Long-term effects of a fatty acid synthase inhibitor on obese mice: food intake, hypothalamic neuropeptides, and UCP3

Short-term treatment of lean and obese mice with the fatty acid synthase (FAS) inhibitor, C75, alters expression of hypothalamic neuropeptides thereby reducing food intake, body weight, and body fat. Here we report the long-term effects of C75 on obese (Ob/Ob) mice. A low dose of C75 administered every third day for 30 days reduced food intake by 62% and body weight by 43% whereas body weight of ad lib-fed controls increased by 11%. Loss of body weight correlated with decreased adipose and liver tissue mass. Decreased food intake correlated with decreased expression of hypothalamic neuropeptide mRNAs for NPY, AgRP, and MCH and an increased expression of neuropeptide mRNAs for alphaMSH (i.e., POMC) and CART. Consistent with increased energy expenditure, C75 treatment caused greater weight loss than pair-fed controls and increased expression of skeletal muscle UCP-3 mRNA. Lowered blood glucose was due largely to restriction of food intake. C75 blocked the normal fasting-induced rise in blood free fatty acids and ketones due either to decreased adipose tissue lipolysis and hepatic ketogenesis or increased fatty acid and ketone utilization by peripheral tissues, notably skeletal muscle.     

Comparison of the obesity phenotypes related to monosodium glutamate effect on arcuate nucleus and/or the high fat diet feeding in C57BL/6 and NMRI mice

In this study, susceptibility of inbred C57BL/6 and outbred NMRI mice to monosodium glutamate (MSG) obesity or diet-induced obesity (DIO) was compared in terms of food intake, body weight, adiposity as well as leptin, insulin and glucose levels. MSG obesity is an early-onset obesity resulting from MSG-induced lesions in arcuate nucleus to neonatal mice. Both male and female C57BL/6 and NMRI mice with MSG obesity did not differ in body weight from their lean controls, but had dramatically increased fat to body weight ratio. All MSG obese mice developed severe hyperleptinemia, more remarkable in females, but only NMRI male mice showed massive hyperinsulinemia and an extremely high HOMA index that pointed to development of insulin resistance. Diet-induced obesity is a late-onset obesity; it developed during 16-week-long feeding with high-fat diet containing 60 % calories as fat. Inbred C57BL/6 mice, which are frequently used in DIO studies, both male and female, had significantly increased fat to body weight ratio and leptin and glucose levels compared with their appropriate lean controls, but only female C57BL/6 mice had also significantly elevated body weight and insulin level. NMRI mice were less prone to DIO than C57BL/6 ones and did not show significant changes in metabolic parameters after feeding with high-fat diet.     

Pharmacological glycerol-3-phosphate acyltransferase inhibition decreases food intake and adiposity and increases insulin sensitivity in diet-induced obesity

Storage of excess calories as triglycerides is central to obesity and its associated disorders. Glycerol-3-phosphate acyltransferases (GPATs) catalyze the initial step in acylglyceride syntheses, including triglyceride synthesis. We utilized a novel small-molecule GPAT inhibitor, FSG67, to investigate metabolic consequences of systemic pharmacological GPAT inhibition in lean and diet-induced obese (DIO) mice. FSG67 administered intraperitoneally decreased body weight and energy intake, without producing conditioned taste aversion. Daily FSG67 (5 mg/kg, 15.3 μmol/kg) produced gradual 12% weight loss in DIO mice beyond that due to transient 9- to 10-day hypophagia (6% weight loss in pair-fed controls). Continued FSG67 maintained the weight loss despite return to baseline energy intake. Weight was lost specifically from fat mass. Indirect calorimetry showed partial protection by FSG67 against decreased rates of oxygen consumption seen with hypophagia. Despite low respiratory exchange ratio due to a high-fat diet, FSG67-treated mice showed further decreased respiratory exchange ratio, beyond pair-fed controls, indicating enhanced fat oxidation. Chronic FSG67 increased glucose tolerance and insulin sensitivity in DIO mice. Chronic FSG67 decreased gene expression for lipogenic enzymes in white adipose tissue and liver and decreased lipid accumulation in white adipose, brown adipose, and liver tissues without signs of damage. RT-PCR showed decreased gene expression for orexigenic hypothalamic neuropeptides AgRP or NPY after acute and chronic systemic FSG67. FSG67 given intracerebroventricularly (100 and 320 nmol icv) produced 24-h weight loss and feeding suppression, indicating contributions from direct central nervous system sites of action. Together, these data point to GPAT as a new potential therapeutic target for the management of obesity and its comorbidities.     

