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Camptothecin effectively treats obesity in mice through GDF15 induction

['Jun Feng Lu', 'Key Laboratory Of Animal Genetics', 'Breeding', 'Reproduction Of Shaanxi Province', 'College Of Animal Science', 'Technology', 'Northwest A F University', 'Yangling', 'Shaanxi', 'Meng Qing Zhu']

Date: 2022-03

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.

Funding: This work was supported by National Natural Science Foundation of China (32070602), National Key Research and Development Program of China (2021YFF1000602), Programs for Shaanxi Science & Technology (2020NY-012), and Special Talent Recruitment Fund of China to Jiang Wei Wu. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

In this study, we perform a series of animal experiments to determine the potential link between CPT and the changes in circulating GDF15 levels, food intake, and body weight. We show that 1 mg kg −1 of CPT administration induces mild circulating GDF15 elevation, which suppresses food intake and reduces body fat mass without causing adverse effects. Using either GDF15 neutralization or GFRAL deficiency in mice, we show that the weight lowering effects of CPT depend on GDF15-GFRAL axis. We also corroborate that the main source of CPT-induced GDF15 derives from activation of hepatic activating transcription factor 4 (ATF4) and C/EBP homologous protein (CHOP). These findings suggest that CPT could be a potential anti-obesity agent when applied at a once daily dose of 1 mg kg −1 of body weight orally.

With advances in bioinformatics and network pharmacology, the Connectivity Map (CMAP), a database collecting gene expression profiles of drug-treated human cell lines [ 19 ], has been widely used for anti-obesity purpose to screen leptin sensitizer [ 20 , 21 ] and thermogenic activators [ 22 ]. In the current work, we screened the CMAP database for small molecules with gene expression signatures of GDF15 and identified Camptothecin (CPT) as a potential GDF15 inducer. CPT is a pentacyclic quinoline alkaloid present in wood, bark, and fruit of the Asian tree Camptotheca acuminate [ 23 ]. The US National Cancer Institute screening program identified CPT as a drug with potential antitumor activity in 1966, mechanistically through inhibition of type 1 DNA topoisomerase (TOP 1) and induction of dsDNA breaks in cancer cells [ 24 ]. Limited Phase II clinical trials of CPT by a single injection every 3 weeks at initial doses ranging between 90 and 180 mg/m 2 in patients with advanced gastrointestinal adenocarcinoma gained partial encouraging objective responses [ 25 ]. However, using these testing doses, frequency, and administration mode, some adverse events including nausea, vomiting, dermatitis, diarrhea, cystitis, leukopenia, thrombocytopenia, and anemia were reported, thus hampering its initial clinical development as a chemotherapeutic drug. Here, by screening of CMAP database, we found a strong correlation between CPT and GDF15 expression, suggesting a potential anti-obesity property of CPT.

Growth differentiation factor 15 (GDF15), also known as macrophage inhibitory cytokine-1, has emerged as a new anti-obesity target [ 9 , 10 ]. It is a stress-responsive cytokine expressed in a variety of tissues and secreted into circulation in response to many stimuli as part of a wide array of disease processes [ 11 ]. Elevating GDF15 levels by transgenic overexpression or pharmacological administration in mice and nonhuman primates lead to a marked fall in body weight [ 12 – 14 ]. Mice lacking GDF15 become more obese on a high-fat diet (HFD) than wild-type (WT) controls [ 15 ]. Recently, with the discovery of GDF15’s specific receptor glial-derived neurotrophic factor (GDNF) receptor alpha-like (GFRAL) [ 13 , 16 , 17 ], enthusiasm is heightened in understanding this hormone’s regulation and exploring its therapeutic application in obesity treatment [ 18 ]. Thus, the aim of this work was to explore a pharmacological GDF15 inducer and test its anti-obesity efficacy in order to provide alternative anti-obesity therapeutics.

Obesity is a global health problem that predisposes people to diseases such as type 2 diabetes, cardiovascular diseases, fatty liver, and even cancer [ 1 , 2 ]. Therefore, it is a great threat to human health and a heavy burden on public health systems. Due to unsatisfactory results of interventions on dietary and physical activity, the use of weight loss drugs has been proposed as a more effective choice for long-term obesity management. In the past several decades, a plethora of efforts have been devoted to explore the anti-obesity targets and develop weight management agents [ 3 ]. Main medications currently approved by FDA for obesity management include orlistat, phentermine, phentermine/topiramate extended release, liraglutide, and semaglutide [ 4 , 5 ]. Some of them have achieved desirable results for a limited population [ 6 ]. However, the problem has not been well resolved because (1) many targets have limitations and functions only for a small population; and (2) adverse effects such as gastrointestinal disorders [ 7 ] and myopathy [ 8 ] have been repeatedly reported in users taken FDA-approved weight control medications. Therefore, exploring new therapeutic targets and understanding their regulations for drug development are current areas of intense interest and active investigation.

