Somatostatin in epithelial cells of intestinal mucosa is present primarily as somatostatin 28

Region-specific antisera to [Tyr14]-SS28(1-14) were used to identify cells containing immunoreactivity to the SS28(1-14) fragment of somatostatin 28 (SS28) in gastric and intestinal mucosal epithelium and in pancreatic islets by immunoperoxidase staining. Radioimmunoassay with iodinated [Tyr14]-SS28(1-14) identified one antiserum (F4) to SS28(1-14) that cross-reacted equally with SS28(1-12), SS28(1-14) and SS28. Two other antisera (F3 and F8) to SS28(1-14) did not cross-react with SS28(1-12) and showed insignificant cross-reactivity to SS28. Immunostaining results showed that F4 stained the same cells that reacted with antiserum AS-10, which is specific for the cyclic tetradecapeptide somatostatin, SS28(15-28). Antisera F3, F4, and F8 all reacted with islet D cells and with somatostatin cells in the antral mucosa. However, only antiserum F4 detected immunoreactivity in mucosal epithelial cells; F3 and F8 did not bind to these cells. After sections of intestine were exposed to trypsin, however, epithelial cells containing immunoreactivity to SS28(1-14) were detected in intestinal mucosa with antisera F3 and F8. These results were obtained for duodenum, jejunum, ileum, and colon, but most of the epithelial cells with immunoreactivity to SS28(1-14) were in the duodenum. Both radioimmunoassay and immunostaining results suggest that F3 and F8 bind to a region of SS28(1-14) that is unavailable to antibodies in the intact SS28 molecule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cysteamine and the endocrine pancreas: Immunocytochemical,immunochemical, and functional aspects

Summary To evaluate the previously reported depletion of pancreatic somatostatin by cysteamine (-mercaptoethylamine), mice were injected subcutaneously with the drug at 300 mg/kg. Immunocytochemical analysis performed on sections from tissue taken at 4 h after the injection revealed an elimination of somatostatin-14-like immunoreactivity without alterations in the somatostatin-28_{(1 – 12)}-like immunoreactivity. In sections from tissues taken at 24 h after injection, no differences between cysteamine-injected animals and controls were observed. Immunochemical analysis of somatostatin-14-like immunoreactivity in pancreatic extracts showed a significant reduction of the concentration (P< 0.001). In contrast, no change in the insulin concentration was observed. Functionally, cysteamine lowered the plasma glucose levels at l h after injection; this effect persisted for 6 h. Plasma insulin levels were likewise reduced transiently by cysteamine. Concomitant administration of somatostatin did not influence these effects of cysteamine. The plasma glucose-lowering effect of cysteamine was seen also in alloxan-diabetic mice. We conclude that cysteamine alters the immunoreactive characteristics of pancreatic somatostatin without affecting the immunoreactivity of insulin, and that cysteamine transiently reduces plasma glucose and insulin levels     

Effects of cysteamine on pro-somatostatin related peptides

Intracerebroventricular (icv) injection of cysteamine to rats produced a marked depletion of somatostatin-14-like immunoreactivity (LI) in all rat brain regions examined. The somatostatin-28 (SS28)-LI and SS28(1-12)-LI were generally not altered by the cysteamine treatment. Following subcutaneous injection of the drug similar depletions of hypothalamic SS14-LI was observed with no change in SS28-LI nor SS28(1-12)-LI. In vitro cysteamine significantly increased the basal release of SS14-LI and markedly potentiated the evoked release of SS14-LI from hypothalamic slices. At 10 mM cysteamine, enhanced release of SS14-LI from hypothalamic slices was still observed despite a marked depletion of tissue content of SS14-LI.     

Prosomatostatin is processed in the Golgi apparatus of rat neural cells.

