重组人GIP衍生物的克隆、表达、纯化及活性研究

葡萄糖依赖性促胰岛素释放肽(glucose-dependent insulinotropic polypeptide,GIP)具有促胰岛素分泌的作用,但因体内半衰期短而限制了其应用。对二肽酶4(dipeptidase IV,DPP IV)识别位点等氨基酸进行改造可有效延长其半衰期。通过人工合成基因或PCR定点突变的方法合成GIP不同突变体基因,将其克隆到大肠杆菌表达载体pET32a(+)中进行诱导表达,通过亲和层析,纯化获得各对应的多肽。经肠激酶酶切后,分别在昆明鼠及糖尿病模型鼠体内研究其生物活性。结果显示突变体mGIP243在昆明鼠体内具有显著的降血糖活性,当给药剂量为48nmol/kg时,其体内降血糖活性可延长至90分钟,相对于将第二位上的丙氨酸突变成丝氨酸的mGIP2和天然GIP半衰期显著延长。GIP类似物mGIP243在2型糖尿病模型鼠中也有降血糖活性,使其可能成为治疗糖尿病的候选药物。     

Regulation of neuropeptide Y (NPY) binding by guanine nucleotides in the rat cerebral cortex

The Na+-motive NADH oxidase activity from Vibrio alginolyticus was extracted with octylglucoside and reconstituted into liposomes by dilution. On the addition of NADH, the reconstituted proteoliposomes generated delta psi (inside positive) and delta pH (inside alkaline) in the presence of a proton conductor CCCP, and accumulated Na+ in the presence of valinomycin. These results indicate that the NADH oxidase activity, reconstituted in opposite orientation, leads to the generation of an electrochemical potential of Na+ by the influx of Na+.     

DPP IV resistance and insulin releasing activity of a novel di-substituted analogue of glucose-dependent insulinotropic polypeptide, (Ser2-Asp13)GIP

Structure-function studies suggest that preservation of the N-terminus and secondary structure of glucose-dependent insulinotropic polypeptide (GIP) is important for biological activity. Therefore, a novel di-substituted analogue of GIP, (Ser(2)-Asp(13))GIP, containing a negatively charged Asp residue in place of an Ala in position 13, was synthesised and evaluated for in vitro biological activity. Incubation with dipeptidyl peptidase IV (DPP IV) showed the half-lives of GIP and (Ser(2)-Asp(13))GIP to be 2.3 and >4h, respectively. Insulin releasing studies in clonal pancreatic BRIN-BD11 cells demonstrated that (Ser(2)-Asp(13))GIP (10(-12)to 10(-7)mol/l) was significantly less potent (60-90%; P<0.05 to P<0.001) than native GIP. The peptide failed to display antagonistic properties as it did not significantly alter insulin secretion when incubated in the presence of GIP (10(-7)mol/l). These results demonstrate that despite increased resistance to DPP IV, substituting Ala in position 13 with a negatively charged Asp, thus producing the di-substituted analogue (Ser(2)-Asp(13))GIP, significantly reduces biological activity, most likely due to modifications within the secondary structure.     

Regional specificity of the gut-incretin response to small intestinal glucose infusion in healthy older subjects

The importance of the region, as opposed to the length, of small intestine exposed to glucose in determining the secretion of the incretin hormones glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) remains unclear. We sought to compare the glycemic, insulinemic and incretin responses to glucose administered to the proximal (12–60 cm beyond the pylorus), or more distal ( > 70 cm beyond the pylorus) small intestine, or both. 10 healthy subjects (9M,1F; aged 70.3 ± 1.4 years) underwent infusion of glucose via a catheter into the proximal (glucose proximally; GP), or distal (glucose distally; GD) small intestine, or both (GPD), on three separate days in a randomised fashion. Blood glucose, serum insulin and plasma GLP-1, GIP and CCK responses were assessed. The iAUC for blood glucose was greater in response to GPD than GP (P < 0.05), with no difference between GD and GP. GP was associated with minimal GLP-1 response (P = 0.05), but substantial increases in GIP, CCK and insulin (P < 0.001 for all). GPD and GD both stimulated GLP-1, GIP, CCK and insulin (P < 0.001 for all). Compared to GP, GPD induced greater GLP-1, GIP and CCK responses (P < 0.05 for all). Compared with GPD, GD was associated with greater GLP-1 (P < 0.05), but reduced GIP and CCK (P < 0.05 for both), responses. We conclude that exposure of glucose to the distal small intestine appears necessary for substantial GLP-1 secretion, while exposure of both the proximal and distal small intestine result in substantial secretion of GIP.     