(A-H) Animal protocol 4 : (A) Schematic diagram of mice treatment. WT and Gfral −/− mice were subjected to oral administration of vehicle or CPT (1 mg kg −1 ) for 30 days. Created with biorender.com . (B) Plasma levels of GDF15 (day 27). (C) Cumulative food intake. (D) Body weight in response to CPT treatment in WT and Gfral −/− mice. (E-F) Weights of adipose tissue and skeletal muscle. (G) GTT and AUC of GTT (day 18). Gfral +/+ (vehicle, n = 7), Gfral +/+ (CPT, n = 7), Gfral −/− (vehicle, n = 5), Gfral −/− (CPT, n = 6). (H) Hepatic TG and TC contents at day 30. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. The underlying data for this figure can be found in S1 Data . (I) Proposed model for the anti-obesity effects of CPT in obese mice. Created with biorender.com . CPT elevates circulating GDF15 via activation of hepatic ISR pathway. This activates the GDF15 receptor GFRAL in the hindbrain AP, which subsequently suppresses food intake and reduces body weight in obese mice. AP, area postrema; ATF4, activating transcription factor 4; AUC, area under the curve; BAT, brown adipose tissue; CHOP, C/EBP homologous protein; CPT, Camptothecin; DIO, diet-induced obese; eWAT, epididymal white adipose tissue; GAS, gastrocnemius; GDF15, growth differentiation factor 15; GFRAL, GDNF receptor alpha-like; GTT, glucose tolerance test; ISR, integrated stress response; iWAT, inguinal white adipose tissue; QUA, quadriceps; TC, total cholesterol; TG, triglyceride; WT, wild-type.

DIO mice (weighing 40 to 42 g) received vehicle or CPT (1 mg kg −1 ); 60 min later, brains were harvested, and tissue sections were subjected to immunofluorescence staining with anti-c-Fos (red) and anti-GFRAL (green). (A) Representative immunofluorescence image of the AP/NTS in CPT- or vehicle-treated DIO mice showing the distribution of GFRAL neurons and the colocalization with the marker of neuronal activation c-Fos. (B) Quantification of CPT-induced c-Fos expression in the AP/NTS (n = 6 per group). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. The underlying data for this figure can be found in S1 Data . AP, area postrema; CPT, Camptothecin; DIO, diet-induced obese; GFRAL, GDNF receptor alpha-like; NTS, nucleus tractus solitarius.

Having shown that CPT exerts beneficial metabolic actions through induction of hepatic Gdf15 expression, we next try to identify the downstream effectors of CPT by determining the expression of GDF15 receptor GFRAL. GFRAL is shown to be exclusively expressed in the area postrema (AP) and nucleus tractus solitarius (NTS) [ 36 ], and administration of recombinant GDF15 is known to rapidly activate GFRAL-positive neurons in these regions [ 41 ]. Immunofluorescence staining of hindbrain sections containing AP and NTS with antibodies against GFRAL and c-Fos showed robust double staining in AP of CPT-treated mice than in vehicle controls, with similar extent of staining in NTS of both mice ( Fig 6A and 6B ), suggesting that CPT induces the release of GDF15 and activates GFRAL-expressing neurons in AP of mice. To further test whether GFRAL is indispensable for the beneficial effects of CPT, GFRAL-deficient mice (Gfral −/− ) were used ( Fig 7A ). Although CPT treatment elevated plasma GDF15 levels in Gfral −/− mice as in Gfral +/+ controls ( Fig 7B ), it failed to reduce food intake, body weight, and fat mass in Gfral −/− mice ( Fig 7C–7E ). The muscle weights (GAS and QUA) were not significantly different among the 4 groups ( Fig 7F ). Also, the beneficial effects of CPT on glucose tolerance ( Fig 7G ) and fatty liver prevention ( Fig 7H ) were not preserved in Gfral −/− mice. Together, these results indicate that GFRAL is the downstream effector of CPT and the GDF15-GFRAL axis mediates the beneficial metabolic actions of CPT in mice.

(A-K) Animal protocol 3 : (A) Schematic diagram of mice treatment. Created with biorender.com . DIO mice were intravenously injected once with adenovirus (AAV8-scramble-shRNA or AAV8-Gdf15-shRNA) through the tail vein (1 × 10 11 vector genomes/mouse). After 21 days, mice were treated daily with either vehicle or CPT for 33 days. Thus, 4 groups of mice (vehicle + AAV8-scramble-shRNA, vehicle + AAV8-Gdf15-shRNA, CPT + AAV8-scramble-shRNA, and CPT + AAV8-Gdf15-shRNA) were studied here. (B) Hepatic Gdf15 mRNA expression and (C) plasma levels of GDF15 after 33 days of treatment. (D) Consecutive body weight. (E) Cumulative food intake. (F-G) Adipose tissue weight. (H) Mass of GAS and QUA. (I) GTT was performed after 42 days of treatment and AUC of GTT. (J) ITT was performed after 49 days of treatment and AUC of ITT. (K) Hepatic TG and TC contents. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. n = 6 per group. The underlying data for this figure can be found in S1 Data . AUC, area under the curve; BAT, brown adipose tissue; CPT, Camptothecin; DIO, diet-induced obese; GAS, gastrocnemius; GDF15, growth differentiation factor 15; GTT, glucose tolerance test; ITT, insulin tolerance test; QUA, quadriceps; TC, total cholesterol; TG, triglyceride.

