Leptin (116-130) amide (mouse)

In vitro pituitary and testicular effects of the leptin-related synthetic peptide leptin(116-130) amide involve actions both similar to and distinct from those of the native leptin molecule in the adult rat

The obese gene (ob) product, leptin, has recently emerged as a key element in body weight homeostasis, neuroendocrine function and fertility. Identification of biologically active, readily synthesized fragments of the leptin molecule has drawn considerable attention, as they may provide a powerful tool for detailed characterization of the biological actions of leptin in different experimental settings. Recently, a fragment of mouse leptin protein comprising amino acids 116-130, termed leptin(116-130) amide, was shown to mimic the effects of the native molecule in terms of body weight gain and food intake, and to elicit LH and prolactin (PRL) secretion in vivo. As a continuation of our previous experimental work, the present study reports on the effects of leptin(116-130) amide on basal and stimulated testosterone secretion by adult rat testis in vitro. In addition, a comparison of the effects of human recombinant leptin and leptin(116-130) amide at the pituitary level on the patterns of LH, FSH, PRL and GH secretion is presented. As reported previously by our group, human recombinant leptin(10(-9)-10(-7)M) significantly inhibited both basal and human chorionic gonadotrophin (hCG)-stimulated testosterone secretion in vitro. Similarly, incubation of testicular tissue in the presence of increasing concentrations of leptin(116-130) amide (10(-9)-10(-5)M) resulted in a dose-dependent inhibition of basal and hCG-stimulated testosterone secretion; a reduction that was significant from a dose of 10(-7)M upwards. In addition, leptin(116-130) amide, at all doses tested (10(-9)-10(-5)M), significantly decreased LH and FSH secretion by incubated hemi-pituitaries from adult male rats. In contrast, in the same experimental protocol, recombinant leptin(10(-9)-10(-7)M) was ineffective in modulating LH and FSH release. Finally, neither recombinant leptin nor leptin(116-130) amide were able to change basal PRL and GH secretion in vitro. Our results confirm the ability of leptin, acting at the testicular level, to inhibit testosterone secretion, and map the effect to a domain of the leptin molecule that lies between amino acid residues 116 and 130. In addition, we provide evidence for a direct inhibitory action of leptin(116-130) amide on pituitary LH and FSH secretion, a phenomenon not observed for the native leptin molecule, in the adult male rat.

The effects of age on uptake, serum half-life and bioavailability of mouse [D-Leu-4]OB3, a synthetic peptide amide with leptin-like activity, in male C57BL/6J mice

We have previously shown that the activity of a synthetic peptide amide corresponding to amino acid residues 116-130 of mouse leptin is contained in a restricted sequence at the amino terminus of the peptide, between residues 116 and 122 (Ser-Cys-Ser-Leu-Pro-Glu-Thr). This peptide was named mouse OB3. The potency of OB3 was improved by replacing the L-leucine residue at position four with its D-isomer. Intraperitoneal administration (i.p.) of mouse OB3 or [D-Leu-4]OB3 to ob/ob and db/db mice reduces food intake, body weight gain and serum glucose levels, and enhances insulin sensitivity. These effects of OB3 and [D-Leu-4]OB3 are very pronounced in young mice and diminish with age. In the present study, we measured uptake, serum half-life, and bioavailability of mouse [D-Leu-4]OB3 in mice of different ages. Groups of male C57BL/6J mice, six and 25 weeks of age, were given a single i.p. injection of 1 mg mouse [D-Leu-4]OB3 in PBS. Five, 10, 20, 40, 60, 120, or 180 min after injection, the mice were anesthetized and exsanguinated. Serum samples were prepared and assayed for mouse [D-Leu-4]OB3 content by competitive ELISA. In six week-old mice, the maximum concentration of mouse [D-Leu-4]OB3 was reached in 10 min, and the serum half-life was approximately 52.5 min. In 25 week-old mice, however, mouse [D-Leu-4]OB3 peaked in 5 min, and the serum half-life was approximately 30.6 min. The relative bioavailability of mouse [D-Leu-4]OB3 in six and 25 week-old mice was determined by measuring the area under the uptake curves. Bioavailability of mouse [D-Leu-4]OB3 was approximately 20% greater in six week-old mice than in 25 week-old mice. The results of this study indicate that at least some pharmacokinetic parameters of peptide uptake change as mice age. They also suggest that differences in uptake, serum half-life, and relative bioavailability of mouse [D-Leu-4]OB3 may contribute, at least in part, to the reduced efficacy of bioactive leptin-related peptides we have consistently observed in ob/ob and db/db mice as they age.

Synthetic leptin c-fragment peptide minimises heat-induced impairment of spermatogenesis in mice via Stat3 signalling