Comparative effects of intraduodenal fat and glucose on the gut-incretin axis in healthy males

BackgroundThe interaction of nutrients with the small intestine stimulates the secretion of numerous enteroendocrine hormones that regulate postprandial metabolism. However, differences in gastrointestinal hormonal responses between the macronutrients are incompletely understood. In the present study, we compared blood glucose and plasma hormone concentrations in response to standardised intraduodenal (ID) fat and glucose infusions in healthy humans.MethodsIn a parallel study design, 16 healthy males who received an intraduodenal fat infusion were compared with 12 healthy males who received intraduodenal glucose, both at a rate of 2 kcal/min over 120 min. Venous blood was sampled at frequent intervals for measurements of blood glucose, and plasma total and active glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP), insulin and glucagon.ResultsPlasma concentrations of the incretin hormones (both total and active GLP-1 and GIP) and glucagon were higher, and plasma insulin and blood glucose concentrations lower, during intraduodenal fat, when compared with intraduodenal glucose, infusion (treatment by time interaction: P < 0.001 for each).ConclusionsCompared with glucose, intraduodenal fat elicits substantially greater GLP-1, GIP and glucagon secretion, with minimal effects on blood glucose or plasma insulin in healthy humans. These observations are consistent with the concept that fat is a more potent stimulus of the ‘gut-incretin’ axis than carbohydrate.     

Glucose-dependent insulinotropic polypeptide analogues and their therapeutic potential for the treatment of obesity-diabetes

Glucose-dependent insulinotropic polypeptide (GIP) is a key incretin hormone, released postprandially into the circulation in response to feeding, producing a glucose-dependent stimulation of insulin secretion. It is this glucose-dependency that has attracted attention towards GIP as a potential therapeutic agent for the treatment of type 2 diabetes. A major drawback to achieving this goal has been the rapid degradation of circulating GIP by the ubiquitous enzyme, dipeptidylpeptidase IV (DPP IV). However, recent studies have described a number of novel structurally modified analogues of GIP with enhanced plasma stability, insulinotropic and antihyperglycaemic activity. The purpose of this article was to provide an overview of the biological effects of several GIP modifications and to highlight the potential of such analogues in the treatment of type 2 diabetes and obesity.     

Comparison of independent and combined chronic metabolic effects of GIP and CB1 receptor blockade in high-fat fed mice

GIP receptor antagonism with (Pro³)GIP protects against obesity, insulin resistance, glucose intolerance and associated disturbances in mice fed high-fat diet. Furthermore, cannabinoid CB₁ receptor antagonism with AM251 reduces appetite and body weight gain in mice. The present study has examined and compared the effects of chronic daily administrations of (Pro³)GIP (25 nmol/kg body weight), AM251 (6 mg/kg body weight) and a combination of both drugs in high-fat fed mice. Daily i.p. injection of (Pro³)GIP, AM251 or combined drug administration over 22 days significantly (P < 0.05 to <0.01) decreased body weight compared with saline-treated controls. This was associated with a significant (P < 0.05 to <0.01) reduction of food intake in mice treated with AM251. Plasma glucose levels and glucose tolerance were significantly (P < 0.05) lowered by 22 days (Pro³)GIP, AM251 or combined drug treatment. These changes were accompanied by a significant (P < 0.05) improvement of insulin sensitivity in all treatment groups. In contrast, AM251 lacked effects on glucose tolerance, metabolic response to feeding and insulin sensitivity in high-fat mice when administered acutely. These data indicate that chemical blockade of GIP- or CB₁-receptor signaling using (Pro³)GIP or AM251, respectively provides an effective means of countering obesity and related abnormalities induced by consumption of high-fat energy-rich diet. AM251 lacks acute effects on glucose homeostasis and there was no evidence of a synergistic effect of combined treatment with (Pro³)GIP.     

