Cyclo (His-Pro) potentiates GABA/ethanol-mediated chloride uptake by neurosynaptosomes

Cyclo (His-Pro) CHP is a cyclic dipeptide endogenous to the brain of a variety of animal species including man. Administration of exogenous peptide to rodents has been shown to exhibit a variety of biologic activities including, modification of pharmacologic actions of alcohol. Since there are many apparent similarities between the actions of GABA and CHP in modulating alcohol pharmacology, we have examined whether CHP can modulate alcohol potentiation of GABA-receptor-mediated 36Cl-influx in neurosynaptosomes. The results show a further dose-dependent potentiation of 36Cl-influx in neurosynaptosomes by CHP in the presence of GABA and alcohol.     

Alterations in pancreatic protein expression in STZ-induced diabetic rats and genetically diabetic mice in response to treatment with hypoglycemic dipeptide Cyclo (His-Pro)

To provide insights into the molecular mechanisms underlying diabetes mellitus, we performed a proteomic study on two diabetic animal models, streptozotocin (STZ)-induced diabetic rats (T1DM) and genetically diabetic (C57BL/6J ob/ob) mice (T2DM). To better understand the recovery process of those diabetic rodents, we examined the effect of hypoglycemic dipeptide Cyclo (His-Pro) (CHP) treatment on the differential expression of pancreatic proteins in both animal models. Oral administration of CHP had an excellent hypoglycemic effect in both animal models, lowering the average plasma glucose level by over 50%. Pancreatic proteins were separated by two-dimensional gel electrophoresis (2-DE) and identified by MALDI-TOF mass spectrometry. This study allowed, for the first time, the identification of 34 proteins that are related to diabetes and potential targets of CHP, a potent anti-diabetic agent for both T1DM and T2DM. The alterations in the expression of these proteins could indicate a tendency for diabetic animals to overcome their diabetic state. These proteins are involved in cellular functions such as metabolism, cellular structure, oxidative stress, as well as signal and energy transduction. Some have already been linked to diabetes, suggesting that the newly identified proteins might also be significant in the etiology of this pathology and should be further investigated. Furthermore, CHP has emerged as a potent tool for both the treatment and study of the molecular mechanisms underlying diabetes. Thus, the findings presented here provide new insights into the study and potential treatment of this pathology.     

Role of endogenous cyclo(His-Pro) in cold-induced hypothermia in the desert rat (Mastomys natalensis)

Central administration of exogenous cyclo(His-Pro) (CHP) is known to produce hypothermia in rodents. In the present study, we examined the role of endogenous CHP in cold-induced hypothermia in the desert rat, Mastomys natalensis. The results of these studies show that a rise in hypothalamic CHP content accompanied a decrease in rectal temperature during cold exposure. Immunoneutralization of endogenous CHP resulted in a significant decline in cold-induced hypothermia. In addition, central administration of cyclo(Ala-Gly), a structural analogue of CHP, also led to a decrease in cold-induced hypothermia. The results of these studies show that changes in endogenous CHP levels may affect body temperature regulation.     

Is all cyclo(His-Pro) derived from thyrotropin-releasing hormone?

Cyclo(His-Pro), or histidyl-proline diketopiperazine, is an endogenous cyclic dipeptide that is ubiquitously distributed in tissues and body fluids of both man and animals. This cyclic dipeptide is not only structurally related to thyrotropin-releasing hormone (TRH, pGlu-His-ProNH₂), but it can also arise from TRH by the action of the enzyme pyroglutamate amino-peptidase (pGlu-peptidase). The data on the distribution of TRH, cyclo(His-Pro), and pGlu-peptidase under normal and abnormal conditions are summarized and potential relationships analyzed. We conclude that all of the cyclo(His-Pro) cannot be derived from TRH. Two additional sources of cyclo(His-Pro) are suggested. It is proposed that 29,247 molecular weight TRH prohormone, prepro TRH, which contains 5 copies of TRH sequence, can be processed to yield cyclo(His-Pro). Thus, both TRH and cyclo(His-Pro) share a common precursor, prepro[TRH/Cyclo(His-Pro)].     

