Characterization and biological actions of N-terminal truncated forms of glucose-dependent insulinotropic polypeptide

The N-terminal domain of glucose-dependent insulinotropic polypeptide (GIP) plays an important role in regulating biological activity. This study examined biological properties of several N-terminal truncated forms of GIP and two novel forms with substitutions at Phe position-6 with Arg or Val. GIP(6-42), GIP(R6-42), GIP(V6-42), GIP(7-42) and GIP(9-42) stimulated cAMP production in BRIN-BD11 cells similar to native GIP, whereas responses to GIP(3-42), GIP(4-42), GIP(5-42) and GIP(8-42) were reduced (P < 0.01 to P < 0.001). GIP-induced cyclic AMP production was significantly inhibited by GIP(3-42), GIP(4-42), GIP(5-42), GIP(6-42), GIP(R6-42), GIP(7-42) and GIP(8-42) (P < 0.001). Compared with native GIP, in vitro insulinotropic activity of GIP(3-42), GIP(4-42), GIP(5-42), GIP(7-42) and GIP(8-42) was reduced (P < 0.05 to P < 0.001), with GIP(4-42), GIP(5-42), GIP(7-42) and GIP(8-42) also potently inhibiting GIP-stimulated insulin secretion (P < 0.001). In ob/ob mice, GIP(4-42) and GIP(8-42) increased (P < 0.05 to P < 0.01) plasma glucose concentrations compared to the glucose-lowering action of native GIP. When GIP(8-42) was co-administered with native GIP it countered the ability of the native peptide to lower plasma glucose and increase circulating insulin concentrations. These data confirm the importance of the N-terminal region of GIP in regulating bioactivity and reveal that sequential truncation of the peptide yields novel GIP receptor antagonists which may have functional significance.     

Peptidomic analysis of skin secretions from the bullfrog Lithobates catesbeianus (Ranidae) identifies multiple peptides with potent insulin-releasing activity

Using a combination of reversed-phase HPLC and electrospray mass spectrometry, peptidomic analysis of norepinephrine-stimulated skin secretions of the American bullfrog Lithobates catesbeianus Shaw, 1802 led to the identification and characterization of five newly described peptides (ranatuerin-1CBb, ranatuerin-2CBc, and -CBd, palustrin-2CBa, and temporin-CBf) together with seven peptides previously isolated on the basis of their antimicrobial activity (ranatuerin-1CBa, ranatuerin-2CBa, brevinin-1CBa, and -1CBb, temporin-CBa, -CBb, and -CBd). The abilities of the most abundant of the purified peptides to stimulate the release of insulin from the rat BRIN-BD11 clonal β cell line were evaluated. Ranatuerin-2CBd (GFLDIIKNLGKTFAGHMLDKIRCTIGTCPPSP) was the most potent peptide producing a significant stimulation of insulin release (119% of basal rate, P < 0.01) from BRIN-BD11 cells at a concentration of 30 nM, with a maximum response (236% of basal rate, P < 0.001) at a concentration of 3 μM. Ranatuerin-2CBd did not stimulate release of the cytosolic enzyme, lactate dehydrogenase at concentrations up to 3 μM, indicating that the integrity of the plasma membrane had been preserved. Brevinin-1CBb (FLPFIARLAAKVFPSIICSVTKKC) produced the maximum stimulation of insulin release (285% of basal rate, P < 0.001 at 3 μM) but the peptide was cytotoxic at this concentration.     

Assessment of the potential of temporin peptides from the frog Rana temporaria (Ranidae) as anti‐diabetic agents

Temporin A (FLPLIGRVLSGIL‐NH₂), temporin F (FLPLIGKVLSGIL‐NH₂), and temporin G (FFPVIGRILNGIL‐NH₂), first identified in skin secretions of the frog Rana temporaria, produced concentration‐dependent stimulation of insulin release from BRIN‐BD11 rat clonal β‐cells at concentrations ≥1 nM, without cytotoxicity at concentrations up to 3 μM. Temporin A was the most effective. The mechanism of insulinotropic action did not involve an increase in intracellular Ca²⁺ concentrations. Temporins B, C, E, H, and K were either inactive or only weakly active. Temporins A, F, and G also produced a concentration‐dependent stimulation of insulin release from 1.1B4 human‐derived pancreatic β‐cells, with temporin G being the most potent and effective, and from isolated mouse islets. The data indicate that cationicity, hydrophobicity, and the angle subtended by the charged residues in the temporin molecule are important determinants for in vitro insulinotropic activity. Temporin A and F (1 μM), but not temporin G, protected BRIN‐BD11 cells against cytokine‐induced apoptosis (P < 0.001) and augmented (P < 0.001) proliferation of the cells to a similar extent as glucagon‐like peptide‐1. Intraperitoneal injection of temporin G (75 nmol/kg body weight) together with a glucose load (18 mmol/kg body weight) in C57BL6 mice improved glucose tolerance with a concomitant increase in insulin secretion whereas temporin A and F administration was without significant effect on plasma glucose levels. The study suggests that combination therapy involving agents developed from the temporin A and G sequences may find application in Type 2 diabetes treatment.     