Differential Pharmacologic Properties of the Two C75 Enantiomers: (+)‐C75 Is a Strong Anorectic Drug; (−)‐C75 Has Antitumor Activity

C75 is a synthetic compound described as having antitumoral properties. It produces hypophagia and weight loss in rodents, limiting its use in cancer therapy but identifying it as a potential anti‐obesity drug. C75 is a fatty acid synthase (FAS) inhibitor and, through its coenzyme A (CoA) derivative, it acts as a carnitine palmitoyltransferase (CPT) 1 inhibitor. Racemic mixtures of C75 have been used in all the previous studies; however, the potential different biological activities of C75 enantiomers have not been examined yet. To address this question we synthesized the two C75 enantiomers separately. Our results showed that (?)‐C75 inhibits FAS activity in vitro and has a cytotoxic effect on tumor cell lines, without affecting food consumption. (+)‐C75 inhibits CPT1 and its administration produces anorexia, suggesting that central inhibition of CPT1 is essential for the anorectic effect of C75. The differential activity of C75 enantiomers may lead to the development of potential new specific drugs for cancer and obesity. Chirality 25:281–287, 2013. © 2013 Wiley Periodicals, Inc.     

Effects of Pravastatin on Obesity,Diabetes, and Adiponectin in Diet‐induced Obese Mice

Objective: The aim of this study was to investigate the in vivo effects of pravastatin on the development of obesity and diabetes in diet‐induced obese (DIO) mice. Methods and Procedures: We examined food intake, body‐weight changes, visceral white adipose tissue (WAT) adiponectin and resistin levels, and energy metabolism. Results: Treatment with 100 mg/kg/day pravastatin for 28 days decreased diet‐induced weight gain and visceral adiposity. In addition, the weight of the WAT, the triglyceride (TG) contents of the liver and muscles, and the levels of serum insulin improved in the pravastatin‐treated DIO mice. Furthermore, pravastatin treatment changed the WAT adiponectin and resistin mRNA expression and serum levels compared with the controls. Finally, pravastatin treatment increased oxygen consumption and decreased the respiratory quotient (RQ). Discussion: Pravastatin treatment prevents the development of obesity and diabetes in DIO mice. The prevention of obesity may be mediated by increased oxygen consumption and a decrease in the RQ. These results provide novel insights into the use of pravastatin as a therapeutic tool for metabolic syndromes.     

Assessment of diet-induced obese rats as an obesity model by comparative functional genomics

Objective: We applied a comparative functional genomics approach to evaluate whether diet‐induced obese (DIO) rats serve as an effective obesity model. Methods and Procedures: Gene‐expression profiles of epididymal fat from DIO and lean rats were generated using microarrays and compared with the published array data of obese and non‐obese human subcutaneous adipocytes. Results: Caloric intake and fuel efficiency were significantly higher in DIO rats, which resulted in increased body weight and adiposity. Circulating glucose, cholesterol, triglyceride, insulin, and leptin levels in DIO rats were significantly higher than those in the lean controls. DIO rats also exhibited impaired insulin sensitivity. A direct comparison of gene‐expression profiles from DIO and lean rats and those from obese and non‐obese humans revealed that global gene‐expression patterns in DIO rat fat resemble those of obese human adipocytes. Differentially expressed genes between obese and non‐obese subjects in both human and rat studies were identified and associated with biological pathways by mapping genes to Gene Ontology (GO) categories. Immune response–related genes and angiogenesis‐related genes exhibited significant upregulation in both obese humans and DIO rats when compared with non‐obese controls. However, genes in fatty acid metabolism and oxidation exhibited a broad downregulation only in obese human adipocytes but not in DIO rat epididymal fat. Discussion: Our study based on gene‐expression profiling suggested that DIO rats in general represent an appropriate obesity model. However, the discrepancies in gene‐expression alterations between DIO rats and obese humans, particularly in the metabolic pathways, may explain the limitations of using DIO rodent models in obesity research and drug discovery.     

