Genetic dissection of ``OLETF', a rat model for non-insulin-dependent diabetes mellitus

To elucidate the genetic factors underlying non-insulin-dependent diabetes mellitus (NIDDM), we performed genome-wide quantitative trait locus (QTL) analysis, using the Otsuka Long-Evans Tokushima Fatty (OLETF) rat. The OLETF rat is an excellent animal model of NIDDM because the features of the disease closely resemble human NIDDM. Genetic dissection with two kinds of F2 intercross progeny, from matings between the OLETF rat and non-diabetic control rats F344 or BN, allowed us to identify on Chromosome (Chr) 1 a major QTL associated with features of NIDDM that was common to both crosses. We also mapped two additional significant loci, on Chrs 7 and 14, in the (OLETF × F344)F₂ cross alone, and designated these three loci as Diabetes mellitus, OLETF type Dmo 1, Dmo2 and Dmo3 respectively. With regard to suggestive QTLs, we found loci on Chrs 10, 11, and 16 that were common to both crosses, as well as loci on Chrs 5 and 12 in the (OLETF × F344)F₂ cross and on Chrs 4 and 13 in the (OLETF × BN)F₂ cross. Our results showed that NIDDM in the OLETF rat is polygenic and demonstrated that different genetic backgrounds could affect ``fitness' for QTLs and produce different phenotypic effects from the same locus. Received: 9 October 1997 / Accepted: 29 January 1998     

Effects of acute exercise on insulin binding to erythrocytes in normal men

Ten normal men were subjected to acute mild and moderate exercise tests, and the insulin binding to erythrocytes was determined before and immediately after exercise and also 10, 30 and 60 minutes after exercise in each test. The insulin binding significantly increased immediately after acute moderate exercise and did not increase immediately after acute mild exercise. In contrast, it decreased below the basal level at 30 and 60 minutes in each test. The changes in insulin receptor binding were due mainly to an alteration in insulin receptor affinity rather than a change in receptor number. These results suggest that isolated erythrocytes may be of some value for study of the effect of physical exercise on the insulin receptor.     

Insulin release from BK virus-induced and in vitro maintained insulinoma cells of Syrian golden hamster

The pancreatic tumor cells (In 111) derived from BK virus-induced insulinoma of Syrian golden hamsters were maintained in culture for several passages and were studied for their insulin secretory ability under various stimulatory conditions. Insulin release was not increased by D-glucose stimulation (27.8 mM), while dibutyryl cyclic AMP (1 mM), theophylline (1 mM), 3-isobutyl-l-methylxanthine (0.1 mM) and elevation of medium calcium from 0.5 to 2.7 mM stimulated insulin release 2.5- to 4-fold. There was a concomitant increase of medium cyclic AMP with addition of theophylline. Streptozotocin (2 mM) treatment for 48 hours significantly reduced insulin release, while alloxan (2 mM), had no inhibitory effect on insulin release. The results indicate that while in vitro-maintained islet tumor cells, In 111, have a cyclic AMP-mediated process involved in insulin secretion analogous to normal beta cells, these cells lack the ability to recognize glucose as an insulin secretagogue probably due to a defect in the cell membrane, though the possibility of alteration in glucose metabolism cannot be fully excluded.     

18-Norspirostanol derivatives from Trillium tschonoskii

Three 18-norspironstanol oligoglycosides partly acylated in their sugar moieties were isolated from the underground parts of Trillium tschonoskii. Their structures were characterized, as 1-O-[2″,3″,4″-tri-O-acetyl-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl]-epitrillenogenin-24-O-acetate, 1-O-[2″,3″,4″-tri-O-acetyl-α-l-rhamno-pyranosyl-(1 → 2)-α-l-arabinopyranosyl]-epitrillenogenin and 1-O-[2″,4″-di-O-acetyl-α-l-rhamnopyranosyl-(1 → 2)-α-l-arabinopyranosyl]-epitrillenogenin-24-O-acetate.     

Neutralizing antibody responses to Japanese encephalitis vaccine in children

Two shots of the current Japanese encephalitis (JE) vaccine were given to children and their immune responses to the Nakayama strain (the vaccine strain) and two wild strains (JaGAr-01 and E-50) of JE virus were examined by neutralizing (N) antibody titrations. Seventy vaccinees had no N antibody to JE virus before the first vaccination and were bled one month after the second vaccination. The N antibody responses to the JaGAr-01 and E-50 strains were found to be similar and to be less than that to the Nakayama strain after the second vaccination: the geometric mean titers (GMT) of N antibodies to the JaGAr-01 and E-50 strains (as logarithms) were 1.87 and 1.75, respectively, while the GMT to the Nakayama strain was 2.89. The seroconversion rates to the Nakayama, JaGAr-01 and E-50 strains were 70/70 (100%), 69/70 (99%) and 68/70 (97%), respectively, after the second vaccination. Twenty-seven of the 70 vacciness were also bled before the second vaccination. Most of them showed a considerably high N antibody response against the Nakayama strain and only one vaccinee failed to show seroconversion after the first vaccination. However, the antibody response to the E-50 strain appeared to be rather low and 9 of 25 vaccinees did not show any seroconversion. Similarly 3 of 25 failed to show any seroconversion against the JaGAr-01 strain. These results indicate that at the initial immunization two shots, at least, of the current JE vaccine are necessary to stimulate effective immune responses to wild strains of JE virus.     

