RESUMEN
Investigations conducted since 2018 have identified several host-defense peptides present in frog skin secretions whose properties suggest the possibility of their development into a new class of agent for Type 2 diabetes (T2D) therapy. Studies in vitro have described peptides that (a) stimulate insulin release from BRIN-BD11 clonal ß-cells and isolated mouse islets, (b) display ß-cell proliferative activity and protect against cytokine-mediated apoptosis and (c) stimulate production of the anti-inflammatory cytokine IL-10 and inhibit production of the pro-inflammatory cytokines TNF-α and IL-1ß. Rhinophrynin-27, phylloseptin-3.2TR and temporin F are peptides with therapeutic potential. Studies in vivo carried out in db/db and high fat-fed mice have shown that twice-daily administration of [S4K]CPF-AM1 and [A14K]PGLa-AM1, analogs of peptides first isolated from the octoploid frog Xenopus amieti, over 28 days lowers circulating glucose and HbA1c concentrations, increases insulin sensitivity and improves glucose tolerance and lipid profile. Peptide treatment produced potentially beneficial changes in the expression of skeletal muscle genes involved in insulin signaling and islet genes involved in insulin secretion in these murine models of T2D. Lead compounds uncovered by the study of frog HDPs may provide a basis for the design of new types of agents that can be used, alone or in combination with existing therapies, for the treatment of T2D.
Asunto(s)
Diabetes Mellitus Tipo 2 , Ratones , Animales , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Insulina/metabolismo , Anuros/metabolismo , Glucosa , CitocinasRESUMEN
PGLa-AM1 (GMASKAGSVL10GKVAKVALKA20AL.NH2) was first identified in skin secretions of the frog Xenopus amieti (Pipidae) on the basis of its antimicrobial properties. PGLa-AM1 and its [A14K] and [A20K] analogues produced a concentration-dependent stimulation of insulin release from BRIN-BD11 rat clonal ß-cells without cytotoxicity at concentrations up to 3 µM. In contrast, the [A3K] analogue was cytotoxic at concentrations ≥ 30 nM. The potency and maximum rate of insulin release produced by the [A14K] and [A20K] peptides were significantly greater than produced by PGLa-AM1. [A14K]PGLa-AM1 also stimulated insulin release from mouse islets at concentrations ≥ 1 nM and from the 1.1B4 human-derived pancreatic ß-cell line at concentrations > 30 pM. PGLa-AM1 (1 µM) produced membrane depolarization in BRIN-BD11 cells with a small, but significant (P < 0.05), increase in intracellular Ca2+ concentrations but the peptide had no direct effect on KATP channels. The [A14K] analogue (1 µM) produced a significant increase in cAMP concentration in BRIN-BD11 cells and down-regulation of the protein kinase A pathway by overnight incubation with forskolin completely abolished the insulin-releasing effects of the peptide. [A14K]PGLa-AM1 (1 µM) protected against cytokine-induced apoptosis (p < 0.001) in BRIN-BD11 cells and augmented (p < 0.001) proliferation of the cells to a similar extent as GLP-1. Intraperitoneal administration of the [A14K] and [A20K] analogues (75 nmol/kg body weight) to both lean mice and high fat-fed mice with insulin resistance improved glucose tolerance with a concomitant increase in insulin secretion. The data provide further support for the assertion that host defense peptides from frogs belonging to the Pipidae family show potential for development into agents for the treatment of patients with Type 2 diabetes.