To investigate whether CPT induces hepatic GDF15 production in vivo, we generated mice with liver-specific Gdf15 knockdown by using AAV8 system carrying a Gdf15 shRNA as illustrated in Fig 5A . As predicted, CPT-induced hepatic Gdf15 mRNA expression and secretion in DIO mice were drastically reduced when receiving AAV8-Gdf15 shRNA ( Fig 5B and 5C ) in contrast to scramble shRNA. Accordingly, the suppression of food intake and the subsequent body weight loss by CPT treatment were reduced upon hepatic Gdf15 knockdown ( Fig 5D and 5E ). The fat-reducing effect of CPT in DIO mice was markedly diminished with AAV-Gdf15 treatment ( Fig 5F ), with no alterations in the weights of BAT ( Fig 5G ) and skeletal muscle (GAS and QUA) ( Fig 5H ). Moreover, the beneficial effects of CPT on glucose homeostasis and insulin sensitivity disappeared upon hepatic Gdf15 knockdown, showing comparable levels of glucose-mediated whole-body glucose disposal as well as insulin-stimulated glucose uptake in CPT-treated mice and vehicle controls ( Fig 5I and 5J ). CPT induced reductions in hepatic TG and TC were markedly diminished in DIO mice with AAV-Gdf15 treatment ( Fig 5K ). We, therefore, conclude that the liver is the main site for CPT-induced GDF15 production.

Since CPT is known to be a specific inhibitor of type 1 DNA topoisomerase (TOP 1) and used to induce dsDNA breaks and cancer cell death [ 38 ], we investigated whether potential dsDNA breaks could account for CPT-induced ISR activation in liver. To this end, we measured the levels of γH2AX, a well-established biomarker of dsDNA breaks [ 39 , 40 ], using an ELISA-based γH2AX pharmacodynamic assay in response to CPT treatment in mouse livers, murine hepatocytes (AML12), and hepatoma cells (Hepa1-6). Similar γH2AX levels were shown in CPT-treated mouse livers and their controls ( S19A Fig ), as well as in 1 μM CPT-treated AML12 hepatocytes and the DMSO-treated controls. However, 1 μM CPT-treated Hepa1-6 cells exhibited higher γH2AX levels than DMSO-treated controls, indicating increased dsDNA breaks ( S19B Fig ). These results indicate that dsDNA breaks do not occur in CPT-treated normal livers and thus are not a contributor of CPT-induced hepatic ISR activation and GDF15 induction.

Based on previous work that GDF15 is a downstream target of the cellular integrated stress response (ISR) pathway [ 37 ], we hypothesized that ATF4 and CHOP, key transcriptional regulators of the ISR, might be involved in this process. In agreement with this, we found markedly increased mRNA and protein expression of ATF4 and CHOP in CPT-treated liver and hepatocytes ( Fig 4D–4G ). Conversely, either siRNA-mediated knockdown of Atf4 or Chop in hepatocytes blunted CPT-induced Gdf15 mRNA expression and GDF15 release into the medium ( Fig 4H–4M ), suggesting an essential role of ISR pathway in CPT-induced hepatic GDF15 production. To examine whether CPT-induced ISR activation is specific to liver, we measured protein expression levels of ATF4 and CHOP in ileum due to its high Gdf15 mRNA expression upon CPT treatment. Unlike in liver, their levels in ileum were similar in the 2 groups of mice ( S18 Fig ). These data indicate that CPT induces hepatic GDF15 production through activation of ISR pathway.

