Proinsulin C-peptide elicits disaggregation of insulin resulting in enhanced physiological insulin effects

Using surface plasmon resonance (SPR) and electrospray mass spectrometry (ESI-MS), proinsulin C-peptide was found to influence insulin-insulin interactions. In SPR with chip-bound insulin, C-peptide mixed with analyte insulin increased the binding, while alone C-peptide did not. A control peptide with the same residues in random sequence had little effect. In ESI-MS, C-peptide lowered the presence of insulin hexamer. The data suggest that C-peptide promotes insulin disaggregation. Insulin/insulin oligomer μM dissociation constants were determined. Compatible with these findings, type 1 diabetic patients receiving insulin and C-peptide developed 66% more stimulation of glucose metabolism than when given insulin alone. A role of C-peptide in promoting insulin disaggregation may be important physiologically during exocytosis of pancreatic β-cell secretory granulae and pharmacologically at insulin injection sites. It is compatible with the normal co-release of C-peptide and insulin and may contribute to the beneficial effect of C-peptide and insulin replacement in type 1 diabetics. Received 3 May 2006; received after revision 9 June 2006; accepted 12 June 2006 Free Online Access     

Separate functional features of proinsulin C-peptide

Proinsulin C-peptide influences a number of physiological parameters in addition to its well-established role in the parent proinsulin molecule. It is of interest as a candidate for future co-replacement therapy with insulin for patients with diabetes mellitus type 1, but specific receptors have not been identified and additional correlation with functional effects is desirable. Based on comparisons of 22 mammalian proinsulin variants, we have constructed analogues for activity studies, choosing phosphorylation of mitogen-activated protein kinases (MAPKs) in Swiss 3T3 fibroblasts for functional measurements. In this manner, we find that effective phosphorylation of MAPKs is promoted by the presence of conserved glutamic acid residues at positions 3, 11 and 27 of C-peptide and by the presence of helix-promoting residues in the N-terminal segment. Previous findings have ascribed functional roles to the C-terminal pentapeptide segment, and all results combined therefore now show the importance of different segments, suggesting that C-peptide interactions are complex or multiple.Received 2 May 2005; received after revision 9 June 2005; accepted 13 June 2005     

Proinsulin C-peptide and its analogues induce intracellular Ca2+ increases in human renal tubular cells

Based on the findings that proinsulin C-peptide binds specifically to cell membranes, we investigated the effects of C-peptide and related molecules on the intracellular Ca²⁺ concentration ([Ca²⁺]_i) in human renal tubular cells using the indicator fura-2/AM. The results show that human C-peptide and its C-terminal pentapeptide (positions 27–31, EGSLQ), but not the des (27–31) C-peptide or randomly scrambled C-peptide, elicit a transient increase in [Ca²⁺]_i. Rat C-peptide and rat C-terminal pentapeptide also induce a [Ca²⁺]_i response in human tubular cells, while a human pentapeptide analogue with Ala at position 1 gives no [Ca²⁺]_i response, and those with Ala at positions 2–5 induce responses with different amplitudes. These results define a species cross-reactivity for C-peptide and demonstrate the importance of Glu at position 1 of the pentapeptide. Preincubation of cells with pertussis toxin abolishes the effect on [Ca²⁺]_i by both C-peptide and the pentapeptide. These results are compatible with previous data on C-peptide binding to cells and activation of Na⁺,K⁺ATPase. Combined, all data show that C-peptide is a bioactive peptide and suggest that it elicits changes in [Ca²⁺]_i via G-protein-coupled pathways, giving downstream enzyme effects. Received 13 May 2002; accepted 16 May 2002     

Cellular internalization of proinsulin C-peptide

Proinsulin C-peptide is known to bind specifically to cell membranes and to exert intracellular effects, but whether it is internalized in target cells is unknown. In this study, using confocal microscopy and immunostained or rhodamine-labeled peptide, we show that C-peptide is internalized and localized to the cytosol of Swiss 3T3 and HEK-293 cells. In addition, transport into nuclei was found using the labeled peptide. The internalization was followed at 37°C for up to 1 h, and was reduced at 4°C and after preincubation with pertussis toxin. Hence, it is concluded to occur via an energy-dependent, pertussis toxin-sensitive mechanism and without detectable degradation within the experimental time course. Surface plasmon resonance measurements demonstrated binding of HEK-293 cell extract components to C-peptide, and subsequent elution of bound material revealed the components to be intracellular proteins. The identification of C-peptide cellular internalization, intracellular binding proteins, absence of rapid subsequent C-peptide degradation and apparent nuclear internalization support a maintained activity similar to that of an intracrine peptide hormone. Hence, the data suggest the possibility of one further C-peptide site of action. Received 31 October 2006; received after revision 27 December 2006; accepted 30 December 2006     

