[\"15138156\"]"},{"id":"25352770-232-240-357807","span":{"begin":4589,"end":4593},"obj":"[\"12958177\"]"},{"id":"25352770-224-232-357808","span":{"begin":4610,"end":4614},"obj":"[\"12588509\"]"},{"id":"25352770-231-239-357809","span":{"begin":4630,"end":4634},"obj":"[\"12591118\"]"},{"id":"25352770-233-241-357811","span":{"begin":4675,"end":4679},"obj":"[\"15271883\"]"},{"id":"25352770-231-239-357812","span":{"begin":4695,"end":4699},"obj":"[\"17447258\"]"},{"id":"25352770-233-241-357813","span":{"begin":4716,"end":4720},"obj":"[\"17434149\"]"},{"id":"25352770-232-240-357814","span":{"begin":4736,"end":4740},"obj":"[\"18802445\"]"},{"id":"25352770-231-239-357815","span":{"begin":4758,"end":4762},"obj":"[\"21060312\"]"},{"id":"25352770-168-176-357816","span":{"begin":4953,"end":4957},"obj":"[\"11223006\"]"},{"id":"25352770-209-217-357817","span":{"begin":5169,"end":5173},"obj":"[\"7891182\"]"},{"id":"25352770-232-240-357818","span":{"begin":5192,"end":5196},"obj":"[\"9243343\"]"},{"id":"25352770-228-236-357819","span":{"begin":5212,"end":5216},"obj":"[\"9419833\"]"},{"id":"25352770-234-242-357820","span":{"begin":5218,"end":5222},"obj":"[\"9527537\"]"},{"id":"25352770-233-241-357821","span":{"begin":5238,"end":5242},"obj":"[\"9883730\"]"},{"id":"25352770-233-241-357822","span":{"begin":5255,"end":5259},"obj":"[\"10612702\"]"},{"id":"25352770-231-239-357823","span":{"begin":5276,"end":5280},"obj":"[\"12588509\"]"},{"id":"25352770-233-241-357824","span":{"begin":5289,"end":5293},"obj":"[\"16765484\"]"},{"id":"25352770-230-238-357825","span":{"begin":5309,"end":5313},"obj":"[\"18802445\"]"},{"id":"25352770-234-242-357826","span":{"begin":5331,"end":5335},"obj":"[\"21060312\"]"},{"id":"25352770-178-186-357827","span":{"begin":5516,"end":5520},"obj":"[\"11564703\"]"},{"id":"25352770-217-225-357828","span":{"begin":6164,"end":6168},"obj":"[\"11850744\"]"},{"id":"25352770-234-242-357829","span":{"begin":6186,"end":6190},"obj":"[\"14700744\"]"},{"id":"25352770-229-237-357830","span":{"begin":6208,"end":6212},"obj":"[\"17008116\"]"},{"id":"25352770-232-240-357831","span":{"begin":6228,"end":6232},"obj":"[\"23318496\"]"},{"id":"25352770-205-213-357832","span":{"begin":6854,"end":6858},"obj":"[\"11156435\"]"},{"id":"25352770-226-234-357833","span":{"begin":6875,"end":6879},"obj":"[\"17182122\"]"},{"id":"25352770-230-238-357834","span":{"begin":6928,"end":6932},"obj":"[\"17182122\"]"},{"id":"25352770-233-241-357835","span":{"begin":7247,"end":7251},"obj":"[\"11564703\"]"},{"id":"25352770-229-237-357836","span":{"begin":7269,"end":7273},"obj":"[\"16762458\"]"},{"id":"25352770-151-159-357837","span":{"begin":7528,"end":7532},"obj":"[\"12958177\"]"},{"id":"25352770-172-180-357838","span":{"begin":7549,"end":7553},"obj":"[\"17182122\"]"},{"id":"25352770-234-242-357839","span":{"begin":7857,"end":7861},"obj":"[\"12958177\"]"},{"id":"25352770-179-187-357840","span":{"begin":8043,"end":8047},"obj":"[\"10905499\"]"},{"id":"25352770-205-213-357841","span":{"begin":8069,"end":8073},"obj":"[\"11522684\"]"},{"id":"25352770-226-234-357842","span":{"begin":8090,"end":8094},"obj":"[\"11791158\"]"},{"id":"25352770-232-240-357844","span":{"begin":8139,"end":8143},"obj":"[\"15823203\"]"},{"id":"25352770-234-242-357845","span":{"begin":8159,"end":8163},"obj":"[\"16210370\"]"},{"id":"25352770-228-236-357846","span":{"begin":8180,"end":8184},"obj":"[\"20151954\"]"},{"id":"25352770-231-239-357847","span":{"begin":8452,"end":8456},"obj":"[\"11522684\"]"},{"id":"25352770-233-241-357849","span":{"begin":8497,"end":8501},"obj":"[\"16210370\"]"},{"id":"25352770-162-170-357850","span":{"begin":8981,"end":8985},"obj":"[\"11564703\"]"},{"id":"25352770-184-192-357851","span":{"begin":9003,"end":9007},"obj":"[\"16762458\"]"},{"id":"25352770-210-218-357852","span":{"begin":9704,"end":9708},"obj":"[\"11850744\"]"},{"id":"25352770-231-239-357853","span":{"begin":9725,"end":9729},"obj":"[\"17182122\"]"},{"id":"25352770-232-240-357854","span":{"begin":10010,"end":10014},"obj":"[\"11850744\"]"},{"id":"25352770-228-236-357855","span":{"begin":10032,"end":10036},"obj":"[\"14700744\"]"},{"id":"25352770-219-227-357856","span":{"begin":10053,"end":10057},"obj":"[\"17182122\"]"}],"text":"Functional implications of CART deletion on energy metabolism from genetic interventions\nIn order to gain further insight into the functional consequences of reduced or absent CART expression, several knockout models have been generated and characterized (Asnicar et al., 2001; Couceyro et al., 2005; Elefteriou et al., 2005; Osei-Hyiaman et al., 2005; Wierup et al., 2005; Moffett et al., 2006; Steiner et al., 2006) (Table 2). The phenotypic effects resulting from CART gene targeting approaches have in part shown inconsistency between studies, however, displayed a general trend in promoting positive energy balance. This in overall terms is also similar with results obtained from our novel CART knockout (KO) model, which was generated from a conditional version that was crossed with an oozyte-specific Cre line. Collective evidence regarding the role of CART in energy homeostasis from a few representative studies conducted by independent research groups (Table 2) as well as in-house results will be discussed.