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The hepatic glucose transporter, GLUT2, facilitates bidirectional glucose transport across the hepatocyte plasma membrane under insulin regulation. We studied the interactions of IR and GLUT2 proteins to determine whether they are physically coupled in a receptor-transporter complex. By comparing endosome and plasma membrane IR and GLUT2 ratios before and after feeding, it was determined that IR and GLUT2 are internalized in a fixed ratio. When solubilized hepatocytes were immunoprecipitated with antibodies against either IR or GLUT2, both proteins co-precipitated. The association of IR and GLUT2 was further assessed by confocal microscopy. Sections of fed liver were incubated with fluorescein-tagged anti-GLUT2 or Texas Red-tagged anti-IR. Colocalization was observed both at the plasma membrane and in the cytosol. Fluorescence-resonance energy transfer studies further confirmed this association. We conclude that IR and GLUT2 form a receptor-transporter complex in hepatocytes, which forms a mechanism of insulin-mediated hepatic glucose regulation., (Copyright 2005 S. Karger AG, Basel.)

Background: Hepatic innervation plays an essential role in insulin extraction and glucose production, but the specific role of hepatic cholinergic innervation remains unclear. We sought to establish a model of isolated hepatic cholinergic denervation (IHCD), and to assess whether glycogen storage or the control of net hepatic glucose production (HGP) was altered by IHCD., Materials and Methods: Sprague-Dawley rats underwent either hepatic vagotomy or sham operation. Liver tissue was stained for vesicular acetylcholine transporter (VAChT) and (nonspecific neural) protein gene product 9. 5 (PGP) for verification of IHCD. Liver glycogen content was quantified in fed and fasted IHCD or sham-operated animals. HGP was determined after single-pass isolated liver perfusion, during which a 30-min 12 ng/ml glucagon infusion was begun after equilibration, and after 10 min, a 200 microU/ml insulin infusion was added., Results: Uniform staining of PGP and absence of VAChT staining in hepatic vagotomized rats demonstrated the validity of our model. Glycogen content of sham-operated livers (n = 8) increased from 6.0 +/- 1.7 in the fasting state to 10.6 +/- 1.8 mg/g liver, after feeding (P < 0.05). IHCD livers (n = 8) showed no comparable increase (3.5 +/- 0.6 to 4.0 +/- 0.7 mg/g liver). Perfusion with glucagon alone resulted in less HGP in IHCD livers (n = 12) compared with sham-operated livers (n = 10) (integrated HGP 3.3 +/- 0.3 mg/g liver min(-1) vs 5.1 +/- 0.5 mg/g liver min(-1), P < 0.05). Insulin infusion revealed impaired responsiveness to insulin after IHCD; the ratio of HGP in the final 10 min of perfusion (glucagon and insulin) to HGP in the initial 10 min (glucagon alone) was 90.3 +/- 2.4% for IHCD livers versus 68.1 +/- 4.4% for sham-operated controls, respectively (P = 0.0002)., Conclusions: Our study shows that IHCD results in significant impairment in liver glycogen storage and impaired hepatic sensitivity to glucagon and, possibly, to insulin. We conclude that hepatic cholinergic integrity is essential to normal hepatic glucose metabolism., (Copyright 2000 Academic Press.)

The docking and fusion of membrane-bound vesicles at the cell plasma membrane are brought about by several participating vesicle membrane, plasma membrane, and soluble cytosolic proteins. An understanding of the interactions between these participating proteins will provide an estimate of the potency and efficacy of secretory vesicle docking and fusion at the plasma membrane in cells of a given tissue. Earlier studies suggest that in chronic pancreatitis, glucose intolerance may be associated with impaired exocytosis/endocytosis of hepatic insulin receptor and glucose transporter proteins. In this study, the binding force profiles between microsome membrane proteins and plasma membrane proteins in liver obtained from normal and pancreatitic rats have been examined using atomic force microscopy. The ability of a VAMP-specific antibody to alter binding between microsome- and plasma membrane-associated membrane proteins was examined. In pancreatitic livers, a significant loss in microsome-plasma membrane binding is observed. Furthermore, our study shows that, in contrast to control livers, the microsome-plasma membrane binding in pancreatitic livers is VAMP-independent, which suggests an absence of VAMP participation in membrane-microsome binding. In confirmation with our earlier findings, these studies suggest altered membrane recycling in liver of rats with chronic pancreatitis.