Asunto(s)
Proteínas Anfibias/uso terapéutico , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Hipoglucemiantes/uso terapéutico , Células Secretoras de Insulina/efectos de los fármacos , Insulina/metabolismo , Proteínas de Xenopus/uso terapéutico , Animales , Calcio/metabolismo , Línea Celular , Proteínas Quinasas Dependientes de AMP Cíclico/efectos de los fármacos , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Regulación hacia Abajo , Humanos , Secreción de Insulina , Células Secretoras de Insulina/metabolismo , Ratones , Pipidae , Ratas , Transducción de SeñalRESUMEN
The frog skin host-defence peptide hymenochirin-1B has been shown to stimulate insulin release in vitro from isolated pancreatic islets and BRIN-BD11 clonal ß-cells. This study examines the effects of 28-day administration of a more potent analogue [P5K]hymenochirin-1B ([P5K]hym-1B) (75 nmol·kg(-1) body weight) to high-fat-fed mice with obesity, glucose intolerance and insulin resistance. Treatment with [P5K]hym-1B significantly decreased plasma glucose concentrations and improved glucose tolerance, insulin secretion, insulin sensitivity and increased the magnitude of the incretin effect (difference in response to oral vs intraperitoneal glucose loads). Responses to established insulin secretagogues were greater in islets isolated from treated animals compared with saline-treated controls. [P5K]hym-1B administration significantly decreased total islet area and ß- and α-cell areas, and resulted in lower concentrations of circulating triglycerides and plasma and pancreatic glucagon. Peptide treatment had no effect on food intake, body weight, indirect calorimetry or circulating concentrations of amylase and marker enzymes of liver and kidney function. RT-PCR demonstrated that the Insr (insulin receptor) gene and genes involved in insulin signalling (Slc2a4, Irs1, Pik3ca, Akt1 and Pkd1) were significantly up-regulated in skeletal muscle from animals treated with [P5K]hym-1B. Expression of the Glp1r (GLP-1 receptor) and Gipr (GIP receptor) genes was significantly elevated in islets from peptide-treated mice. These data suggest that [P5K]hym-1B shows potential for development into an agent for treating patients with type 2 diabetes.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Glucemia/efectos de los fármacos , Forma de la Célula/efectos de los fármacos , Dieta Alta en Grasa , Intolerancia a la Glucosa/tratamiento farmacológico , Hipoglucemiantes/farmacología , Resistencia a la Insulina , Insulina/sangre , Islotes Pancreáticos/efectos de los fármacos , Animales , Biomarcadores/sangre , Glucemia/metabolismo , Modelos Animales de Enfermedad , Metabolismo Energético/efectos de los fármacos , Regulación de la Expresión Génica/efectos de los fármacos , Intolerancia a la Glucosa/sangre , Intolerancia a la Glucosa/genética , Intolerancia a la Glucosa/patología , Resistencia a la Insulina/genética , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/patología , Lípidos/sangre , Ratones , Músculo Esquelético/efectos de los fármacos , Músculo Esquelético/metabolismo , Transducción de Señal/efectos de los fármacos , Factores de Tiempo , Técnicas de Cultivo de TejidosRESUMEN
The frog skin host-defense peptide tigerinin-1R stimulates insulin release in vitro and improves glucose tolerance and insulin sensitivity in animal models of type 2 diabetes. This study extends these observations by investigating the molecular mechanisms of action underlying the beneficial metabolic effects of the analogue [Arg4]tigerinin-1R in mice with diet-induced obesity, glucose intolerance and insulin resistance. The study also investigates the electrophysiological effects of the peptide on KATP and L-type Ca2+ channels in BRIN-BD11 clonal ß cells. Non-fasting plasma glucose and glucagon concentrations were significantly (p<0.05) decreased and plasma insulin increased by twice daily treatment with [Arg4]tigerinin-1R (75 nmol/kg body weight) for 28 days. Oral and intraperitoneal glucose tolerance were significantly (p<0.05) improved accompanied by enhanced secretion and action of insulin. The peptide blocked KATP channels and, consistent with this, improved beta cell responses of isolated islets to a range of secretagogues. Peptide administration resulted in up-regulation of key functional genes in islets involved insulin secretion (Abcc8, Kcnj11, Cacna1c and Slc2a2) and in skeletal muscle involved with insulin action (Insr, Irs1, Pdk1, Pik3ca, and Slc2a4). These observations encourage further development of tigerinin-1R analogues for the treatment of patients with type 2 diabetes.
