Matthew C. Leinung

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 ip 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 ...

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.

BENEFITS OF CONTINUOUS GLUCOSE MONITOR USE IN CLINICAL PRACTICE

OBJECTIVE To determine the benefits of personal continuous glucose monitoring (CGM) outside of a controlled clinical trial in a single ambulatory diabetes clinic. METHODS In this retrospective study, we reviewed medical records of all patients who began CGM in our university-based clinical practice between July 2006 and October 2008. Data pertaining to 1 year before initiation of CGM through January 2009 were collected. All patient visits were performed by any 1 of 7 board-certified endocrinologists and/or 5 certified diabetes educators. A severe hypoglycemic event was considered to have occurred if the patient reported requiring assistance or losing consciousness, or if there was documentation from another source (eg, an emergency department visit). Analysis of the effect of CGM on hemoglobin A1c and occurrence of severe hypoglycemia was performed. RESULTS A total of 117 patients initiated CGM between July 2006 and October 2008 and used CGM for at least 2 months (total experience on CGM, 1136 patient-months; average 9.7 months per patient). Mean age was 44.5 +/- 12.8 years (range, 14.3-71.7 years), and average duration of diabetes mellitus was 23.9 years. All patients were using insulin pumps before initiation of CGM, including 10 patients with type 2 diabetes. Sixty-eight patients (58%) had preexisting hypoglycemia unawareness. Average hemoglobin A1c level for 1 year before CGM initiation was 7.6 +/- 1.1%, and with CGM use it dropped to 7.2 +/- 0.8% (P<.001). Forty-two patients had severe hypoglycemic events in the year before CGM use or during CGM use. Overall, CGM use was associated with a significant decrease in the rate of severe hypoglycemic episodes (odds ratio, 0.40; 95% confidence interval, 0.24-0.65). CONCLUSIONS Personal CGM, in a real-world setting, improves glucose control and reduces the rate of severe hypoglycemic episodes.

Leptin and the treatment of obesity: its current status

Leptin, the protein product of the ob gene, is primarily an adipocyte-secreted hormone, whose functional significance is rapidly expanding. Although early research efforts were focused on defining leptins role in reversing obesity in rodents, there is now substantial evidence indicating that its influence extends to several hypothalamic-pituitary-endocrine axes, including gonadal, adrenal, thyroid, growth hormone, and pancreatic islets. A role for leptin in hematopoiesis, angiogenesis, immune function, osteogenesis, and wound healing has also been documented. The results of recent clinical trials with recombinant human leptin indicated that its effectiveness in restoring energy balance and correcting obesity-related endocrinopathies in genetically obese rodent models extended only partially to the management of human obesity. New efforts in drug development have focused on leptin-related synthetic peptide agonists as potential anti-obesity pharmacophores.

[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.

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.

Intranasal administration of mouse [D-Leu-4]OB3, a synthetic peptide amide with leptin-like activity, enhances total uptake and bioavailability in Swiss Webster mice when compared to intraperitoneal, subcutaneous, and intramuscular delivery systems.

Using a synthetic peptide strategy, we localized an active domain in mouse leptin to a sequence between amino acids 106 and 140. Intraperitoneal (ip) administration of a number of synthetic peptide amides encompassed by this domain reduced body weight gain, food and water intake, blood glucose levels, and increased insulin sensitivity in genetically obese mice. In the present study, we examined the pharmacokinetics of mouse [D-Leu-4]OB3, our most potent peptide, in male Swiss Webster mice following ip, subcutaneous (sc), and intramuscular (im) injection, and after intranasal administration with Intravail, a new class of patented transmucosal delivery enhancement agents. Total uptake (1,072,270, 1,182,498; 1,481,060; ng/ml/min), serum half-life (48.8; 34.0; 30.0 min) and relative bioavailability (1.0, 1.1; 1.4;) of mouse [D-Leu-4]OB3 were similar when the peptide was given by ip, sc, or im injection, respectively. Total uptake and relative bioavailability were enhanced following intranasal delivery (4,336,963 ng/ml/min and 4.0, respectively), and serum half-life was 41.1 min. These results indicate that intranasal delivery of mouse [D-Leu-4]OB3 with Intravail is a more effective method of peptide administration than injection methods, and suggest that it may have potential as a novel, non-invasive approach to the treatment of obesity and its associated metabolic dysfunctions in humans.