Proteolytic processing of somatostatin precursor produces several peptides including somatostatin-14 (S-14), somatostatin-28 (S-28), and somatostatin-28 (1-12) (S-28(1-12)). The subcellular sites at which these cleavages occur were identified by quantitative evaluation of these products in enriched fractions of the biosynthetic secretory apparatus of rat cortical or hypothalamic cells. Each of the major cellular compartments was obtained by discontinuous gradient centrifugation and was characterized both by specific enzyme markers and electron microscopy. The prosomatostatin-derived fragments were measured by radioimmunoassay after chromatographic separation. Two specific antibodies were used, allowing the identification of either S-28(1-12) or S-14 which results from peptide bond hydrolysis at a monobasic (arginine) and a dibasic (Arg-Lys) cleavage site, respectively. These antibodies also revealed prosomatostatin-derived forms containing at their COOH terminus the corresponding dodeca- and tetradecapeptide sequences. Whereas the reticulum-enriched fractions contained the highest levels of prosomatostatin, the proportion of precursor was significantly lower in the Golgi apparatus. In the latter fraction, other processed forms were also present, i.e. S-14 and S-28(1-12) together with the NH2-terminal domain (1-76) of prosomatostatin (pro-S(1-76). Inhibition of the intracellular transport either by monensin or by preincubation at reduced temperature resulted in an increase of prosomatostatin-derived peptides in the Golgi-enriched fractions. Finally, immunogold labeling using antibodies raised against S-28(1-12) and S-14 epitopes revealed the presence of these forms almost exclusively in the Golgi-enriched fraction mainly at the surface of saccules and vesicles. Together these data demonstrate that in rat neural cells, prosomatostatin proteolytic processing at both monobasic and dibasic sites is initiated at the level of the Golgi apparatus.     

Ultrastructural studies on calcitonin gene-related peptide-, tachykinins- and somatostatin-immunoreactive neurones in rat dorsal root ganglia: Evidence for the colocalization of different peptides in single secretory granules

Summary Calcitonin gene-related peptide (CGRP)-, tachykinins- and somatostatin-immunoreactive neurones in rat dorsal root ganglia have been studied by means of single and double immunogold labelling techniques. Peptide-immunoreactive neurones are generally B- or C-type cells of small size, with well developed rough endoplasmic reticulum and scanty neurofilaments. In neurones classifiable as A₂-type cells, i.e. larger neurones with a lighter cytoplasm due to the presence of poorly developed Nissl bodies and numerous neurofilaments, only CGRP immunoreactivity was detected. Immunolabelled structures were identified as large (60–100 nm diameter), electron-dense, membranebounded p-type granules. They were observed only in neuronal cell bodies or in the intraganglionic portions of the axons. No granules immunoreactive to the antisera applied in this study were observed in non-neuronal cells. Immunostaining experiments with different combinations of the antisera revealed, in some cells, the presence of double immunolabelled granules; in particular localization of CGRP and tachykinins, CGRP and somatostatin, and tachykinins and somatostatin to single secretory granules was demonstrated. The finding that more than one peptide is localized to the same secretory granule supports the postulate that peptides are co-released upon nerve stimulation providing morphological support for physiological and pharmacological data demonstrating an interaction between different peptides in the modulation of synaptic activity.     

Reduced somatostatin-like immunoreactivity in the brain of LEC rats with hepatic encephalopathy.

Somatostatin-14-like immunoreactivity (S14LI) and somatostatin-28(1-12)-like immunoreactivity (S28(1-12)LI) in the brain of LEC (Long Evans Cinnamon) rats with hepatic encephalopathy were measured. Significant reduction of both S14LI and S28(1-12)LI was observed in the hypothalamus, medulla oblongata, striatum and spinal cord. Both of the immunoreactivities in the hypothalamus of these rats were approx. 50% of those in LEC rats without hepatic encephalopathy. The amounts of reduction of S14LI significantly correlated with those of reduction of S28(1-12)LI. No significant difference in gel chromatographic profiles of S14LI and S28(1-12)LI was observed between LEC rats with and without hepatic encephalopathy. These results suggest that the reduction of somatostatin-like immunoreactivity in LEC rats with hepatic encephalopathy may be caused by a decrease in production of prosomatostatin rather than altered degradation.     

Electron-microscopic immunocytochemical study of the endocrine pancreas of sea bass (Dicentrarchus labrax)

Insulin (B)-, somatostatin 25 (SST-25) (D1)-, somatostatin 14 (SST-14) (D2)-, glucagon (A)-, and glucagon PP/PYY/NPY (PP-like)-immunoreactive cells in islets of sea bass (Dicentrarchus labrax) were characterized according to their ultrastructure and immunogold labeling. Cells labeled with antisera to bonito and salmon insulin had numerous secretory granules with a small halo and round core, and a few with wide halo and round or crystalloid core. Gold particles were found throughout the granule in tissue labeled with the former but only in the core in tissue labeled with the latter. D1 cells had large granules with a medium electron-dense content and some with a darker core. D2 had smaller medium or high electron-dense secretory granules than D1 cells, located mainly in cell periphery. Glucagon-immunoreactive cells contained some granules with a polygonal core that was heavily labeled and other granules with a round core with no or hardly any labeling. Glucagon and PP-like immunoreactivity were co-localized in secretory granules, in which the gold particles showed no different distribution with the various antisera used. PYY-immunoreactive granules were also found in nerve endings. All the pancreatic endocrine cell types showing involutive characteristics are found.     