Asunto(s)
Proteínas Anfibias/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Resistencia a la Insulina , Obesidad/metabolismo , Animales , Glucemia/análisis , Dieta Alta en Grasa/efectos adversos , Prueba de Tolerancia a la Glucosa , Insulina/análisis , Insulina/metabolismo , Masculino , RatonesRESUMEN
AIMS: We have previously described the insulinotropic activities of [I10W]tigerinin-1R (RVCSAIPLPWCH.NH2) in vitro. In this study, we investigated the effects of the peptide on nutrient homoeostasis in mice with diet-induced obesity and insulin resistance. METHODS: Male NIH Swiss mice were maintained on a high-fat diet for 12 weeks prior to the study. Twice-daily intraperitoneal injections of [I10W]tigerinin-1R (75 nmol/kg body weight) were administered for 28 days. Body weight, energy intake, body fat content, and plasma concentrations of triglyceride, cholesterol, non-fasting glucose and insulin were monitored. Effects of the peptide on glycaemic control were measured by glucose tolerance and insulin sensitivity tests. Pancreatic hormone content and insulin secretory responses of islets isolated from treated and untreated mice were examined. Immunohistochemical analysis was performed to study possible changes in islet morphology. RESULTS: Administration of [I10W]tigerinin-1R to high-fat-fed mice produced significant (P < 0.05) decreases in plasma glucose, glucagon and triglyceride concentrations and an increase in plasma insulin compared to high-fat-fed controls. No changes in body weight or energy intake were observed with peptide treatment, but glycaemic control was significantly improved in response to oral or intraperitoneal glucose. Insulin sensitivity and secretory responses of islets to established insulin secretagogues were also significantly improved in peptide-treated mice. Total body fat, pancreatic insulin and glucagon contents, islet, beta and alpha cell areas were all significantly decreased in treated mice. CONCLUSIONS: This study shows that [I10W]tigerinin-1R improves insulin sensitivity, islet function and glycaemic control in high-fat-fed mice and has potential as a template for development of novel anti-diabetic agents.
Asunto(s)
Adiposidad/efectos de los fármacos , Hipoglucemiantes/uso terapéutico , Resistencia a la Insulina , Células Secretoras de Insulina/efectos de los fármacos , Péptidos/uso terapéutico , Triglicéridos/sangre , Animales , Péptidos Catiónicos Antimicrobianos , Glucemia/metabolismo , Composición Corporal , Dieta Alta en Grasa/efectos adversos , Glucagón/sangre , Prueba de Tolerancia a la Glucosa , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/ultraestructura , Lípidos/sangre , Masculino , Ratones , Obesidad/tratamiento farmacológicoRESUMEN
Hymenochirin-1b (Hym-1B; IKLSPETKDNLKKVLKGAIKGAIAVAKMV.NH2) is a cationic, α-helical amphibian host-defense peptide with antimicrobial, anticancer, and immunomodulatory properties. This study investigates the abilities of the peptide and nine analogues containing substitutions of Pro(5), Glu(6), and Asp(9) by either L-lysine or D-lysine to stimulate insulin release in vitro using BRIN-BD11 clonal ß cells or isolated mouse islets and in vivo using mice fed a high-fat diet to produce obesity and insulin resistance. Hym-1B produced a significant and concentration-dependent increase in the rate of insulin release from BRIN-BD11 cells without cytotoxicity at concentrations up to 1 µM with a threshold concentration of 1 nM. The threshold concentrations for the analogues were: [P5K], [E6K], [D9K], [P5K, E6K] and [E6K, D9k] 0.003 nM, [E6K, D9K] and [D9k] 0.01 nM, [P5K, D9K] 0.1 nM and [E6k] 0.3 nM. All peptides displayed cytotoxicity at concentrations ≥1 µM except the [P5K] and [D9k] analogues which were non-toxic at 3 µM. The potency and maximum rate of insulin release from mouse islets produced by the [P5K] peptide were significantly greater than produced by Hym-1B. Neither Hym-1B nor the [P5K] analogue at 1 µM concentration had an effect on membrane depolarization or intracellular Ca(2+). The [P5K] analogue (1 µM) produced a significant increase in cAMP concentration in BRIN-BD11 cells and stimulated GLP-1 secretion from GLUTag cells. Down-regulation of the protein kinase A pathway by overnight incubation with forskolin completely abolished the insulin-releasing effects of [P5K]hym-1B. Intraperitoneal administration of the [P5K] and [D9k] analogues (75 nmol/kg body weight) to high-fat-fed mice with insulin resistance significantly enhanced glucose tolerance with a concomitant increase in insulin secretion. We conclude that [P5K]hym-1B and [D9k]hym-1B show potential for development into anti-diabetic agents.