The physiological and nutritional importance of dietary fibre

Summary Fibrous material is an integral part of the daily diet, and it exerts direct physiological effects throughout the gastrointestinal tract, in addition to affecting metabolic activities more indirectly. The interplay of these effects is responsible for the presumed desirable influence of fibre on weight regulation, carbohydrate and lipid metabolism, and on colon function. Numerous mechanisms of action have been identified which are related to the type and the physicochemical nature of the fibre. This review concentrates mainly on the serum cholesterol-lowering effect of dietary fibre, its colonic fermentation, and finally on some possible adverse effects that one should be aware of when consuming high amounts of dietary fibre.     

The physiological and nutritional importance of dietary fibre.

Fibrous material is an integral part of the daily diet, and it exerts direct physiological effects throughout the gastrointestinal tract, in addition to affecting metabolic activities more indirectly. The interplay of these effects is responsible for the presumed desirable influence of fibre on weight regulation, carbohydrate and lipid metabolism, and on colon function. Numerous mechanisms of action have been identified which are related to the type and the physicochemical nature of the fibre. This review concentrates mainly on the serum cholesterol-lowering effect of dietary fibre, its colonic fermentation, and finally on some possible adverse effects that one should be aware of when consuming high amounts of dietary fibre.     

C-peptide fragments stimulate glucose utilization in diabetic rats

Studies of C-peptide cellular effects show that not only the full-length native peptide but also specific C-terminal fragments are biologically active in in vitro systems. In the present study, the effect of five C-peptide fragments and the native peptide on whole-body glucose turnover was studied in streptozotocin diabetic rats using the insulin clamp technique. Insulin was infused intravenously at 18 pmol kg^–1 min^–1 for 90 min and blood glucose concentration was clamped at 8 and 4 mM in diabetic and non-diabetic animals. A steady state was reached during the last 30 min of the study period. Rat C-peptide II and fragments comprising residues 27–31 and 28–31 were effective in augmenting glucose turnover in diabetic rats (+100% to 150%), while no significant effects were seen for segments 1–26, 11–19 and 11–15. The metabolic clearance rate for glucose during infusion of C-peptide or fragments 27–31 and 28–31 in diabetic rats was similar to that seen in non-diabetic animals. We conclude that C-terminal tetra- and pentapeptides, but not fragments from the middle segment of C-peptide, are as effective as the full-length peptide in stimulating whole-body glucose turnover in diabetic rats.Received 18 December 2003; received after revision 19 January 2004; accepted 21 January 2004     

Proinsulin C-peptide and its C-terminal pentapeptide: degradation in human serum and Schiff base formation with subsequent CO<Subscript>2</Subscript> incorporation

Processing of human proinsulin C-peptide and its C-terminal pentapeptide in blood serum was studied using reverse-phase HPLC and electrospray mass spectrometry. The results reveal degradation of both peptides, with a longer half-life for intact C-peptide than for the C-terminal pentapeptide. Products from C-peptide degradation were not distinguishable from the peptide background, suggesting endopeptidase degradation of C-peptide. In contrast, a set of products from the C-terminal pentapeptide were identifiable and corresponded to successive losses from the N terminus, showing that the pentapeptide is degraded by aminopeptidase in serum. Consistent with this finding, a slower degradation was found for the N-acetyl-protected pentapeptide. Removal of serum proteins by acetone precipitation produced N-terminally carbamate-modified C-peptide via a Schiff base intermediate (a ketimine with acetone), to which CO(2) was added and acetone removed, generating a cyclic side chain via anhydride formation. The modification was not seen with the pyroglutamate form of C-peptide, with the N-terminally acetylated C-peptide, or with a control peptide having N-terminal Phe, but was found with human C-peptide, its N-terminal tetrapeptide, and a rat C-peptide fragment (all with N-terminal Glu). Hence, the modification appears to require N-terminal Glu, but this is not the only prerequisite since the C-terminal pentapeptide and another control peptide (also starting with Glu) were not modified. A peptide aldimine Schiff base leading to CO(2) incorporation was detected with formaldehyde in NaHCO(3). The observation that C-peptide forms Schiff bases with ketones/aldehydes, enhancing covalent attachment of CO(2), may have biological implications.     