The 3D structure of rat DPPIV/CD26 as obtained by cryo-TEM and single particle analysis

We present the three-dimensional structure of rat DPPIV/CD26, as determined by cryo-TEM and single particle analysis at a resolution of approximately 14A. The reconstruction confirms that the protein exists as a dimer, as predicted earlier. Since there are structural analogies to the serine peptidase POP, docking calculations of the two structures were performed. Although the docking showed a similar spatial organization (catalytic domain, beta-propeller, distal opening, central cavity), the detailed comparison revealed clear discrepancies. The most marked difference is a second (lateral) opening in DPPIV/CD26, which would enable direct access to the catalytic site. We therefore assume that substrate selectivity and binding rate are most probably driven by different mechanisms in DPPIV/CD26 and POP.     

Glucose-dependent insulinotropic peptide receptor expression in the hippocampus and neocortex of mesial temporal lobe epilepsy patients and rats undergoing pilocarpine induced status epilepticus

The glucose-dependent insulinotropic peptide receptor (GIPR) has been implicated with neuroplasticity and may be related to epilepsy. GIPR expression was analyzed by immunohistochemistry in the hippocampus (HIP) and neocortex (Cx) of rats undergoing pilocarpine induced status epilepticus (Pilo-SE), and in three young male patients with left mesial temporal lobe epilepsy related to hippocampal sclerosis (MTLE-HS) treated surgically. A combined GIPR immunohistochemistry and Fluoro-Jade staining was carried out to investigate the association between the GIPR expression and neuronal degeneration induced by Pilo-SE. GIPR was expressed in the cytoplasm of neurons from the HIP CA subfields, dentate gyrus (DG) and Cx of animals and human samples. The GIPR expression after the Pilo-SE induction increases significantly in the HIP after 1 h and 5 days, but not after 12 h or 50 days. In the Cx, the GIPR expression increases after 1 h, 12 h and 5 days, but not 50 days after the Pilo-SE. The expression of GIPR 12 h after Pilo-SE was inversely proportional to the Fluoro-Jade staining intensity. In the human tissue, GIPR expression patterns were similar to those observed in chronic Pilo-SE animals. No Fluoro-Jade stained cells were observed in the human sample. GIPR is expressed in human HIP and Cx. There was a time and region dependent increase of GIPR expression in the HIP and Cx after Pilo-SE that was inversely associated to neuronal degeneration.     

On the origin of serum CD26 and its altered concentration in cancer patients

Dipeptidyl peptidase IV (DPP-IV), assigned to the CD26 cluster, is expressed on epithelial cells and lymphocytes and is a multifunctional or pleiotropic protein. Its peptidase activity causes degradation of many biologically active peptides, e.g. some incretins secreted by the enteroendocrine system. DPP-IV has, therefore, become a novel therapeutic target for inhibitors that extend endogenously produced insulin half-life in diabetics, and several reviews have appeared in recent months concerning the clinical significance of CD26/DPP-IV. Biological fluids contain relatively high levels of soluble CD26 (sCD26). The physiological role of sCD26 and its relation, if any, to CD26 functions, remain poorly understood because whether the process for CD26 secretion and/or shedding from cell membranes is regulated or not is not known. Liver epithelium and lymphocytes are often cited as the most likely source of sCD26. It is important to establish which tissue or organ is the protein source as well as the circumstances that can provoke an abnormal presence/absence or altered levels in many diseases including cancer, so that sCD26 can be validated as a clinical marker or a therapeutic target. For example, we have previously reported low levels of sCD26 in the blood of colorectal cancer patients, which indicated the potential usefulness of the protein as a biomarker for this cancer in early diagnosis, monitoring and prognosis. Through this review, we envisage a role for sCD26 and the alteration of normal peptidase capacity (in clipping enteroendocrine or other peptides) in the complex crosstalk between the lymphoid lineage and, at least, some malignant tumours.     