Asunto(s)
Proteínas Anfibias/farmacología , Péptidos Catiónicos Antimicrobianos/farmacología , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/farmacología , Insulina/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos/síntesis química , Anuros , Calcio/metabolismo , Línea Celular , AMP Cíclico/biosíntesis , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Dieta Alta en Grasa , Péptido 1 Similar al Glucagón/metabolismo , Glucosa/metabolismo , Resistencia a la Insulina , Secreción de Insulina , Islotes Pancreáticos/metabolismo , L-Lactato Deshidrogenasa/metabolismo , Potenciales de la Membrana/efectos de los fármacos , Ratones , Ratones Endogámicos C57BL , Técnicas de Cultivo de Órganos , Proteína Quinasa C/metabolismo , Ratas , Relación Estructura-ActividadRESUMEN
The frog skin host-defense peptide esculentin-2CHa (GFSSIFRGVA10KFASKGLGK D20LAKLGVDLVA30CKISKQC) displays antimicrobial, antitumor, and immunomodulatory properties. This study investigated the antidiabetic actions of the peptide and selected analogues. Esculentin-2CHa stimulated insulin secretion from rat BRIN-BD11 clonal pancreatic ß-cells at concentrations greater than 0.3 nM without cytotoxicity by a mechanism involving membrane depolarization and increase of intracellular Ca2+. Insulinotropic activity was attenuated by activation of KATP channels, inhibition of voltage-dependent Ca2+ channels and chelation of extracellular Ca2+. The [L21K], [L24K], [D20K, D27K] and [C31S,C37S] analogues were more potent but less effective than esculentin-2CHa whereas the [L28K] and [C31K] analogues were both more potent and produced a significantly (P < 0.001) greater maximum response. Acute administration of [L28K]esculentin-2CHa (75 nmol/kg body weight) to high fat fed mice with obesity and insulin resistance enhanced glucose tolerance and insulin secretion. Twice-daily administration of this dose of [L28K]esculentin-2CHa for 28 days had no significant effect on body weight, food intake, indirect calorimetry or body composition. However, mice exhibited decreased non-fasting plasma glucose (P < 0.05), increased non-fasting plasma insulin (P < 0.05) as well as improved glucose tolerance and insulin secretion (P < 0.01) following both oral and intraperitoneal glucose loads. Impaired responses of isolated islets from high fat fed mice to established insulin secretagogues were restored by [L28K]esculentin-2CHa treatment. Peptide treatment was accompanied by significantly lower plasma and pancreatic glucagon levels and normalization of α-cell mass. Circulating triglyceride concentrations were decreased but plasma cholesterol and LDL concentrations were not significantly affected. The data encourage further investigation of the potential of esculentin-2CHa related peptides for treatment of patients with type 2 diabetes.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/farmacología , Resistencia a la Insulina , Células Secretoras de Insulina/efectos de los fármacos , Obesidad/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos/uso terapéutico , Glucemia/metabolismo , Dieta Alta en Grasa/efectos adversos , Células Secretoras de Insulina/metabolismo , Masculino , Ratones , Obesidad/tratamiento farmacológico , Obesidad/etiologíaRESUMEN