Camptothecin effectively treats obesity in mice through GDF15 induction

Elevated circulating levels of growth differentiation factor 15 (GDF15) have been shown to reduce food intake and lower body weight through activation of hindbrain receptor glial-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) in rodents and nonhuman primates, thus endogenous induction of this peptide holds promise for obesity treatment. Here, through in silico drug-screening methods, we found that small molecule Camptothecin (CPT), a previously identified drug with potential antitumor activity, is a GDF15 inducer. Oral CPT administration increases circulating GDF15 levels in diet-induced obese (DIO) mice and genetic ob/ob mice, with elevated Gdf15 expression predominantly in the liver through activation of integrated stress response. In line with GDF15’s anorectic effect, CPT suppresses food intake, thereby reducing body weight, blood glucose, and hepatic fat content in obese mice. Conversely, CPT loses these beneficial effects when Gdf15 is inhibited by a neutralizing antibody or AAV8-mediated liver-specific knockdown. Similarly, CPT failed to reduce food intake and body weight in GDF15’s specific receptor GFRAL-deficient mice despite high levels of GDF15. Together, these results indicate that CPT is a promising anti-obesity agent through activation of GDF15-GFRAL pathway.

Elevated circulating levels of growth differentiation factor 15 (GDF15) have been shown to reduce food intake and lower body weight in rodents and nonhuman primates. This study reveals that the small molecule Camptothecin induces endogenous GDF15, suppressing food intake and reducing body weight in obese mice, suggesting a promising candidate for anti-obesity treatment.     

Loss of Kupffer cells in diet-induced obesity is associated with increased hepatic steatosis,STAT3 signaling,and further decreases in insulin signaling

While adipose tissue-associated macrophages contribute to development of chronic inflammation and insulin resistance of obesity, little is known about the role of hepatic Kupffer cells in this environment. Here we address the impact of Kupffer cell ablation using clodronate-encapsulated liposome depletion in a diet-induced obese (DIO) and insulin resistant mouse model. Hepatic expression of macrophage markers measured by realtime RT-PCR remained unaltered in DIO mice despite characteristic expansion of adipose tissue-associated macrophages. DIO mouse livers displayed increased expression of alternative activation markers but unaltered proinflammatory cytokine expression when compared to lean mice. Kupffer cell ablation reduced hepatic anti-inflammatory cytokine IL-10 mRNA expression in lean and DIO mice by 95% and 84%, respectively. Despite decreased hepatic IL-6 gene expression after ablation in lean and DIO mice, hepatic STAT3 phosphorylation, Socs3 and acute phase protein mRNA expression increased. Kupffer cell ablation in DIO mice resulted in additional hepatic triglyceride accumulation and a 30–40% reduction in hepatic insulin receptor autophosphorylation and Akt activation. Implicating systemic loss of IL-10, high-fat-fed IL-10 knockout mice also displayed increased hepatic STAT3 signaling and hepatic triglyceride accumulation. Insulin signaling was not altered, however. In conclusion, Kupffer cells are a major source of hepatic IL-10 expression, the loss of which is associated with increased STAT3-dependent signaling and steatosis. One or more additional factors appear to be required, however, for the Kupffer cell-dependent protective effect on insulin receptor signaling in DIO mice.     

