Top PDF Role of insulin-degrading enzyme (IDE) in diabetes mellitus and insulin resistance

Liver homogenates were collected and diluted 1:100 in duplicate in hydrolysis buffer up to a final volume of 50 µL. To one of the duplicates 2 µL of hydrolysis enzyme mix were added; while, to the other duplicates 2 µL of ultrapure water were added. These last samples were used as controls of hepatic glucose content in each liver. After that, the microplate was incubated for 30 min at room temperature. Then, 50 µL of master reaction mix, which contains the development buffer (46 µL), the development enzyme mix (2 µL) and the fluorescent peroxidase substrate (2 µL), was added to each well and incubated for 30 min at room temperature. During the reaction incubation, the plate was protected from light. Finally, the microplate was read spectrophotometrically at a wavelength of 562 nm using a microplate reader (Heales, China). The intensity of this colored product is directly proportional to the concentration of glycogen present in the original specimen. A standard curve of known concentration can be established accordingly. The unknown concentration in samples can be determined by reference to this standard curve.

IDE is a homodimeric protein, with each monomer being composed of two ~55 kDa homologous N- and C-terminal bowl-shaped domains (IDE-N and IDE-C) that are connected by a short linker to form the final 110 kDa protein [155]. Overall each monomer of IDE resembles a clamshell that can adopt “open” and “closed” conformation, with IDE-N and IDE-C forming each of the two halves. When in the closed conformation, the two halves form a large internal chamber that completely encapsulates and entraps insulin and other substrates. In the open conformation, substrates can enter into the catalytic chamber, and proteolyzed fragments can exit. The active site of IDE is bipartite, comprised of regions within both IDE-N (which contains the active-site Zn atom) and IDE-C that is fully formed only when the protease is in the closed position [156]. As a consequence, IDE can only process substrates that are small enough and appropriately shaped to fit inside its internal chamber. These features prohibit IDE from engulfing peptides greater than ~80 amino acids in length [154, 155].

Whereas the liver-speci fi c IR knockout mice (LIRKO) share with L- IDE-KO mice elevated plasma glucose levels, glucose intolerance and hepatic insulin resistance, they differ by developing chronic hyperinsulinemia in part, resulting from a failure to clear circulating insu- lin by receptor-mediated endocytosis [42,43]. This suggests that Ide de fi - ciency per se causes insulin resistance and glucose intolerance independently of hyperinsulinemia. From this point of view, our data identify a role for IDE in the molecular pathways underlining hepatic insu- lin resistance, independently of their effect on glucagon, amylin and A β 40. In summary, L-IDE-KO mice provides an in vivo evidence that the ab- sence of hepatic IDE causes insulin resistance, higher blood glucose levels, and glucose intolerance, through molecular mechanisms involving impaired hepatic insulin signaling and upregulation of gluconeogenic gene transcription. In agreement with previous studies [16], this appears to occur via a mechanism that does not implicate its role in hepatic insulin degradation. With the important caveat that non-proteolytic functions of IDE might be operative in the observed phenotype, these results suggest that pharmacological inhibition of IDE are contraindicated in the treatment of type 2 diabetes.

Furthermore, although beta-cells from OHAs-treated DM2 subjects show reductions in IDE protein levels, as reported by Steneberg and colleagues, insulin-treated subjects showed no changes relative to controls. Insulin- treated subjects, however, showed significant increases in IDE protein level in beta-cells relative to OHAs- treated patients. These results nicely correlate with those in hyperinsulinemic islets of db/db and HFD mice. At the same time, this finding suggested that insulin treatment stimulates elevated IDE protein levels, which we confirmed through multiple experiments in INS1E cells and rodent and human islets. IDE activity inhibition using Phenantroline suggests a physiological role of IDE on insulin secretion, in accordance with Steneberg et al (Steneberg et al., 2013). Interestingly, the result of GSIS after insulin stimulus may suggest an IDE-independent