Focus on cyclo(His-Pro): history and perspectives as antioxidant peptide

Cyclo(His-Pro) is an endogenous cyclic dipeptide structurally related to tyreotropin-releasing hormone that was originally discovered in brain. In the central nervous system it has been described to exert multiple biological activities, which seem to be related to a presynaptic dopaminergic mechanism and include among the others a leptin-like function. It can be found in several body fluids and in the gastrointestinal tract where it has been suggested to act as a gut peptide with influence on the entero-insular axis. The oral administration of cyclo(His-Pro) and zinc was described to improve with a synergistic mechanism the glycaemic control in diabetes. The most intriguing function of this cyclic dipeptide is related with its neuroprotective role that was first reported in traumatic injuries of the spinal cord, and then confirmed in other models of experimental injuries of the nervous system. The mechanism that lies behind the neuroprotective activity of cyclo(His-Pro) remain poorly understood. Recent in vitro studies on rat pheochromocytoma PC12 cells have shown that it is a protective factor against stress stimuli and there is early pre-clinical evidence strongly suggesting that it enhances the expression of small heat shock proteins and antioxidant protection at the cellular level. Future research is underway to better characterize the possible use of this cyclic dipeptide in the therapy of neurodegenerative and metabolic disorders.     

Specific binding to adrenal particulate fraction of cyclo(histidyl-proline), a TRH metabolite

Histidyl-proline diketopiperazine (cyclo(His-Pro), a metabolite of the neuropeptide thyrotropin releasing hormone, has been shown to possess intrinsic biological activities. The binding of this peptide to various tissue particulate preparations was investigated. While the peptide showed no apparent binding to particulate fractions derived from brain, pituitary, and some other tissues, binding to adrenal and liver was demonstrated. The binding of cyclo(His-Pro) to bovine adrenal cortical particles was further characterized. Binding at equilibrium was greater at 4 degrees C than at 37 degrees C. The binding was dependent on tissue concentration, showed a pH optimum between 7 and 8, and was inactivated by treatment of the particulate fraction with trypsin or by boiling. The interaction of cyclo(His-Pro) with the tissue was not associated with any metabolism of the peptide. Kinetic studies of association of cyclo(His-Pro) with adrenal cortical particles indicated a single class of binding sites with a KD of approximately 900 nM and a maximum number of sites of 92 pmoles/mg protein. The binding was stereospecific and the histidine moiety of the peptide was the major determinant of the binding. A variety of catechols, serotonin and histamine competed with cyclo(His-Pro) for binding with IC50's ranging from 17-450 muM. Cyclo(His-Pro) did not affect monoamine oxidase or adenylate cyclase activity in adrenal cortical particulate preparations.     

Unresponsiveness of GH cells to cyclo(histidyl-proline), a metabolite of thyrotropin releasing hormone

Cyclo(Histidyl-Proline) is a metabolite of thyrotropin-releasing hormone. It has been suggested that this peptide plays a role in regulating prolactin secretion in GH cells. An investigation of the effect of cyclo(His-Pro) on GH cells indicated that it does not affect basal prolactin release or accumulation or the levels stimulated by TRH. cAMP levels in GH cells are elevated by TRH or VIP, but not influenced by cyclo(His-Pro). cGMP levels in GH cells are not affected by either TRH or cyclo(His-Pro). While there is specific binding of TRH to receptors in GH cells, no such receptors for cyclo(His-Pro) are detectable. It is suggested that GH cells are unresponsive to cyclo(His-Pro).     

Chronic Alcohol Consumption Increases Cyclo (His-Pro)-Like Immunoreactivity in the Rat Brain

Abstract: Administration of histidyl-proline diketopiperazine (cyclo (HisPro)) to rats attenuates ethanol-induced sleep. To understand the role played by cyclo (His-Pro) in the pathophysiology of prolonged alcohol consumption, we have measured the distribution of this peptide in brains of control and alcohol-treated rats. The data show that prolonged alcohol consumption increases the concentration of cyclo (His-Pro) in hypothalamic as well as extrahypothalamic brain. These changes may reflect a physiologic adaptation of the brain during alcohol consumption.     

