Oral delivery of mouse [d-Leu-4]-OB3, a synthetic peptide amide with leptin-like activity, in male C57BL/6J wild-type and ob/ob mice: effects on energy balance, glycaemic control and serum osteocalcin levels.

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 bioavailability than commonly used injections methods of delivery. The absorption profile associated with intranasal delivery of mouse [d-Leu-4]-OB3 showed an early peak representing absorption across the nasal mucosa, and a later peak suggesting a gastrointestinal site of uptake. AIM AND METHODS: In the present study, we examined the effects of orally administered (by gavage) mouse [d-Leu-4]-OB3 on energy balance, glycaemic control and serum osteocalcin levels in male C57BL/6J wild-type and ob/ob mice allowed food and water ad libitum or calorie restricted by 40% of normal intake. RESULTS: In wild-type mice fed ad libitum, oral delivery of mouse [d-Leu-4]-OB3 reduced body weight gain, food intake and serum glucose, by 4.4, 6.8 and 28.2% respectively. Serum osteocalcin levels and water intake were essentially the same in control and treated wild-type mice. In ob/ob mice fed ad libitum, mouse [d-Leu-4]-OB3 reduced body weight gain, food intake, water intake and serum glucose by 11.6, 16.5, 22.4 and 24.4% respectively. Serum osteocalcin in ob/ob mice treated with mouse [d-Leu-4]-OB3 was elevated by 62% over controls. Calorie restriction alone caused significant weight loss in both wild-type (9.0%) and ob/ob (4.8%) mice, and mouse [d-Leu-4]-OB3 did not further enhance this weight loss. As expected, serum glucose levels in wild-type and ob/ob mice were significantly reduced by calorie restriction alone. Mouse [d-Leu-4]-OB3 further reduced serum glucose in wild-type mice and normalized levels in ob/ob mice. Calorie restriction alone reduced serum osteocalcin levels by 44.2% in wild-type mice and by 19.1% in ob/ob mice. Mouse [d-Leu-4]-OB3 prevented this decrease in groups of mice. CONCLUSIONS: The results of this study suggest that oral delivery of mouse [d-Leu-4]-OB3 in Intravail is possible and may have potential not only as an alternative therapy in the treatment of human obesity and some of its associated metabolic dysfunctions, but also may help to prevent and/or reverse at least some of the bone loss which accompanies osteoporosis, anorexia nervosa and other wasting diseases.

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.

Inhibitory effects of leptin-related synthetic peptide 116-130 on food intake and body weight gain in female C57BL/6J ob/ob mice may not be mediated by peptide activation of the long isoform of the leptin receptor.

We recently reported that intraperitoneal administration of leptin-related synthetic peptide 116-130 [LEP-(116-130)] resulted in reduced food intake and significant weight loss in homozygous female C57BL/6J ob/ob mice. In this study, we used two in vitro bioassays to show that the interaction of LEP-(116-130) with the long form of the leptin receptor (OB-Rb), the receptor isoform that is predominantly expressed in the hypothalamus, is not required for the observed in vivo effects of the peptide on energy balance. LEP-(116-130) was unable to compete the binding of alkaline phosphatase-leptin fusion protein to OB-R. Moreover, LEP-(116-130) was unable to activate signal transduction by OB-Rb in vitro. In homozygous female C57BLKS/J-m db/db mice that do not express OB-Rb, intraperitoneal administration of LEP-(116-130) reduced body weight gain and blood glucose levels but not food intake, which further supports a mechanism of action that does not require peptide interaction with OB-Rb.

Design of a synthetic leptin agonist: effects on energy balance, glucose homeostasis, and thermoregulation.

We have previously reported that a synthetic peptide amide corresponding to amino acid residues 116-130 of mouse leptin, LEP-(116-130), reduces body weight gain, food intake, and blood glucose levels in ob/ob and db/db mice. In the present study we show that the activity of LEP-(116-130) resides in a restricted sequence between amino acid residues 116-122. A synthetic peptide corresponding to this sequence (Ser-Cys-Ser-Leu-Pro-Gln-Thr) has been named OB3. Single point D-amino acid substitution was used to study the structure-function relationship of each residue in OB3. D-Amino acid analogs of OB3 were synthesized by the solid phase method, purified to 98+%, and administered (1 mg/day, ip) for 7 days to female C57BL/6J ob/ob mice. The effects of the peptides on body weight gain, food and water intake, glucose homeostasis, and thermoregulation were assessed. In most cases, the efficacy of OB3 on all parameters tested was reduced by substitution of an L-amino acid with its corresponding D-isoform. A statistically significant increase (2.6-fold) in the weight-reducing effect of OB3, however, was observed by inversion of the configuration of the leucine residue at position 4 (Leu-4) of OB3 by substitution with its D-amino acid isoform [D-Leu-4]. Compared with OB3, mice treated with [D-Leu-4]-OB3 consumed 7.9% less food and 16.5% less water. Blood glucose was normalized to levels comparable to those in wild-type control mice within 2 days after initiation of [D-Leu-4]-OB3 treatment. Unlike native leptin, however, neither OB3 nor any of its D-amino acid-substituted analogs had any apparent effect on thermogenesis. Our results indicate that synthetic peptide strategies may be useful in the development of potent and stabile pharmacophores with potential therapeutic significance in the treatment of human obesity and its related metabolic dysfunctions.