The frog skin host-defense peptide CPF-SE1 has previously been shown to stimulate the in vitro release of insulin from clonal BRIN-BD11 ß-cells. In this study, the in vivo effects of the peptide were investigated in male NIH Swiss mice maintained on a high-fat diet to induce obesity and insulin resistance. Insulin-secretory responses of islets isolated from treated and untreated mice and changes in islet morphology were also examined. Total body fat, plasma glucagon, triglyceride and cholesterol concentrations were measured at the end of the treatment period. Acute intraperitoneal administration of CPF-SE1 (75 nmol body weight) to high-fat fed mice together with glucose (18 mmol/kg body weight) improved glucose tolerance and insulin responses compared to high-fat fed controls. Long term administration of CPF-SE1 (twice-daily administration of 75 nmol/kg body weight for 28 days) did not affect body weight or energy intake but decreased circulating glucose and increased insulin concentrations. Insulin sensitivity and insulin-secretory responses of islets to secretagogues were also significantly improved at 28 days in peptide-treated mice. In addition, significant decreases in plasma glucagon concentrations, pancreatic insulin and glucagon content, islet and beta cell area, body fat and plasma triglyceride levels were observed in CPF-SE1 treated with mice. In conclusion, CPF-SE1 improves beta cell function, insulin sensitivity and glycaemic control whilst reducing total body fat and circulating triglyceride levels. The peptide shows potential for development into an agent for treatment of patients with metabolic syndrome and type 2 diabetes.
Asunto(s)
Anuros , Dieta Alta en Grasa/efectos adversos , Resistencia a la Insulina , Islotes Pancreáticos/efectos de los fármacos , Lípidos/sangre , Péptidos/farmacología , Piel/química , Secuencia de Aminoácidos , Animales , Glucemia/metabolismo , Composición Corporal/efectos de los fármacos , Peso Corporal/efectos de los fármacos , Ingestión de Energía/efectos de los fármacos , Glucagón/sangre , Glucagón/metabolismo , Prueba de Tolerancia a la Glucosa , Insulina/sangre , Insulina/metabolismo , Secreción de Insulina , Islotes Pancreáticos/metabolismo , Islotes Pancreáticos/fisiología , Masculino , Ratones , Datos de Secuencia Molecular , Péptidos/químicaRESUMEN
AIMS: This paper investigates the anti-diabetic effects of tigerinin-1R (RVCSAIPLPICH.NH2), a previously described amphibian host defence peptide, in mice with diet-induced obesity-diabetes. METHODS: Proteolytic degradation of synthetic tigerinin-1R was investigated by reversed-phase HPLC and MALDI-TOF mass spectrometry. Changes in glycaemic responses and metabolic parameters were measured in mice with high fat diet-induced obesity-diabetes treated with twice-daily with of tigerinin-1R (75 nmol/kg bw) for 15 days. Indirect calorimetry and body composition were measured by CLAMS and DEXA whole body scanning. Insulin secretory responses of islets isolated from treated and untreated mice were examined. RESULTS: Tigerinin-1R was resistant to in vitro degradation by plasma enzymes. Twice-daily injection of tigerinin-1R for 15 days had no significant effect on food intake or body weight. Non-fasting glucose levels were significantly lowered, and insulin levels were elevated compared to saline treated controls. Glycaemic responses to both oral and intraperitoneal glucose administration were significantly improved by tigerinin-1R treatment. Plasma insulin was also significantly elevated. The peptide had no significant effect on insulin sensitivity but the beta cell responses of islets isolated from treated mice to a range of nutrients and peptidergic secretagogues were significantly improved. Oxygen consumption, CO2 production, respiratory exchange ratio, energy expenditure and body composition were not significantly altered by treatment with tigerinin-1R. CONCLUSION: Tigerinin-1R significantly improves glucose homeostasis and may have potential as a novel antidiabetic agent.