\nTable 2 Summary of the metabolic phenotypes of germline CART knockout mouse models in various studies when compared with wild-type controls.\nSchematic representation of the gene targeting strategies adopted for generating CART knockout mouse models, depicting the wild-type allele of the mouse CART gene, the CART targeting vector, and the mutant CART allele. The three exons of the mouse CART gene are indicated by boxes labeled E1–E3. In the targeting constructs, the yellow boxes labeled “neo” or “HIS/neo” indicate the neomycin or histidinol/neomycin resistance selection cassettes respectively for targeted disruption of the CART gene. Homologous recombination between the targeting vectors and the complementary CART genomic loci (dotted lines) in mouse embryonic stem cells generates genetic ablations of exons E1 and E2, or all of exons E1–E3 of the CART coding region respectively. The targeting constructs were introduced into 129 SvJ mouse embryonic stem cells and subsequently injected into C57BL/6 blastocysts. M, male; F, female; HFD, high fat diet; NSD, no significant difference. In all models investigated, the expected increase in feeding due to depletion of the anorectic CART peptide was not observed under standard chow feeding conditions. However, when exposed to a high caloric diet, lack of CART led to altered feeding behavior and body weight (Asnicar et al., 2001; Couceyro et al., 2005; Wierup et al., 2005; Moffett et al., 2006). An increase in body weight has also been shown in CART KO mice fed on regular chow, although manifestation of such trait appears to require a longer time frame, where in one study, a statistically significant increase was absent at 17 weeks of age (Asnicar et al., 2001), whilst an independent group adopting the identical mouse model reported notably higher body weight for the knockout mice at 20 weeks (Moffett et al., 2006). In a different CART knockout mouse line, again altered weight was undetectable prior to 40 weeks of age (Couceyro et al., 2005; Wierup et al., 2005). Similarly, when fed a high fat diet, a significantly higher food consumption was observed in the CART KO compared to wild-type (WT) mice (Asnicar et al., 2001), in some but not all studies addressed. Importantly, despite a lack of consistent alteration in food intake, all studies reported an elevated body weight gain in CART knockout animals regardless of dietary options, with an even more prominent effect shown with a high caloric diet (Asnicar et al., 2001; Wierup et al., 2005; Moffett et al., 2006).\nInterestingly, fasting-induced food intake experiments in our novel CART KO model suggest that lack of CART may be beneficial for body weight conservation during starvation. In our study, chow-fed CART KO animals showed a slightly lower food consumption compared with WT during a refeeding period following a 24-h fast, yet demonstrated a similar degree and pace of recovery in body weight, implying that less food might be required for returning to the pre-fast weight. On the other hand, the same CART KO mice on HFD experienced a less dramatic drop in body weight upon fasting, and accordingly showed a more effective weight regain to baseline during refeeding. Such improved reactions to food deprivation may be correlated to the observed difference in growth and possibly attributable to events in the interaction between CART and an improved stress response in CART KO mice, consistent with a role of the CART system in stress and anxiety-like responses (Koylu et al., 2000; Stanley et al., 2001, 2004; Kong et al., 2003; Larsen et al., 2003; Vrang et al., 2003; Dominguez et al., 2004a; Smith et al., 2004; Gozen et al., 2007; Hunter et al., 2007; Rogge et al., 2008; Nakhate et al., 2011). For example, i.c.v. CART has been reported to substantially influence the plasma levels of various stress hormones, such as adrenocorticotropic hormone and corticosterone (Stanley et al., 2001). Furthermore, anatomical implication has also been provided by the expression of CART transcript and peptide at various levels of the HPA axis as well as other stress-related areas in the CNS (Douglass et al., 1995; Couceyro et al., 1997; Koylu et al., 1997, 1998; Elias et al., 1998; Broberger, 1999; Larsen et al., 2003; Vrang, 2006; Rogge et al., 2008; Nakhate et al., 2011).