In vivo effects of leptin-related synthetic peptides on body weight and food intake in female ob/ob mice: localization of leptin activity to domains between amino acid residues 106-140.

In C57BL/6J ob/ob mice, a single base mutation of the ob gene in codon 105 results in the replacement of arginine by a premature stop codon and production of a truncated inactive form of leptin. These observations suggest that leptin activity may be localized, at least in part, to domains distal to amino acid residue 104. To investigate this possibility, we synthesized six overlapping peptide amides corresponding to residues 106-167 of leptin, and examined their effects on body weight and food intake in female C57BL/6J ob/ob mice. When compared with vehicle-injected control mice, weight gain by mice receiving 28 daily 1-mg i.p. injections of LEP-(106-120), LEP-(116-130), or LEP-(126-140) was significantly (P < 0.01) reduced with no apparent toxicity. Weight gain by mice receiving LEP-(136-150), LEP-(146-160), or LEP-(156-167) was not significantly different from that of vehicle-injected control mice. The effects of LEP-(106-120), LEP-(116-130), or LEP-(126-140) were most pronounced during the first week of peptide treatment. Within 7 days, mice receiving these peptides lost 12.3%, 13.8%, and 9.8%, respectively, of their initial body weights. After 28 days, mice given vehicle alone, LEP-(136-150), LEP-(146-160), or LEP-(156-167) were 14.7%, 20.3%, 25.0%, and 24.8% heavier, respectively, than they were at the beginning of the study. Mice given LEP-(106-120) or LEP-(126-140) were only 1.8% and 4.2% heavier, respectively, whereas mice given LEP-(116-130) were 3.4% lighter. Food intake by mice receiving LEP-(106-120), LEP-(116-130), or LEP-(126-140), but not by mice receiving LEP-(136-150), LEP-(146-160), or LEP-(156-167), was reduced by 15%. The results of this study indicate 1) that leptin activity is localized, at least in part, in domains between residues 106-140; 2) that leptin-related peptides have in vivo effects similar to those of native leptin; and 3) offer hope for development of peptide analogs of leptin having potential application in human or veterinary medicine.

Long term follow-up of transsphenoidal surgery for the treatment of Cushing's disease in childhood.

Transsphenoidal removal of pituitary adenomas has become the accepted treatment for Cushing's disease. As Cushing's disease is rare in childhood, few reports describe extended follow-up after transsphenoidal surgery for this disease in children. Twenty-two patients less than 19 yr of age were diagnosed with Cushing's disease by standard testing. All patients underwent transsphenoidal surgery (TSS) as primary treatment at Mayo Clinic between 1975 and 1990. Follow-up was available in all patients and averaged 6.7 yr. Adenomas were identified pathologically in 19 patients and visualized by the surgeon in the remainder. There was 1 macroadenoma. Ten patients are considered cured with a mean follow-up of 5.5 yr. Five patients had evidence of persistent disease within 6 months of TSS and required further treatment. Seven patients had remission lasting greater than 6 months with subsequent relapse (mean time to recurrence, 7.0 yr). In children treated with TSS for Cushing's disease, the rate of cure with extended follow-up is only approximately 50%. This rate is much lower than that in adults and may indicate that Cushing's disease is more aggressive in childhood. As TSS is associated with low morbidity, we feel it is still the treatment of choice for Cushing's disease in children.

Pituitary adenomas in childhood and adolescence.