- Grarup N., Rose C.S., Andersson E.A., Andersen G., Nielsen A.L., Albrechtsen A., Clausen J.O., Rasmussen S.S, Jørgensen T., Sandbæk A., Lauritzen T., Schmitz O., Hansen T., Pedersen O., Studies of Association of Variants Near the HHEX, CDKN2A/B, and IGF2BP2 Genes With Type 2 Diabetes and Impaired Insulin Release in 10,705 Danish Subjects. Validation and Extension of Genome- Wide Association Studies. Diabetes, 2007. 56: p. 3105–3111.

Alzheimer´s disease is a chronic neurodegenerative disorder affecting millions of people worldwide, characterized by a progressive decline in cognitive functions. Factors involved in the pathogenesis of Alzheimer´s disease include metabolic alterations such as insulin resistance and hyperglycemia, both of which are also hallmarks of type-2 diabetes mellitus. The accumulation of β -amyloid peptides in the brain of Alzheimer´s patients is responsible in part for the neurotoxicity underlying the loss of synaptic plasticity that triggers a cascade of events leading to cell death. A large number of studies revealed the key role of the hippocampus and cerebral cortex in the memory and learning deficits of Alzheimer´s disease. Although ample evidence suggests a link between altered insulin action, the dysregulation of glucose metabolism, and β -amyloid accumulation in animal models and humans with Alzheimer´s, no supporting evidence was available. In this article, we review the potential toxic effects of β -amyloid in the hypothalamus, a brain center involved in the control of insulin action and glucose metabolism. Furthermore, we discuss our recent studies unraveling a novel neurotoxic action of β -amyloid that perturbs hypothalamic glucoregulation, leading to increased hepatic glucose production and hyperglycemia. These findings provide evidence for a link between β -amyloid toxicity and altered glucose metabolism. (REV INVES CLIN. 2016;68:53-8)

The hydrogel samples in the collapsed state had a volume swelling ratio of 1.5. At high pH, the samples reached a volume swelling ration between 15 and 25 (Figure 1). In synthesis 3, the use of the enzyme solution itself decreased slightly the pH of the reactive mixture resulting in a high proportion of solvent, but only similar to the volume fraction at equilibrium (Table 5.1). When the hydrogel was synthesized with TEGDMA as a crosslinking agent and excess of solvent, the material showed less swelling at high pH, as noticed from curves for synthesis 1 and 2. The solvent may participate in the polymerization reaction either growing or terminating chains [3]. The excess of solvent may have hindered the crosslinking of the monomer allowing for more space between equally charged groups (in a loose network) and decreasing the stretching electrostatic repulsive forces that caused the volume change of the sample [4, 5]. The opposite occurred when PEGDMA 1000 was used in synthesis 3 and 4, possible because the greater length of the crosslinking agent may have acted as a higher resistance against the action of repulsive forces, and been dominant over the latter.

The hydrogel samples in the collapsed state had a volume swelling ratio of 1.5. At high pH, the samples reached a volume swelling ration between 15 and 25 (Figure 1). In synthesis 3, the use of the enzyme solution itself decreased slightly the pH of the reactive mixture resulting in a high proportion of solvent, but only similar to the volume fraction at equilibrium (Table 5.1). When the hydrogel was synthesized with TEGDMA as a crosslinking agent and excess of solvent, the material showed less swelling at high pH, as noticed from curves for synthesis 1 and 2. The solvent may participate in the polymerization reaction either growing or terminating chains [3]. The excess of solvent may have hindered the crosslinking of the monomer allowing for more space between equally charged groups (in a loose network) and decreasing the stretching electrostatic repulsive forces that caused the volume change of the sample [4, 5]. The opposite occurred when PEGDMA 1000 was used in synthesis 3 and 4, possible because the greater length of the crosslinking agent may have acted as a higher resistance against the action of repulsive forces, and been dominant over the latter.