An inversion supergene in Drosophila underpins latitudinal clines in survival traits

Chromosomal inversions often contribute to local adaptation across latitudinal clines, but the underlying selective mechanisms remain poorly understood. We and others have previously shown that a clinal inversion polymorphism in Drosophila melanogaster, In(3R)Payne, underpins body size clines along the North American and Australian east coasts. Here, we ask whether this polymorphism also contributes to clinal variation in other fitness‐related traits, namely survival traits (lifespan, survival upon starvation and survival upon cold shock). We generated homokaryon lines, either carrying the inverted or standard chromosomal arrangement, isolated from populations approximating the endpoints of the North American cline (Florida, Maine) and phenotyped the flies at two growth temperatures (18 °C, 25 °C). Across both temperatures, high‐latitude flies from Maine lived longer and were more stress resistant than low‐latitude flies from Florida, as previously observed. Interestingly, we find that this latitudinal pattern is partly explained by the clinal distribution of the In(3R)P polymorphism, which is at ~ 50% frequency in Florida but absent in Maine: inverted karyotypes tended to be shorter‐lived and less stress resistant than uninverted karyotypes. We also detected an interaction between karyotype and temperature on survival traits. As In(3R)P influences body size and multiple survival traits, it can be viewed as a ‘supergene’, a cluster of tightly linked loci affecting multiple complex phenotypes. We conjecture that the inversion cline is maintained by fitness trade‐offs and balancing selection across geography; elucidating the mechanisms whereby this inversion affects alternative, locally adapted phenotypes across the cline is an important task for future work.     

Specific binding of the C-peptide of proinsulin to cultured B-cells from a transplantable rat islet cell tumor

Specific binding of the C-peptide of proinsulin was evaluated using a transplantable NEDH rat islet cell tumour predominantly composed of insulin-secreting B-cells. Cultured tumour B-cells exhibited greater than 90% viability assessed by trypan blue exclusion, and retained the ability to form tumours with accompanying hypoglycaemia and hyperinsulinaemia after reimplantation. During binding experiments with synthetic rat C-peptide I and iodinated tyrosylated rat C-peptide I, turnout B-cells exhibited 54±6% specific binding. Displacement of tracer increased with increasing concentrations of unlabelled rat C-peptide I (0.25–1,000 ng/ml), and the specificity of binding was substantiated by reduced displacement with human C-peptide. Scatchard analysis of specific C-peptide binding revealed a curvilinear plot with upward concavity. The demonstration of specific C-peptide binding to insulin-secreting B-cells provides evidence for a physiological role of proinsulin C-peptide.     

Development of glucose intolerance and impaired plasma insulin response to glucose in obese hyperglycaemic (ob/ob) mice

The development of glucose intolerance in Aston ob/ob mice showed a gross exaggeration of the age-related changes of glucose tolerance in lean (+/+) mice. Intraperitoneal glucose tolerance in ob/ob mice was poor at 5 weeks, improved by 10 weeks, but markedly worsened by 20 weeks. A 24 hour fast further exaggerated the glucose intolerance of ob/ob mice. Unlike lean mice, tolerance improved in ob/ob mice at 40 weeks. Alterations of insulin sensitivity and the plasma insulin response to glucose accounted in part for these observations. Insulin sensitivity deteriorated until 20 weeks, but improved at 40 weeks in both fed and 24 hour fasted ob/ob mice. A positive plasma insulin response to glucose was lost after 5 weeks in fed ob/ob mice. The severity of this abnormality corresponded with the extent of the basal hyperinsulinaemia. A 24 hour fast reduced plasma insulin concentrations and restored a positive plasma insulin response to glucose in ob/ob mice. The results suggest that the plasma insulin response to glucose in ob/ob mice is related to the secretory activity of the B-cells prior to stimulation. Furthermore, it is evident that factors in addition to insulin insensitivity and the impaired plasma insulin response to glucose contribute to the development of glucose intolerance in these mice.