A clinicopathological study of 56 pediatric patients with non-ACTH-secreting pituitary adenomas removed by a transsphenoidal neurosurgical approach was undertaken to better define the clinical presentation, to assess demographic factors, to determine the immunohistochemical staining characteristics of the tumors, and to evaluate the outcome of transsphenoidal surgical treatment and other adjuvant therapies. A separate analysis of prolactinoma patients was performed. All tumors were confirmed histologically and immunophenotyped for pituitary hormones. Forty-one patients had tumors that stained for PRL alone, eight patients had tumors that stained for PRL and GH, six patients had plurihormonal adenomas, and one patient had a tumor that stained for glycoprotein hormones. No tumors contained GH alone. Macroadenomas exceeded microadenomas (1.4:1). There were no male patients with microadenomas of any type. Females outnumbered males (3.3:1). Patients presented most frequently with headache, menstrual dysfunction (in females), galactorrhea, and hypopituitarism. All but one of the patients with hypopituitarism at presentation had macroadenomas. Tumor staining characteristics did not always correlate well with clinical status, especially with regard to GH-containing tumors. Pediatric pituitary tumors did not appear to be more invasive or more aggressive than adult pituitary tumors, contrary to some previous reports. The patients with microadenomas had a 70% operative cure rate and a 65% long term cure rate; the recurrence rate for microadenoma patients was 25%. Macroadenoma patients had a 33% operative cure rate, a 55% long term cure rate, and a recurrence rate of 33%. Thus, microadenoma and macroadenoma patients had similar long term cure rates, but macroadenoma patients required more aggressive adjuvant therapy (second surgery, radiation, or bromocriptine) and had higher rates of hypopituitarism (52% of macroadenoma patients vs. 0% of microadenoma patients required long term hormone replacement).

Cushing's disease in children.

As with adults, the diagnosis of Cushing's disease in childhood can be difficult. The clinical presentation in children is distinctive in terms of the effects on growth and pubertal development. The biochemical diagnosis can usually be made with standard dexamethasone suppression testing, similar to such testing in adults. The clinical course of Cushing's disease in children tends to be more aggressive than in adults, as evidenced by higher rates of Nelson's syndrome after bilateral adrenalectomy and higher rates of recurrence after removal of corticotrope adenomas. TSS is well tolerated in children and has fewer side effects than other modalities. Currently, pituitary irradiation is best reserved as adjunctive therapy. Newer stereotactic computer assisted linear accelerator techniques (e.g., proton knife) may provide increased effectiveness with fewer side effects and be particularly useful in childhood Cushing's disease. Appropriate treatment of children with Cushing's disease can be compatible with satisfactory psychosocial adjustment and fertility in adulthood.

Diagnosis of corticotropin-producing bronchial carcinoid tumors causing Cushing's syndrome.

Cushing's syndrome due to ectopic production of adrenocorticotropic hormone (corticotropin) has been recognized for many years. Traditionally, clinicians have thought that most cases were due to lung carcinomas and that the clinical manifestations differed from those for pituitary-dependent Cushing's syndrome. We report two cases of corticotropin-producing bronchial carcinoid tumors that were clinically and biochemically indistinguishable from pituitary-dependent Cushing's syndrome. Review of the literature revealed that bronchial carcinoid tumors are the most common cause of Cushing's syndrome due to ectopic secretion of corticotropin. On biochemical and anatomic studies, they are frequently indistinguishable from pituitary-dependent Cushing's syndrome and thus may be difficult to diagnose. Inferior petrosal sinus sampling for corticotropin and computerized imaging of the chest may be the best aids in making the diagnosis.

Epitope mapping of secreted mouse leptin utilizing peripherally administered synthetic peptides.

We have recently reported that intraperitoneal (i.p.) administration of synthetic peptide amides corresponding to amino acids 106-140 of mouse leptin significantly reduced food intake and body weight gain in female C57BL/6J ob/ob mice. These results suggested that leptin activity was localized in domains toward its C-terminus between residues 106-140. In the present study, 14 overlapping peptides encompassing the complete sequence of secreted mouse leptin were synthesized, and their effects on body weight and food intake in female C57BL/6 J ob/ob mice were assessed. When given as seven daily 1-mg i.p. injections, only peptides corresponding to amino acids 106-120, 116-130 and 126-140 caused significant reductions in body weight and food intake. These results confirmed our earlier study and suggest that in contrast to the domain encompassed by amino acids 106-140, the N-terminal of mouse leptin between amino acids 21-105 may not contain functional epitopes that can be utilized as lead compounds in the development of peripherally administered bioactive peptide analogs or nonpeptide mimetics of leptin, which may have potential usefulness in treatment of the energy imbalance associated with obesity.

[D-LEU-4]-OB3, a synthetic leptin agonist, improves hyperglycemic control in C57BL/6J ob/ob mice.