A marked change in insulin binding to rat thymic lymphocytes by fractionation of the cells into two subsets

As compared with the pre-separated thymic lymphocyte (TL) population, a marked reduction of insulin binding was found in the two fractions [supernatant lymphocytes (SL) and precipitate lymphocytes (PL)] separated by low-speed differential centrifugation from the TL population. The reduction in insulin binding to SL or PL appeared to be due to a decrease in the affinity rather than a decrease in the number of insulin receptors. The reduced insulin binding was enhanced to near the level of the TL population when SL and PL were mixed and then incubated for 90 min at 37 degrees C. Moreover, when the cell-free supernatant from the mixed and incubated fraction was added to SL and PL respectively, the insulin binding to each fraction was found to be enhanced.     

Construction of a non-support bioreactor: optical resolution of 2-(4-chlorophenoxy)propanoic acid in an organic solvent system

Summary A non-support bioreactor, a novel column reactor packed with a free non-supported enzyme was constructed by applying the insolubility of the enzyme in organic solvents. Stereoselective esterification of 2-(4-chlorophenoxy)propanoic acid by lipase OF 360 from Candida cylindracea with n-tetradecanol was selected as a model reaction. Non-supported lipase revealed threefold higher activity than Celite-adsorbed lipase by maintaining high stereoselectivity in a batch reaction. In continuous operation, a non-support bioreactor produced the ester with fourfold higher productivity to that of a column reactor packed with Celite-adsorbed lipase (an adsorbed bioreactor). However, the optical purity of the remaining (S)-acid was low even when the conversion ration was kept at approximately 50%. Lipase recovered from the non-support bioreactor after continuous operation retained the original stereoselectivity in a batch reaction. Therefore, semi-continuous operation was conducted by recycling the substrate solution at a high flow rate. The non-support reactor showed high stereoselectivity and ten times the productivity compared with the adsorbed bioreactor. The reason for this high performance is discussed. Offprint requests to: A. Tanaka     

Widespread Distribution of the 14-3-3 Protein in Vertebrate Brains and Bovine Tissues: Correlation with the Distributions of Calcium-Dependent Protein Kinases

A highly specific antiserum was prepared against bovine brain 14-3-3 protein, a protein kinase-dependent activator of tyrosine and tryptophan hydroxylases. The immunoassay using this antiserum proved the presence of 14-3-3 protein in various bovine tissues and in brains of various vertebrate species. The quantitative analysis indicated that the tissue distribution of 14-3-3 protein is more closely related to the known distributions of the Ca2(+)-dependent protein kinases, i.e., Ca2+/calmodulin- and Ca2+/phospholipid-dependent protein kinases, rather than those of tyrosine and tryptophan hydroxylases. This result, together with the available data on this protein, suggests potential roles of the 14-3-3 protein in more diverse kinase-mediated processes than the predicted role in monoamine synthesis.     

Increased insulin-insensitive glucose transport in polymorphonuclear leukocytes from non-insulin-dependent diabetic patients.

We studied the transport rate of a non-metabolizable hexose analogue, 3-O-methyl-D-glucose, in polymorphonuclear leukocytes (insulin-insensitive cells) from patients with untreated non-insulin-dependent diabetes mellitus. The mean glucose transport rate was significantly elevated in the diabetic patients compared with healthy controls (13.3 +/- 3.7 vs 10.4 +/- 2.5 fl/cell.sec, mean +/- SD, p less than 0.01). In the diabetic subjects, glucose transport rates were positively correlated with HbA1c levels (r = 0.563, p less than 0.01) but had no relations with ambient plasma glucose concentrations. Short-term incubation with 20 mM D-glucose had no effect on glucose transport in those cells. When glucose transport rates, HbA1c and fasting plasma glucose levels were simultaneously measured at weekly intervals over a four-week period in three diabetic subjects, the alterations in transport rates generally paralleled the changes observed in HbA1c levels rather than plasma glucose concentrations. It can be concluded that unlike insulin-sensitive cells such as adipocytes and muscle, glucose transport in human polymorphonuclear leukocytes, which are insulin insensitive cells, is increased in patients with non-insulin-dependent diabetes mellitus. Long-term, not short-term, derangement of glucose metabolism seems to be associated with increased glucose transport rate found in those patients.     

Co-purification of alanine-glyoxylate aminotransferase with 2-aminobutyrate aminotransferase in rat kidney

Alanine-glyoxylate aminotransferase and 2-aminobutyrate aminotransferase were co-purified from rat kidney to a single protein (about 500-fold purified from the homogenate). The activity ratios of alanine-glyoxylate aminotransferase to 2-aminobutyrate aminotransferase were constant during co-purification steps suggesting the 2-aminobutyrate aminotransferase activity was catalysed by only alanine-glyoxylate aminotransferase. The molecular weight of the enzyme was estimated to be approx. 213 000, 220 000 and 236 000 by analytical ultracentrifugation, Sephadex G-150 gel filtration and sucrose density gradient centrifugation, respectively. From the polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, the enzyme consisted of four apparently similar subunits having a molecular weight of approx. 56 000. The enzyme was almost specific to L-alanine and L-2-aminobutyrate as amino donor and to glyoxylate, pyruvate and 2-oxobutyrate as amino acceptor. The enzyme was identified with rat liver alanine-glyoxylate aminotransferase isoenzyme 2 but not with rat liver alanine-glyoxylate aminotransferase isoenzyme 1 from Ouchterlony double diffusion analysis. Absorption spectra and some kinetic properties of the enzyme were clarified.