\nAmongst a multitude of potential factors contributing to the enhanced body weight in CART KO models, the gain in fat mass has been considered the most important (Asnicar et al., 2001). This is concordant with results from our novel CART KO model, where body composition analysis by dual-energy X-ray absorptiometry (DXA) revealed a pronounced increase in adiposity independent of diet. The gain in whole body fat mass was further confirmed by tissue dissection showing significantly elevated fat masses in all white adipose tissues, including the inguinal, epididymal, mesenteric and retroperitoneal regions. This also suggests an important function of CART in lipid metabolism, where CART has been linked to inhibition of lipogenesis and stimulation of lipid substrate mobilization and utilization (Rohner-Jeanrenaud et al., 2002; Wortley et al., 2004; Vasseur et al., 2007; Banke et al., 2013).\nIn addition to fat mass, lean mass constitutes another major determinant of energy homeostasis that directly influences energy expenditure. As opposed to the increase in fat mass, whole body lean mass of our CART-deficient mice was distinctly lower compared to WT controls regardless of dietary treatments. Similarly, the reduction was consistent across both periods of DXA measurements at 10 and 14 weeks of age. Intriguingly, research investigating the effects of chronic central CART infusion in genetically normal DIO rats reported a diminished body weight gain primarily due to a loss of lean mass (Larsen et al., 2000; Qing and Chen, 2007), while fat mass was unaffected (Qing and Chen, 2007).\nConsistency exists between results from our CART KO model and previous studies focusing on the metabolic characterization of CART knockout mice, where no significant difference in total energy expenditure or physical activity could be detected when compared with WT controls regardless of diets (Asnicar et al., 2001; Moffett et al., 2006), even after the correction of the potentially confounding effects of lean mass on energy expenditure. However, overexpression studies have provided indications of both supporting and opposing roles of CART in regulating energy expenditure (Kong et al., 2003; Qing and Chen, 2007). An increase in energy expenditure was suggested in rats subject to chronic overexpression of CART through intra-arcuate targeted gene transfer, where the animals showed exaggerated weight loss and a downregulation of endogenous arcuate CART mRNA levels upon fasting and food restriction (Kong et al., 2003). Despite discordance between various animal studies, evidence exists for the involvement of CART in the regulation of energy expenditure and body weight in humans (Challis et al., 2000; del Giudice et al., 2001; Yamada et al., 2002; Dominguez et al., 2004a; Guerardel et al., 2005; Yanik et al., 2006; Rigoli et al., 2010). Specifically, a missense mutation in the pro-CART transcripts was discovered to co-segregate with a severe obese phenotype and was also associated with decreased resting energy expenditure in members of an Italian family over three generations (del Giudice et al., 2001; Dominguez et al., 2004a; Yanik et al., 2006).\nIn spite of a lack of direct indication endorsing a function of CART in modulating energy expenditure, comparing the respiratory exchange ratio (RER) between CART knockout animals and WT counterparts may shed light on any potential effects of CART ablation on energy metabolism through the fuel source preferences. Although no measurable difference was described in knockout animals in the respiratory quotient derived from RER as reported by previous studies (Asnicar et al., 2001; Moffett et al., 2006), a notably lower RER has been detected in our new CART knockout mice. Consistency in the lower RER particularly during the dark photoperiod was shown across both nutritional statuses, signifying that fat was preferentially metabolized over carbohydrates to supply energy for the body. A possible explanation for this could be the higher fat content in knockout animals, which may lead to the predominant fuel source based on the relative higher availability of fat than carbohydrates. On the other hand, a suppressed average respiratory quotient was demonstrated in both normal and DIO rats chronically overexpressing central CART compared to vehicle-treated controls (Rohner-Jeanrenaud et al., 2002; Qing and Chen, 2007). The reduction was exaggerated during the dark phase, under both regular feeding and fasting-refeeding conditions, indicating a stimulatory role of CART in promoting lipid oxidation and limiting fat storage, hence inhibiting excessive body fat accrual (Rohner-Jeanrenaud et al., 2002; Wortley et al., 2004; Qing and Chen, 2007).\nTaken together, results from the literature as well as in-house studies of CART knockout models generally support the property of CART as a satiety factor and an anorexigenic signal in the brain, as evident in the elevation in body weight gain attributable mostly to the increased fat mass consistent across studies, although controversy exists for the corresponding food intake data. As for the aspect of energy intake, RER was demonstrated to be reduced both under the conditions of CART overexpression and ablation, suggesting fat was metabolized as the primary fuel for energy supply. A possible reason could be that although CART may intrinsically promote the utilization of fat as the predominant fuel source for reducing energy intake, the effectiveness of CART deletion on the disturbance of lipid metabolism hence accumulation of adiposity may have surmounted the simultaneous CART deficiency-induced enhancement of energy intake, resulting in a net reduction in RER based on the readily available fat depots."}