Differential gene expression in pancreatic tissues of streptozocin-induced diabetic rats and genetically-diabetic mice in response to hypoglycemic dipeptide cyclo (His-Pro) treatment

Diabetic studies are mostly interested in gene expression in the pancreas, the site of insulin secretion that regulates blood glucose levels. However, a single gene approach has been ruled out for many years in discovering new genes or the molecular networks involved in the induction process of diabetes. To understand the molecular mechanisms by which cyclo (His-Pro) (CHP) affects amelioration of diabetes mellitus, we performed gene expression profiling in the pancreatic tissues of two diabetic animal models, streptozocin (STZ)-induced diabetic rats (T1DM) and genetically-diabetic (C57BL/6J ob/ob) mice (T2DM). To understand the healing process of these diabetic rodents, we examined the effects of CHP on various gene expression in pancreatic tissues of both animal models. Our microarray analysis revealed that a total of 1,175 genes were down-regulated and 629 genes were up-regulated in response to STZ treatment, and the altered expression levels of numerous genes were restored to normal state upon CHP treatment. In particular, 476 genes showed significantly altered gene expression upon CHP treatment. In a functional classification, 7,198 genes were counted as differentially expressed in pancreatic tissues of STZ- and CHP-treated rats compared with control, whereas 1,534 genes were restored to normal states by CHP treatment. Microarray data demonstrated for the first time that overexpression of the genes encoding IL-1 receptor, lipid metabolic enzymes (e.g. Mte1, Ptdss1, and Sult2a1), myo-inositol oxygenase, glucagon, and somatostatin as well as down-regulation of olfactory receptor 984 and mitochondrial ribosomal protein, which are highly linked to T1DM etiology. In genetically-diabetic mice, 4,384 genes were altered in gene expression by more than 2-fold compared to the control mice, when counted differentially expressed. In genetically-diabetic mice, 4,384 genes altered in expression by higher than 2-fold were counted as differentially expressed genes in pancreatic tissues of CHP-treated mice. On the other hand, 2,140 genes were up-regulated and 2,244 genes were down-regulated by CHP treatment. The results of the microarray analysis revealed that up-regulation of IL-2, IL12a, and leptin receptor and down-regulation of PIK3 played important physiological roles in the onset of T2DM. In conclusion, we hypothesize that CHP accelerates alterations of gene expression in ameliorating diabetes and antagonizes those that induces the disease.     

[3H]cyclo(Histidyl-Proline) in rat tissues: Distribution,clearance and binding

Cyclo(Histidyl-Proline), a metabolite of TRH, has been demonstrated to have a number of biological activities. The clearance, distribution and binding of the peptide in the rat was studied. Cyclo(His-Pro) was cleared from the circulation biphasically ($\text{tl}\text{2}\text{= 1.25 and 33 min}$). Unmetabolized cyclo(His-Pro) appeared rapidly in urine. Accumulation of [³H]cyclo(His-Pro) in adrenal, liver and kidney was demonstrated. Membrane preparations from adrenal and liver, but not from kidney, brain, pituitary, and other tissues were shown to bind cyclo(His-Pro) specifically.     

Liver tryptophan pyrrolase. A major determinant of the lower brain 5-hydroxytryptamine concentration in alcohol-preferring C57BL mice.

The lower brain 5-hydroxytryptamine concentration in alcohol-preferring C57BL, compared with -non-preferring CBA, mice is caused by a decrease in circulating tryptophan availability to the brain secondarily to a higher liver tryptophan pyrrolase activity associated with a higher circulating corticosterone concentration. Activity or expression of liver tryptophan pyrrolase and/or their induction by glucocorticoids may be important biological determinants of predisposition to alcohol consumption.     

Effect of asymmetric dimethylarginine on atherogenesis and erythrocyte deformability in apolipoprotein E deficient mice

Previous investigations have shown that the level of asymmetric dimethylarginine (ADMA) was increased in hypercholesterolemic animal and humans, and the decreased erythrocyte deformability has been suggested to be a factor contributing to atherogenesis. In the present study, we investigated the effect of ADMA, endogenous or exogenous, on atherogenesis and erythrocyte deformability in apolipoprotein E deficient (ApoE-/-) mice. On a regular chow diet, ApoE-/- mice or C57BL/6 J mice at 12 weeks of age were treated with ADMA (5 mg/kg/day) for 4 weeks. Atherosclerotic lesion area, erythrocyte deformability, plasma lipids and asymmetric dimethylarginine (ADMA) level were determined. Plasma concentrations of triglyceride (TG), low-density lipoprotein-cholesterol (LDL-C), total cholesterol (TC), ADMA, and atherosclerotic lesion area were significantly increased, and the level of plasma high-density lipoprotein-cholesterol (HDL-C), erythrocyte deformability in ApoE-/- mice were markedly decreased compared with that of C57BL/6J mice (P<0.05 or P<0.01). Exogenous ADMA treatment increased the plasma TG level, produced atherosclerotic lesions, and decreased erythrocyte deformability in C57BL/6J mice (P<0.05 or P<0.01). Treatment with exogenous ADMA further increased the plasma TG level and lesion areas, and decreased erythrocyte deformability in ApoE-/- mice. In vitro, exogenous ADMA caused a decrease of erythrocyte deformability in a concentration-dependent manner, and the effect of ADMA was reversed by L-arginine. The present results suggest that endogenous ADMA is an important contributor to the development of atherosclerosis and that reduction of erythrocyte deformability and impaired endothelial function induced by ADMA may be an important factor facilitating atherosclerotic lesions.     

