Immunocytochemical localization of glucose 6-phosphatase and cytosolic phosphoenolpyruvate carboxykinase in gluconeogenic tissues reveals unsuspected metabolic zonation

Abstract

Immunohistochemical analysis was used to define the precise cell-specific localization of Glucose-6-phosphatase (Glc6Pase) and cytosolic form of the phosphoenolpyruvate carboxykinase (PEPCK-C) in the digestive system (liver, small intestine and pancreas) and the kidney. Co-expression of Glc6Pase and PEPCK-C was shown to take place in hepatocytes, in proximal tubules of the cortex kidney and at the top of the villi of the small intestine suggesting that these tissues are all able to perform complete gluconeogenesis. On the other hand, intrahepatic bile ducts, collecting tubes of the nephron and the urinary epithelium in the calices of the kidney, as well as the crypts of the small intestine, express Glc6Pase without significant levels of PEPCK-C. In such cases, the function of Glc6Pase could be related to the transepithelial transport of glucose characteristic of these tissues, rather than to the neoformation of glucose. Lastly, PEPCK-C expression in the absence of Glc6Pase was noted in both the exocrine pancreas and the endocrine islets of Langerhans. Possible roles of PEPCK-C in exocrine pancreas might be the provision of gluconeogenic intermediates for further conversion into glucose in the liver, whereas PEPCK-C would be instrumental in pyruvate cycling, which has been suggested to play a regulatory role in insulin secretion by the beta-cells of the islets.

A link between hepatic glucose production and peripheral energy metabolism via hepatokines

“…Histological analyses showed differential zonation of triglycerides in livers of L-G6pc −/− and control mice. Control mice accumulated lipid droplets in the perivenous region, the preferential site for glycolysis and lipogenesis [28], while L-G6pc −/− mice accumulated fat in the periportal region, dedicated to gluconeogenesis, which corresponds to G6Pase histological localization [25]. In accordance to what we previously observed after 9 months of HF/HS diet [29], L-G6pc −/− mice showed reduced hepatic triglyceride accumulation compared to control mice (Figure S2B).…”

“…G6P and glycogen determination were carried out on frozen tissue homogenates [24]. G6Pase activity was determined as described previously [25].…”

“…Protein extracts were obtained by homogenization of samples in a lysis buffer (50 mM Tris pH 7.5, 5 mM MgCl 2 , 100 mM KCl, 1 mM EDTA, 10% glycerol, 1 mM DTT, 1% NP40, protease and phosphatase inhibitors). Proteins were separated by SDS-PAGE and immune-blotted using antibodies against G6PC [25], phospho-AMPKα (Thr172; 2535, Cell Signaling), AMPKα (2603, Cell Signaling), phospho-ACC (3661, Cell Signaling), ACC (3676, Cell Signaling), phospho-p38 MAPK (Thr180/Tyr182, 92115, Cell Signaling), p38 MAPK (92125, Cell Signaling), and AGF (sc-160959, Santa Cruz). The intensity of the bands was determined by densitometry with the system VersaDoc™ (Biorad) and analyzed using the software Quantity One ® (Biorad).…”

Control of Blood Glucose in the Absence of Hepatic Glucose Production During Prolonged Fasting in Mice

“…G6Pase and PEPCK-c activities were assayed at maximal velocity, as described previously (19). Immunoblotting was carried out using purified anti-rat G6PC (20), anti-PEPCK (Santa Cruz Biotechnology), and antiglutaminase (kindly provided by Dr. N. Curthoys) antibodies. Membranes were reprobed with anti-β-actin monoclonal antibody for standardization.…”

Targeted deletion of liver glucose-6 phosphatase mimics glycogen storage disease type 1a including development of multiple adenomas

“…The frozen tissues were sampled, and G6Pase and PEPCK activities determined as previously described [10,[28][29][30]. Immunoblotting was carried out using purified anti-rat G6PC (use at 1:1,500) or anti-PEPCK (use at 1:5,000; Santa Cruz Biotechnology) antibodies [31]. Total RNAs were isolated from tissues with TRIzol reagent (Invitrogen).…”