tein levels, and hypertension, whereas in prospective studies, lower IGF-I levels predict the future development of isch- emic heart disease (4). It has been suggested that IGF-I may play a relevant role in the higher brain functions underlying cognition and may serve a homeostatic role during brain aging (23, 26, 49). There is compelling evidence to suggest that inflammation significantly contributes to neurodegen- erative changes (51). IGF-I acts to antagonize the interferon- ␥-induced microglial activation. Our group has previously reported the antiinflammatory proprieties of these doses of IGF-I in cirrhotic rats (16, 18). It has also been reported that IGF-I modulates local cerebral glucose use and ATP levels (48). Type 2 diabetes animal models associated with insulin resistance show reduced insulin brain uptake and content. Recent data point to changes in the insulin receptor cascade in obesity related insulin resistance, suggesting that brain insulin receptors also become less sensitive to insulin, which could reduce synaptic plasticity (52). There is also some indication that reduced S to insulin or IGF-I in the brain, as observed in aging, obesity, and diabetes, decreases the clear- ance of Abeta amyloid. Such a decrease involves the insulin receptor cascade and can also increase amyloid toxicity (52). Another point that deserves particular mention is that very low doses of IGF-I are able to induce many beneficial activ- ities in aging, promoting similar effects to those found in other conditions of IGF-I deficiency, such as liver cirrhosis (9 –19). In type 2 diabetes mellitus, the use of IGF-I at daily doses of 24 ␮ g/100 g body wt induced decreases in fasting and postprandial blood glucose levels. This hypoglycemic effect, together with the reduction in insulin levels, can at- tenuate the anabolic effect of IGF-I when administrated at these doses (53). In the present study, the neuroprotective, hepatoprotective, and metabolic activities of IGF-I were ob- served at doses as low as 2.25 ␮ g/100 g bw ⫺1 䡠 d ⫺1 (12- to 14-fold inferior to those used in the clinical trial mentioned previously). Hypoglycemia was not observed with low doses in cirrhosis (10 –14). In the present work these doses were able to improve the alterations of glucose metabolism ob- served in O (insulin resistance, hyperglycemia with hyperinsulinemia).

In chronic hepatitis C, insulin resistance (IR) and type 2 diabetes mellitus (DM) are more prevalent than in healthy controls or in chronic hepatitis B patients. HCV infection promotes IR mainly through increased TNF- α and cytokine suppressor (SOCS-3) production. Both events inhibit insulin receptor and IRS-1 (insulin receptor substrate) tyrosine phosphorylation. Hepatic steatosis is also 2.5 fold more frequent in hepatitis C vi- rus (HCV) infected patients as compared to the general population. Metabolic factors play a crucial role in the etiology of hepatic steatosis genotype non-3 related, which are also the genotypes with a greater association to IR. However, genotype 3, and particularly 3a, has a greater direct steatogenic capacity, and consequently, in those patients, the association with metabolic factors is weaker. Instead, in genotype 3, steatosis associates with viral factors like viral load. Those metabolic fac- tors influence not only the natural history of HCV in- fection, as well as associate to an accelerated hepatic fi- brosis progression, to a worse prognosis when hepatic cirrhosis is present, namely an increased risk of hepa- tocellular carcinoma, and to a lower sustained viral re- sponse rate. On the other hand, in patients who achieve viral eradication, IR and hepatic steatosis may re- gress, and return if viral infection recurs, which once again indicates an intrinsic steatosis and IR promoter action by HCV.