Somatostatin-28- and somatostatin- 14-like immunoreactivities in the rat pituitary gland

Summary Endogenous SS14- as well as SS28-like immuno-reactive materials were detected in both male and female rats by radioimmunoassay and by immunocytochemistry on ultrathin frozen sections. The content of somatostatin-like immunoreactivity was 0.39±0.08 pg per mg adenohypophysis. Immunoreactivity was localized by immunocyto-chemistry in three pituitary cell types: somatotrophs, lactotrophs and thyrotrophs, but not in corticotrophs and gonadotrophs. In these three pituitary cell types the SS28- and the SS14-like immunoreactive materials were localized in the cytoplasm and in the nucleus. In the cytoplasm the immunoreactivity was seen in the cytoplasmic matrix and in the secretory granules. In the nucleus it was present mainly in the euchromatin close to the heterochromatin. In somatotrophs and lactotrophs, SS14- and SS28-like immunoreactive materials have been detected at the plasma membrane level. These results suggest that (1) endogenous SS14 and SS28 are present in adenohypophysis in somatotrophs, lactotrophs and thyrotrophs, and (2) the two peptides act on both the cytoplasmic components and the nucleus.     

Immunochemical characterisation of somatostatin in ruminants

Following development and validation of a radioimmunoassay for somatostatin, the immunoreactivity of this peptide in the plasma of ruminants was measured and the levels in sheep were 9-31 pM (mean 18 +/- 7 pM, n = 48), in lambs 10-54 pM (mean 25 +/- 10 pM, n = 18) and in calves 5-35 pM (mean 12 +/- 6 pM, n = 22). Somatostatin-like immunoreactivity was present in sheep in high concentrations in the antrum (2342 +/- 280 pmol/g wet weight), duodenum (446 +/- 73 pmol/g) and pancreas (832 +/- 208 pmol/g). Lower concentrations (6-150 pmol/g) were found in other regions of the gastrointestinal tract. Molecular sieve chromatography on Bio-Gel P-10 showed that while most of the somatostatin in the antrum was somatostatin-14, in the duodenum about 30% of the total immunoreactivity was somatostatin-28.     

Immunohistochemical studies for multiple peptide-immunoreactivities and co-localization of Met-enkephalin-Arg6-Gly7-Leu8, neuropeptide Y and somatostatin in human adrenal medulla and pheochromocytomas

Three normal human adult adrenal medullas and 12 cases of pheochromocytomas were studied for immunohistochemical localization of various peptides. Met-enkephalin-Arg6-Gly7-Leu8 (MEAGL) was present in all cases of pheochromocytomas. The normal adrenal medulla showed cells immunoreactive for MEAGL, neuropeptide tyrosine (NPY) and proopiomelanocortin derived N-terminal fragment (NTF). MEAGL and NPY were co-localized in some adrenal medullary cells. Pheochromocytomas showed striking multiple immunoreactivities regardless of histologic types, pleomorphic or organoid. Ten cases showed immunoreactivities for more than two peptides. All cases showed immunoreactivity for MEAGL and 9 cases showed NPY positive cells. Some tumor cells contain both MEAGL and NPY in the cytoplasm. Six cases were positive for somatostatin. Some tumor cells were shown to contain both MEAGL and SS. The appearance of SS and other peptides was considered to be related to the neoplastic transformation of the adrenal medulla.     