Histidyl-proline diketopiperazine cyclo (His-Pro): identification and characterization in rat pancreatic islets

Measurements of cyclo (His-Pro) in the pancreas were carried out in the rat by a specific radioimmunoassay. Cyclo (His-Pro)-like immunoreactivity was identified in pancreatic islets with a mean concentration of 2023 pg/mg protein, 88-fold higher than that of the whole pancreas. Cyclo (His-Pro) immunoreactivity from pancreatic extracts was indistinguishable immunologically and chromatographically from synthetic cyclo (His-Pro). Insulin-induced hypoglycemia caused a significant, 53% decrease in pancreatic cyclo (His-Pro) concentrations, and FLA-63, a dopamine beta-oxidase inhibitor, also reduced islet cyclo (His-Pro) concentrations 51%. These data indicate that cyclo (His-Pro) is present in rat pancreatic islets and may play a potential role in modulating pancreatic responses to nutrient and pharmacologic stimuli.     

Use of genetic analysis to test the potential role of serotonin in alcohol preference.

The hypothesis that alcohol preference in mice is influenced by brain serotonin levels was tested using genetic analysis. Alcohol preference and static serotonin content were assessed in C57BL/Ibg (alcohol-preferring) and DBA/2 (alcohol-avoiding) mice, as well as in Fl and F2 generations obtained by crossbreeding. The two parental strains showed dissimilar alcohol preferences but identical concentrations of brain serotonin. Serotonin concentration segregated independently of alcohol preference in the F1 and F2 generations. These data provides strong evidence against the hypothesis that brain serotonin content influences alcohol preference. However, they do not preclude the possibility that differential alcohol influences on serotonin metabolism or turnover rate may result in differing preference for a alcohol.     

Identification of two forms of an endogenous murine retroviral env gene linked to the Rmcf locus.

The Rmcf gene restricts the replication of recombinant murine mink cell focus-inducing (MCF) viruses in cell cultures derived from mice carrying the resistance allele (Rmcfr) and may play a role in resistance to retrovirus-induced leukemias in vivo. We have characterized the endogenous gp70 expressed by Rmcfr and Rmcfs mice with a panel of type-specific monoclonal antibodies which discriminate xenotropic and MCF gp70. Embryo and tail skin cultures derived from Rmcfr mice (DBA/2 and CBA/N) expressed gp70 bearing a determinant unique to MCF viruses, whereas cultures from Rmcfs mice expressed either no detectable gp70 (NFS/N and IRW) or a gp70 serologically related to a subgroup of xenotropic viruses (C57BL/6, CBA/J, and A/WySn). Studies of progeny embryos derived from a (C57BL/6 X DBA/2) X C57BL/6 backcross established that the Rmcf resistance allele was linked to the expression of the MCF gp70 and that the gene encoding the xenotropic gp70 expressed by C57BL/6 Rmcfs mice was allelic with the MCF gp70 from Rmcfr mice. These data indicate that the Rmcf locus contains an endogenous gp70 gene having two allelic forms, one of which inhibits exogenous MCF infection in vitro by a mechanism of viral interference.     