We have recently shown that the activity of a synthetic peptide corresponding to amino acid residues 116-130 of secreted mouse leptin is contained in a restricted sequence at the amino terminus of the peptide, between residues 116-122 (Ser-Cys-Ser-Leu-Pro-Gln-Thr, OB3). Substitution of the Leu residue at position 4 of OB3 with its D-isomer ([D-Leu-4]-OB3) enhanced the ability of OB3 (1 mg/day, ip, 7 days) to reduce body weight gain, food and water intake, and serum glucose in female C57BL/6J ob/ob mice. In the present study, we have utilized a pair-feeding approach to demonstrate that the antihyperglycemic action of [D-Leu-4]-OB3 is not solely due to its effects on caloric intake. One group of female C57BL/6J ob/ob mice (n=6) was fed ad libitum, and two additional groups (n=6 per group) were allowed 3.0 g food/mouse daily, an amount previously determined to satisfy [D-Leu-4]-OB3-treated mice. At the end of the 7-day test period, vehicle-injected mice fed ad libitum were approximately 10% heavier than their initial body weights, while pair-fed mice injected with vehicle and [D-Leu-4]-OB3-treated mice lost 5% of their initial body weights. After 1 day of treatment, blood glucose was reduced by 20% in pair-fed vehicle-injected mice, and by 40% in mice given [D-Leu-4]-OB3. Food restriction reduced blood glucose throughout the 7-day study, but not to levels seen in wild-type nonobese C57BL/6J mice of the same sex and age. After 2 days of treatment with [D-Leu-4]-OB3, however, blood glucose was reduced to levels comparable to those seen in wild-type nonobese mice. [D-Leu-4]-OB3 also lowered serum insulin levels by 53% when compared to mice fed ad libitum. Neither pair-feeding nor [D-Leu-4]-OB3 treatment had any apparent effect on thermogenesis. These results suggest that [D-Leu-4]-OB3 exerts its effects on serum glucose not only by suppressing caloric intake, but also through a separate effect on glucose metabolism which may involve increased tissue sensitivity to insulin.

Thyroid storm.

Despite earlier recognition and treatment of hyperthyroidism, thyroid storm remains a life-threatening, although fortunately rare, medical emergency. Prompt recognition and aggressive treatment employing a multifaceted approach are generally effective at correcting the homeostatic decompensation that is the hallmark of thyroid storm. Research is furthering understanding of the cellular actions of thyroid hormone and may lead to additional, even more effective treatment modalities in the future.

Comparison of diabetes care provided by an endocrinology clinic and a primary-care clinic.

OBJECTIVE: To compare the quality of ambulatory diabetes care provided by physicians in an endocrinology clinic with that in a primary-care clinic. METHODS: We conducted a retrospective study of the medical records of patients with diabetes treated for 2 to 4 years in an endocrinology clinic and a primary-care clinic at an academic medical center. Adherence to American Diabetes Association (ADA) clinical practice recommendations and hemoglobin A(1c) (HbA(1c)) levels were assessed in randomly chosen patients-a total of 68 patients from the primary-care clinic and 105 patients from the endocrinology clinic, with total patient-years of follow-up of 241 and 370, respectively. RESULTS: In six of seven areas assessed, the endocrinology clinic was significantly more compliant with ADA recommendations than was the primary-care clinic: queries about hypoglycemia (88% versus 20%); frequency of glycated hemoglobin determinations (3.3 versus 2.1 per patient/yr); yearly lipid panel (44% versus 25%); and yearly ophthalmologic (90% versus 50%), neurologic (56% versus 37%), and foot (88% versus 59%) examinations (all P<0.001). The rate of yearly proteinuria evaluations was similar in the two clinics (66% versus 65%). On assessment of all patients, the mean HbA(1c) level was significantly lower in the endocrinology clinic (8.29%) than in the primary-care clinic (8.73%) (P = 0.01). CONCLUSION: Adherence to ADA clinical practice recommendations was significantly better in the endocrinology clinic than in the primary-care clinic. This finding and the significantly lower levels of HbA(1c) in patients in the endocrinology clinic setting would be expected to translate into improved long-term patient outcome.

Induction of adrenal suppression by megestrol acetate in patients with AIDS.