3D structure of the CD26-ADA complex obtained by cryo-EM and single particle analysis

The specific binding of adenosine deaminase to the multifunctional membrane glycoprotein dipeptidyl peptidase IV is thought to be immunologically relevant for certain regulatory and co-stimulatory processes. In this study we present the 3D structure of the complete CD26-ADA complex obtained by single particle cryo-EM at 22A resolution. ADA binding occurs at the outer edges of the beta-propeller of CD26. Docking calculations of available CD26 and ADA crystal data into the obtained EM density map revealed that the ADA-binding site is stretched across CD26 beta-propeller blades 4 and 5 involving the outermost distal hydrophobic amino acids L294 and V341 but not T440 and K441 as suggested by antibody binding. Though the docking of the ADA orientation appears less significant due to the lack of distinct surface features, non-ambiguous conclusions can be drawn in the combination with earlier indirect non-imaging methods affirming the crucial role of the ADA alpha2-helix for binding.     

Comparison of the subchronic antidiabetic effects of DPP IV-resistant GIP and GLP-1 analogues in obese diabetic (ob/ob) mice.

Glucagon-like peptide-1 (GLP-1) and glucose-dependent insulinotropic polypeptide (GIP) are the two key incretin hormones released from the gastrointestinal tract that regulate blood glucose homeostasis through potent insulin secretion. The rapid degradation of GIP and GLP-1 by the ubiquitous enzyme dipeptidyl peptidase IV (DPP IV) renders both peptides noninsulinotropic. However, DPP IV stable agonists, such as N-AcGIP and (Val8)GLP-1, have now been developed. The present study has examined and compared the metabolic effects of subchronic administration of daily i.p. injections of N-AcGIP, (Val8) GLP-1 and a combination of both peptides (all at 25 nmol/kg bw) in obese diabetic (ob/ob) mice. Initial in vitro experiments confirmed the potent insulinotropic properties of N-AcGIP and (Val8)GLP-1 in the clonal pancreatic BRIN BD11 cell line. Subchronic administration of N-AcGIP, (Val8)GLP-1 or combined peptide administration had no significant effects on the body weight, food intake and plasma insulin concentrations. However, all treatment groups had significantly (p < 0.05) decreased plasma glucose levels and improved glucose tolerance by day 14. The effectiveness of the peptide groups was similar, and glucose concentrations were substantially reduced following injection of insulin to assess insulin sensitivity compared to control. These results provide evidence for an improvement of glucose homeostasis following treatment with enzyme-resistant GIP and GLP-1 analogues.     

Comparison of the activity levels and localization of dipeptidyl peptidase IV in normal and tumor human lung cells

Dipeptidyl peptidase IV (DPPIV) was studied in three human lung cells - P (fetal lung-derived cells), A549 (lung adenocarcinoma) and SK-MES-1 (squamous cell carcinoma) using a fluorescent cytochemical procedure developed on the basis of the substrate 4-(glycyl-l-prolyl hydrazido)-N-hexyl-1,8-naphthalimide. The observed differences in the enzyme expression were confirmed by measuring the enzyme hydrolysis of glycyl-l-prolyl-para-nitroanilide. The surface and total dipeptidyl peptidase activities of P cells were correspondingly 7-8 and 3-10 times higher than those of SK-MES-1 and A549 cells. The ratio surface per total activity showed that in P (95%) and A549 (93%) cells the enzyme is associated with the plasmalemma while in SK-MES-1 cells (35%) it is bound to intracellular membranes. In order to compare the results from cell cultures with those in human tumor, the enzyme activity was investigated in cryo-sections of three cases of diagnosed squamous lung carcinoma. DPPIV activity was restricted to the connective tissue stroma surrounding the DPPIV-negative tumor foci.     

