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Appetite suppression and weight reduction by a centrally active aminosterol - MSI-1436
Diabetes, July, 2002 by Rexford S. Ahima, Hiralben R. Patel, Nobuhiko Takahashi, Yong Qi, Stanley M. Hileman, Michael A. Zasloff
Obesity is highly prevalent in the U.S. and other developed countries and is increasing worldwide (1,2). This epidemic has serious public health consequences because obesity is associated with excess mortality and morbidity from type 2 diabetes, cardiovascular disease, and other complications (1,2). Diet and exercise remain the cornerstone of obesity management; however, it is likely that many patients will require drug treatment to reduce body weight and prevent complications (3). Here, we describe the anti-obesity action of a novel aminosterol. MSI-1436 is a spermine metabolite of cholesterol that was originally isolated from the dogfish shark (Squalus acanthias) liver during a search for naturally occurring antimicrobial compounds (4,5). MSI-1436 is structurally similar to squalamine (MSI-1256) except for a spermine side-chain at C-3 on the cholesterol A-ring (4,5). The bioactivity of MSI-1436 is also dependent on a seven a-OH and sulfated moiety at C-25 (5). Unexpectedly, MSI-1436 was shown to inhibit feeding and decrease body weight in a highly specific manner in normal and obese rodents (5).
MSI-1436 is distributed to the brain and several peripheral tissues (5). A single or intermittent treatment with MSI-1436 results in a prolonged reduction in food intake and body weight and has been partly attributed to its long half-life (~7 days in rodents) (5). However, it is unclear whether MSI-1436-induced weight loss is due to appetite suppression alone. Moreover, although it had been suggested that MSI-1436 is more potent when administered into the cerebral ventricle, its targets in the central nervous system are unknown. The objective of this study was to investigate the contributions of energy intake and expenditure to the sustained effect of MSI-1436 on body weight and determine whether the biological activity of MSI-1436 in the brain is mediated by well-known hypothalamic neuronal pathways that mediate feeding behavior and energy balance.
RESEARCH DESIGN AND METHODS
Experiments were performed in accordance with guidelines and regulations of the National Institutes of Health and Institutional Animal Care and Use Committee of the University of Pennsylvania.
Determine the effect of MSI-1436 on food intake and energy expenditure. Male 12-week-old C57B1/6J mice (Jackson Laboratories, Bar Harbor. ME) were housed individually in a 12:12 h light-dark cycle (lights on at 0600; temperature 22[degrees]C) and allowed normal laboratory diet and water ad libitum. Synthetic MSI-1436 and squalamine (MSI-1256) were provided by Genaera (Plymouth Meeting, PA). Our preliminary studies confirmed that squalamine did not affect food intake or body weight (5). In contrast, intraperitoneal (IP) injection at MSI-1436 during the light or dark cycle reduced food intake and body weight in a dose dependent manner (data not shown). We selected a lower dose of MSI-1436 that did not suppress drinking as previously described (5). MSI-1436 (5 mg/kg IP x three doses) was administered at 0900-1000 at 3-day intervals to a group of mice (n = 6). Control mice (n = 6) were treated with vehicle (100 [micro]l endotoxin free [H.sub.2]O IP). A third group of mice (n = 6) was pair-fed to the daily intake of MSI-1436 mice. Indirect calorimetry was performed after the final treatment. The trace were acclimatized to the test cage for 2 days, and energy expenditure was measured at 15-min intervals for 24 h on the third day (Oxymax Equalflow System; Columbus Instruments, Columbus, Oil) (6). The following settings weir used per cycle: air flow 500 ml/min, sample flow 400 ml/min, settle time 120 s. measuring time 60 s, temperature 22 [degrees] C, respiratory exchange ratio (RER) = volume of carbon dioxide generated (VC[O.sub.2]) divided by oxygen consumption (V[O.sub.2]). Heal (kcal/ h) = 3.S15 + 1.232 x RER. Total mad ambulatory activity were measured simultaneously with photodetectors (Optovarimex System; Columbus Instruments). Rectal temperature was measured with a thermistor (Physitemp Instruments, Clifton, NJ).