OBJECTIVE: To investigate the development of secondary adrenal suppression in a patient with the acquired immunodeficiency syndrome (AIDS) who was receiving megestrol acetate. DESIGN AND PATIENTS: Case report of one patient abruptly withdrawn from long-term therapy with megestrol acetate; prospective study of four patients with AIDS who were starting therapy with megestrol acetate for cachexia. SETTING: Outpatient clinic of a university hospital. INTERVENTIONS: Study patients received megestrol acetate, 80 mg three times daily. MEASUREMENTS: Study patients had cosyntropin-stimulation testing and oral glucose tolerance testing before and after starting therapy with megestrol acetate. RESULTS: The patient described in the case report developed symptoms of adrenal insufficiency after withdrawal of megestrol acetate after 4 years of treatment. His basal cortisol and adrenocorticotropic hormone (ACTH) levels were low. He showed an abnormally diminished response to a short cosyntropin-stimulation test but did respond to a 3-day cosyntropin-stimulation test. The morning cortisol levels of the study patients decreased significantly (from 11.0 +/- 1.8 micrograms/dL to 1.5 +/- 0.9 micrograms/dL; P < 0.01), and the ACTH levels of these patients decreased to below normal (from 16.6 +/- 5.5 pg/mL to 6.3 +/- 3.3 pg/mL; P = 0.02) during treatment with megestrol acetate. Cortisol levels after administration of cosyntropin decreased significantly (from 27.3 +/- 3.3 pg/mL to 9.3 +/- 6.3 pg/mL; P = 0.01) during treatment with megestrol acetate. The results of oral glucose tolerance testing in two patients were consistent with the development of insulin resistance, and daily insulin requirements increased 10-fold in a patient who had preexisting diabetes. CONCLUSIONS: Prolonged administration of megestrol acetate can induce clinically significant secondary adrenal suppression, and abrupt withdrawal of megestrol acetate after prolonged administration can cause adrenal insufficiency.

Synthetic analogs of the carboxyl-terminus of beta-thyrotropin: the importance of basic amino acids in receptor binding activity.

Previously, using a synthetic peptide strategy, we determined that four distinct regions of human beta-thyrotropin (beta TSH) were responsible for interaction of TSH with the TSH receptor. The most potent of these four regions was the carboxyl-terminus of the subunit, represented by the peptide sequence beta 101-112, which inhibited binding of radiolabeled beta TSH to receptor in radioreceptor assay with an IC50 of approximately 100 microM. In the current studies, we systematically substituted the native amino acids in region beta 101-112 with alanine, and we have determined which residues within this span are important to the binding activity of TSH to its receptor. Substitution of Lys101, Asn103, Tyr104, Cys105, Lys107, and Lys110 with alanine each caused a significant fall in activity as compared to the native sequence, whereas substitution at the remaining positions had little or no effect. Because three of these residues are positively charged at physiologic pH, we hypothesized that this charge may be important to the binding activity of the sequence. We modified the charge characteristics of the region by synthesizing two series of analogs in which the residues identified in the alanine substitution studies were substituted with Arg, D-Lys, and D-Arg at each position. In addition, a series of analogs containing basic residues, either added to or substituted for nonbasic residues in the sequence beta 101-112, was synthesized. Substitution of Arg, D-Lys, and D-Arg for Lys101, Lys107, and Lys110 had little effect on activity; however, inclusion of additional basic residues in the beta 101-112 sequence significantly enhanced the inhibitory activity of the region.(ABSTRACT TRUNCATED AT 250 WORDS)

Further characterization of the receptor-binding region of the thyroid-stimulating hormone alpha subunit: a comprehensive synthetic peptide study of the alpha-subunit 26-46 sequence.

Previously, using a synthetic peptide strategy, we determined that the region of the common glycoprotein hormone alpha subunit between residues 26 and 46 is a site of interaction of the hormone with the thyroid membrane-bound receptor for thyroid-stimulating hormone (TSH). We have undertaken to identify further the specific residues within this 21-amino acid span that are critical in hormone receptor binding. We synthesized three nested sets of peptide, two in which we systematically truncated the amino-terminal region of the sequence and another in which we truncated the carboxyl-terminal region, and we synthesized a fourth nested set in which we systematically substituted alanine for the native residues from the region of highest activity. Each peptide was tested in a TSH radioreceptor assay for its ability to inhibit binding of 125I-labeled bovine TSH to porcine thyroid membranes. Removal either by truncation or alanine substitution, of several specific residues resulted in a significant reduction in the ability of the sequence to interact with receptor; these residues included Cys31, Cys32, Phe33, Arg35, Arg42, Lys44, and Lys45, suggesting that they are crucial for binding activity. Loss of activity also occurred with substitution for Gly30 and Ser34, but the reduction was less pronounced. Amino-terminal truncation of the sequence through Arg35 (leaving the alpha-subunit peptide 36-46) resulted in greater than 98% loss of activity of the sequence. We conclude that two distinct receptor binding regions lie within the alpha-subunit 26-46 sequence. The first lies between residues Gly30 and Arg35 and includes Cys31, Cys32, and Phe33 as important constituents, and the second region lies between residues Arg42 and Lys45 and includes Lys44 as an important residue and Ser43 as a less important component.