Mammalian spermatogenesis is a temperature-sensitive process, and an increase in testicular temperature impairs spermatogenesis. Leptin modulates testicular activity, but the effect of leptin or its synthetic analogue on heat-induced testicular impairment is unclear. We investigated the effects of synthetic leptin peptide (116-130 amides) on testicular activity in heat-stressed mice model. 15 adult mice (25.54 ± 1.43 g) were selected for the study. Ten mice were subjected to a single heat stress treatment (HS) at 43 °C for 15 min by submerging the lower half of the body in a thermostatic water bath. After heat treatment, mice were divided into two groups, the heat-stressed HS group (n = 5) and the second group as HSL, treated with leptin peptide (116-130 amide) for 14 days. The HS group showed a significant (p < 0.05) decline in the GSI (0.25 ± 0.018), Johnsenscore (4.5 ±.19), seminiferous tubule diameter (160.75 ± 10.18 μm), germinal epithelium height, (GEH) (37.5 ± 1.59 μm) compared to the CN (GSI-0.37 ± 0.015; Johnsen score-7.9 ± 0.20; GEH- 73.25 ± 1.29 μm; tubule diameter-230.25 ± 1.39 μm) and the HSL groups (GSI-0.38 ± 0.014; Johnsen' score-8.0 ± 0.32; GEH- 37.5 ± 1.59 μm; tubule diameter-160.75 ± 10.18 μm) groups. Heat treatment significantly (p < 0.05) increased the intra-testicular levels of leptin (HS-20.11 ± 2.1 pg/mg protein; CN-10.50 ± 0.17 pg/mg protein; HSL-12.99 ± 0.52 pg/mg protein) with a reduced level of pStat3, suggesting leptin resistance during testicular hyperthermia. Furthermore, heat treatment was associated with significantly (p < 0.05) decreased germ cell proliferation and reduced circulating testosterone levels (HS-2.69 ± 2.01 ng/mL; CN-7.69 ± 0.32 ng/mL; HSL-5.36 ± 0.73 ng/mL). However, the circulating androstenedione levels showed a significant (p < 0.05) increase in the HS group (0.75 ± 0.03 ng/mL) compared to the CN (0.51 ± 0.02 ng/mL) and HSL (0.57 ± 0.07 ng/mL) groups. Immunolocalisation of 3β-HSD showed moderate to faint staining in the Leydig cells in the HS group compared to the CN and HSL groups. Treatment with leptin peptide resulted in decrease in the intra-testicular leptin levels with increased phosphorylation of Stat3, suggesting improved leptin resistance, which was positively associated with increased germ cell proliferation, elevated testosterone levels, and improved testicular histoarchitecture. Testicular hyperthermia may cause leptin resistance and impaired leptin signalling, decreased testosterone biosynthesis and suppressed spermatogenesis, which could be a manifestation of leptin resistance. Treatment with leptin peptide improves leptin signalling and testicular activity in heat-stressed mice, but the underlying mechanism is still unclear.

Intranasal delivery of mouse [D-Leu-4]-OB3, a synthetic peptide amide with leptin-like activity, improves energy balance, glycaemic control, insulin sensitivity and bone formation in leptin-resistant C57BLK/6-m db/db mice

Background: We have recently shown that intranasal administration of mouse [D-Leu-4]-OB3 reconstituted in Intravail(?) to male Swiss Webster mice resulted in significantly higher uptake and bioavailability when compared with commonly used injection methods of delivery.
Aim and methods: In this study, we examined the effects of intranasal delivery of mouse [D-Leu-4]-OB3 in Intravail(?) on energy balance, glucose regulation, insulin secretion and serum levels of osteocalcin, a specific and sensitive marker of bone formation. Genetically obese C57BLK/6-m db/db mice were allowed food and water ad libitum and given either Intravail(?) alone or mouse [D-Leu-4]-OB3 in Intravail(?) for 14 days by intranasal instillation.
Results: Mouse [D-Leu-4]-OB3 reduced body weight gain, daily food intake, daily water intake and serum glucose by 11.5, 2.2, 4.0 and 61.9%, respectively. Serum insulin levels in db/db mice given mouse [D-Leu-4]-OB3 were approximately threefold lower than those in mice receiving Intravail(?) alone. Mouse [D-Leu-4]-OB3 elevated serum osteocalcin in db/db mice by 28.7% over Intravail(?) treated control mice.
Conclusions: The results of our study indicate that intranasal delivery of biologically active mouse [D-Leu-4]-OB3 in Intravail(?) is feasible and has significant effects on regulating body weight gain, food and water intake, serum glucose, insulin sensitivity and bone formation in leptin-resistant C57BLK/6-m db/db mice.

Oral delivery of mouse [D-Leu-4]-OB3, a synthetic peptide amide with leptin-like activity, in male Swiss Webster mice: a study comparing the pharmacokinetics of oral delivery to intraperitoneal, subcutaneous, intramuscular, and intranasal administration

We have recently shown that intranasal administration of mouse [D-Leu-4]-OB3 reconstituted in Intravail to male Swiss Webster mice resulted in significantly higher bioavailability than commonly used injection methods of delivery. The absorption profile associated with intranasal delivery of mouse [D-Leu-4]-OB3 showed an early peak representing rapid uptake across the nasal mucosa, and a later peak suggesting a gastrointestinal site of absorption. In the present study, we show that gastrointestinal absorption of mouse [D-Leu-4]-OB3 does occur, and that reformulation of mouse [D-Leu-4-OB3 with Intravail significantly enhances its uptake. The pharmacokinetics of orally delivered (by gavage) mouse [D-Leu-4]-OB3 in the absence or presence of Intravail were examined, and compared to previously reported pharmacokinetic parameters of mouse [D-Leu-4]-OB3 following intraperitoneal (ip), subcutaneous (sc), intramuscular (im), and intranasal administration. When compared to oral delivery in PBS, Intravai significantly enhanced the total uptake (552,710 ng/ml/min vs.137,585 ng/ml/min) and relative bioavailability (4.0 vs. 1.0) of mouse [D-Leu-4-OB3. The relative oral bioavailabilities of mouse [D-Leu-4]-OB3 when compared to ip, sc, im, and intranasal delivery were 52.2%, 47.3%, 37.8% and 12.9%, respectively. The results of this study indicate that oral delivery of mouse [D-Leu-4]-OB3 in Intravail is an effective method of administration achieving relatively high serum levels of the bioactive peptide when compared to commonly used methods of injection. In addition to intranasal administration, oral delivery of mouse [D-Leu-4]-OB3 in Intravail may have potential as a novel, non-invasive approach to the treatment of obesity and its associated metabolic dysfunctions in humans.