El estudio comenzó con la caracterización de los islotes del modelo de DM2 conocido como ratón db/db, que confirmó lo que ya venía publicándose en la bibliografía [14, 15, 16, 17] y en otros experimentos llevados a cabo en nuestro laboratorio [18]: el porcentaje de células β pancreáticas en el islote disminuye en páncreas de diabéticos (db/db), en los que además cualitativamente existe una mayor desestructuración. Hay que explicar que en nuestro estudio, esta diferencia no fue significativa en las muestras de islotes de 12 semanas pero sí en las de 26 semanas, lo que podemos achacar a que en el periodo de 12 semanas la diabetes era de reciente instauración (diabetes franca en ratones db/db a partir de 9 semanas). No obstante, pese a que no viéramos diferencias cuantitativas en este grupo, sí que existían a nivel cualitativo: los islotes de animales control mostraban características más homogéneas y tinciones de insulina de mayor intensidad, mientras que en los islotes de los ratones db/db la tinción de insulina es menos intensa y la estructura del islote más desorganizada, no tiñéndose todas las células β pancreáticas homogéneamente.

Methods: Prospective, randomized, controlled, double-blind placebo study. We included 81 patients with ED and 20 men without ED (control group). Exclusion criteria: pharmacologic, anatomic or endocrine ED (hypogonadism or hyperprolactinemia), DM2, prior prostatic surgery or chronic illnesses. The erectile function was rated according the International Index of Erectile Function 5. IR was measerud by HOMA index. Thirty patients with ED, IR and poor response to sildenafil were randomized to receive metformin or placebo.

± 0,08mmol/L, DRS: 7,85 ± 0,15mmol/L (p<0,05). Por otra parte, los niveles plasmáticos de insulina obteni- dos en estas condiciones fueron similares a los obser- vados al final del período de oscuridad (datos no mostrados). La velocidad de infusión de glucosa (VIG) que cuantifica la acción de la insulina in vivo fue significativamente menor (p<0,05) en los anima- les alimentados con DRS comparados a los del grupo que recibió la DC, lo que demuestra un deterioro de la sensibilidad a la insulina en estos animales (Figura 1). No se observaron cambios en los niveles de he- matocrito desde el inicio y durante el clamp en nin- guno de los grupos dietarios (datos no mostrados).

This study has shown that T2DM patients with MO have a higher prevalence of steatosis and that severe steato- sis was also seen more often in this patient subgroup. Sim- ilarly, NASH and Fb were seen significantly more often and more severe in patients with diabetes. These findings are also described in other studies that have identified an association between T2DM and more aggressive and pro- gressive forms of NAFLD, emphasizing the greater risk of severe NAFLD in these patients. 23,33,38-42 T2DM and

Ability of alternative indices of insulin sensivity to predict cardiovascular risk: comparison with the “minimal model”: Insulin Resistance Atherosclerosis Study (IRAS) Investi[r]

El propósito de este trabajo es revisar las estrategias farmacológicas específicamente destinadas a disminuir la insulinorresistencia. Se plantean cuatro objetivos, siendo el primero la inhibición de la producción hepática de glucosa. Aquí se incluye a la metformina, biguanida que reduce la gluconeogénesis desde lactato y aumen- ta la actividad de la AMPK (enzima que estimula la oxidación de ácidos grasos no esterificados y disminuye la gluconeogénesis y síntesis de colesterol). También se encuentran el BAY-27-9955 y NNC-25-2504L (antagonis- tas del glucagon), y los inhibidores de la glucógeno fosforilasa hepática, glucógeno sintasa-kinasa-3, piruvato- deshidrogensa-kinasa, fructosa-1,6-difosfatasa y glucosa-6-fosfatasa. Los insulinosensibilizadores, segundo obje- tivo, incluyen a tiazolidinodionas (agonistas PPARγ) que aumentan la exposición de GLUT 4, lipogénesis y dis - minuyen la gluconeogénesis; y a drogas en desarrollo: nuevos agonistas PPAR, activadores RxR, agonistas β3 e inhibidores de la PTB-1B. El tercer objetivo son los modificadores del metabolismo lipídico, y contiene a los inhibidores de la 11β-HSD1. Los activadores del receptor insulínico, dependientes e independientes de insuli- na, constituyen el cuarto objetivo. La importancia del desarrollo de nuevos fármacos radica en que el control de la insulinorresistencia retrasaría la aparición de diabetes 2, e implicaría un menor riesgo cardiovascular.