过表达PINK1抵抗鱼藤酮引起多巴胺神经元损伤的研究

目的:明确在C57BL/6小鼠纹状体过表达野生型的人源帕金森相关蛋白PINK1能否减轻由侧脑室注射鱼藤酮引起多巴胺神经元损伤。方法:通过向C57BL/6小鼠(雄性,7周龄,18~20g)左侧纹状体中注射带有GFP人源野生型PINK1及突变体PINK1^G309D的慢病毒包装颗粒,两周后向小鼠左侧侧脑室中定位注射鱼藤酮,通过蛋白质印迹,免疫组化和行为学的方法检测PINK1对鱼藤酮引起多巴胺神经元损伤的影响。结果:蛋白质印迹和免疫组化的实验都证明了在C57BL/6小鼠纹状体过表达野生型的PINK1对于鱼藤酮引起多巴胺能神经元的减少有明显的抑制作用(P<0.01),但对鱼藤酮引起的行为学损伤没有明显的改善作用。     

Distribution and characterization of cyclo(His-Pro)-like immunoreactivity in the rat gastrointestinal tract

The distribution of cyclo(His-Pro), thyrotropin-releasing hormone (TRH) and $\text{pyroglutamate aminopeptidase}$ activity was examined in the rat gastrointestinal (GI) tract. Cyclo(His-Pro)-like immunoreactivity was present in the following order of distribution (fmoles/mg protein): caecum > colon = jejunum = ileum > stomach = duodenum = rectum, and was immunologically and chromatographically identical with the authentic cyclo(His-Pro). Cyclo(His-Pro) concentrations showed significantly positive correlations with TRH concentrations, but not with $\text{pyroglutamate aminopeptidase}$ activities, in most tissues of the GI tract, suggesting a precursor role of TRH for gut cyclo(His-Pro). These data suggest that cyclo(His-Pro) may be involved in regulating rat GI functions.     

GABA(A) receptor beta(2) subunit mRNA content is differentially regulated in ethanol-dependent DBA/2J and C57BL/6J mice

Chronic ethanol treatment is known to alter gene expression and function of γ-aminobutyric acid type-A (GABA_A) receptors. Here we focus on the β₂ subunit which is widely expressed in the mammalian brain, and plays a key role in the GABA binding site. Previous studies using rodent models of ethanol dependence show either increased or no change of β₂ subunit mRNA and peptide content following chronic ethanol administration. In humans, polymorphism at the β₂ subunit is associated with ethanol dependence in some, but not all, populations. In the present study we measured mRNA content in the cerebellum and cerebral cortex using ethanol-naive and ethanol-dependent DBA/2J and C57BL/6J mice. The DBA/2J strain displays severe ethanol withdrawal severity, while the C57BL/6J strain shows milder withdrawal reactions. RNase protection analysis demonstrated that the DBA/2J strain is more sensitive to ethanol-induced increases in β₂ subunit mRNA content in the cerebellum, showing significant increases at lower blood ethanol concentrations than C57BL/6J mice. The ethanol-induced regulation in C57BL/6J mice appears to be more complex, with decreases in β₂ subunit mRNA content at low blood ethanol concentrations, and increases at higher concentrations. These data suggest that differences between C57BL/6J and DBA/2J mice in the degree of physical dependence (withdrawal) on ethanol may be related to differential sensitivity to ethanol regulation of β₂ subunit expression.     

Interleukin 1beta (IL1B) signaling is a critical component of radiation-induced skin fibrosis

Interleukin 1 beta (IL1B), a potent pro-inflammatory cytokine, is directly up-regulated by radiation and is known to regulate other inflammation-related molecules, such as the matrix metalloproteinases (MMPs) and their endogenous inhibitors (TIMPs). However, the nature of the interaction of IL1B with MMPs and TIMPs in radiation-induced skin fibrosis is unknown. We examined the response of primary dermal keratinocytes, fibroblasts and endothelial cells to single-fraction radiation (10 Gy) and compared the results to a temporal sequence of histology from irradiated C57BL/6 and IL1R1 knockout mice. These studies showed that keratinocytes are the major IL1-producing cells in vitro and that radiation induces an immediate and chronic elevation in the expression of IL1B mRNA in the skin of C57BL/6 mice. This elevation was principally early and was less pronounced in the IL1R1 knockout strain, which also demonstrated reduced late radiation fibrosis. Radiation also increased expression of MMP mRNA in C57BL/6 mice. Finally, exogenous IL1B protein induced robust endogenous IL1B mRNA expression, along with a brisk increase in MMPs and collagen III, but only in the C57BL/6 mice. In conclusion, these data suggest that IL1B plays a critical role in radiation-induced fibrosis and that the increased MMPs fail to block the IL1-related collagen accumulation.