Light and electron microscopical immunocytochemical localization of pancreastatin-like immunoreactivity in porcine tissues

Summary Pancreastatin is a 49 amino acid comprising peptide isolated from porcine pancreas that is derived by proteolytic processing from chromogranin A. Using an antibody against the synthetic C-terminal fragment pancreastatin (33–49), we examined the light and electron microscopical immunocytochemical localization of this peptide in porcine tissues. Pancreastatin-like immunoreactivity (PLI) was found in pancreatic somatostatin-, insulin- and glucagon cells in varying intensities; pancreatic polypeptide cells were always negative. At the electron microscopical (EM) level the immunoreactivity was confined to the electron dense core of the secretory granules in the case of somatostatin and insulin cells or to the less electron dense halo of the glucagon granules. In the antrum PLI positive cells represented gastrin (G), somatostatin (D) and enterochromaffin (EC) cells, in the duodenum in addition to EC- and G-cells a small number of PLI positive cells showed a positive immunoreaction for glucagon-like peptide (GLP) I and secretin in serial sections. Both norepinephrine and epinephrine containing cells of the adrenal medulla exhibited a strong reaction for PLI. In the pituitary several cell populations stained with varying intensities, including gonadotrophs and thyrotrophs. PLI is present in a distinct and characteristic subpopulation of neuroendocrine cells in various organs. The subcellular localization may indicate a function in the granular concentration, packaging and storage of peptides and amines in the brain-gut endocrine system.     

Cell-specific processing of preprosomatostatin in cultured neuroendocrine cells

We have previously found that preprosomatostatin is processed accurately to both somatostatin-14 and somatostatin-28 in pituitary gonadotrophs of transgenic mice. The foreign somatostatin peptides have been shown to enter the regulated secretory pathway of these cells. To determine whether accurate preprosomatostatin processing can occur in any neuroendocrine cell, we introduced preprosomatostatin cDNA expression vectors into several different neuroendocrine cell lines. We found that prosomatostatin was cleaved efficiently to somatostatin-14 and somatostatin-28 in RIN 5F and AtT20 cells, but not in GH4 or PC12 cells. The ability of a particular cell type to process prosomatostatin did not correlate with cellular storage capacity and was independent of the level of biosynthesis of the precursor. These data suggest that prosomatostatin processing requires specific pathways which are present in some neuroendocrine cells, but not in others.     

Ultrastructural distribution of somatostatin-14 and-28 in rat adrenal cells

Summary Previous studies have shown that somatostatin modulates angiotensin-induced aldosterone secretion by adrenal glomerulosa cells. This effect is mediated through specific receptors which do not show any preference for somatostatin-14 (S14) or the N-extended form somatostatin-28 (S28). The study of the distribution of ¹²⁵I-Tyr [Tyr⁰, DTrp⁸] S14-and ¹²⁵I-Tyr[Leu⁸, DTrp²², Tyr²⁵] S28-binding in frozen sections of the rat adrenal by autoradiography indicated that both peptides bind to similar loci. High concentrations of binding sites were observed in the zona glomerulosa, and low concentrations were detected in the medulla. At the ultrastructural level, immunocytochemistry after cryoultramicrotomy revealed endogenous S14-and S28-like immunoreactive material in zona glomerulosa and in medulla. In glomerulosa cells, immunoreactive material was localized at the plasma membrane level, in the cytoplasmic matrix, in the mitochondria, and in the nucleus. S14-and S28-like materials were detected in both epinephrine and norepinephrine-storing cells of the adrenal medulla. In these cells, the distribution of either immunoreactive product was similar; it was observed in cytoplasmic matrix, secretory granules and nucleus, but not at the plasma membrane level. In situ hybridization does not reveal somatostatin mRNA in zona glomerulosa or medulla. These results demonstrate that S14 and S28 bind to, and are taken up by zona glomerulosa and adrenal medullary cells, but are not produced by these cells.     

Seven peptides derived from pro-somatostatin in rat brain

Acid extracts of murine hypothalamic and extra-hypothalamic rat brains were analyzed by specific radioimmunoassays for the presence of somatostatin-14, somatostatin-28 and somatostatin-28(1–12)-like immunoreactivity. Seven molecular forms were observed after gel permeation chromatography. In addition to somatostatin-14, somatostatin-28 and somatostatin-28(1–12), there were two peptides of 4,400 and 7,500 mol. wt. which contained the somatostatin-28(1–12) sequence with an extension towards the NH₂-terminus of pro-somatostatin. Moreover, two other peptides of 6,000 and 9,500 mol. wt. were detected containing the whole somatostatin-28 structure. These results imply that the processing of brain pro-somatostatin involves a minimum of four cleavage sites and yields at least seven peptides.     