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,"span":{"begin":8180,"end":8184},"obj":"20151954"},{"id":"25352770-11522684-357847","span":{"begin":8452,"end":8456},"obj":"11522684"},{"id":"25352770-16210370-357849","span":{"begin":8497,"end":8501},"obj":"16210370"},{"id":"25352770-11564703-357850","span":{"begin":8981,"end":8985},"obj":"11564703"},{"id":"25352770-16762458-357851","span":{"begin":9003,"end":9007},"obj":"16762458"},{"id":"25352770-11850744-357852","span":{"begin":9704,"end":9708},"obj":"11850744"},{"id":"25352770-17182122-357853","span":{"begin":9725,"end":9729},"obj":"17182122"},{"id":"25352770-11850744-357854","span":{"begin":10010,"end":10014},"obj":"11850744"},{"id":"25352770-14700744-357855","span":{"begin":10032,"end":10036},"obj":"14700744"},{"id":"25352770-17182122-357856","span":{"begin":10053,"end":10057},"obj":"17182122"}],"text":"Functional implications of CART deletion on energy metabolism from genetic interventions\nIn order to gain further insight into the functional consequences of reduced or absent CART expression, several knockout models have been generated and characterized (Asnicar et al., 2001; Couceyro et al., 2005; Elefteriou et al., 2005; Osei-Hyiaman et al., 2005; Wierup et al., 2005; Moffett et al., 2006; Steiner et al., 2006) (Table 2). The phenotypic effects resulting from CART gene targeting approaches have in part shown inconsistency between studies, however, displayed a general trend in promoting positive energy balance. This in overall terms is also similar with results obtained from our novel CART knockout (KO) model, which was generated from a conditional version that was crossed with an oozyte-specific Cre line. Collective evidence regarding the role of CART in energy homeostasis from a few representative studies conducted by independent research groups (Table 2) as well as in-house results will be discussed.\nTable 2 Summary of the metabolic phenotypes of germline CART knockout mouse models in various studies when compared with wild-type controls.\nSchematic representation of the gene targeting strategies adopted for generating CART knockout mouse models, depicting the wild-type allele of the mouse CART gene, the CART targeting vector, and the mutant CART allele. The three exons of the mouse CART gene are indicated by boxes labeled E1–E3. In the targeting constructs, the yellow boxes labeled “neo” or “HIS/neo” indicate the neomycin or histidinol/neomycin resistance selection cassettes respectively for targeted disruption of the CART gene. Homologous recombination between the targeting vectors and the complementary CART genomic loci (dotted lines) in mouse embryonic stem cells generates genetic ablations of exons E1 and E2, or all of exons E1–E3 of the CART coding region respectively. The targeting constructs were introduced into 129 SvJ mouse embryonic stem cells and subsequently injected into C57BL/6 blastocysts. M, male; F, female; HFD, high fat diet; NSD, no significant difference. In all models investigated, the expected increase in feeding due to depletion of the anorectic CART peptide was not observed under standard chow feeding conditions. However, when exposed to a high caloric diet, lack of CART led to altered feeding behavior and body weight (Asnicar et al., 2001; Couceyro et al., 2005; Wierup et al., 2005; Moffett et al., 2006). An increase in body weight has also been shown in CART KO mice fed on regular chow, although manifestation of such trait appears to require a longer time frame, where in one study, a statistically significant increase was absent at 17 weeks of age (Asnicar et al., 2001), whilst an independent group adopting the identical mouse model reported notably higher body weight for the knockout mice at 20 weeks (Moffett et al., 2006). In a different CART knockout mouse line, again altered weight was undetectable prior to 40 weeks of age (Couceyro et al., 2005; Wierup et al., 2005). Similarly, when fed a high fat diet, a significantly higher food consumption was observed in the CART KO compared to wild-type (WT) mice (Asnicar et al., 2001), in some but not all studies addressed. Importantly, despite a lack of consistent alteration in food intake, all studies reported an elevated body weight gain in CART knockout animals regardless of dietary options, with an even more prominent effect shown with a high caloric diet (Asnicar et al., 2001; Wierup et al., 2005; Moffett et al., 2006).