Increase in DPP-IV in the intestine, liver and kidney of the rat treated with high fat diet and streptozotocin

High fat diet or insulin deficiency is commonly seen in Type II diabetes, while the mechanism remains unclear. To test our hypothesis that DPP-IV contributes to Type II diabetes, we examined the expression and activity of DPP-IV in rats (n=8 to each group) treated for 12 weeks with 3 separate diets: a) normal control; b) a high fat diet; and c) a high fat diet plus streptozotocin, a chemical for induction of insulin-deficient diabetes. Compared to rats on the normal diet, the rats with a high fat diet significantly increased DPP-IV's expression and activity about 142-152% in the intestine (P<0.05) and 153-240% in kidneys (P<0.05), but there was no change in the liver. Administration of streptozotocin to the rats treated with the high fat diet showed an insufficient insulin secretion and higher blood glucose in response to glucose/insulin tolerance test, and an increase in expression of DPP-IV and activity by 188-242% in the intestine (P<0.01); 191-225% in liver (P<0.01); and 211-321% in the kidneys (P<0.01). Immunohistochemistry studies confirmed the above results, showing increased DPP-IV immunostaining localized primarily in intestinal epithelium, hepatocytes and renal tubular cells. This study, for the first time reports an increase in DPP-IV associated with a high fat diet, as well as in the combination of a high fat diet with an insulin deficiency. Since both high fat diet and insulin deficiency are closely linked with etiology of Type II diabetes, the evidence in this study suggests a role of DPP-IV in development of Type II diabetes.     

The response of plasma gastric-inhibitory polypeptide (GIP) to slow and fast glucose ingestion in Billroth II resected patients and normal controls

Eight Billroth II resected patients and 8 normal controls were given two oral glucose loads, one ingested within 2 min, and the other ingested slowly over 80 min. In the Billroth II resected group, the integrated plasma GIP release was significantly higher after the fast than after the slow glucose ingestion. In this group the integrated plasma GIP release was also significantly higher than in the control group, but only after the fast glucose ingestion. These findings indicate that the rate of glucose delivery into the intestine may be of importance in the plasma GIP response to oral glucose.     

Characterization of the cellular and metabolic effects of a novel enzyme-resistant antagonist of glucose-dependent insulinotropic polypeptide

A novel N-terminally substituted Pro(3) analogue of glucose-dependent insulinotropic polypeptide (GIP) was synthesized and tested for plasma stability and biological activity both in vitro and in vivo. Native GIP was rapidly degraded by human plasma with only 39 +/- 6% remaining intact after 8 h, whereas (Pro(3))GIP was completely stable even after 24 h. In CHL cells expressing the human GIP receptor, (Pro(3))GIP antagonized the cyclic adenosine monophosphate (cAMP) stimulatory ability of 10(-7) M native GIP, with an IC(50) value of 2.6 microM. In the clonal pancreatic beta cell line BRIN-BD11, (Pro(3))GIP over the concentration range 10(-13) to 10(-8) M dose dependently inhibited GIP-stimulated (10(-7) M) insulin release (1.2- to 1.7-fold; P < 0.05 to P < 0.001). In obese diabetic (ob/ob) mice, intraperitoneal administration of (Pro(3))GIP (25 nmol/kg body wt) countered the ability of native GIP to stimulate plasma insulin (2.4-fold decrease; P < 0.001) and lower the glycemic excursion (1.5-fold decrease; P < 0.001) induced by a glucose load (18 mmol/kg body wt). Collectively these data demonstrate that (Pro(3))GIP is a novel and potent enzyme-resistant GIP receptor antagonist capable of blocking the ability of native GIP to increase cAMP, stimulate insulin secretion, and improve glucose homeostasis in a commonly employed animal model of type 2 diabetes.     

