Research Article: The Activation by Glucose of Liver Membrane Nitric Oxide Synthase in the Synthesis and Translocation of Glucose transporter-4 in the Production of Insulin in the Mice Hepatocytes

Date Published: December 3, 2013

Publisher: Public Library of Science

Author(s): Suman Bhattacharya, Rajeshwary Ghosh, Smarajit Maiti, Gausal Azam Khan, Asru K. Sinha, Ranjit Ray.

http://doi.org/10.1371/journal.pone.0081935

Abstract

Glucose has been reported to have an essential role in the synthesis and secretion of insulin in hepatocytes. As the efflux of glucose is facilitated from the liver cells into the circulation, the mechanism of transportation of glucose into the hepatocytes for the synthesis of insulin was investigated.

Grated liver suspension (GLS) was prepared by grating intact liver from adult mice by using a grater. Nitric oxide (NO) was measured by methemoglobin method. Glucose transporter-4 (Glut-4) was measured by immunoblot technique using Glut-4 antibody.

Incubation of GLS with different amounts of glucose resulted in the uptake of glucose by the suspension with increased NO synthesis due to the stimulation of a glucose activated nitric oxide synthase that was present in the liver membrane. The inhibition of glucose induced NO synthesis resulted in the inhibition of glucose uptake. Glucose at 0.02M that maximally increased NO synthesis in the hepatocytes led to the translocation and increased synthesis of Glut-4 by 3.3 fold over the control that was inhibited by the inhibition of NO synthesis. The glucose induced NO synthesis was also found to result in the synthesis of insulin, in the presence of glucose due to the expression of both proinsulin genes I and II in the liver cells.

It was concluded that glucose itself facilitated its own transportation in the liver cells both via Glut-4 and by the synthesis of NO which had an essential role for insulin synthesis in the presence of glucose in these cells.

Partial Text

Glucose has been reported to have a critically important role both in the synthesis and in the secretion of insulin in the islets of Langerhans [1,2] which is currently believed to be the only site of production and secretion of the hypoglycaemic protein. We, on the other hand, have recently reported that a glucose dependent synthesis and secretion of insulin also occurred in the hepatocytes in adult mice [3]. It should be mentioned here that various etiological and demographical studies have previously suggested that the liver could have a significant contribution in both the long and short term of glucose homeostasis [4], and the chronic hepatitis is reported to lead to diabetes mellitus [5]. It has also been reported that rat hepatic stem cells cultured in high glucose concentration were capable of producing insulin and the hypoglycaemic hormone was ubiquitously present in extra pancreatic tissues of rat and humans [6,7]. Although insulin is essential for the synthesis of hepatic glycogen synthesis, as much as 50% of the pancreatic insulin was reported to be destroyed in the liver suggesting that liver itself might have synthesized insulin essential for the glycogen synthesis.

These results demonstrated that glucose which has an essential role in the synthesis and secretion of insulin both in the pancreatic β cells [1,2] and in the hepatocytes in the adult mice liver [3] had also a critically important of role both in the synthesis (Figure 5) and in the translocation of Glut-4 (Figure 4) for its own transportation from the external medium into the liver cells even in the presence of opposing effect of Glut-2 which is known to efflux the sugar from the liver cells into the circulation [13]. The effects of glucose on the synthesis and translocation of Glut-4 was found to be related to the stimulation of a constitutive form of NOS by glucose itself in the liver cells membranes. The existence of the glucose activated NOS in the liver cells, that could be critically important in the hepatic insulin synthesis has never been realized before. We however reported before the glucose induced NO synthesis was also involved in the glucose transportation in the islets of Langerhans for the synthesis and secretion of the hypoglycaemic protein [12]. As described in the Figure-5 the glucose induced increase of Glut-4 synthesis was maximally stimulated at 0.02M glucose and the increase of glucose concentration greater than 0.02M in the reaction mixture actually resulted in the reduction of Glut-4 synthesis in the liver cells. It was also noted that either 0.02M glucose or the use of NO itself instead of glucose was found to stimulate the Glut-4 synthesis in the reaction mixture. Furthermore the addition of 0.1mM NAME to the reaction mixture containing 0.02M glucose inhibited both NO and Glut-4 synthesis. The glucose induced synthesis of NO in the liver cell membrane was found to stimulate the actual synthesis of Glut-4 in the liver cells as demonstrated by the in vitro translation of mRNA of Glut-4 (Figure 6) and not merely due to the release of preformed Glut-4 from the liver cells by NO.

It can be concluded from the results as described above on the glucose induced NO synthesis that the activation of GANOS in the liver cell membrane would play a critically important role both in the transportation of glucose and in the synthesis of bioactive insulin from proinsulin genes products in the liver cells. The presence of glucose alone in the liver cells was not sufficient by itself for the synthesis of insulin, and the activation of the liver membrane nitric oxide synthase by glucose played an essential role in the glucose induced synthesis of insulin, at least in the mice hepatocytes.

 

Source:

http://doi.org/10.1371/journal.pone.0081935