\nInterestingly, fasting-induced food intake experiments in our novel CART KO model suggest that lack of CART may be beneficial for body weight conservation during starvation. In our study, chow-fed CART KO animals showed a slightly lower food consumption compared with WT during a refeeding period following a 24-h fast, yet demonstrated a similar degree and pace of recovery in body weight, implying that less food might be required for returning to the pre-fast weight. On the other hand, the same CART KO mice on HFD experienced a less dramatic drop in body weight upon fasting, and accordingly showed a more effective weight regain to baseline during refeeding. Such improved reactions to food deprivation may be correlated to the observed difference in growth and possibly attributable to events in the interaction between CART and an improved stress response in CART KO mice, consistent with a role of the CART system in stress and anxiety-like responses (Koylu et al., 2000; Stanley et al., 2001, 2004; Kong et al., 2003; Larsen et al., 2003; Vrang et al., 2003; Dominguez et al., 2004a; Smith et al., 2004; Gozen et al., 2007; Hunter et al., 2007; Rogge et al., 2008; Nakhate et al., 2011). For example, i.c.v. CART has been reported to substantially influence the plasma levels of various stress hormones, such as adrenocorticotropic hormone and corticosterone (Stanley et al., 2001). Furthermore, anatomical implication has also been provided by the expression of CART transcript and peptide at various levels of the HPA axis as well as other stress-related areas in the CNS (Douglass et al., 1995; Couceyro et al., 1997; Koylu et al., 1997, 1998; Elias et al., 1998; Broberger, 1999; Larsen et al., 2003; Vrang, 2006; Rogge et al., 2008; Nakhate et al., 2011).\nAmongst a multitude of potential factors contributing to the enhanced body weight in CART KO models, the gain in fat mass has been considered the most important (Asnicar et al., 2001). This is concordant with results from our novel CART KO model, where body composition analysis by dual-energy X-ray absorptiometry (DXA) revealed a pronounced increase in adiposity independent of diet. The gain in whole body fat mass was further confirmed by tissue dissection showing significantly elevated fat masses in all white adipose tissues, including the inguinal, epididymal, mesenteric and retroperitoneal regions. This also suggests an important function of CART in lipid metabolism, where CART has been linked to inhibition of lipogenesis and stimulation of lipid substrate mobilization and utilization (Rohner-Jeanrenaud et al., 2002; Wortley et al., 2004; Vasseur et al., 2007; Banke et al., 2013).\nIn addition to fat mass, lean mass constitutes another major determinant of energy homeostasis that directly influences energy expenditure. As opposed to the increase in fat mass, whole body lean mass of our CART-deficient mice was distinctly lower compared to WT controls regardless of dietary treatments. Similarly, the reduction was consistent across both periods of DXA measurements at 10 and 14 weeks of age. Intriguingly, research investigating the effects of chronic central CART infusion in genetically normal DIO rats reported a diminished body weight gain primarily due to a loss of lean mass (Larsen et al., 2000; Qing and Chen, 2007), while fat mass was unaffected (Qing and Chen, 2007).\nConsistency exists between results from our CART KO model and previous studies focusing on the metabolic characterization of CART knockout mice, where no significant difference in total energy expenditure or physical activity could be detected when compared with WT controls regardless of diets (Asnicar et al., 2001; Moffett et al., 2006), even after the correction of the potentially confounding effects of lean mass on energy expenditure. However, overexpression studies have provided indications of both supporting and opposing roles of CART in regulating energy expenditure (Kong et al., 2003; Qing and Chen, 2007). An increase in energy expenditure was suggested in rats subject to chronic overexpression of CART through intra-arcuate targeted gene transfer, where the animals showed exaggerated weight loss and a downregulation of endogenous arcuate CART mRNA levels upon fasting and food restriction (Kong et al., 2003). Despite discordance between various animal studies, evidence exists for the involvement of CART in the regulation of energy expenditure and body weight in humans (Challis et al., 2000; del Giudice et al., 2001; Yamada et al., 2002; Dominguez et al., 2004a; Guerardel et al., 2005; Yanik et al., 2006; Rigoli et al., 2010). Specifically, a missense mutation in the pro-CART transcripts was discovered to co-segregate with a severe obese phenotype and was also associated with decreased resting energy expenditure in members of an Italian family over three generations (del Giudice et al., 2001; Dominguez et al., 2004a; Yanik et al., 2006).