A small synthetic peptide sequence of human growth hormone (hGH), AOD-9401, has lipolytic and antilipogenic activity similar to that of the intact hormone. Here we report its effect on lipid metabolism in rodent models of obesity and in human adipose tissue to assess its potential as a pharmacological agent for the treatment of human obesity. C57BL/6J (ob/ob) mice were orally treated with either saline (n = 8) or AOD-9401 (n = 10) for 30 days. From day 16 onward, body weight gain in AOD-9401-treated animals was significantly lower than that of saline-treated controls. Food consumption did not differ between the two groups. Analyses of adipose tissue ex vivo revealed that AOD-9401 significantly reduced lipogenic activity and increased lipolytic activity in this tissue. Increased catabolism was also reflected in an acute increase in energy expenditure and glucose and fat oxidation in ob/ob mice treated with AOD-9401. In addition, AOD-9401 increased in vitro lipolytic activity and decreased lipogenic activity in isolated adipose tissue from obese rodents and humans. Together, these findings indicate that oral administration of AOD-9401 alters lipid metabolism in adipose tissue, resulting in a reduction of weight gain in obese animals. The marked lipolytic and antilipogenic actions of AOD-9401 in human adipose tissues suggest that this small synthetic hGH peptide has potential in the treatment of human obesity.
human growth hormone peptide; lipolysis; lipogenesis; energy expenditure
OBESITY is a major public health concern in most developed countries. For example, in Australia almost one in five adults is obese, making them highly susceptible to diabetes, coronary heart disease, and high blood pressure, as well as reduced psychological health (1). Obesity is normally treated by diet and exercise, but attempts to sustain significant weight loss by dieting and exercise nearly always meet with failure (6). There is a great need to develop better pharmacotherapy for obesity (18). Here, we begin an assessment of the potential use of AOD-9401, a fragment of human growth hormone (hGH), in the treatment of obesity.
The lipolytic/antilipogenic property of hGH is well known (11, 19). For example, it is well documented that hGH is a potent inhibitor of lipoprotein lipase and can increase circulating free fatty acids, ultimately reducing fat cell mass (20). The association of circulating hGH with fat mass is well characterized in adult GH-deficient patients, where a strong correlation between excess abdominal adiposity and reduced circulating GH levels exists that can be normalized after GH replacement therapy (10). However, clinical applications of hGH for long-term obesity treatment have not been successful because of its diabetogenic and other unwanted side effects (3). Advances in peptide synthesis technology have made it possible to produce specific and discrete functional domains of hGH (9), and there is now considerable evidence supporting the concept of discrete structural domains within hGH responsible for the different metabolic functions of the intact hGH (4). For example, Ng et al. (13) have reported that the insulin-like actions of hGH may reside in the amino-terminal region of the molecule [hGH-(6---13)]. This was later confirmed to be within hGH-(1---43), another well-known hypoglycemic fragment that exists in the circulation of humans and results from posttranslational cleavage in vivo (20, 29). On the other hand, the carboxy terminus of the hGH molecule [hGH-(177---191), or AOD-9401] has been identified as the lipid mobilizing domain of the intact hormone. This fragment inhibits the activity of acetyl-CoA carboxylase in adipocytes and hepatocytes, and it acts to reduce glucose incorporation into lipid in both isolated cells and tissues (30). It has been suggested to be the lipolytic domain of the hGH molecule. Previous studies in our laboratory have shown that weight loss can be induced by chronic intraperitoneal treatment of AOD-9401 (12).
This study aims to extend these findings by examining whether oral administration of this fragment of hGH can also reduce body weight and affect lipid metabolism. This study had three parts. First, we investigated the effectiveness of oral administration of AOD-9401 on body weight reduction, energy balance, lipolysis, and lipogenesis in obese C57BL/6J (ob/ob) mice. Next, we examined the in vitro antilipogenic, lipolytic, and fat oxidation activity of AOD-9401 in peripheral adipose tissues from obese rodents. Finally, to assess the feasibility of human treatment, the in vitro action of AOD-9401 on lipolysis and lipogenesis in adipose tissue from obese human adipose tissue was examined.