The mice were killed by C[O.sub.2] inhalation, and blood was obtained by cardiac puncture. Glucose and triglycerides (Sigma, St. Louis, MO) and nonesterified fatty acids (NEFAs) (Wake Chemicals, Richmond, VA) were measured with enzyme assays. Plasma insulin, leptin, corticosterone, and thyroxine were measured by radioimmunoassay as previously described (7,8). Uncoupling protein (UCP)-1 mRNA was measured in brown adipose tissue (BAT) by Northern blot analysis using a cDNA probe (provided by Mitch Lazar, University of Pennsylvania). The rest of the carcass was dried to a constant weight at 60 [degrees] C to determine water content and digested in ethanol-KOH, and fat (triglyceride) content was measured with a colorimetric assay (Sigma) (8).
We determined whether MSI-1436 could prevent the hyperphagia normally associated with fasting. Male 12-week-old C57BI/6J mice were fasted for 48 h and received a single injection of MSI-1436 (5 mg/kg IP) or vehicle (n = 6/group) after the fast. They were housed in individual cages and allowed ad libitum access to normal diet and water. Food intake and body weight were measured daily. Feeding frequency and duration were monitored using infrared detectors (Vitalview System; Mini Mitter, Bend, OR). The data on feeding frequency and duration were collated in 1-h bins and analyzed using Actiview software (Mini Mitter).
Determine the dose response to intracerebroventricular versus systemic MSI-1436 treatment. Male Sprague-Dawley rats (256-300 g) were obtained from Taconic Farms (Germantown, NY), housed ill a 12:12 h light-dark cycle (lights on at 0600; temperature 22 [degrees] C), and allowed normal laboratory food and water ad libitum. The animals were anesthetized with sodium pentobarbital, and a 22-gauge stainless steel guide cannula with obturator (Plastics One, Roanoke, VAt was implanted unilaterally in the lateral cerebral ventricle using the following coordinates: 0.8 mm posterior to bregma, 1.5 mm lateral to the midline, and 4 mm below the skull. The intracerebroventricular (ICV) cannula was attached to the cranium with stainless steel screws and dental cement. Cannula placement was verified with the drinking response to ICV angiotensin II injection and histologically after the experiment. Only data from rats with correctly positioned cannulas were included in the analysis.
The animals were housed individually after surgery and handled daily to habituate, and two sham injections were performed at 3-day intervals. Experiments were performed after restoration of body weight (~1 week). MS1-1436 (2, 10, or 30 [micro]g) or vehicle (5 [micro]l endotoxin-free [H.sub.2]O) was administered ICV over 1 min to unanasthetized rats (n = 6) with an injector protruding 1 mm below the guide cannula into the cerebral ventricle. Other rats (n = 6/group) received a single intraperitoneal injection of MSI-1436 (5 mg/kg) or vehicle (200 [micro]l endotoxin-free [H.sub.2]O). The animals were presented with a preweighed amount of pellet food, and food consumption and body weight were raeasured dally. The effect of MSI-1436 on conditioned taste aversion was investigated in other rats using the two bottle paradigm (9). The animals (n = 4 per group) were treated with a single injection of MSI-1436 (30 [micro]g ICV), MSI-1436 (10 mg/kg IP), vehicle (ICV or IP), or LiCl (100 mg/kg). Saccharin preference ratio was determined as the volume of 0.1% saccharin divided by water consumed in 24 h (9).
Determine the effect of MSI-1436 on Fos immunoreactivity and response to paraventricular hypothalamic nucleus injection. Expression of the immediate-early gene c-fos has been used to map neuronal pathways for feeding regulation (10-13). Fos immunostaining was performed Oil coronal brain sections from pathogen-free male Sprague-Dawley rats (Taconic Farms) as previously described (10). The animals were treated with MSI-1436 (5 mg/kg in 200 [micro]l endotoxin-free [H.sub.2]O IP or 20 [micro]g in 5 [micro]l endotoxin-free [H.sub.2]O ICV; n = 4/group). Control rats (n = 4) were treated with vehicle (IP or ICV). The animals were anesthetized with sodium pentobarbital 2 h later (1000-1200) and perfused transcardially with PBS followed by 10% neutral-buffered formalin. Brains were cryoprotected in 20% sucrose/PBS, and free-floating coronal sections (40 [micro]m) were cut on a sliding microtome and processed for Fos immunoreactivity (10). The sections were examined with a Nikon E600 microscope equipped with a digital earners and image analysis system (SPOT Diagnostic Instruments. Sterling Heights, MI). Fos-positive cells/region/hemisphere with distinct nuclear staining were counted by an observer blinded to the experimental protocol using software provided by the manufacturer (Phase 3 Imaging, Glenmills, PA).