\nIn spite of a lack of direct indication endorsing a function of CART in modulating energy expenditure, comparing the respiratory exchange ratio (RER) between CART knockout animals and WT counterparts may shed light on any potential effects of CART ablation on energy metabolism through the fuel source preferences. Although no measurable difference was described in knockout animals in the respiratory quotient derived from RER as reported by previous studies (Asnicar et al., 2001; Moffett et al., 2006), a notably lower RER has been detected in our new CART knockout mice. Consistency in the lower RER particularly during the dark photoperiod was shown across both nutritional statuses, signifying that fat was preferentially metabolized over carbohydrates to supply energy for the body. A possible explanation for this could be the higher fat content in knockout animals, which may lead to the predominant fuel source based on the relative higher availability of fat than carbohydrates. On the other hand, a suppressed average respiratory quotient was demonstrated in both normal and DIO rats chronically overexpressing central CART compared to vehicle-treated controls (Rohner-Jeanrenaud et al., 2002; Qing and Chen, 2007). The reduction was exaggerated during the dark phase, under both regular feeding and fasting-refeeding conditions, indicating a stimulatory role of CART in promoting lipid oxidation and limiting fat storage, hence inhibiting excessive body fat accrual (Rohner-Jeanrenaud et al., 2002; Wortley et al., 2004; Qing and Chen, 2007).\nTaken together, results from the literature as well as in-house studies of CART knockout models generally support the property of CART as a satiety factor and an anorexigenic signal in the brain, as evident in the elevation in body weight gain attributable mostly to the increased fat mass consistent across studies, although controversy exists for the corresponding food intake data. As for the aspect of energy intake, RER was demonstrated to be reduced both under the conditions of CART overexpression and ablation, suggesting fat was metabolized as the primary fuel for energy supply. A possible reason could be that although CART may intrinsically promote the utilization of fat as the predominant fuel source for reducing energy intake, the effectiveness of CART deletion on the disturbance of lipid metabolism hence accumulation of adiposity may have surmounted the simultaneous CART deficiency-induced enhancement of energy intake, resulting in a net reduction in RER based on the readily available fat depots."}

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"id":"25352770-16210370-38284834","span":{"begin":8159,"end":8163},"obj":"16210370"},{"id":"25352770-20151954-38284835","span":{"begin":8180,"end":8184},"obj":"20151954"},{"id":"25352770-11522684-38284836","span":{"begin":8452,"end":8456},"obj":"11522684"},{"id":"25352770-16210370-38284838","span":{"begin":8497,"end":8501},"obj":"16210370"},{"id":"25352770-11564703-38284839","span":{"begin":8981,"end":8985},"obj":"11564703"},{"id":"25352770-16762458-38284840","span":{"begin":9003,"end":9007},"obj":"16762458"},{"id":"25352770-11850744-38284841","span":{"begin":9704,"end":9708},"obj":"11850744"},{"id":"25352770-17182122-38284842","span":{"begin":9725,"end":9729},"obj":"17182122"},{"id":"25352770-11850744-38284843","span":{"begin":10010,"end":10014},"obj":"11850744"},{"id":"25352770-14700744-38284844","span":{"begin":10032,"end":10036},"obj":"14700744"},{"id":"25352770-17182122-38284845","span":{"begin":10053,"end":10057},"obj":"17182122"}],"text":"Functional implications of CART deletion on energy metabolism from genetic interventions\nIn order to gain further insight into the functional consequences of reduced or absent CART expression, several knockout models have been generated and characterized (Asnicar et al., 2001; Couceyro et al., 2005; Elefteriou et al., 2005; Osei-Hyiaman et al., 2005; Wierup et al., 2005; Moffett et al., 2006; Steiner et al., 2006) (Table 2). The phenotypic effects resulting from CART gene targeting approaches have in part shown inconsistency between studies, however, displayed a general trend in promoting positive energy balance. This in overall terms is also similar with results obtained from our novel CART knockout (KO) model, which was generated from a conditional version that was crossed with an oozyte-specific Cre line. Collective evidence regarding the role of CART in energy homeostasis from a few representative studies conducted by independent research groups (Table 2) as well as in-house results will be discussed.\nTable 2 Summary of the metabolic phenotypes of germline CART knockout mouse models in various studies when compared with wild-type controls.