MATERIALS AND METHODS
Chemical synthesis of hGH functional domain (AOD-9401). AOD-9401, a synthetic fragment of hGH consisting of the amino acid residues 177-191, was prepared with solid-phase synthesis procedure and purified with reverse-phase HPLC methodology in our laboratories at Monash University (9). The structure of the peptide analog was verified with mass spectrometry and amino acid analysis.
In vitro tests for nonenzymic and enzymic degradation of AOD-9401. AOD-9401 was tested for its in vitro stability against potential gastrointestinal degradation, according to the standard protocols of enzyme digestion for protein (26). The procedure of De Laureto et al. (4) was used to evaluate the rate of degradation by measuring the residual peptide with RP-HPLC techniques as well as amino acid analysis.
Experimental animals and oral treatment. Eighteen male C57BL/6J (ob/ob) mice aged 10-12 wk and weighing 46.4 * 1.1 (SE) g were used in this study. The animals were divided into saline (n = 8) or AOD-9401 treatment groups (n = 10) and were matched for body weight and coïtus. Animals were housed in a normal 12:12-h light-dark cycle at a constant room temperature of 23°C in the Departmental Animal House at Monash University. Animals were fed ad libitum a standard laboratory nonpurified diet (Clark King, Melbourne, Australia) and allowed free access to water at all times. The mice were given daily oral doses of either AOD-9401 (500 µg/kg body wt) dissolved in 0.3 ml saline or only saline of equivalent volume for 30 days. Accurate dosing was facilitated by a stainless steel gavage needle (7.5 × 0.1 cm diameter). The dose was administered slowly to avoid reflux.
Measurements of body weight gain, food consumption, energy expenditure, and physical activity. The body weights of the mice were measured before treatment and then every 2 days until the end of the study. Food consumption was measured every 2 days after treatment started and averaged to give a daily measurement. Grams of nonpurified diet consumed were multiplied by 2.85 to give caloric intake in kilocalories per day and then multiplied by 4.184 to convert to kilojoules per day. Energy expenditure was measured at the end of the treatment period with an indirect calorimeter (Columbus Instruments, Columbus, Ohio).
Gravimetrically determined standard gas mixtures of 20.48% O2 and 0.5% CO2 were used to calibrate the machine before use (BOC Gases Australia, Preston, Victoria, Australia). After gavage, mice were fasted for 2 h and then placed in a 20 × 13 × 11-cm Perspex box through which fresh air was drawn at a rate of 0.65 l/min. Mean rates of CO2 produced and O2 consumed were calculated every 3 min over a 30-min period. Rates of energy expenditure (kcal/min) were calculated using data from the final 15 min, after assuming a urinary nitrogen excretion of 0.84 mg · min1 · kg body wt1 (27). For the last 3 days of the treatment period, voluntary physical activity levels were also measured in these mice by use of 15-cm-diameter running wheels. Continuous 24-h monitoring of the use of the running wheels was made using a computerized meter (7).
Acute effect of AOD-9401 on fat oxidation in vivo. The acute effect of AOD-9401 on the rates of fat and glucose oxidation was assessed at the conclusion of the 30-day oral treatment period in the obese ob/ob mice after food intake, physical activity, and resting energy expenditure studies had been completed. On the morning of the last day of the study, a group of three saline-treated mice and four AOD-9401-treated mice were food-deprived for 1 h; then basal fat oxidation, glucose oxidation, and energy expenditure were measured for 10 min with the indirect calorimetry procedure described previously. The mice were then given an intraperitoneal injection of saline (in the saline-treated group) or AOD-9401 (250 µg/kg in the AOD-treated group), and rates of fat oxidation, glucose oxidation, and energy expenditure were measured for a further 18 min.