The final step in the exocytotic process is the docking and fusion of membrane-bound secretory vesicles at the cell plasma membrane. This docking and fusion is brought about by several participating vesicle membrane, plasma membrane and soluble cytosolic proteins. A clear understanding of the interactions between these participating proteins giving rise to vesicle docking and fusion is essential. In this study, the binding force profiles between synaptic vesicle membrane and plasma membrane proteins have been examined for the first time using the atomic force microscope. Binding force contributions of a synaptic vesicle membrane protein VAMP1, and the plasma membrane proteins SNAP-25 and syntaxin, are also implicated from these studies. Our study suggests that these three proteins are the major, if not the only contributors to the interactive binding force that exist between the two membranes., (Copyright 1998 Academic Press.)

In the last decade, several monomeric and heterotrimeric guanine nucleotide binding proteins have been identified to associate with secretory vesicles and to be implicated in exocytosis. Vesicle volume also has been proposed to play a regulatory role in secretory vesicle fusion at the plasma membrane. However, the molecular mechanism of function of the guanine nucleotide binding proteins and of the regulation of secretory vesicle volume in the exocytotic process remains unclear. In this study, we report association of the secretory vesicle membrane with the alpha subunit of a heterotrimeric GTP binding protein G(alpha i3) and implicate its involvement in vesicle swelling. Using an atomic force microscope in combination with confocal microscopy, we were able to study the dynamics of isolated zymogen granules, the secretory vesicles in exocrine pancreas. Exposure of zymogen granules to GTP resulted in a 15-25% increase in vesicle height as measured by the atomic force microscope and a similar increase in vesicle diameter as determined by confocal microscopy. Mas7, an active mastoparan analog known to stimulate Gi proteins, was found to stimulate the GTPase activity of isolated zymogen granules and cause swelling. Increase in vesicle size in the presence of GTP, NaF, and Mas7 were irreversible and KCl-sensitive. Ca2+ had no effect on zymogen granule size. Taken together, the results indicate that G(alpha i3) protein localized in the secretory vesicle membrane mediates vesicle swelling, a potentially important prerequisite for vesicle fusion at the cell plasma membrane.

The tyrosine kinase pp60src is known to phosphorylate synaptophysin and in doing so may regulate neurotransmitter release. The tyrosyl phosphorylated state of synaptophysin is dependent on pp60src kinase and the unknown protein tyrosine phosphate phosphohydrolase (PTPase, EC 3.1.3.48). Here we report the protein tyrosine phosphate phosphohydrolase SH-PTP1, to associate with synaptic vesicles and interact with synaptophysin. These studies identify SH-PTP1 as a new member of the secretory machinery at the nerve terminal and suggest its involvement in neurotransmission.

The dynamics at the plasma membrane resulting from secretory vesicle docking and fusion and compensatory endocytosis has been difficult to observe in living cells primarily due to limited resolution at the light microscopic level. Using the atomic force microscope, we have been able to image and record changes in plasma membrane structure at ultrahigh resolution after stimulation of secretion from isolated pancreatic acinar cells. "Pits" measuring 500-2000 nm and containing 3-20 depressions measuring 100-180 nm in diameter were observed only at the apical region of acinar cells. The time course of an increase and decrease in "depression" size correlated with an increase and decrease of amylase secretion from live acinar cells. Depression dynamics and amylase release were found to be regulated in part by actin. No structural changes were identified at the basolateral region of these cells. Our results suggest depressions to be the fusion pores identified earlier in mast cells by freeze-fracture electron microscopy and by electrophysiological measurements. The atomic force microscope has enabled us to observe plasma membrane dynamics of the exocytic process in living cells in real time.