(A) Gdf15 mRNA expression in iWAT, BAT, skeletal muscle, liver, kidney, and gut of DIO mice after 30 days of treatment (n = 6). (B) GDF15 release in medium of cultured hepatocytes (AML12) treated with indicated doses of CPT for 24 h (n = 6). (C) The induction of Gdf15 mRNA expression by 1 μM CPT treatment for 24 h in AML 12 cells (n = 6). (D) mRNA expression levels of Atf4 and Chop in livers of DIO mice after 30 days of treatment (n = 6). (E) mRNA expression levels of Atf4 and Chop in 1 μM CPT-treated AML12 cells for 24 h (n = 5). (F-G) Immunoblot analysis of ATF4 and CHOP relative to β-actin in livers of DIO mice treated with 1 mg kg −1 of CPT for 30 days and in AML 12 cells treated with 1 μM CPT for 24 h (n = 3), respectively. (H) siRNA knockdown of Atf4 blunts 1 μM CPT-induced ATF4 expression (n = 3). (I-J) GDF15 release and relative Gdf15 expression in control siRNA and Atf4 siRNA transfected AML 12 cells treated with 1 μM CPT for 24 h (n = 5). (K) siRNA knockdown of CHOP blunts CPT-induced CHOP expression (n = 3), (L-M) GDF15 release and relative Gdf15 expression in control siRNA and Chop siRNA transfected AML 12 cells treated with 1 μM CPT for 24 h (n = 5). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001. The underlying data for this figure can be found in S1 Data . The original blot for this figure can be found in S1 Raw Image . ATF4, activating transcription factor 4; BAT, brown adipose tissue; CHOP, C/EBP homologous protein; CPT, Camptothecin; DIO, diet-induced obese; GDF15, growth differentiation factor 15; ISR, integrated stress response; iWAT, inguinal white adipose tissue.

GDF15 is a circulating protein that could be potentially produced by multiple tissues under different conditions [ 34 , 35 ]. To clarify which organs of CPT-mediated GDF15 production were mainly derived from, we examined Gdf15 gene expression in a tissue panel including iWAT, BAT, skeletal muscle, liver, kidney, heart, and gut, which have been reported as potential sources of circulating GDF15 [ 36 ], obtained from DIO mice treated with either CPT or vehicle for 30 days. Compared with controls, markedly increased Gdf15 mRNA expression was observed in CPT-treated livers ( Fig 4A ). To further determine whether hepatocytes are capable of responding to CPT with an increase in GDF15, we incubated mouse hepatocytes (AML12) with CPT and found induction of GDF15 release into the medium in a dose-dependent manner ( Fig 4B ) and a clear elevation of Gdf15 mRNA expression in hepatocytes ( Fig 4C ), demonstrating a direct link between CPT treatment and hepatic GDF15 secretion.

(A-J) Animal protocol 2 : (A) Ob/ob mice were treated with either 5 mg kg −1 GDF15-neutralizing antibody or control IgG every other day for 27 days through subcutaneous injection, 24 h before oral CPT (1 mg kg −1 day −1 ) or vehicle. Created with biorender.com . (B) Effect of CPT on circulating levels of GDF15 after 21 days of treatment. (C) Cumulative food intake. (D) Consecutive body weight of mice. (E) Adipose tissue weights. (F) Liver weight. (G) BAT weight. (H) Mass of GAS and QUA. (I) GTT and AUC of GTT (day 18). (J) Hepatic TG and TC contents. Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, n = 6 per group. The underlying data for this figure can be found in S1 Data . AUC, area under the curve; BAT, brown adipose tissue; CPT, Camptothecin; eWAT, epididymal white adipose tissue; GAS, gastrocnemius; GDF15, growth differentiation factor 15; GTT, glucose tolerance test; IgG, immunoglobulin G; iWAT, inguinal white adipose tissue; QUA, quadriceps; TC, total cholesterol; TG, triglyceride.

CPT treatment elevates circulating GDF15 in mice, to further test whether GDF15 is indispensable for CPT-mediated metabolic benefits, the action of GDF15 was inhibited by a GDF15-neutralizing antibody ( Fig 3A and 3B ). The efficiency of this antibody was validated in a cohort of WT mice received either exogenous GDF15 alone or along with coadministration of this neutralizing antibody ( S17A and S17B Fig ). CPT-consuming ob/ob mice treated with anti-GDF15 showed similar amount of food intake to vehicle control mice treated with anti-GDF15, whereas the food intake reduction seen in CPT-consuming ob/ob mice treated with immunoglobulin G (IgG) was maintained ( Fig 3C ), suggesting that GDF15 directly mediates the anorectic effect of CPT. Consistent with this, the body weight gain in CPT-treated mice (3.68 ± 0.62 g, 8.02% ± 1.35%) was not different from that of vehicle controls (5.90 ± 0.60 g, 12.91% ± 1.41% of body weight) upon GDF15 neutralization. In contrast, the weight loss seen in CPT-treated mice was preserved with IgG treatment compared with their corresponding controls, reaching 4.90% ± 0.68% below starting body weight ( Fig 3D ). The significant reductions in fat mass and liver weight seen with CPT treatment were not observed in the anti-GDF15 group ( Fig 3E and 3F ). The weights of brown adipose tissue (BAT), GAS, and QUA were not altered by GDF15 neutralization ( Fig 3G and 3H ). Moreover, GDF15 neutralization abrogated the beneficial effects of CPT on glucose homeostasis ( Fig 3I ) and prevention of hepatic steatosis ( Fig 3J ). Together, these results indicate that GDF15 mediates the beneficial metabolic effects of CPT in mice.