Liver enlargement induced by oxythioquinox: effect of the fat content of the diet (author's transl)]

The relationship between the level of lipid calories in the diet and the effects of oxythioquinox, administered at 200 mg/kg fresh food, for 35 days, was studied on the liver enlargment of rat. The results show that the level lipid calories itself have no effect since, in the treated animals, the liver enlargement is due part to an increase of the water content and part to an increase of the size of the cells.     

Partition coefficients of drugs in bilayer lipid membranes

The oil/water partition coefficient of drugs is widely accepted as a key parameter in drug design. The coefficients are usually determined using a bulk octanol phase to represent the lipid. The physiologically and pharmacologically relevant structure is, of course, the bilayer lipid membrane, but until now there has been no convenient means of measuring the partition coefficients of small molecules into a single bilayer. This paper demonstrates that the partition coefficient may be calculated from the change in membrane refractive index which occurs when a drug molecule partitions into the membrane. The refractive index is determined by an integratedoptics technique ideally suited to an ultra-thin structure such as a lipid bilayer.     

Antimicrobial peptides: natural templates for synthetic membrane-active compounds

The innate immunity of multicellular organisms relies in large part on the action of antimicrobial peptides (AMPs) to resist microbial invasion. Crafted by evolution into an extremely diversified array of sequences and folds, AMPs do share a common amphiphilic 3-D arrangement. This feature is directly linked with a common mechanism of action that predominantly (although not exclusively) develops upon interaction of peptides with cell membranes of target cells. This minireview reports on current understanding of the modes of interaction of AMPs with biological and model membranes, especially focusing on recent insights into the folding and oligomerization requirements of peptides to bind and insert into lipid membranes and exert their antibiotic effects. Given the potential of AMPs to be developed into a new class of anti-infective agents, emphasis is placed on how the information on peptide-membrane interactions could direct the design and selection of improved biomimetic synthetic peptides with antibiotic properties.     

Hypertrophie du foie provoquée par l'oxythioquinox: Influence du taux de lipides de la ration alimentaire

Summary The relationship between the level of lipid calories in the diet and the effects of oxythioquinox, administered at 200 mg/kg fresh food, for 35 days, wa studied on the liver enlargement of rat. The results show that the level lipid calories itself have no effect since, in the treated animals, the liver enlargement is due part to an increase of the water content and part to an increase of the size of the cells.     

Ligand-dependent localization and function of ORP–VAP complexes at membrane contact sites

Oxysterol-binding protein/OSBP-related proteins (ORPs) constitute a conserved family of sterol/phospholipid-binding proteins with lipid transporter or sensor functions. We investigated the spatial occurrence and regulation of the interactions of human OSBP/ORPs or the S. cerevisiae orthologs, the Osh (OSBP homolog) proteins, with their endoplasmic reticulum (ER) anchors, the VAMP-associated proteins (VAPs), by employing bimolecular fluorescence complementation and pull-down set-ups. The ORP–VAP interactions localize frequently at distinct subcellular sites, shown in several cases to represent membrane contact sites (MCSs). Using established ORP ligand-binding domain mutants and pull-down assays with recombinant proteins, we show that ORP liganding regulates the ORP–VAP association, alters the subcellular targeting of ORP–VAP complexes, or modifies organelle morphology. There is distinct protein specificity in the effects of the mutants on subcellular targeting of ORP–VAP complexes. We provide evidence that complexes of human ORP2 and VAPs at ER–lipid droplet interfaces regulate the hydrolysis of triglycerides and lipid droplet turnover. The data suggest evolutionarily conserved, complex ligand-dependent functions of ORP–VAP complexes at MCSs, with implications for cellular lipid homeostasis and signaling.     

Antiperoxide effect of S-allyl cysteine sulfoxide,an insulin secretagogue,in diabetic rats

Treatment of alloxan diabetic rats with the antioxidant S-allyl cysteine sulfoxide (SACS) isolated from garlic (Allium sativum Linn)., ameliorated the diabetic condition almost to the same extent as did glibenclamide and insulin. In addition, SACS controlled lipid peroxidation better than the other two drugs. Furthermore, SACS significantly stimulated in vitro insulin secretion from B cells isolated from normal rats. Hence it can be surmised that the beneficial effects of SACS could be due to both its antioxidant and its secretagogue actions. The former effect is more predominant and the latter is only secondary. These effects highlight the therapeutic value of garlic, which is a component of many diets.     

