Lamin Marenah

A stable analogue of glucose-dependent insulinotropic polypeptide, GIP(LysPAL16), enhances functional differentiation of mouse embryonic stem cells into cells expressing islet-specific genes and hormones

Abstract Embryonic stem (ES) cells can be differentiated into insulin-producing cells by conditioning the culture media. However, the number of insulin-expressing cells and amount of insulin released is very low. Glucose-dependent insulinotropic polypeptide (GIP) enhances the growth and differentiation of pancreatic β-cells. This study examined the potential of the stable analogue GIP(LysPAL16) to enhance the differentiation of mouse ES cells into insulin-producing cells using a five-stage culturing strategy. Semi-quantitative PCR indicated mRNA expression of islet development markers (nestin, Pdx1, Nkx6.1, Oct4), mature pancreatic β-cell markers (insulin, glucagon, Glut2, Sur1, Kir6.1) and the GIP receptor gene GIP-R in undifferentiated (stage 1) cells, with increasing levels in differentiated stages 4 and 5. IAPP and somatostatin genes were only expressed in differentiated stages. Immunohistochemical studies confirmed the presence of insulin, glucagon, somatostatin and IAPP in differentiated ES cells. After supplementation with GIP(LysPAL16), ES cells at stage 4 released insulin in response to secretagogues and glucose in a concentration-dependent manner, with 35–100% increases in insulin release. Cellular C-peptide content also increased by 45% at stages 4 and 5. We conclude that the stable GIP analogue enhanced differentiation of mouse ES cells towards a phenotype expressing specific β-cell genes and releasing insulin.

Insulin releasing properties of the temporin family of antimicrobial peptides

Temporin-1Vb, -1Oe, -1DRb, and -1TGb (10(-8) -10(-6)M) produced significant (p<0.05) and concentration-dependent stimulatory effects on insulin secretion from clonal rat BRIN-BD11 cells without increased release of lactate dehydrogenase. Temporin-1Va and temporin-1Vc (10(-8) - 10(-6)M) also stimulated insulin-release but were cytotoxic at 10(-6)M. Temporin-1DRa was without effect. The temporins at 10(-7) M had no effect on intracellular calcium concentrations suggesting that they stimulate insulin release via a K(ATP) channel- independent pathway.

Isolation and characterisation of an unexpected class of insulinotropic peptides in the skin of the frog Agalychnis litodryas

Skin secretions of the frog Agalychnis litodryas were evaluated for the isolation and characterisation of novel insulinotropic peptides. Crude secretions obtained from young adult frogs by mild electrical stimulation of the dorsal skin surface were purified by reverse-phase high-performance liquid chromatography (HPLC) yielding 70 fractions. In acute 20-min incubations with glucose responsive BRIN-BD11 cells, fractions 39-42 (band 1) and fractions 44-46 (band 2) significantly stimulated insulin release by 2-3.5-fold compared with 5.6 mM glucose alone. Pooled fractions in band 1 and band 2 were rechromatographed to reveal 20 homogenous peptide peaks, which elicited significant 1.5-4-fold increases in insulin release. Mass spectrometry analyses indicated molecular masses of between 1649.2 and 4988.9 Da. The two peptides with the greatest insulin-releasing activity were directly subjected to N-terminal amino acid sequence analysis. The sequence of the 3020 Da peptide, called frog skin insulinotropic peptide or FSIP, was determined as AVWKDFLKNIGKAAGKAVLNSVTDMVNE, which has 79% homology with the C-terminal of the 75 amino acid dermaseptin BIV precursor. A partial N-terminal sequence was determined for the 2546.2 Da peptide as MLADVFEKIMGD... These data indicate that the skin secretions of A. litodryas frogs contain biologically active peptides which merit further evaluation as a new class of insulin secretagogues.

Skin secretion of the toad Bombina variegata contains multiple insulin-releasing peptides including bombesin and entirely novel insulinotropic structures

Abstract Skin secretions of the toad Bombina variegata were evaluated for the isolation and characterisation of insulinotropic peptides. Crude secretions obtained from young adult toads by mild electrical stimulation of the dorsal skin surface were purified by reverse phase HPLC yielding 44 peaks. In acute incubations with glucose-responsive BRIN-BD11 cells, peaks 21, 22, 23, 24 and 25 showed a 1.5-3.5-fold increase in insulin release compared with 5.6 mM glucose alone (p<0.001; n=3). Structural analyses of the purified insulinreleasing peaks were performed by automated Edman degradation and mass spectrometry. Peptides represented by peaks 21, 22 and 23 had molecular masses of 1641.7 Da, 1662.6 Da and 1619.8 Da respectively. These peptides were unblocked by removal of pyroglutamic acid from the Nterminus prior to Edman degradation, revealing lengths of 14 amino acids. Peak 21 yielded a primary structure of PyrQRLGHQWAVGHLM, which a data base search revealed as an analogue of bombesin (His6 bombesin), while peak 23 was an exact match of bombesin (PyrQRLGNQWAVGHLM) originally isolated from Bombina bombina. Peak 22 indicated a primary structure of PyrDSFGNQWARGHFM (72% homology with bombesin). Peaks 24 and 25 revealed entirely novel insulinotropic peptides with molecular masses and primary structures of 1650.5 Da and 2300.0 Da and GKPFYPPPIYPEDM (GM-14) and IYNAICPCKHCNKCKPGLLAN (IN-21) respectively. Preliminary studies on the mechanisms underlying the insulinotropic actions of peaks 21, 22, 23 and 24 suggest possible involvement of a cAMPdependent, G protein-insensitive pathway. These data indicate that Bombina variegata skin secretions contain peptides with insulinreleasing activity, which may have mammalian counterparts and prove useful for possible exploitation as antidiabetic agents from natural resources.