Given that exogenous injection of GDF15 in mice was reported to produce food aversion and visceral malaise under certain circumstances [ 31 ], we tested whether CPT-induced endogenous GDF15 elevation and food intake suppression could be of relevance to these abnormal behaviors using conditioned taste aversion (CTA) test in mice and pica test in rats as described [ 31 – 33 ]. Despite high levels of GDF15, CPT-treated mice did not show obvious food avoidance ( S15A–S15C Fig ). In the pica test, CPT-treated rats showed similar kaolin intake to vehicle control rats for the entire testing period (CPT: 0.53 ± 0.17 g kaolin per day; vehicle: 0.33 ± 0.12 g) ( S16A and S16B Fig ), suggesting absence of sickness behaviors indicative of nausea or malaise upon low-dose CPT (1 mg kg −1 CPT) treatment. The mean daily food intake was lower in CPT-treated rats than in vehicle controls (CPT: 20.01 ± 0.58 g; vehicle: 22.14 ± 0.64 g per day; P < 0.05) ( S16C and S16D Fig ). At the end of the experiment, CPT-treated rats showed pronounced reductions in body weight gain compared to vehicle controls (CPT: −6.53 ± 1.57 g; vehicle: 24.82 ± 3.18 g; P < 0.01) ( S16E Fig ). Together, these results indicate that (1) 1 mg kg −1 day −1 of CPT-induced GDF15 is not sufficient to cause food aversion and visceral malaise; and/or (2) the effects of endogenous GDF15 might be different from that of exogenous.

To test whether reduced food intake solely or along with altered energy expenditure (EE) contributes to the body weight control effect of CPT in mice, new cohorts of DIO mice treated with either CPT or vehicle were raised. We first applied a 9-day pair-feeding regime to mice to investigate whether this could abrogate the weight difference between CPT-treated mice and controls. In this experiment, the average daily food intake of CPT-treated mice was the same as that of the pair-fed vehicle groups. We observed similar body weight in CPT-treated mice to their pair-fed vehicle controls ( S13A–S13C Fig ), suggesting that food suppression is the main contributing factor responsible for body weight loss. Furthermore, we undertook indirect calorimetry in CPT-treated and vehicle control mice under ad libitum to establish whether there are additional effects on EE. CPT-treated mice showed comparable EE to controls when data were analyzed by analysis of covariance (ANCOVA) with body weight as the covariate ( S14A–S14C Fig ), as were of body temperature and heat production ( S14D and S14E Fig ). Collectively, these results show that the anti-obesity effect of CPT is due to the suppression of food intake, which is consistent with the reported anorectic effects of GDF15.

Given the documented adverse effects of CPT in clinical trials, we carefully examined most of these aspects in mice at the end of the 30-day treatment. CPT-treated mice showed similar counts of leucocyte and platelet, and comparable levels of hemoglobin to their vehicle controls ( S1 Table ), indicating absence of leukopenia, thrombocytopenia, and anemia. We did not observe abnormalities in hair, skin, mouth, and bladder in CPT-treated mice ( S1 Table ), indicating absence of alopecia, dermatitis, stomatitis, and cystitis. Moreover, CPT-treated mice showed similar levels of creatinine (CREA) and blood urea nitrogen (BUN) to their controls ( S12A–S12D Fig ). These results suggest that oral CPT (1 mg kg −1 ) does not cause apparent adverse effects in mice. Together, our results demonstrate that CPT ameliorates obesity and associated metabolic abnormalities in mice, which may be due to elevations of circulating GDF15.

The metabolic benefits of CPT on glucose homeostasis were revealed by glucose tolerance test (GTT) and insulin tolerance test (ITT). CPT-treated DIO mice showed improved responsiveness to insulin stimulation than the corresponding controls ( Fig 2I ), suggesting improved insulin sensitivity. Similarly, we observed faster glucose disposal capacity in CPT-treated DIO mice than in controls ( Fig 2J ), indicating enhanced glucose tolerance upon CPT treatment. CPT treatment improved glucose tolerance ( S10A Fig ) of ob/ob mice without altering their insulin sensitivity ( S10B Fig ). We next asked whether CPT itself is improving glucose metabolism or this effect is attributed to CPT-induced weight loss. To test it, we undertook further GTT in a cohort of weight-matched DIO mice receiving a single dose of CPT (1 mg kg −1 ). In these mice, CPT had no significant effect upon glucose disposal ( S11 Fig ). Thus, the beneficial effect of CPT on glucose metabolism is a secondary response to weight loss. Collectively, these results demonstrate that CPT ameliorates obesity, thereby reducing hyperlipidemia, fatty liver, and hyperglycemia in obese mice.