Absence of a relationship between immunoreactive-gastrin and somatostatin-like immunoreactivity secretion in the perfused rat stomach

The possible interaction between somatostatin-like immunoreactivity (SLI) and immunoreactive-gastrin release was studied in an isolated perfused rat stomach preparation. Gastrin release was abolished by antrectomy but basal and gastric inhibitory polypeptide-stimulated SLI levels were unchanged from control experiments, implicating the corpus as the major source of SLI released into the vasculature. Perfused stomachs of vagotomized rats exhibited basal hypergastrinaemia with no alteration in basal or stimulated SLI release, suggesting an uncoupling of SLI and gastrin release. This study indicated that SLI released into the vasculature originated in the acid secretory region of the stomach and therefore may be involved in the regulation of acid secretion at the level of the parietal cell mass.     

Absence of down-regulation of the insulin receptor by insulin. A possible mechanism of insulin resistance in the rat.

Insulin resistance occurs in rat adipocytes during pregnancy and lactation despite increased or normal insulin binding respectively; this suggests that a post-receptor defect exists. The possibility has been examined that, although insulin binding occurs normally, internalization of insulin or its receptor may be impaired in these states. Insulin produced a dose-dependent reduction in the number of insulin receptors on adipocytes from virgin rats maintained in culture medium, probably due to internalization of the hormone-receptor complex. In contrast, adipocytes from pregnant and lactating rats did not exhibit this 'down-regulation' phenomenon. Down regulation was, however, apparent in all groups when the experiments were performed in Tris buffer (where receptor recycling is inhibited), suggesting that in pregnant and lactating rats insulin receptors are rapidly recycled back to the plasma membrane, whereas in virgin rats this recycling process is less effective. Internalization of insulin was also determined by using 125I-labelled insulin. Adipocytes from pregnant and lactating rats appeared to internalize similar amounts of insulin to virgin rats. In the presence of the lysosomal inhibitor chloroquine, adipocytes from pregnant rats internalized more insulin than virgin or lactating rats. These results suggest that adipocytes from pregnant and lactating rats internalize insulin and its receptor normally, whereas intracellular processing of the insulin receptor may differ from that in virgin rats. In addition the rate of lysosomal degradation of insulin may be altered in adipocytes from pregnant rats.     

Asymmetric distribution and down-regulation of the muscarinic acetylcholine receptor in rat cerebral cortex

The distribution and down-regulation of the muscarinic acetylcholine receptor (mAChR) were studied in dissociated cells from right (RCC) and left (LCC) cerebral cortex. For this purpose [³H]quinuclidinyl benzilate (QNB) and [³H]pirenzepine (Pz), two muscarinic antagonists, were used. The mAChR binding sites detected with [³H]QNB were asymmetrically distributed between the two hemispheres, the majority being found in the RCC. Asymmetry was also evident in the distribution of the mAChR subtypes (M₁ and M₂) detected with [³H]Pz. Under basal conditions the RCC had roughly 50% more M₁ subtype than the LCC. The pharmacological and kinetic parameters were similar for both antagonists in RCC and LCC, indicating that the observed lateralization was due to a different density of the receptor rather than to different kinetics of binding of the two radioligands. After sustained stimulation with the agonist carbamoylcholine, the receptor sites detected with [³H]Pz, i.e. the M₁ subtype of mAChR, decreased at a higher rate in the RCC (44%) than in the LCC (25% of controls), demonstrating that the down-regulation process is more active in the right than in the left cortex, and thus implying that there is better coupling between the stimulated mAChR and its effector system in the former.