Carboxypeptidase-beta from Bubalus bubalus pancreas: purification, properties and MALDI-TOF monitored activation of proinsulin

Carboxypeptidase-B [E.C 3.4.17.2] catalyzes the hydrolysis of peptides and esters at C-terminus of arginine and lysine residues. Our study describes the large scale purification, N-terminal sequence analysis and physiochemical properties of pancreatic enzyme from river buffalo [Bubalus bubalis]. The enzyme was purified up to 71 folds by anionexchange chromatography with 21% final recovery. Purified enzyme displayed two bands on SDS-PAGE with molecular weights of 9 kDa and 26 kDa respectively, the N-terminal sequence of later was EFLDKLDFYV. The enzyme has shown optimum activity at pH 9.0 and 40°C. The K[M], Kcat and K[cat]/K[M] values of purified carboxypeptidase-B with Hippuryl-LArg are 30 micro M, 72sec[-1] and 2.4x10[5] M[-1] sec[-1] respectively. A computer based model for the structure of enzyme was proposed by chromatographic studies of component fragments and N-terminal sequence. The enzyme purified in the present study was free of carboxypeptidase A and endoprotease contamination. It was efficiently used in the processing of recombinant buffalo proinsulin, in combination with trypsin. Activation of proinsulin was monitored by MALDI-TOF analysis of peptides before and after the action of enzymes

Specific insulin and proinsulin secretion in glucokinase-deficient individuals

Glucokinase (GCK) is an enzyme that regulates insulin secretion, keeping glucose levels within a narrow range. Mutations in the glucokinase gene cause a rare form of diabetes called maturity-onset diabetes of the young (MODY). An early onset (less than 25 years), autosomal dominant inheritance and low insulin secretion stimulated by glucose characterize MODY patients. Specific insulin and proinsulin were measured in serum by immunofluorimetric assays (IFMA) during a 75-g oral glucose tolerance test (OGTT). Two kindreds (SA and LZ) were studied and compared to non-diabetic unrelated individuals (control group 1) matched for age and body mass index (BMI). In one kindred, some of these subjects were also obese (BMI>26 kg/m2), and other family members also presented with obesity and/or late-onset NIDDM. The MODY patients were also compared to a group of five of their first-degree relatives with obesity and/or late-onset NIDDM. The proinsulin profile was different in members of the two MODY kindreds. Fasting proinsulin and the proinsulin/insulin ratio were similar in MODY members of kindred LZ and subjects from control group 1, but were significantly lower than in MODY members of kindred SA (P<0.02 and P<0.01, for proinsulin and proinsulin/insulin ratio, respectively). Moreover, MODY members of family SA had higher levels of proinsulin and proinsulin/insulin ratio, although not significantly different, when compared to their first-degree relatives and to subjects from control group 2. In conclusion, we observed variable degrees of proinsulin levels and proinsulin/insulin ratio in MODY members of two different kindreds. The higher values of these parameters found in MODY and non-MODY members of kindred SA is probably related to the obesity and late-onset NIDDM background present in this family.

Etiopatogenia do diabetes mellitus do tipo 2 / Etiopathogeny of type 2 diabetes mellitus

O diabetes mellitus do tipo 2, na sua forma habitual, de início tardio, é caracterizado por resistência e deficiência insulínica, que contribuem de maneiras diferentes para a patogenia da doença. A alteração funcional da célula beta se manifesta precocemente, com aumento da relação pró-insulina/insulina. Graus variados de deficiência insulínica (absoluta ou relativa) São observados nos vários grupos étnicos estudados (caucasianos, mexicano-americanos, nipo-americanos, nipo-brasileiros, índios americanos, afro-americanos e asiáticos) onde a grande prevalência do diabetes do tipo 2 está associada à maior ingestão calórica e à redução da atividade física. A obesidade resultante tem um padrão central, visceral-abdominal. Nestas populações, a resistência insulínica é fator inicial e predominante, estanto associada à hiperinsulinemia, e à dislipidemia e ao maior risco de acometimento por doenças cardiovasculares. O papel da obesidade e dos ácidos graxos livres na produção hepática de glicose e na resistência à insulina são abordados, assim como o diagnóstico diferencial com outros tipos de diabetes.

Transferencia de insulinas en 198 pacientes de seis países latinoamericanos / Transference of insulins in 198 patients of six Latinoamerican countries

Es un estudio multicéntrico, comparativo, ciego, aleatorio y prospectivo que evaluó la eficacia y seguridad de la transferencia de insulinas de origen animal a insulina humana biosintética en 198 pacientes de seis países latinoamericanos. Todos los pacientes recibían insulinas de origen animal al menos dos meses antes de su inclusión al estudio. Este tuvo una duración de seis semanas, con evaluaciones al inicio, y a las dos y seis semanas. La eficacia se determinó por cambios inicial-final en la glucemia de ayunas y hemoglobina glucosilada, y la seguridad por la aparición de efectos adversos. De los 198 pacientes, 94 se asignaron al grupo de insulina bovina y 104 al de humana. Al ingreso, ambos grupos fueron similares en sus características. El único cambio al final del estudio fue una disminución en la glucemia en ayunas en el grupo de insulina humana (bovina 212 ñ 95.3 vs 193 ñ 78.8 mg/dL, p= 0.18; humana 198 ñ 86.8 vs 169 ñ 71.7, p= 0.025). El resto de los parámetros no tuvieron modificaciones estadísticamente significativas, aunque la tendencia fue hacia la mejoría en las cifras de hemoglobina glucosilada en ambos grupos. Se presentaron episodios de hipoglucemia leve con mayor frecuencia en el grupo de insulina humana, y hubo un episodio de hipoglucemia grave sin síntomas premonitorios con insulina humana. Concluimos que la transferencia de insulina de origen animal a insulina humana biosintética, en pacientes diabéticos insulinorrequirientes latinoamericanos, es eficaz y razonablemente segura (con ajuste de las dosis de insulina al hacer el cambio)

Effect of (-)epicatechin on cAMP content, insulin release and conversion of proinsulin to insulin in immature and mature rat islets in vitro.

(-)Epicatechin, an active principle in the water extract of the bark of Pterocarpus marsupium increases the cAMP content of the islets which is associated with the increased insulin release, conversion of proinsulin to insulin and cathepsin B activity. The response of the islets to the (-)epicatechin stimulation is more pronounced in immature (one month old) than in mature (12 month old) rats.