Skeletal muscle lipid oxidation and obesity: influence of weight loss and exercise

Obesity is associated with a decrement in the ability of skeletal muscle to oxidize lipid. The purpose of this investigation was to determine whether clinical interventions (weight loss, exercise training) could reverse the impairment in fatty acid oxidation (FAO) evident in extremely obese individuals. FAO was assessed by incubating skeletal muscle homogenates with [1-(14)C]palmitate and measuring (14)CO(2) production. Weight loss was studied using both cross-sectional and longitudinal designs. Muscle FAO in extremely obese women who had lost weight (decrease in body mass of approximately 50 kg) was compared with extremely obese and lean individuals (BMI of 22.8 +/- 1.2, 50.7 +/- 3.9, and 36.5 +/- 3.5 kg/m(2) for lean, obese, and obese after weight loss, respectively). There was no difference in muscle FAO between the extremely obese and weight loss groups, and FAO was depressed (-45%; P < or = 0.05) compared with the lean subjects. Muscle FAO also did not change in extremely obese women (n = 8) before and 1 yr after a 55-kg weight loss. In contrast, 10 consecutive days of exercise training increased (P < or = 0.05) FAO in the skeletal muscle of lean (+1.7-fold), obese (+1.8-fold), and previously extremely obese subjects after weight loss (+2.6-fold). mRNA content for PDK4, CPT I, and PGC-1alpha corresponded with FAO in that there were no changes with weight loss and an increase with physical activity. These data indicate that a defect in the ability to oxidize lipid in skeletal muscle is evident with obesity, which is corrected with exercise training but persists after weight loss.     

Effect of Diet‐induced Obesity on Acute Pancreatitis Induced by Administration of Interleukin‐12 Plus Interleukin‐18 in Mice

Obesity is associated with increased severity of acute pancreatitis (AP). We recently developed a model of AP induced by administration of interleukin (IL)‐12+IL‐18, two cytokines that are elevated in patients with AP. In this model, severe AP develops in obese leptin‐deficient ob/ob mice compared to lean littermates. In the present report, we evaluated the pancreatic response of diet‐induced obesity (DIO) mice to IL‐12+IL‐18. Body weight loss and adipose tissue necrosis were more severe and prolonged in cytokine‐injected DIO compared to lean mice. Edematous AP developed in lean mice, whereas DIO mice developed necrotizing AP. Obese DIO mice developed more severe hypocalcemia, increased liver damage and a heightened acute‐phase response compared to lean mice, although leukopenia and thrombocytopenia were of comparable severity in lean and DIO mice. Serum levels of IL‐6, IL‐10, and IL‐22 were significantly higher in DIO compared to lean mice, whereas interferon‐γ and tumor necrosis factor‐α did not differ between the two groups. In conclusion, obesity induced by high‐fat diet is associated with increased disease severity and duration in the model of AP induced by administration of IL‐12+IL‐18.     

Leptin resistance of adipocytes in obesity: role of suppressors of cytokine signaling

Liver-derived hyperleptinemia induced in normal rats by adenovirus-induced gene transfer causes rapid disappearance of body fat, whereas the endogenous adipocyte-derived hyperleptinemia of obesity does not. Here we induce liver-derived hyperleptinemia in rats with adipocyte-derived hyperleptinemia of acquired obesity caused by ventromedial hypothalamus lesioning (VMH rats) or by feeding 60% fat (DIO rats). Liver-derived hyperleptinemia in obese rats caused only a 5-7% loss of body weight, compared to a 13% loss in normoleptinemic lean animals; but in actual grams of weight lost there was no significant difference between obese and lean groups, suggesting that a subset of cells remain leptin-sensitive in obesity. mRNA and protein of a putative leptin-resistance factor, suppressor of cytokine signaling (SOCS)-1 or -3, were both increased in white adipose tissues (WAT) of VMH and DIO rats. Since transgenic overexpression of SOCS-3 in islets reduced the lipopenic effect of leptin by 75%, we conclude that the increased expression of SOCS-1 and -3 in WAT of rats with acquired obesity could have blocked leptin's lipopenic action in the leptin-resistant WAT population.     

