Dynamin Is Functionally Coupled to Insulin Granule Exocytosis

The insulin granule integral membrane protein marker phogrin-green fluorescent protein was co-localized with insulin in Min6B1 beta-cell secretory granules but did not undergo plasma membrane translocation following glucose stimulation. Surprisingly, although expression of a dominant-interfering dynamin mutant (Dyn/K44A) inhibited transferrin receptor endocytosis, it had no effect on phogringreen fluorescent protein localization in the basal or secretagogue-stimulated state. By contrast, co-expression of Dyn/K44A with human growth hormone as an insulin secretory marker resulted in a marked inhibition of human growth hormone release by glucose, KCl, and a combination of multiple secretagogues. Moreover, serial pulse depolarization stimulated an increase in cell surface capacitance that was also blocked in cells expressing Dyn/K44A. Similarly, small interference RNA-mediated knockdown of dynamin resulted in marked inhibition of glucose-stimulated insulin secretion. Together, these data suggest the presence of a selective kiss and run mechanism of insulin release. Moreover, these data indicate a coupling between endocytosis and exocytosis in the regulation of beta-cell insulin secretion

Human growth hormone responses to sprinting

A number of studies have shown exercise to stimulate human growth hormone (hGH) secretion, although most of these have considered prolonged submaximal or resistance exercise. Only a few have studied maximal sprint exercise, and these studies have demonstrated considerably elevated circulating hGH concentrations during recovery. However, there is little agreement in the literature regarding the regulation of hGH secretion during and after exercise. This thesis describes a series of experiments considering the hGH response to sprint exercise, with the intention of gaining a better understanding of some of the mechanisms involved in regulating the exercise-induced hGH release. [Continues.

Genetic polymorphisms in the locus control region and promoter of GH1 are related to serum IGF-I levels and height in patients with isolated growth hormone deficiency and healthy controls

Background/Aims: Expression of the human growth hormone (GH) gene (GH1) is regulated by a locus contro

Thiol-Disulfide Exchange in Human Growth Hormone

The biopharmaceutical industry has been growing at a tremendous rate, with sales of $63.6 billion 2012 in the US [1]. Nevertheless, the successful development of many protein drugs has been impeded by physical and chemical instabilities arising from their inherent chemical complexity and often leading to protein aggregation. The formation of non-native disulfide bonds is a common route to covalent aggregation of therapeutic proteins and other biologics [2, 3]. Disulfide bonds participate in hydrolytic and oxidative degradation reactions that form non-native disulfide bonds and other reactive species. The mechanisms responsible for protein aggregation are poorly understood and formulations are currently optimized on a trial and error basis. This approach contributes to high development costs and increases the time to market. The main goal of our research is to elucidate the mechanisms of thiol-disulfide exchange and disulfide scrambling in therapeutic proteins. To accomplish this goal, model peptides derived from human growth hormone (hGH) and intact hGH were used to investigate reaction mechanisms and kinetics in solution and solid-state environments. The results will be useful in the rational development of stable, safe and efficacious protein formulations that contain free cysteines and disulfides

LC-MS characterization and cell-binding properties of chelate modified somatropin

Somatropin, a recombinant protein containing 191 amino acids, is derived from the endogenous human growth hormone, somatotropin. This protein is clinically used in children and adults with inadequate endogenous growth hormone to stimulate a normal bone and muscle growth. In addition, somatropin is recently being investigated for the diagnosis and radiotherapy of certain hormonal cancers. In some of these cancers, over-expression of the human growth hormone receptor (hGHR) is described. The modification of the protein with a chelating agent like NOTA (1,4,7-triazacyclononane-1,4,7-triacetic acid) allows the inclusion of metals coupled to the protein. The NOTA unit is selectively introduced on a lysine side chain. As site-specific labelling is necessary to avoid active region interactions (1-16, 41-68, 103-119 and 167-175), characterization of the chelate-modified somatropin is indispensable. Therefore, we have applied an enzymatic digestion procedure using trypsin, chymotrypsin and a combination of both enzymes. The resulting peptides were then monitored using HPLC-MSn, allowing the investigation of the exact amino acid modifications. The use of a mixture of trypsin and chymotrypsin gave an enhanced information efficiency. Moreover, the intact protein, without enzymatic degradation, was analysed on a protein HPLC column using UV detection for quantification and ESI-MS/MS for characterization. Based upon the HPLC-MSn results of the digested somatropin, the chelating molecule is mainly bound to a specific lysine amino acid that is located away from the receptor binding site. Therefore, the cell-binding functionality of the characterized NOTA-somatropin is measured, using a HepG2 cell line

Human growth hormone alters carbohydrate storage in blood and liver in both genders of an Indian bird, Acridotheres tristis (Linn.)

Background: Growth hormone (GH) is a peptide hormone that plays vital roles in cell growth and metabolism. Aim: The study investigates the effect of GH on carbohydrate metabolism using Indian bird, Acridotheres tristis. Methods: Three different doses (0.4, 0.6, and 0.8mg/100g body weight) of human growth hormone (HGH) given once to both genders of a bird Acridotheres tristis to observe the effect on blood glucose and hepatic glycogen content in the body. Glucose and glycogen were quantitatively assayed. Results: Their effect was recorded for different time intervals (1, 4, 12, 24, 72, 96, and 144 h). Hypoglycaemic condition was recorded within an hour of hormone treatment in male and female birds. The lowest dose (0.4mg/100g body weight) was more effective than other two doses. Simultaneous depletion of hepatic glycogen was also recorded, although initially increase in glycogen level was also noticed in both genders. It was noticed that the highest dose (0.8mg/100g body weight) was most responsive. Conclusion: The effect of human growth hormone was not dose and time dependent in both male and female birds. HGH is thus hypoglycaemic and hepatic glycogenolytic in nature in A. tristis.Key words: Human growth hormone, blood glucose, hepatic glycogen, hypoglycaemia, glycogenolysis, bir

Immunofunctional assay of human growth hormone (hGH) in serum: A possible consensus for quantitative hGH measurement

Confirmation of the diagnosis of GH deficiency in adults and children involves provocative testing for human (h) GH. Different commercially available immunoassays yield largely discrepant results in the measurement of GH levels in human serum. These discrepancies result in doubtful relevance of cut-off levels proposed for GH provocative testing. We have developed an immunofunctional assay method that allows quantitation of only those GH forms in circulation that possess both binding sites of the hormone for its receptor and thus can initiate a biological signal in target cells. An anti-hGH monoclonal antibody recognizing binding site 2 of hGH is immobilized and used to capture hGH from the serum sample. Biotin-labeled recombinant GH-binding protein in a second incubation step forms a complex with those hGH molecular isoforms that have both binding sites for the receptor. The signal is detected after a short third incubation step with labeled streptavidin. The assay is sensitive (detection range, 0.1-100 micrograms/L) and has average inter- and intraassay precisions of 10.3% and 7.3% respectively. Endogenous GH-binding protein does not interfere with the hGH result; placental lactogen slows no detectable cross-reaction in this immunofunctional assay. The degree of immunofunctionally active hGH forms in serum samples, calculated by comparison of immunofunctional assay and RIA results, varied between 52-93%. We propose this immunofunctional assay for GH measurement as a new reference method for hGH quantitation in serum. The immunofunction assay translates only hGH forms into an assay signal that are capable of dimerizing GH receptors and, thus, of initiating a biological effect in target cells