Reduced somatostatin-28-(1-12)-like immunoreactivity in cerebral cortex of dogs with an Eck fistula and somatostatin molecular forms in brain

Somatostatin-28-(1-12)-like immunoreactivity (S28(1-12)LI) in brains of Eck fistula dogs, prepared as an experimental model of hepatic encephalopathy, was measured. Significant reductions of S28(1-12)LI were observed in all cortical regions of Eck fistula dogs. The reductions of S28(1-12)LI were significantly correlated with decreases in somatostatin-14-like immunoreactivity (S14LI) in the cortical region. The ratios of S28(1-12)LI to S14LI in all cortical regions were not different between Eck fistula and normal dogs. Additionally, no difference in gel chromatographic profiles of S28(1-12)LI and S14LI was observed between Eck fistula and normal dogs. These results imply that reduced somatostatin immunoreactivity in hepatic encephalopathy may be caused not by altered degradation but by reduced production of prosomatostatin. Our S28(1-12)LI assay system could detect prosomatostatin(1-76) and S28(1-12) and the S14LI system prosomatostatin, S28 and S14. S28(1-12)LI/S14LI ratios in cortex were 0.64-0.83 and these were significantly different from those (1.02-1.36) in thalamus, midbrain and medulla. Relative proportions of prosomatostatin (20%) and S28 (23-24%) in cortex were larger than those (6-7% and 5-7%, respectively) in thalamus, midbrain and medulla. The differential distribution of these molecular forms suggests that processing of prosomatostatin in cortex may be different from that in thalamus, midbrain and medulla.     

Somatostatin Precursor in the Rat Striatum: Changes After Local Injection of Kainic Acid

The molecular forms of somatostatin contained in the rat striatum were separated by size-exclusion HPLC. Three major peaks of somatostatin-like immunoreactivity (SLI) were resolved. Two peaks cochromatographed with synthetic somatostatin-14 (SS-14) and somatostatin-28 (SS-28), respectively. One peak exhibited a higher molecular weight (about 10,000) and may contain a proform of somatostatin. Local injection of the neurotoxin kainic acid (1 microgram) into the left striatum resulted in a persistent decrease (65-85%) of all three forms of somatostatin. In the contralateral--not injected--striatum a decrease of SLI was also observed which was maximal (45%) after 2 days and was largely abolished after 7 days. This decrease of SLI in the contralateral striatum, however, was due mainly to a decrease of SS-14 and SS-28 but not of the putative proform. Our data suggest that kainic acid causes a destruction of somatostatin-containing perikarya in the injected striatum, whereas in the contralateral striatum increased release with subsequent inactivation of SS-14 and SS-28 takes place. The putative somatostatin proform may serve as neurochemical marker for somatostatin-containing perikarya in the striatum.     

Immunochemical and immunohistochemical studies,using antisera against porcine 25 kDa amelogenin, 89 kDa enamelin and the 13–17 kDa nonamelogenins,on immature enamel of the pig and rat

Summary Enamel proteins were extracted from the newly formed layer of immature porcine enamel, and the 25 kDa amelogenin, 89 kDa enamelin and 13–17 kDa nonamelogenins were purified. Specific antisera were raised against these proteins. Antibodies specific to the C-terminal region (residues 149–173) of the 25 kDa amelogenin were generated by absorption of the anti-25 kDa amelogenin serum with 20 kDa amelogenin, which contains residues 1–148 of the antigen. Immunoelectrotransfer blotting of the extracted porcine enamel proteins showed that the anti-25 kDa amelogenin serum recognized the 25 kDa and other low and high molecular weight amelogenins. The C-terminal specific anti-25 kDa amelogenin serum reacted only with amelogenins having molecular weights over 23 kDa. The anti-89 kDa enamelin serum recognized the 89 kDa enamelin and lower molecular weight proteins, but neither the amelogenins nor the 13–17 kDa nonamelogenins. The antiserum against the 13–17 kDa nonamelogenins showed no cross reactivity to the 89 kDa enamelin, but recognized higher molecular weight nonamelogenins. In immunohistochemical preparations of the porcine tooth germs, the 25 kDa amelogenin-like immunoreactivity over immature enamel decreased in a gradient from the enamel surface to the middle layer. In the inner layer immunoreactivity was concentrated over the prism sheaths. The C-terminal specific 25 kDa amelogenin-like immunoreactivity was intense at the outer layer of immature enamel and decreased sharply toward the middle layer. Prism sheaths were intensely stained by the antiserum to the 13–17 kDa nonamelogenins. The 89 kDa enamelin-like immunoreactivity over enamel prisms was intense at the outer layer and decreased toward the middle layer. Staining by the anti-89 kDa enamelin serum of prism sheaths was faint. In immature rat incisor enamel, the C-terminal specific 25 kDa amelogenin antiserum demonstrated a staining pattern similar to that in the immature enamel of the pig. Distinct 13–17 kDa nonamelogenin-like and 89 kDa enamelin-like immunoreactivities were found especially in the layer adjacent to the Tomes' process. We conclude that some enamel proteins are degraded soon after their secretion from the secretory ameloblast in the rat and the pig. The specific enamel proteins which reacted with the antiserum to the 13–17 kDa nonamelogenins seem to be involved with the formation of prism sheaths in immature porcine enamel, but not in rat incisor enamel.     

