The male β3-KO mice and wild-type (WT) that were used in this study were offspring of animals provided by Dr. Bradford Howell (Beth Israel Hospital, Harvard Medical School, Boston, MA). The animals were bred and housed in the central animal house facility (Monash University). For chronic studies, animals were housed in the Departmental Animal Facility (Biochemistry, Monash University). For acute energy expenditure studies, animals were transported to the Department of Medicine (University of Melbourne), acclimatized, and killed following experimentation. The WT andβ 3-KO mice genotype were verified by breeding records and RT-PCR analysis performed in the laboratory of Professor Roger Summers.
A similar clinical study was conducted in obese subjects (METAOD002). In that double-blind placebo-controlled, 4 sequence, 4 period William’s Latin Square design study 23 subjects participated. The subjects were 19 to 50 years old and had a BMI ≥ 35 kg/m2 (range 36 to 67 kg/m2). Each subject received 4 single doses (25, 50 and 100 µg/kg AOD9604 or placebo; single IV infusion doses over 20 minutes), separated by a 7-day washout period.
The PCR reaction mixture contained 1 U Taq polymerase (Life Technologies, Inc.), the supplied buffer [20 mM Tris-HCl (pH 8.4) and 50 mM KCl], 200 μM dNTPs, 2 mM Mg-acetate, 2.5 pmol of forward primer, 2.5 pmol labeled reverse primer, and cDNA in a vol of 10 μl. The PCR reactions were carried out in a Hybaid PCR Sprint machine (Hybaid, Ltd., Middlesex, UK). Following the initial heating of the samples at 95 C for 2 min, each cycle of amplification consisted of 30 sec at 95 C, 30 sec at 64 C, and 30 sec at 72 C. It was found that 24 cycles were optimum for the amplification process.
The effects of hGH and AOD9604 on fat metabolism may be mediated by an alteration in the expression of a lipolytic/antilipogenic gene. Theβ 3-AR is a major lipolytic receptor identified in rodent fat cells (18) that mediates its effects through G protein coupling to adenylate cyclase, generation of cAMP, and stimulation of PKA (19). This enzyme then phosphorylates proteins in the lipolytic cascade, including hormone-sensitive lipase (20). In BAT, the β3-AR stimulates uncoupling of the electron transport chain, enhancing the ability of mitochondria to generate heat in preference to ATP through the dissipation of the electron gradient (21). Mice that lack this receptor have lower rates of resting energy expenditure (0.0041 vs. 0.0047 kcal/min, P < 0.02) and lower rates of fat oxidation (0.00019 vs. 0.00030 g/min, P < 0.02) than control mice (data not shown).
The lateral and medial condyles of the femur and tibia were fixed with 10% neutral buffered formalin and decalcified with 20% ethylenediaminetetraacetic acid (EDTA). Calcified condyles were embedded in paraffin, and standard frontal sections of 5 μm were prepared and stained with haematoxylin and eosin in the cartilage of the lateral part of the femoral condyle, according to gross morphological observations . If the staining was not adequate, the specimen was cut at the next cartilage surface. Cartilage degradation features were analyzed using the scoring system modified by Mankin et al. . Histopathological evidence of cartilage degeneration was evaluated by structural scoring (0, normal; 1, surface irregularities; 2, pannus and surface irregularities; 3, clefts to transitional zones; 4, clefts to radial zones; 5, clefts to calcified zones; and 6, complete disorganization) and cell status (0, normal; 1, diffuse hypercellularity; 2, cloning; and 3, hypocellularity) of the articular cartilage. Total score ranged from 0 (normal) to 9 (complete disorganization and hypocellularity of the articular cartilage). All sections were graded by two independent pathologists who did not have any information about the injection solutions.
Both human GH (hGH) and a lipolytic fragment (AOD9604) synthesized from its C-terminus are capable of inducing weight loss and increasing lipolytic sensitivity following long-term treatment in mice. One mechanism by which this may occur is through an interaction with the beta-adrenergic pathway, particularly with the beta(3)-adrenergic receptors (beta(3)-AR). Here we describe how hGH and AOD9604 can reduce body weight and body fat in obese mice following 14 d of chronic ip administration. These results correlate with increases in the level of expression of beta(3)-AR RNA, the major lipolytic receptor found in fat cells. Importantly, both hGH and AOD9604 are capable of increasing the repressed levels of beta(3)-AR RNA in obese mice to levels comparable with those in lean mice. The importance of beta(3)-AR was verified when long-term treatment with hGH and AOD9604 in beta(3)-AR knock-out mice failed to produce the change in body weight and increase in lipolysis that was observed in wild-type control mice. However, in an acute experiment, AOD9604 was capable of increasing energy expenditure and fat oxidation in the beta(3)-AR knock-out mice. In conclusion, this study demonstrates that the lipolytic actions of both hGH and AOD9604 are not mediated directly through the beta(3)-AR although both compounds increase beta(3)-AR expression, which may subsequently contribute to enhanced lipolytic sensitivity.
Results: After a single injection of CJC 1295, there were dose dependent increases in mean plasma GH concentrations by 2- to 10-fold for 6 d or more and in mean plasma IGF-I concentrations by 1.5- to 3-fold for 9–11 d. The estimated half-life of CJC 1295 was 5.8–8.1 d. After multiple CJC 1295 doses, mean IGF-I levels remained above baseline for up to 28 d. No serious adverse reactions were reported.
The first was a double-blind, placebo-controlled, parallel group, multi-center study (5 Australian hospitals) (METAOD005). In this study 300 healthy obese males and females (BMI ≥ 35 kg/m2; Median BMI: 40 kg/m2, range: 35 to 67 kg/m2; 30 to 65 years old; 54% males and 46% females) were randomized to a 14-week period of daily oral dosing. The treatment period comprised a 2-week single-blind placebo run-in period followed by 12 weeks administration of either placebo or AOD9604 (1, 5, 10, 20 or 30 mg AOD9604 or placebo once daily; n =50 per group).
Mean gross morphological and histopathological scores in Group 1 were significantly higher than those in Groups 2, 3, and 4 (p<0.05). Mean gross morphological and histopathological scores in Group 4 were significantly lower than those in Groups 2 and 3 (p<0.05). However, there are no differences between the mean gross morphological and histopathological scores of Groups 2 and 3 (Figures 4 and 5).
GHRP + GHRH (twice per day) ◦Inject your GHRP + GHRH peptides together in the same syringe (ensuring you have not consumed any food/beverages for at least 1 hour before, an optimal time would be first thing in the morning). ◦Ingest a protein only or protein and carbohydrate meal afterward to create an insulin spike. ◦Do weight training in the hours afterwards. ◦at least 1 hour after your dinner (or last meal of the day), take your second GHRP + GHRH injection. ◦If you are trying to control your body fat then have a protein only meal 20-30 minutes afterwards, otherwise a protein/carbohydrate meal will create a better insulin spike.
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