Isolation of adipose tissues. Groups of mice were killed with a lethal dose of pentobarbitone (0.2 ml) 24 h after the last treatment with oral AOD-9401 on day 30. Energy expenditure measurements after the intraperitoneal AOD-9401 dosing were not conducted on these mice. Epididymal fat pads from male mice were isolated as in our previous studies (14). The tissues were washed in room-temperature saline before being weighed into ~200-mg pieces for ex vivo assays. For in vitro assays, male C57BL/6J (ob/ob) mice were used. Male Zucker rats (200-300 g, 12 wk of age) were used to assess adipose tissue fat oxidation rates in vitro in response to AOD-9401. Human subcutaneous abdominal adipose tissue was obtained with consent from an overweight female patient (age: 42 yr; body mass index: 28.4) who had no other known medical complications and who had undergone fat-reduction surgery for cosmetic reasons.
Assay for lipogenic activity in adipose tissue. The rate of incorporation of exogenous [14C]glucose into total lipid in adipose tissue was used as an index of lipogenic activity. Tissues were placed in 2 ml of Krebs-Ringer bicarbonate (KRB) buffer (pH 7.4) containing 2% defatted BSA and 0.1 mg/ml glucose and then were gassed with 95% O2-5% CO2 in a shaking water bath, with temperature controlled at 37°C. After 30 min of preincubation, the tissues were transferred to another 2 ml of fresh medium containing [14C]glucose (final specific activity of 0.05 µCi/µmol) for a further 90 min (same conditions as above). Tissues were then removed and rinsed thoroughly in saline, and lipid was extracted with a chloroform-methanol (2:1) mixture. 14C radioactivity was counted on a Wallac 1410 liquid scintillation counter (Turku, Finland). The rates of total lipid synthesis were expressed as picomoles of glucose incorporated into fat per milligram of tissue per hour.
Assay for lipolytic activity in adipose tissue. The rate of lipolytic activity was measured by the release of glycerol into the incubation medium. Tissue pieces were placed in 2 ml KRB buffer with 2% BSA and 0.1 mg/ml glucose and incubated for 60 min (same conditions as above). The tissues were then removed and discarded, and the amount of glycerol present in the incubation medium was enzymatically assayed using glycerol phosphate oxidase reactions (Sigma Diagnostics, catalog no. GPO-337; St Louis, MO). Glycerol was determined with a spectrophotometer and converted to micromoles of glycerol released per gram of tissue per hour.
Plasma measurements. Blood samples were collected from the tail vein of anesthetized animals in capillary tubes after chronic treatment. Plasma was stored at 20°C until used. Glucose was measured using a 2300 STAT glucose analyzer. Free fatty acids (FFAs) were determined by the method developed from Noma (15). Triglycerides (TGs) were measured with a kit according to the recommendations of the manufacturer (Sigma).
In vitro FFA oxidation assay. FFA oxidation was determined by measuring the converted [14C]O2 from [1-14C]palmitic acid (23). [14C]O2, a final product of -oxidation of FFA, was trapped by hyamine hydroxide and measured by a liquid scintillation counter. Adipose tissues removed from laboratory animals were sliced into segments of ~200 mg each. The tissues were placed in 25-ml vials containing 2 ml of Krebs-Ringer phosphate buffer and 4% defatted BSA and then were preincubated at 37°C for 30 min under an atmosphere of carbogen (95% O2-5% CO2). Tissues were then transferred to Konte flasks containing fresh incubation medium, with 0.15 mM sodium palmitate and 0.20 µCi/µmol of 14C specific activity and different concentrations of hGH-(177-191) peptide. A filter paper roll was placed in a well inside the flask and then was sealed with a rubber septum stopper. Flasks were incubated at 37°C for 1 h, and the reaction was terminated by injecting 250 µl of 4.5 M H2SO4 with a needle through the rubber septum into the medium of a flask. Hyamine hydroxide (250 µl) was then injected into the filter paper roll in the center well. Incubation proceeded for another 60 min to ensure the complete absorption of released [14C]O2 by the paper roll. The filter paper rolls were then carefully removed and transferred to scintillation vials, and the 14C radioactivity was measured by a liquid scintillation counter. The rate of [14C]palmitic acid oxidation to [14C]O2 was calculated and expressed as micromoles per gram of tissue per hour.
Statistical analysis. The Student's t-test was used to analyze the results. All data are expressed as means * SE. P values of <0.05 were accepted as statistically significant.