\nSchematic representation of the gene targeting strategies adopted for generating CART knockout mouse models, depicting the wild-type allele of the mouse CART gene, the CART targeting vector, and the mutant CART allele. The three exons of the mouse CART gene are indicated by boxes labeled E1–E3. In the targeting constructs, the yellow boxes labeled “neo” or “HIS/neo” indicate the neomycin or histidinol/neomycin resistance selection cassettes respectively for targeted disruption of the CART gene. Homologous recombination between the targeting vectors and the complementary CART genomic loci (dotted lines) in mouse embryonic stem cells generates genetic ablations of exons E1 and E2, or all of exons E1–E3 of the CART coding region respectively. The targeting constructs were introduced into 129 SvJ mouse embryonic stem cells and subsequently injected into C57BL/6 blastocysts. M, male; F, female; HFD, high fat diet; NSD, no significant difference. In all models investigated, the expected increase in feeding due to depletion of the anorectic CART peptide was not observed under standard chow feeding conditions. However, when exposed to a high caloric diet, lack of CART led to altered feeding behavior and body weight (Asnicar et al., 2001; Couceyro et al., 2005; Wierup et al., 2005; Moffett et al., 2006). An increase in body weight has also been shown in CART KO mice fed on regular chow, although manifestation of such trait appears to require a longer time frame, where in one study, a statistically significant increase was absent at 17 weeks of age (Asnicar et al., 2001), whilst an independent group adopting the identical mouse model reported notably higher body weight for the knockout mice at 20 weeks (Moffett et al., 2006). In a different CART knockout mouse line, again altered weight was undetectable prior to 40 weeks of age (Couceyro et al., 2005; Wierup et al., 2005). Similarly, when fed a high fat diet, a significantly higher food consumption was observed in the CART KO compared to wild-type (WT) mice (Asnicar et al., 2001), in some but not all studies addressed. Importantly, despite a lack of consistent alteration in food intake, all studies reported an elevated body weight gain in CART knockout animals regardless of dietary options, with an even more prominent effect shown with a high caloric diet (Asnicar et al., 2001; Wierup et al., 2005; Moffett et al., 2006).\nInterestingly, fasting-induced food intake experiments in our novel CART KO model suggest that lack of CART may be beneficial for body weight conservation during starvation. In our study, chow-fed CART KO animals showed a slightly lower food consumption compared with WT during a refeeding period following a 24-h fast, yet demonstrated a similar degree and pace of recovery in body weight, implying that less food might be required for returning to the pre-fast weight. On the other hand, the same CART KO mice on HFD experienced a less dramatic drop in body weight upon fasting, and accordingly showed a more effective weight regain to baseline during refeeding. Such improved reactions to food deprivation may be correlated to the observed difference in growth and possibly attributable to events in the interaction between CART and an improved stress response in CART KO mice, consistent with a role of the CART system in stress and anxiety-like responses (Koylu et al., 2000; Stanley et al., 2001, 2004; Kong et al., 2003; Larsen et al., 2003; Vrang et al., 2003; Dominguez et al., 2004a; Smith et al., 2004; Gozen et al., 2007; Hunter et al., 2007; Rogge et al., 2008; Nakhate et al., 2011). For example, i.c.v. CART has been reported to substantially influence the plasma levels of various stress hormones, such as adrenocorticotropic hormone and corticosterone (Stanley et al., 2001). Furthermore, anatomical implication has also been provided by the expression of CART transcript and peptide at various levels of the HPA axis as well as other stress-related areas in the CNS (Douglass et al., 1995; Couceyro et al., 1997; Koylu et al., 1997, 1998; Elias et al., 1998; Broberger, 1999; Larsen et al., 2003; Vrang, 2006; Rogge et al., 2008; Nakhate et al., 2011).\nAmongst a multitude of potential factors contributing to the enhanced body weight in CART KO models, the gain in fat mass has been considered the most important (Asnicar et al., 2001). This is concordant with results from our novel CART KO model, where body composition analysis by dual-energy X-ray absorptiometry (DXA) revealed a pronounced increase in adiposity independent of diet. The gain in whole body fat mass was further confirmed by tissue dissection showing significantly elevated fat masses in all white adipose tissues, including the inguinal, epididymal, mesenteric and retroperitoneal regions. This also suggests an important function of CART in lipid metabolism, where CART has been linked to inhibition of lipogenesis and stimulation of lipid substrate mobilization and utilization (Rohner-Jeanrenaud et al., 2002; Wortley et al., 2004; Vasseur et al., 2007; Banke et al., 2013).