IFN-alpha induces apoptosis of adipose tissue cells

Interferon alpha (IFN-alpha) is produced in response to viral infections and used clinically in the therapy of a variety of cancers and viral infections. IFN-alpha treatment is often associated with severe weight reduction. To elucidate the mechanism of IFN-associated weight loss, we studied its effect on adipocytes in vitro and in vivo. Diet-induced obese (DIO) C57BL/6 mice were treated continuously for 8 days with human IFN-alpha A/D (100 U/g body weight) or with vehicle alone. The body weight and adipose cell size of IFN-alpha A/D-treated DIO mice were significantly lower (P<0.05 and P<0.001, respectively) as compared with those of control DIO mice. PI3K and Bcl-2 were down-regulated whereas Bax expression was elevated in adipose tissue following IFN treatment as compared to adipose tissue of control DIO mice. Treatment of differentiated 3T3-F442A adipocytes with IFN-alpha A/D (250 U/ml, 36 h) significantly increased the number of apoptotic cells from 15.8% in control cells to 56+/-6%. In conclusion, weight loss following IFN-alpha therapy is due at least in part to increased apoptosis of adipocytes.     

Novel effect of C75 on carnitine palmitoyltransferase I activity and palmitate oxidation

C75 is a potential drug for the treatment of obesity. It was first identified as a competitive, irreversible inhibitor of fatty acid synthase (FAS). It has also been described as a malonyl-CoA analogue that antagonizes the allosteric inhibitory effect of malonyl-CoA on carnitine palmitoyltransferase I (CPT I), the main regulatory enzyme involved in fatty acid oxidation. On the basis of MALDI-TOF analysis, we now provide evidence that C75 can be transformed to its C75-CoA derivative. Unlike the activation produced by C75, the CoA derivative is a potent competitive inhibitor that binds tightly but reversibly to CPT I. IC50 values for yeast-overexpressed L- or M-CPT I isoforms, as well as for purified mitochondria from rat liver and muscle, were within the same range as those observed for etomoxiryl-CoA, a potent inhibitor of CPT I. When a pancreatic INS(823/13), muscle L6E9, or kidney HEK293 cell line was incubated directly with C75, fatty acid oxidation was inhibited. This suggests that C75 could be transformed in the cell to its C75-CoA derivative, inhibiting CPT I activity and consequently fatty acid oxidation. In vivo, a single intraperitoneal injection of C75 in mice produced short-term inhibition of CPT I activity in mitochondria from the liver, soleus, and pancreas, indicating that C75 could be transformed to its C75-CoA derivative in these tissues. Finally, in silico molecular docking studies showed that C75-CoA occupies the same pocket in CPT I as palmitoyl-CoA, suggesting an inhibiting mechanism based on mutual exclusion. Overall, our results describe a novel role for C75 in CPT I activity, highlighting the inhibitory effect of its C75-CoA derivative.     

Chronic injection of pansomatostatin agonist ODT8-SST differentially modulates food intake and decreases body weight gain in lean and diet-induced obese rats

The aim of this study was to investigate the central actions of the stable pansomatostatin peptide agonist, ODT8-SST on body weight. ODT8-SST or vehicle was acutely (1μg/rat) injected or chronically infused (5μg/rat/d, 14d) intracerebroventricularly and daily food intake, body weight and composition were monitored. In lean rats, neither acute nor chronic ODT8-SST influenced daily food intake while body weight was reduced by 2.2% after acute injection and there was a 14g reduction of body weight gain after 14d compared to vehicle (p<0.01). In diet-induced obese (DIO) rats, chronic ODT8-SST increased cumulative 2-week food intake compared to vehicle (+14%, p<0.05) and also blunted body weight change (-11g, p<0.05). ODT8-SST for 14d reduced lean mass (-22g and -25g respectively, p<0.001) and total water (-19g and -22g respectively, p<0.001) in lean and DIO rats and increased fat mass in DIO (+16g, p<0.001) but not lean rats (+1g, p>0.05) compared to vehicle. In DIO rats, ODT8-SST reduced ambulatory (-27%/24h, p<0.05) and fine movements (-38%, p<0.01) which was associated with an increased positive energy balance compared to vehicle (+50g, p<0.01). Chronic central somatostatin receptor activation in lean rats reduces body weight gain and lean mass independently of food intake which is likely related to growth hormone inhibition. In DIO rats, ODT8-SST reduces lean mass but promotes food intake and fat mass, indicating differential responsiveness to somatostatin under obese conditions.     