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.

Actions of l-thyroxine and Nano-diamino-tetrac (Nanotetrac) on PD-L1 in cancer cells

The PD-1 (programmed death-1)/PD-L1 (PD-ligand 1) checkpoint is a critical regulator of activated T cell-cancer cell interactions, defending tumor cells against immune destruction. Nano-diamino-tetrac (NDAT; Nanotetrac) is an anticancer/anti-angiogenic agent targeted to the thyroid hormone-tetrac receptor on the extracellular domain of integrin αvβ3. NDAT inhibits the cancer cell PI3-K and MAPK signal transduction pathways that are critical to PD-L1 gene expression. We examined actions in vitro of thyroid hormone (l-thyroxine, T4) and NDAT on PD-L1 mRNA abundance (qPCR) and PD-L1 protein content in human breast cancer (MDA-MB-231) cells and colon carcinoma (HCT116 and HT-29) cells. In MDA-MB-231 cells, a physiological concentration of T4 (10-7M total; 10-10M free hormone) stimulated PD-L1 gene expression by 38% and increased PD-L1 protein by 2.7-fold (p<0.05, all changes). NDAT (10-7M) reduced PD-L1 in T4-exposed cells by 21% (mRNA) and 39% (protein) (p<0.05, all changes). In HCT116 cells, T4 enhanced PD-L1 gene expression by 17% and protein content by 24% (p<0.05). NDAT reduced basal PD-L1 mRNA by 35% and protein by 31% and in T4-treated cells lowered mRNA by 33% and protein by 66%. In HT-29 cells, T4 increased PD-L1 mRNA by 62% and protein by 27%. NDAT lowered basal and T4-stimulated responses in PD-L1 mRNA and protein by 35-40% (p<0.05). Activation of ERK1/2 was involved in T4-induced PD-L1 accumulation. We propose that, by a nongenomic mechanism, endogenous T4 may clinically support activity of the defensive PD-1/PD-L1 checkpoint in tumor cells. NDAT non-immunologically suppresses basal and T4-induced PD-L1 gene expression and protein accumulation in cancer cells.

Endocrine treatment of transsexual persons: extensive personal experience.

OBJECTIVE The Endocrine Societys recently published clinical practice guidelines for the treatment of transsexual persons acknowledged the need for further information on transsexual health. We report here the experience of one provider with the endocrine treatment of transsexual persons over the past 2 decades. METHODS Data on demographics, clinical response to treatment, and psychosocial status were collected on all transsexual persons receiving cross-sex hormone therapy since 1991 at the endocrinology clinic at Albany Medical Center, a tertiary care referral center serving upstate New York. RESULTS Through 2009, a total 192 male-to-female (MTF) and 50 female-to-male (FTM) transsexual persons were seen. These patients had a high prevalence of mental health and psychiatric problems (over 50%), with low rates of employment and high levels of disability. Mental health and psychiatric problems were inversely correlated with age at presentation. The prevalence of sex reassignment surgery was low (31% for MTF). The number of persons seeking treatment has increased substantially in recent years. Cross-sex hormone therapy achieves very good results in FTM persons and is most successful in MTF persons when initiated at younger ages. CONCLUSION Transsexual persons seeking hormonal therapy are being seen with increasing frequency. The dysphoria present in many transsexual persons is associated with significant mood disorders that interfere with successful careers. Starting therapy at an earlier age may lessen the negative impact on mental health and lead to improved social outcomes. However, significant barriers exist, such as insufficient insurance coverage, which limit comprehensive care.