\nIn addition to fat mass, lean mass constitutes another major determinant of energy homeostasis that directly influences energy expenditure. As opposed to the increase in fat mass, whole body lean mass of our CART-deficient mice was distinctly lower compared to WT controls regardless of dietary treatments. Similarly, the reduction was consistent across both periods of DXA measurements at 10 and 14 weeks of age. Intriguingly, research investigating the effects of chronic central CART infusion in genetically normal DIO rats reported a diminished body weight gain primarily due to a loss of lean mass (Larsen et al., 2000; Qing and Chen, 2007), while fat mass was unaffected (Qing and Chen, 2007).\nConsistency exists between results from our CART KO model and previous studies focusing on the metabolic characterization of CART knockout mice, where no significant difference in total energy expenditure or physical activity could be detected when compared with WT controls regardless of diets (Asnicar et al., 2001; Moffett et al., 2006), even after the correction of the potentially confounding effects of lean mass on energy expenditure. However, overexpression studies have provided indications of both supporting and opposing roles of CART in regulating energy expenditure (Kong et al., 2003; Qing and Chen, 2007). An increase in energy expenditure was suggested in rats subject to chronic overexpression of CART through intra-arcuate targeted gene transfer, where the animals showed exaggerated weight loss and a downregulation of endogenous arcuate CART mRNA levels upon fasting and food restriction (Kong et al., 2003). Despite discordance between various animal studies, evidence exists for the involvement of CART in the regulation of energy expenditure and body weight in humans (Challis et al., 2000; del Giudice et al., 2001; Yamada et al., 2002; Dominguez et al., 2004a; Guerardel et al., 2005; Yanik et al., 2006; Rigoli et al., 2010). Specifically, a missense mutation in the pro-CART transcripts was discovered to co-segregate with a severe obese phenotype and was also associated with decreased resting energy expenditure in members of an Italian family over three generations (del Giudice et al., 2001; Dominguez et al., 2004a; Yanik et al., 2006).\nIn spite of a lack of direct indication endorsing a function of CART in modulating energy expenditure, comparing the respiratory exchange ratio (RER) between CART knockout animals and WT counterparts may shed light on any potential effects of CART ablation on energy metabolism through the fuel source preferences. Although no measurable difference was described in knockout animals in the respiratory quotient derived from RER as reported by previous studies (Asnicar et al., 2001; Moffett et al., 2006), a notably lower RER has been detected in our new CART knockout mice. Consistency in the lower RER particularly during the dark photoperiod was shown across both nutritional statuses, signifying that fat was preferentially metabolized over carbohydrates to supply energy for the body. A possible explanation for this could be the higher fat content in knockout animals, which may lead to the predominant fuel source based on the relative higher availability of fat than carbohydrates. On the other hand, a suppressed average respiratory quotient was demonstrated in both normal and DIO rats chronically overexpressing central CART compared to vehicle-treated controls (Rohner-Jeanrenaud et al., 2002; Qing and Chen, 2007). The reduction was exaggerated during the dark phase, under both regular feeding and fasting-refeeding conditions, indicating a stimulatory role of CART in promoting lipid oxidation and limiting fat storage, hence inhibiting excessive body fat accrual (Rohner-Jeanrenaud et al., 2002; Wortley et al., 2004; Qing and Chen, 2007).\nTaken together, results from the literature as well as in-house studies of CART knockout models generally support the property of CART as a satiety factor and an anorexigenic signal in the brain, as evident in the elevation in body weight gain attributable mostly to the increased fat mass consistent across studies, although controversy exists for the corresponding food intake data. As for the aspect of energy intake, RER was demonstrated to be reduced both under the conditions of CART overexpression and ablation, suggesting fat was metabolized as the primary fuel for energy supply. A possible reason could be that although CART may intrinsically promote the utilization of fat as the predominant fuel source for reducing energy intake, the effectiveness of CART deletion on the disturbance of lipid metabolism hence accumulation of adiposity may have surmounted the simultaneous CART deficiency-induced enhancement of energy intake, resulting in a net reduction in RER based on the readily available fat depots."}