(A-J) Animal protocol 1 : DIO mice orally received vehicle or CPT (1 mg kg −1 day −1 ) for 30 days. (A) Plasma levels of GDF15 at indicated time points. (B) Cumulative food intake. (C) Consecutive body weight. (D) Tissue weights of iWAT and eWAT in CPT-treated mice and corresponding controls. (E) Plasma levels of TG and TC. (F) Liver weight. (G) Hepatic TG and TC contents. (H) Plasma levels of ALT and AST. (I) ITT and AUC of ITT (day 15). (J) GTT and AUC of GTT (day 21). Data are presented as mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001, n = 6 per group. The underlying data for this figure can be found in S1 Data . AUC, area under the curve; ALT, alanine aminotransferase; AST, aspartate aminotransferase; CPT, Camptothecin; DIO, diet-induced obese; eWAT, epididymal white adipose tissue; GDF15, growth differentiation factor 15; GTT, glucose tolerance test; ITT, insulin tolerance test; iWAT, inguinal white adipose tissue; TC, total cholesterol; TG, triglyceride.

To determine whether long-term CPT treatment could sustain high circulating levels of GDF15 and thereby alleviates obesity in mice, we performed longitudinal study in which both DIO mice and ob/ob mice orally received 1 mg kg −1 day −1 of CPT for 30 days. We observed significantly increased circulating levels of GDF15 in CPT-treated DIO mice (1.6- to 1.9- fold, Fig 2A ) and ob/ob mice (1.6- to 1.8- fold, S5A Fig ) at the 4 indicated time points (1 w, 2 w, 3 w, and 4 w). In line with this, CPT treatment reduced accumulative food intake in DIO mice starting from day 6 (12.01%) till the end of the observation period ( Fig 2B ), and in ob/ob mice starting from day 9 ( S5B Fig ). CPT treatment reduced the body weight of DIO mice from 43.63 ± 0.54 g to 39.00 ± 0.20 g (on day 1 versus day 30, P < 0.001) ( Fig 2C ), equivalent to a weight loss of 10.58% ± 0.91% relative to their initial weights. The body weight of vehicle-treated control mice increased by 3.39 g ± 0.53 g, corresponding to 8.25% ± 1.32% weight gain relative to their initial weights. Body weight reduction was also shown in CPT-treated ob/ob mice (6.03% ± 0.34% of weight loss) ( S5C Fig ). In contrast to obese mice, a 30-day testing of 1 mg kg −1 day −1 of CPT in lean mice showed similar food intake ( S6A Fig ) and body weight ( S6B Fig ) to their vehicle controls. The nonelevated GDF15 levels ( S6C Fig ) might be a main cause for absence of impact on body weight in CPT-treated lean mice.

To test the effect of CPT on GDF15 induction in vivo, both HFD-induced obese mice (DIO) and genetic ob/ob mice were orally treated at an acute single dose of either vehicle or CPT (1 mg kg −1 of body weight). The human equivalent dose (HED) of CPT used in mice were 3.01 mg/m 2 for a 60-kg person based on body surface area normalization [ 27 ], which were approximately 30- to 60-fold lower than those tested in Phase II clinical trials for advanced gastrointestinal adenocarcinoma patients (90 to 180 mg/m 2 ). The plasma concentration time profile for 1 mg kg −1 of CPT in DIO mice was shown in S2 Fig . CPT concentrations were approximately 9.5 ng ml −1 at 1 h, and then decreased to 1 ng ml −1 at 12 h. Consistent with the above screening results, 1 mg kg −1 of CPT administration resulted in a 2.2- and 2.0-fold increase in circulating GDF15 levels after 1 h in DIO mice ( Fig 1D ) and ob/ob mice ( S3 Fig ), respectively. Of note, we ( S4 Fig ) and others [ 28 ] showed that CPT-11 was unable to induce GDF15 secretion in mice. These data support that CPT induces circulating GDF15 secretion in obese mice.

(A) Schematic diagram depicting the workflow of obtaining gene expression signatures from the CMAP database to identify GDF15 inducer. (B) Left panel: the dot distribution of enrichment scores of the PC3 and MCF7 cell line. The expression levels of Gdf15 induced by different small molecules range from 0 to 1 enrichment score. In this study, we choose a list of small molecules with an enrichment score over 0.6. Each point represents an individual small molecule. The red dot indicated by the arrow represents CPT. Right panel: top 10 small molecules. (C) Left panel: scatter plot showing the Log 2 fold changes of gene abundance upon 2 μM CPT treatment in 293T cell line. Differentially abundant genes (n = 828, P < 0.05) are indicated [red, up-regulated (n = 398) and blue down-regulated (n = 430)]. The circled red dot indicated by the arrow represents Gdf15. Significantly abundant genes were evaluated by Student t test (FDR = 0.05). Right panel: heatmap showing the top 50 up-regulated (red) and down-regulated (blue) genes obtained from 293T cells treated with vehicle or 2 μM CPT. (D) Plasma levels of GDF15 measured 1 h, 2 h, 6 h, 12 h, or 24 h in DIO mice receiving a single oral dose of 1 mg kg −1 CPT, * P < 0.05, ** P < 0.01, *** P < 0.001. n = 5 per group. The underlying data for this figure can be found in S1 Data . CMAP, Connectivity Map; CPT, Camptothecin; FDR, false discovery rate; GDF15, growth differentiation factor 15.