Adiposity elevates plasma MCP-1 levels leading to the increased CD11b-positive monocytes in mice

Obesity is currently considered as an epidemic in the western world, and it represents a major risk factor for life-threatening diseases such as heart attack, stroke, diabetes, and cancer. Taking advantage of DNA microarray technology, we tried to identify the molecules explaining the relationship between obesity and vascular disorders, comparing mRNA expression of about 12,000 genes in white adipose tissue between normal, high fat diet-induced obesity (DIO) and d-Trp34 neuropeptide Y-induced obesity in mice. Expression of monocyte chemoattractant protein-1 (MCP-1) mRNA displayed a 7.2-fold increase in obese mice as compared with normal mice, leading to substantially elevated MCP-1 protein levels in adipocytes. MCP-1 levels in plasma were also increased in DIO mice, and a strong correlation between plasma MCP-1 levels and body weight was identified. We also showed that elevated MCP-1 protein levels in plasma increased the CD11b-positive monocyte/macrophage population in DIO mice. Furthermore, infusion of MCP-1 into lean mice increased the CD11b-positive monocyte population without inducing changes in body weight. Given the importance of MCP-1 in activation of monocytes and subsequent atherosclerotic development, these results suggest a novel role of adiposity in the development of vascular disorders.     

Expression of FAS within hypothalamic neurons: a model for decreased food intake after C75 treatment

We previously demonstrated that C75, a specific and potent inhibitor of fatty acid synthase (FAS), reduced food intake and decreased body weight in mice. In the present study, we determined that these effects were not due to conditioned taste aversion. To investigate the mechanism of C75 action, we examined FAS brain expression. FAS was expressed in a number of brain regions, including arcuate and paraventricular nuclei (PVN) within regions that comprise the arcuate-PVN pathway in mouse and human. Although C75 and fasting significantly downregulated liver FAS, FAS levels remained high in hypothalamus, indicating that FAS levels were regulated differently in brain from those in liver. Double fluorescence in situ for FAS and neuropeptide Y (NPY) showed that FAS co-localized with NPY in neurons in the arcuate nucleus. NPY immnuoreactivity after C75 treatment was decreased in axon terminals that innervate the PVN and lateral hypothalamus. Collectively, these results demonstrate that FAS is present and active in neurons and suggests that C75 may alter food intake via interactions within the arcuate-PVN pathway mediated by NPY.     

The role of hypothalamic malonyl-CoA in energy homeostasis

Energy balance is monitored by hypothalamic neurons that respond to peripheral hormonal and afferent neural signals that sense energy status. Recent physiologic, pharmacologic, and genetic evidence has implicated malonyl-CoA, an intermediate in fatty acid synthesis, as a regulatory component of this energy-sensing system. The level of malonyl-CoA in the hypothalamus is dynamically regulated by fasting and feeding, which alter subsequent feeding behavior. Fatty acid synthase (FAS) inhibitors, administered systemically or intracerebroventricularly to lean or obese mice, increase hypothalamic malonyl-CoA leading to the suppression of food intake. Conversely, lowering malonyl-CoA with an acetyl-CoA carboxylase (ACC) inhibitor or by the ectopic expression of malonyl-CoA decarboxylase in the hypothalamus increases food intake and reverses inhibition by FAS inhibitors. Physiologically, the level of hypothalamic malonyl-CoA appears to be determined through phosphorylation/dephosphorylation of ACC by AMP kinase in response to changes in the AMP/ATP ratio, an indicator of energy status. Recent evidence suggests that the brain-specific carnitine:palmitoyl-CoA transferase-1 (CPT1c) may be a regulated target of malonyl-CoA that relays the "malonyl-CoA signal" in hypothalamic neurons that express the orexigenic and anorexigenic neuropeptides that regulate food intake and peripheral energy expenditure. Together these findings support a role for malonyl-CoA as an intermediary in the control of energy homeostasis.