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Klinische Studie 2003 – MSI-1436 reduziert das Hungergefühl und das Übergewicht – vor erst bei Nagern
An der University of Pennsylvania und Georgetown haben Wissenschaftler die Substanz Squalus acanthias entdeckt. Ihre Eigenschaft: Reduktion von Appetit und Körpergewicht bei Nagern.
Bei diesem aus der im Cholesterin der Hai-Leber vorkommenden Substanz entwickelte das team den Wirkstoff MSI-1436. Dieser weist eine appetit-suppressive Wirkung auf. Seine Entdeckung verdanken die Forscher dem Zufall. Vorgesehen war, so Michael Zasloff, Leiter der Forschungsgruppe, die Hai-Leber auf natürliche Antibiotika hin zu untersuchen. "Das erstaunliche an dieser Substanz ist, dass die Versuchstiere Fett und nicht Muskelmasse abbauten", so Zasloff. Die metabolischen Stoffelwechselvorgänge hätten sich dabei nicht verlangsamt. Dies jedoch ist bei den meisten Diäten der Fall, da der Körper eine reduzierte Kalorienaufnahme mit einem verlangsamten Metabolismus kompensiert. "Die Personen neigen dann zu Muskelabbau anstatt Fett abzubauen", erklärt der Wissenschaftler. 2003 soll diese Substanz erstmals in die Testphase gehen.
Das MSI-1436 ist einer anderen Substanz der Hai-Leber ähnlich. "Squalamin" findet derzeit auch im klinischen Test für den Einsatz der Krebsbehandlung Anwendung. Auch diese hormonartige Substanz wurde von Zasloff entdeckt. Das körpereigene Antibiotikum "Squalamin" des Dornhais schützt ihn vor Bakterien, Pilzen und Parasiten, mit unter ist die Substanz sogar dafür verantwortlich, dass Haie sehr selten an Krebs erkranken.
Diabetes, July, 2002 by Rexford S. Ahima, Hiralben R. Patel, Nobuhiko Takahashi, Yong Qi, Stanley M. Hileman, Michael A. Zasloff
Obesity is highly prevalent in the U.S. and other developed countries and is increasing worldwide (1,2). This epidemic has serious public health consequences because obesity is associated with excess mortality and morbidity from type 2 diabetes, cardiovascular disease, and other complications (1,2). Diet and exercise remain the cornerstone of obesity management; however, it is likely that many patients will require drug treatment to reduce body weight and prevent complications (3). Here, we describe the anti-obesity action of a novel aminosterol. MSI-1436 is a spermine metabolite of cholesterol that was originally isolated from the dogfish shark (Squalus acanthias) liver during a search for naturally occurring antimicrobial compounds (4,5). MSI-1436 is structurally similar to squalamine (MSI-1256) except for a spermine side-chain at C-3 on the cholesterol A-ring (4,5). The bioactivity of MSI-1436 is also dependent on a seven a-OH and sulfated moiety at C-25 (5). Unexpectedly, MSI-1436 was shown to inhibit feeding and decrease body weight in a highly specific manner in normal and obese rodents (5).
MSI-1436 is distributed to the brain and several peripheral tissues (5). A single or intermittent treatment with MSI-1436 results in a prolonged reduction in food intake and body weight and has been partly attributed to its long half-life (~7 days in rodents) (5). However, it is unclear whether MSI-1436-induced weight loss is due to appetite suppression alone. Moreover, although it had been suggested that MSI-1436 is more potent when administered into the cerebral ventricle, its targets in the central nervous system are unknown. The objective of this study was to investigate the contributions of energy intake and expenditure to the sustained effect of MSI-1436 on body weight and determine whether the biological activity of MSI-1436 in the brain is mediated by well-known hypothalamic neuronal pathways that mediate feeding behavior and energy balance.
RESEARCH DESIGN AND METHODS
Experiments were performed in accordance with guidelines and regulations of the National Institutes of Health and Institutional Animal Care and Use Committee of the University of Pennsylvania.