GDF15 has become a keen target of interest for anti-obesity therapies [ 26 ]. Screening of small molecules that could induce GDF15 expression in vivo holds promise for clinical applications. For this purpose, we used CMAP, which comprises a database of whole-genome gene expression profiles derived from human cell lines treated with more than 6,000 small molecules [ 19 ], to screen for small molecules that could elevate the expression of Gdf15 ( Fig 1A ). A list of 109 CMAP molecules was selected by analyzing signatures of the cell line MCF7 and PC3. The distribution of individual small molecules in the selected library is shown based on their enrichment scores ( S1 Fig ). Combined distribution of the enrichment scores of the selected molecules for both MCF7 and PC3 ranked CPT as the second top compound in the database for Gdf15 expression (the first compound has been prohibited by Therapeutic Goods Administration) ( Fig 1B ). Of note, an FDA-approved CPT analog Irinotecan (CPT-11) is included in the CMAP database. Nonetheless, its enrichment scores rank far below 100 in terms of Gdf15 expression. Analysis of the expression profile of CPT-treated 293T cell line from the available GEO dataset (GSE2451) revealed Gdf15 as the second up-regulated gene among the top 50 differentially expressed genes as shown by volcano analysis ( Fig 1C , left) and heatmap ( Fig 1C , right), exhibiting more than 600-fold increase compared with controls. Together, these results suggest that CPT is a potential GDF15 inducer.

Discussion

In this study, by screening of CMAP database, we discover that the small molecule CPT acts as an inducer of GDF15. In vivo testing show that CPT induces hepatic expression and secretion of GDF15 in mice, which reduces food intake and thereby normalizing body weight and reversing metabolic dysfunctions in obese mice (Fig 7I). In contrast, antibody neutralization of GDF15 or deficiency of GFRAL in mice abolishes the metabolic benefits of CPT, corroborating that GDF15-GFRAL axis is an essential signaling node for CPT. This work reveals important physiological effects of CPT that could provide therapeutic benefits for obesity. Our strategy from virtual screening to validation, and from animal physiology to mechanistic elucidation, represents an illustrative approach potentially valuable for the next generation of translational medicine.

One may question the translational potential of CPT as a treatment for obesity due to its widely documented adverse effects in patients with adenocarcinoma of gastrointestinal origin [25]. This is less likely to happen because (1) we directly showed that the recommended anti-obesity dose of CPT (1 mg kg−1 day−1) did not provoke the documented adverse effects in obese mice, as judged by normal levels of hemoglobin, counts of white blood cell and platelet, as well as absence of diarrhea and alopecia. (2) The immune system, energy metabolism, and drug response in obese people are different from those in cancer patients [42]. For example, we show that the beneficial effects of CPT on body weight control appear not mechanistically linked to dsDNA breaks, whereas it is considered as the mechanism of action for CPT’s anticancer property [24]. This is an important consideration if one worries that CPT might cause dsDNA breaks as a treatment for obesity. (3) About 1 mg kg−1 day−1 of CPT recommended for anti-obesity purposes is equivalent to approximately one-thirtieth of the lowest dose tested in cancer patients during Phase II clinical trials, thus is much lower than those tested for anticancer purposes. These represent a major asset in support of the potential advancement of this compound to the investigational new anti-obesity drug status.

The genetic leptin-deficient ob/ob mice were used in this study. CPT-elevated circulating GDF15, although relatively mild in ob/ob mice compared to that in HFD-induced DIO mice, is able to reduce food intake and body weight in the absence of leptin. Our observations are consistent with findings that overexpression of GDF15 reduced food intake and body weight in ob/ob and db/db mice [43]. Similar findings that exogenous GDF15 reversed hyperphagia and obesity were also shown in leptin receptor-deficient ZF rats [41]. These results suggest that GDF15 functions independently of leptin signaling pathway although both hormones regulate appetite. Thus, GDF15 may have potential clinical implications for the treatment of hereditary obesity, especially in the circumstance of leptin receptor deficiency, which cannot be improved by leptin administration.

It is worth noting that only obese mice respond to CPT. This obesity-dependent effect is widely shown in mice [20,44] and is also supported by our previous work on small compounds Urolithin A [45] and Neohesperidin [46]. The underlying mechanisms of these phenomena might be different and need further investigation. In regard to CPT, low levels of GDF15 might be the reason for the absence of impact on food intake and body weight in lean mice. It is well established that the effect of GDF15 on food intake is dose dependent [37]. CPT-induced GDF15 elevations were shown in obese mice (Fig 2A, S5A Fig) but not in lean mice (S6C Fig). Thus, the mechanism underlying the obesity-dependent effect of CPT is highly probably due to serum levels of GDF15, which is relatively low and not sufficient to suppress food intake and body weight in lean mice.