Intracellular osmotic action

Water often acts as a critical reactant in cellular reactions. Its role can be detected by modulating water activity with osmotic agents. We describe the principles behind this 'osmotic stress' strategy, and survey the ubiquity of water effects on molecular structures that have aqueous, solute-excluding regions. These effects are seen with single-functioning molecules such as membrane channels and solution enzymes, as well as in the molecular assembly of actin, the organization of DNA and the specificity of protein/DNA interactions.     

Lipid peroxidation,low-level chemiluminescence and regulation of secretion in the mammary gland

In mammary explants of lactating mice, changes in the intensity of chemiluminescence (CL) were observed after the addition to the incubation medium of hormones and mediators that are involved in the regulation of secretion: oxytocin, acetylcholine, epinephrine and norepinephrine. A 15-min period of treatment with oxytocin, epinephrine or norepinephrine changed the level of thiobarbituric acid-reactive substances (TBARS). Two mammary explants, one of which was treated with oxytocin, acetylcholine, epinephrine or norepinephrine, were found to interact even when separated by a quartz glass wall. Analysis of the level of TBARS formation in these two explants showed that the observed interactions might be connected with light emission resulting from lipid peroxidation (LP) processes. The possible role of LP and low-level CL in the regulation of mammary gland secretion is discussed.     

Opposite actions of testosterone and progesterone on UCP1 mRNA expression in cultured brown adipocytes

The brown adipose tissue (BAT) thermogenic response to diet-induced obesity and cold has been found to be gender dependent. In the present work, we aimed to investigate the effects of the main physiological male and female sex hormones, i.e. testosterone, progesterone and 17-β-estradiol, on the expression of uncoupling protein 1 (UCP1) – the main mediator of BAT thermogenesis – and on UCP2 and lipid accumulation in rodent brown adipocytes differentiated in culture. Testosterone-treated cells showed fewer and smaller lipid droplets than control cells and a dose-dependent inhibition of UCP1 mRNA expression, under adrenergic stimulation by norepinephrine (NE). These effects were reverted by the androgen receptor antagonist flutamide, suggesting they are dependent, at least in part, on the androgen receptor. Progesterone- and 17-β-estradiol-treated cells showed more and larger lipid droplets and progesterone stimulated NE-induced UCP1 mRNA expression at the lower concentration tested, but not at higher concentrations, suggesting that for brown adipocytes, this hormone is dose dependent. 17-β-Estradiol did not have any remarkable effect either on UCP1 or UCP2 mRNA expression. Interestingly, the specific progesterone receptor antagonist RU486 induced UCP1 and UCP2 mRNAs, including UCP1 mRNA expression in non-NE-treated brown adipocytes, suggesting a profound effect of this anti-progestagen on brown adipocyte thermogenic capacity. Thus, are conclude that testosterone, 17-β-estradiol, progesterone and RU486 have distinct actions on brown adipocytes, thus modulating UCP1 and UCP2 mRNA expression and/or lipid accumulation, and that sex hormones are factors that may explain in part the gender-dependent BAT thermogenic response. Received 24 June 2002; received after revision 20 August 2002; accepted 26 August 2002 RID="*" ID="*"Corresponding author.     

Degradation of proinsulin C-peptide in kidney and placenta extracts by a specific endoprotease activity

Degradation of proinsulin C-peptide in mouse kidney and human placenta extracts was studied using reverse-phase high-performance liquid chromatography and nano-electrospray mass spectrometry. In total, 15 proteolytic cleavage sites were identified in human and mouse C-peptides. Early sites included the peptide bonds N-terminal of Val/Leu10, Leu12, Leu21, Leu24 and Leu26 in different combinations for the two tissues and two peptides. Notably, these cleavages were N-terminal of a hydrophobic residue, and all but one N-terminal of Leu. A late degradation product of the human peptide detected in the kidney extract was the C-terminal hexapeptide, containing just one residue more than the biologically active C-terminal pentapeptide of C-peptide. We conclude that the degradation of C-peptide in kidney and placenta follows similar patterns, dominated by endopeptidase cleavages N-terminal of Leu.