Novel insulin-releasing peptides in the skin of Phyllomedusa trinitatis frog include 28 amino acid peptide from dermaseptin BIV precursor

Objective: The granular glands of amphibians have long been known to produce many biologically active compounds. The aim of this study was to isolate and characterize insulinotropic peptides from the skin of Phyllomedusa trinitatis frog. Methods and Results: Crude secretions obtained by mild electrical stimulation of the dorsal skin surface were purified by reverse phase HPLC yielding 80 fractions. In acute incubations with glucose-responsive BRIN-BD11 cells, fractions 39-40 (band 1) and fractions 43-46 (band 2) significantly stimulated insulin release by 1.5 to 2.5-fold. Pooled fractions in bands 1 and 2 were rechromatographed to 4 homogeneous peaks, each with insulin-releasing activity. Mass spectrometry analysis was successfully completed for 3 peptides, indicating 2996.4, 3379.9, and 8326.4 Da. The sequence of the 2996.4 Da peptide was determined as ALWKDILKNVGKAAGKAVLNTVTDMVNQ. This 28-amino-acid peptide has 100% homology with the C-terminal of the 75-amino-acid dermaseptin BIV precursor of a family of structurally related antimicrobial peptides in the skin of the Phyllomedusinae subfamily. Conclusion: These data demonstrate that the defensive skin secretions of P. trinitatis contain biologically active peptides, which may have mammalian counterparts and merit further investigation as insulin secretagogues.

Isolation and structural characterisation of a novel 13-amino acid insulin-releasing peptide from the skin secretion of Agalychnis calcarifer

Abstract We describe the isolation and characterisation of an insulinotropic peptide from the skin secretions of Agalychnis calcarifer frogs. Peptides in crude secretions obtained by mild electrical stimulation from the dorsal skin surface were purified by reversed-phase HPLC, yielding fractions in two zones with insulin-releasing activity (p<0.001). The peaks showing greatest in vitro insulin-releasing activity were subsequently purified to homogeneity, revealing a novel insulinotropic 13-amino-acid (1653.2 Da) peptide with the primary structure RRKPLFPLIPRPK (RK-13). A database search for RK-13 showed 53.8% similarity with the N-terminal region of proline-arginine-rich antimicrobial peptide (PR-39). Synthetic RK-13 stimulated insulin release in a dose-dependent, glucose-sensitive manner, exerting its effects through a cyclic AMP-protein kinase A pathway independent of pertussis toxin-sensitive G proteins. Unlike PR-39, RK-13 lacks antimicrobial effects on the growth of yeast, and Gram-positive and Gram-negative bacteria. Our data indicate that skin secretions of Agalychnis calcarifer frogs contain insulin-releasing peptides, including RK-13, which merit further investigation as insulin secretagogues.

Isolation and structural characterization of novel Rugosin A-like insulinotropic peptide from the skin secretions of Rana saharica frog

Skin secretions of Rana saharica were evaluated for the isolation and characterization of novel insulinotropic peptides. Crude secretions obtained from young adult frogs by mild electrical stimulation of the dorsal skin surface were purified by reversed-phase HPLC yielding 80 fractions. In acute incubations with glucose-responsive BRIN-BD11cells, fractions 36-43, 46-54 and 57-63 showed the significant 2-8-fold increase in insulin-releasing activity (P<0.001) compared with 5.6mM of glucose alone. A pool of fractions 36-43 was subsequently rechromatographed to 28 homogenous peaks out of which 7 were capable of subsequent 1.5-3-fold increase in insulin release (P<0.001). Structural analysis of the non-toxic peptides with greatest insulin-releasing activity was performed by mass spectrometry and Edman degradation. Mass spectrometry analysis of two peaks indicated the molecular masses of 1892.6 and 2930.8Da. The sequence of the 1892.6-Da peptide was determined as KGAAKGLLEVASCKLSKSC, which has 68% homology with Rugosin A originally isolated from the skin secretion of Rana rugosa. A partial N-terminal sequence was determined for the 2930.8-Da peptide as AVITGACERDVQCGGGTCCAVSLI.... These data indicate that the skin secretions of Rana saharica frogs contain novel peptides with insulin-releasing activity.