We provide a mechanistic basis underlying the metabolic benefits of CPT. Using GDF15 neutralization or its receptor GFRAL deficiency in mice, we corroborated that GDF15-GFRAL signaling is indispensable for the metabolic actions of CPT. About 1 mg kg−1 day−1 of CPT moderately increases circulating levels of GDF15 (approximately 400 pg ml−1) and progressively reduces food intake without causing obvious adverse effects, suggesting that endogenous induction of GDF15 to a concentration range between 250 and 600 pg ml−1 is able to control body weight. We, therefore, recommend the range of plasma GDF15 levels for anti-obesity purposes.

Since CPT was originally tested as an anticancer drug, and some platinum-based anticancer drugs such as cisplatin and bleomycin have been shown to induce GDF15 in subjects with testicular cancer [47], one may deduce that a large number of the anticancer drugs could induce GDF15. This assumption is refuted by studies from Breen and colleagues, showing that clinically approved anticancer drugs including gemcitabine, methotrexate, paclitaxel, pemetrexed, and even the clinically approved CPT analogy CPT-11 were unable to induce GDF15 [28]. Therefore, induction of GDF15 by anticancer drugs seems not category based but individually different.

Although multiple organs and tissues contribute to the GDF15 rise in plasma [48–50], our data showed that the liver is the main source of circulating GDF15 following CPT treatment. Hepatic GDF15 production has been related to the increased levels of circulating GDF15 following sustained caloric excess as well as a consequence of acute nutritional stress [37], which indicates that the liver plays a key role in the production of GDF15. Furthermore, elegant work from the O’Rahilly lab showed that intestine and kidney are main sources for metformin-induced GDF15 in mice [51]. Recently, it has been shown that prolonged endurance exercise increases Gdf15 expression in liver, skeletal muscle, and cardiac muscle in mice [52]. Whether the functions of hepatic GDF15 are different from those of intestinal, renal, or muscle sources merits further investigation.

GDF15 has been repeatedly shown to cause anorexia and food aversion [31,53–55] as part of the biological role of the peptide [37]. However, the roles of GDF15 in these aspects are far from conclusive. One recent study showed that recombinant human GDF15 (pharmacological), but not similar concentrations of endurance exercise induced physiological GDF15, suppresses feeding in mice, suggesting an origin-related effect of GDF15 on appetite regulation [52]. This study also indicates that the functional difference between acutely administered exogenous GDF15 and endogenous GDF15 induced by exercise or drugs may be due to the speed of GDF15 elevation. The former often leads to a fast rise in plasma GDF15, whereas the latter is relatively slow. Here, by performing CTA test in mice and pica test in rats, we observed absence of sickness behaviors indicative of nausea or malaise upon low-dose CPT (1 mg kg−1) treatment. Concentration is another key factor affecting the effects of GDF15. Low levels of GDF15 (<200 pg ml−1) is not sufficient to suppress appetite and cause food aversion [51,52] in normal mice. High levels of GDF15 were reported in cancer patients with cachexia, manifesting great loss of adipose tissue and skeletal muscle [56,57]. So far, the relationship between GDF15 and these side effects is associative rather than causative. Pathological levels of GDF15 might contribute to these adverse effects. In line with this, Hsu and colleagues observed great loss of adipose tissue and skeletal muscle when GDF15 levels were at approximately 900 pg ml−1 induced by 10 mg kg−1 of Cisplatin in mice [41], whereas skeletal muscle mass and strength were not affected or even improved when GDF15 levels were maintained approximately 400 pg ml−1 with 1 mg kg−1 of CPT, suggestive of concentration-dependent side effects of GDF15. Of note, the effect of GDF15 on skeletal muscle has been investigated in mice with GDF15 overexpressing locally in the tibialis anterior. In this way, Gdf15 mRNA expression is increased in the skeletal muscle, manifesting muscle wasting in normal mice [58]. However, Gdf15 mRNA is not increased in the skeletal muscle of CPT-treated mice as shown in Fig 4A. Thus, the effect of GDF15 on skeletal muscle might be concentration dependent and/or source related.

In addition to the anti-obesity property of CPT-induced GDF15 elevation, GDF15 is also implicated in life span [59], atherosclerotic cardiovascular disease [60], inflammation and immunology [61], mitochondrial disease, cancer, rheumatoid arthritis, chronic renal, and cardiac failure [43,62]. Therefore, our findings open avenues of CPT research in these fields. In summary, CPT ameliorates obesity by acting as a GDF15 inducer, providing a convincing argument that CPT may have therapeutic benefits for obesity and its associated metabolic disorders. Further study is needed to evaluate the efficacy and safety of CPT in advanced models to increase the translational impact.

[END]

[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001517

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