Takeshi Ohara

Accumulation of Pyrraline-modified Albumin in Phagocytes due to Reduced Degradation by Lysosomal Enzymes

Previous studies suggested that the interaction between proteins modified by advanced glycation end products (AGEs) and cells, such as macrophages, may be involved in diabetic angiopathy. Pyrraline is one of the AGEs and known to be elevated in plasma of diabetic rats and humans, and is present in vascular lesions of diabetic and elderly subjects. We examined whether modification of albumin by pyrraline influences its degradation by macrophage-like cell line, P388D1 cells. Degradation of pyrraline-modified albumin by these cells was diminished, causing accumulation of the albumin in these cells. The susceptibility of pyrraline-modified albumin to lysosomal proteolytic enzymes was reduced by approximately 40% in vitro, while lysosomal activity in the cells per se was not affected. This phenomenon was also observed when human monocytes were used instead of P388D1 cells. Our results suggest that accumulation of pyrraline-modified albumin in P388D1 cells is due to the reduced susceptibility of the protein to lysosomal enzymatic degradation. Such alterations in the interaction between AGEs-modified protein and phagocytes may contribute to angiopathy in elderly subjects and patients with diabetes.

Na+K+-ATPase activity and its αII subunit gene expression in rat skeletal muscle: Influence of diabetes, fasting, and refeeding ☆

We have examined the effects of diabetes, fasting, and refeeding on Na+/K(+)-adenosine triphosphatase (ATPase) activity and its catalytic alpha II subunit gene expression in skeletal muscle. Two hypoinsulinemic states, streptozotocin-induced diabetes and 48-hour fasting caused a significant decrease (P less than .05) in skeletal muscle Na+/K(+)-ATPase activity and a marked increase (P less than .01) in the levels of alpha II subunit mRNA. A decrease in enzyme activity was observed on the 2nd and the 14th day of diabetes, whereas an increase in alpha II mRNA levels was found only on the 14th day. The levels of alpha I mRNA were not affected, while the levels of mRNA of the structural beta subunit were decreased on the 14th day of diabetes. Correction of hyperglycemia with insulin restored enzyme activity and alpha II isoform mRNA levels toward normal in diabetic animals. Refeeding for 48 or 72 hours restored these parameters to normal in skeletal muscle of previously fasting rats. These observations suggest that a decrease in muscle Na+/K(+)-ATPase activity may lead to a compensatory increase in its alpha II subunit gene expression. The levels of insulin and not of glycemia appear to be critical in modulating Na+/K(+)-ATPase activity and gene expression.

Acceleration of Fructose Mediated Collagen Glycation

The effect of fructose on the formation of advanced Maillard reaction products which show fluorescence and have crosslinking was investigated. Type I collagen was added to various concentrations of glucose and fructose which were then incubated at 37°C for 4 weeks. The level of furosine and the fluorescence intensity both increased in direct proportion to glucose and fructose levels and to the duration of incubation. Incubation with fructose produced less furosine but more intense fluorescence than incubation with glucose. Furthermore, collagen was significantly less soluble after incubation with fructose than after incubation with glucose. These results suggest that fructose in the polyol pathway plays an important role in the formation of advanced Maillard products.

Tissue glycogen content and glucose intolerance

Insulin stimulates glycogen synthesis in the the liver and skeletal muscle. After a mixed meal, the secretion of insulin from pancreatic β cells thus results in about 20% and 30% of the carbohydrate intake being stored in the form of glycogen in the liver and skeletal muscle, respectively (1, 2). Defects in this process can therefore be a major contributor to postprandial hyperglycemia. Indeed, the glycogen contents of the liver and skeletal muscle are reduced in individuals with type 2 diabetes (3, 4). Glycogen metabolism is controlled predominantly by the coordinated action of two enzymes, glycogen synthase and glycogen phosphorylase, both of which are regulated by phosphorylation and allosteric modulators. Insulin promotes the net dephosphorylation of both glycogen synthase and glycogen phosphorylase through the inhibition of protein kinases and the activation of protein phosphatases. Among the protein kinases, glycogen synthase kinase–3 (GSK-3) is thought to be an important target for insulin in its stimulation of glycogen synthase activity (5, 6). Among the protein phosphatases, protein phosphatase 1 (PP1) has been implicated in this action of insulin (6). PP1 is an abundant protein serine-threonine phosphatase that is expressed in all compartments of eukaryotic cells. The catalytic subunit of PP1 thus interacts with a wide variety of targeting subunits that localize it to specific sites within the cell. A family of proteins that target PP1 to glycogen and thereby regulate its activity has been identified. These proteins include GM (PPP1R3), GL (PPP1R4), PTG (protein targeting to glycogen or PPP1R5), and PPP1R6.

Fructose-related glycation

We investigated in vitro the effect of the polyol pathway on the formation of advanced Maillard reaction products which have fluorescence and cross-links. Bovine serum albumin supplemented with various concentrations of glucose, fructose or sorbitol was incubated for 14 days. The fluorescence intensity was higher after incubation with fructose than after incubation with glucose. However, no significant increase in fluorescence intensity was found after incubation with sorbitol. These results suggest that in the polyol pathway fructose plays an important role in the formation of advanced Maillard products.