Determine the effect of MSI-1436 on food intake and energy expenditure. Male 12-week-old C57B1/6J mice (Jackson Laboratories, Bar Harbor. ME) were housed individually in a 12:12 h light-dark cycle (lights on at 0600; temperature 22[degrees]C) and allowed normal laboratory diet and water ad libitum. Synthetic MSI-1436 and squalamine (MSI-1256) were provided by Genaera (Plymouth Meeting, PA). Our preliminary studies confirmed that squalamine did not affect food intake or body weight (5). In contrast, intraperitoneal (IP) injection at MSI-1436 during the light or dark cycle reduced food intake and body weight in a dose dependent manner (data not shown). We selected a lower dose of MSI-1436 that did not suppress drinking as previously described (5). MSI-1436 (5 mg/kg IP x three doses) was administered at 0900-1000 at 3-day intervals to a group of mice (n = 6). Control mice (n = 6) were treated with vehicle (100 [micro]l endotoxin free [H.sub.2]O IP). A third group of mice (n = 6) was pair-fed to the daily intake of MSI-1436 mice. Indirect calorimetry was performed after the final treatment. The trace were acclimatized to the test cage for 2 days, and energy expenditure was measured at 15-min intervals for 24 h on the third day (Oxymax Equalflow System; Columbus Instruments, Columbus, Oil) (6). The following settings weir used per cycle: air flow 500 ml/min, sample flow 400 ml/min, settle time 120 s. measuring time 60 s, temperature 22 [degrees] C, respiratory exchange ratio (RER) = volume of carbon dioxide generated (VC[O.sub.2]) divided by oxygen consumption (V[O.sub.2]). Heal (kcal/ h) = 3.S15 + 1.232 x RER. Total mad ambulatory activity were measured simultaneously with photodetectors (Optovarimex System; Columbus Instruments). Rectal temperature was measured with a thermistor (Physitemp Instruments, Clifton, NJ).
The mice were killed by C[O.sub.2] inhalation, and blood was obtained by cardiac puncture. Glucose and triglycerides (Sigma, St. Louis, MO) and nonesterified fatty acids (NEFAs) (Wake Chemicals, Richmond, VA) were measured with enzyme assays. Plasma insulin, leptin, corticosterone, and thyroxine were measured by radioimmunoassay as previously described (7,8). Uncoupling protein (UCP)-1 mRNA was measured in brown adipose tissue (BAT) by Northern blot analysis using a cDNA probe (provided by Mitch Lazar, University of Pennsylvania). The rest of the carcass was dried to a constant weight at 60 [degrees] C to determine water content and digested in ethanol-KOH, and fat (triglyceride) content was measured with a colorimetric assay (Sigma) (8).
We determined whether MSI-1436 could prevent the hyperphagia normally associated with fasting. Male 12-week-old C57BI/6J mice were fasted for 48 h and received a single injection of MSI-1436 (5 mg/kg IP) or vehicle (n = 6/group) after the fast. They were housed in individual cages and allowed ad libitum access to normal diet and water. Food intake and body weight were measured daily. Feeding frequency and duration were monitored using infrared detectors (Vitalview System; Mini Mitter, Bend, OR). The data on feeding frequency and duration were collated in 1-h bins and analyzed using Actiview software (Mini Mitter).
Determine the dose response to intracerebroventricular versus systemic MSI-1436 treatment. Male Sprague-Dawley rats (256-300 g) were obtained from Taconic Farms (Germantown, NY), housed ill a 12:12 h light-dark cycle (lights on at 0600; temperature 22 [degrees] C), and allowed normal laboratory food and water ad libitum. The animals were anesthetized with sodium pentobarbital, and a 22-gauge stainless steel guide cannula with obturator (Plastics One, Roanoke, VAt was implanted unilaterally in the lateral cerebral ventricle using the following coordinates: 0.8 mm posterior to bregma, 1.5 mm lateral to the midline, and 4 mm below the skull. The intracerebroventricular (ICV) cannula was attached to the cranium with stainless steel screws and dental cement. Cannula placement was verified with the drinking response to ICV angiotensin II injection and histologically after the experiment. Only data from rats with correctly positioned cannulas were included in the analysis.