Degradation and conversion of somatostatin in normal and diabetic rats in vivo and in vitro

Plasma somatostatin-like immunoreactivity in the portal and jugular veins of streptozotocin diabetic rats was compared with that in normal control rats. In the diabetic group, somatostatin levels in the portal (p less than 0.05) and jugular (p less than 0.01) veins were both elevated compared with those in the control group. Moreover, the degree of elevation was greater in the jugular vein than in the portal vein. To further investigate the role of the liver in the clearance of somatostatin-28 in vivo, 2 micrograms of somatostatin-28 was administered as a bolus into the external jugular vein of intact and functionally hepatectomized rats. The mean half-time of somatostatin-28 was significantly longer in intact diabetic rats than in controls (p less than 0.05). The functional hepatectomy did not cause a significant difference in the half-time in diabetic rats but made it longer in control rats. These results suggest that the longer half-time of somatostatin-28 in diabetic rats in vivo is due to its slower hepatic clearance. The hepatic clearance of somatostatin-28 and somatostatin-14 was further studied in vitro using a recirculating liver perfusion method. The hepatic clearance of 1.2 nM of either somatostatin-28 or somatostatin-14 was significantly lower in diabetic rats than in controls (p less than 0.01). This indicates that elevated plasma somatostatin levels in diabetic rats are caused at least in part by decreased hepatic clearance of somatostatin.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of hypercalcemia on parafollicular cells in the rat thyroid gland

Hypercalcemia was induced in rats by the administration of A.T.10. We then determined the levels of total and ionized calcium and calcitonin in the serum, as well as performed ultrastructural observations and histochemical investigations of the calcitonin and neuron-specific enolase immunoreactivities in the stimulated parafollicular cells. The main aim of the study was to apply histochemical procedures to determine the immunoreactions of calcitonin gene-related peptide (CGRP), somatostatin and secretory protein-I in stimulated parafollicular cells. Immunoreactions of CGRP and calcitonin decreased strikingly in A.T.10-treated animals, whereas no visible changes were noted in somatostatin immunoreactivity. In the case of secretory protein-I, an insignificant increase of its immunoreactivity was observed in the treated animals. The cytophysiological significance of these results is discussed.     

The effect of albumin fusion structure on the production and bioactivity of the somatostatin-28 fusion protein in Pichia pastoris

Somatostatin, a natural inhibitor of growth hormone (GH), and its analogs have been used in clinical settings for the treatment of acromegaly, gigantism, thyrotropinoma, and other carcinoid syndromes. However, natural somatostatin is limited for clinical usage because of its short half-life in vivo. Albumin fusion technology was used to construct long-acting fusion proteins and Pichia pastoris was used as an expression system. Three fusion proteins (SS28)₂-HSA, (SS28)₃-HSA, and HSA-(SS28)₂, were constructed with different fusion copies of somatostatin-28 and fusion orientations. The expression level of (SS28)₃-HSA was much lower than (SS28)₂-HSA and HSA-(SS28)₂ due to the additional fusion of the somatostatin-28 molecule. MALDI-TOF mass spectrometry revealed that severe degradation occurred in the fermentation process. Similar to the standard, somatostatin-14, all three fusion proteins were able to inhibit GH secretion in blood, with (SS28)₂-HSA being the most effective one. A pharmacokinetics study showed that (SS28)₂-HSA had a prolonged half-life of 2 h. These results showed that increasing the number of small protein copies fused to HSA may not be a suitable method for improving protein bioactivity.