En el estudio PREDIMERC (Prevalencia de Diabetes Mellitus y Riesgo Cardiovascular) realizado en población residente en la Comunidad de Madrid en el año 2007, se seleccionó una muestra aleatoria y representativa de 2268 personas de 30– 74 años de edad (1085 hombres y 1183 mujeres). El objetivo fue determinar la prevalencia de diabetes y los principales factores de riesgo cardiovasculares en la población mediante una encuesta poblacional y con medidas objetivas de la glucemia, los lípidos en sangre, la presión arterial, el peso y la talla. De todos los factores de riesgo estudiados, se destaca la alta prevalencia de las variables ligadas al comportamiento o estilo de vida, como el tabaquismo y sedentarismo. La obesidad fue un factor importante por ser un factor de riesgo cardiovascular en sí mismo y por la asociación que presentó con el resto de los factores estudiados 32 .

 Se caracteriza por destrucción especifica de las células beta del páncreas ) DM II ( se caracteriza por alteración en la respuesta a la insulina por parte de células diana debido a mutaciones en el gen receptor de la hormona) la glucosa puede penetrar enle hígado en ausencia de la insulina pero en la diabetes la actividad de la enzima glucosinasa hepática esta muy disminuida mientras que la actividad de la enzima glucosa 6dosfatasa aumenta mucho, como la glucosa no penetra en los tejidos extra hepáticos, la degradación de glucosa hasta piruvato, la vía de oxidacion directa, la síntesis de glucógeno muscular y hepático se hallan relativamente anuladas en paceintes diabéticos. La gluconeogénesis y la glucogenólisis están aumentadas por la acción del glucagón que se encuentra elevado en el paciente diabético, produciendo hiperglicemia.

Introducción: Recientemente han surgido nuevas evidencias que relacionan el metabolismo óseo con el energético. La osteocalcina es una proteína de la matriz ósea no colágena, sintetizada por los osteoblastos que modula localmente la mineralización ósea, tradicionalmente usada como marcador de formación ósea. Se ha demostrado tanto en modelos “in vitro” como en animales de experimentación que la osteocalcina tiene acción hormonal. Esta proteína tiene la propiedad de regular la insulinosensibilidad, la insulinosecreción y la proliferación de las células beta pancreáticas.

Figure 4. Prolonged leptolide administration improves insulin sensitivity in a preclinical model of insulin resistance. C57BL6J male mice were fed an HFD for ten weeks. The last four weeks, mice were injected intraperitoneally with leptolide or saline once a day. Afterwards, insulin sensitivity and plasma levels of insulin and triglycerides were assessed. (A) Glucose tolerance test of mice treated with vehicle or 0.1 mg/kg leptolide. (B) Area under the curve of the ip-GTT. Glucose tolerance improved in leptolide- compared to vehicle-treated mice. (C) Insulin tolerance test of mice treated with vehicle or 0.1 mg/kg leptolide. (D) Area under the curve of the ip-ITT. Insulin sensitivity improved in leptolide- compared to vehicle-treated mice. (E) Glucose decay after first 30 min of insulin injection during ip-ITT. (F) Insulin sensitivity index (%S). (G) HOMA index. Insulin sensitivity and HOMA indexes showed improved insulin sensitivity in HFD + leptolide mice. (H) Fasting body weight. (I) Fasting and (J) non-fasting plasma insulin levels. Insulin levels were non-significantly decreased. (K) Fasting plasma triglycerides levels were decreased in mice treated with leptolide. (L) Liver TG (triglyceride) content was decreased in parallel with plasma triglyceride levels. Values are the means ± S.E.M. of n = 12 per group. * p < 0.05 versus vehicle by Student’s t-test; $ p < 0.05 versus SD by ANOVA.