The animals were housed individually after surgery and handled daily to habituate, and two sham injections were performed at 3-day intervals. Experiments were performed after restoration of body weight (~1 week). MS1-1436 (2, 10, or 30 [micro]g) or vehicle (5 [micro]l endotoxin-free [H.sub.2]O) was administered ICV over 1 min to unanasthetized rats (n = 6) with an injector protruding 1 mm below the guide cannula into the cerebral ventricle. Other rats (n = 6/group) received a single intraperitoneal injection of MSI-1436 (5 mg/kg) or vehicle (200 [micro]l endotoxin-free [H.sub.2]O). The animals were presented with a preweighed amount of pellet food, and food consumption and body weight were raeasured dally. The effect of MSI-1436 on conditioned taste aversion was investigated in other rats using the two bottle paradigm (9). The animals (n = 4 per group) were treated with a single injection of MSI-1436 (30 [micro]g ICV), MSI-1436 (10 mg/kg IP), vehicle (ICV or IP), or LiCl (100 mg/kg). Saccharin preference ratio was determined as the volume of 0.1% saccharin divided by water consumed in 24 h (9).
Determine the effect of MSI-1436 on Fos immunoreactivity and response to paraventricular hypothalamic nucleus injection. Expression of the immediate-early gene c-fos has been used to map neuronal pathways for feeding regulation (10-13). Fos immunostaining was performed Oil coronal brain sections from pathogen-free male Sprague-Dawley rats (Taconic Farms) as previously described (10). The animals were treated with MSI-1436 (5 mg/kg in 200 [micro]l endotoxin-free [H.sub.2]O IP or 20 [micro]g in 5 [micro]l endotoxin-free [H.sub.2]O ICV; n = 4/group). Control rats (n = 4) were treated with vehicle (IP or ICV). The animals were anesthetized with sodium pentobarbital 2 h later (1000-1200) and perfused transcardially with PBS followed by 10% neutral-buffered formalin. Brains were cryoprotected in 20% sucrose/PBS, and free-floating coronal sections (40 [micro]m) were cut on a sliding microtome and processed for Fos immunoreactivity (10). The sections were examined with a Nikon E600 microscope equipped with a digital earners and image analysis system (SPOT Diagnostic Instruments. Sterling Heights, MI). Fos-positive cells/region/hemisphere with distinct nuclear staining were counted by an observer blinded to the experimental protocol using software provided by the manufacturer (Phase 3 Imaging, Glenmills, PA).
--------------------------------------------------------------
Klinische Studie 2003 – MSI-1436 reduziert das Hungergefühl und das Übergewicht – vor erst bei Nagern
An der University of Pennsylvania und Georgetown haben Wissenschaftler die Substanz Squalus acanthias entdeckt. Ihre Eigenschaft: Reduktion von Appetit und Körpergewicht bei Nagern.
Bei diesem aus der im Cholesterin der Hai-Leber vorkommenden Substanz entwickelte das team den Wirkstoff MSI-1436. Dieser weist eine appetit-suppressive Wirkung auf. Seine Entdeckung verdanken die Forscher dem Zufall. Vorgesehen war, so Michael Zasloff, Leiter der Forschungsgruppe, die Hai-Leber auf natürliche Antibiotika hin zu untersuchen. "Das erstaunliche an dieser Substanz ist, dass die Versuchstiere Fett und nicht Muskelmasse abbauten", so Zasloff. Die metabolischen Stoffelwechselvorgänge hätten sich dabei nicht verlangsamt. Dies jedoch ist bei den meisten Diäten der Fall, da der Körper eine reduzierte Kalorienaufnahme mit einem verlangsamten Metabolismus kompensiert. "Die Personen neigen dann zu Muskelabbau anstatt Fett abzubauen", erklärt der Wissenschaftler. 2003 soll diese Substanz erstmals in die Testphase gehen.
Das MSI-1436 ist einer anderen Substanz der Hai-Leber ähnlich. "Squalamin" findet derzeit auch im klinischen Test für den Einsatz der Krebsbehandlung Anwendung. Auch diese hormonartige Substanz wurde von Zasloff entdeckt. Das körpereigene Antibiotikum "Squalamin" des Dornhais schützt ihn vor Bakterien, Pilzen und Parasiten, mit unter ist die Substanz sogar dafür verantwortlich, dass Haie sehr selten an Krebs erkranken.
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