Lipase Activity In Cells Research Articles

Detection of Lipase Activity in Cells by a Fluorescent Probe Based on Formation of Self-Assembled Micelles.

SummaryReliable and sensitive detection of lipase activity is essential for the early diagnosis and monitoring of acute pancreatitis or progression of digestive diseases. However, the available fluorescent probes for detection of lipase activity are only implemented in a hexane-water two-phase system due to the nature of heterogeneous catalysis of lipase, thus limiting their applications in direct imaging of lipase activity in living cells and tissues. Here we designed and synthesized a “turn on” fluorescent probe CPP based on self-assembled micelles for hydrolysis of lipase. The CPP probe exhibits high selectivity and excellent sensitivity for the detection of lipase in such a homogeneous system and is successfully applied for monitoring lipase activity in pancreatic AR42J cells, tissues, and serums. Taken together, the fluorescent CPP probe not only provides a tool for diagnostic potential in pancreatic disease but also demonstrates an application potential for micelle self-assembly-based development of biological probes.

Negative feedback regulation of Gq signaling by protein kinase C is disrupted by diacylglycerol kinase ζ in COS-7 cells

Cellular response to Gq-linked agonists is shaped by regulatory inputs which determine signal strength and duration. Stimulation of phospholipase C-β (PLC-β) lipase activity results in an increase in the levels of diacylglycerol (DAG) and activation of protein kinase C (PKC) activity. PKC has been implicated in the feedback regulation of Gq signaling through actions on PLC-β and phospholipase D (PLD) lipase activity. As PKC activity is modulated by multiple layers of regulation, the physiological impact of PKC on Gq signaling is unclear. PKC signaling can be terminated by diacylglycerol kinases (DGKs) which are regulated in a cell-specific manner. The present studies investigated the contribution of the ubiquitously expressed DGKζ isoform in the regulation of PKC signaling and Gq response in transfected COS-7 cells. Genetic depletion of DGKζ protein with antisense oligonucleotides dramatically reduced DAG metabolism. The sustained increase in PKC signaling was associated with a pronounced inhibition of carbachol-stimulated lipase activity in cells co-transfected with m1 muscarinic receptor, Gαq and either with or without PLC-β1. The data also reveal that sustained activation of PKC alone does not increase cellular PLD1 activity. Therefore, G12-activated RhoA is physiologically important for adequate stimulation of PLD1 activity. These data show that the impact of PKC on Gq signal transduction is determined by the background of cell-specific processes.

Effects of Diet and Ketones on Rat Pancreatic Lipase in Cultured Acinar Cells

The activity, synthesis rate and mRNA level of pancreatic lipase increase with dietary fat intake. Ketones, intermediates of lipid metabolism, have been proposed to mediate this change. Therefore, we investigated their direct effect on cultured pancreatic acinar cells and examined their possible interactive effects with glucose and dietary fat. beta-Hydroxybutyrate (0.01 to 2 mmol/L) did not affect lipase activity in cells isolated from rats fed a commercial nonpurified (NP) diet and cultured in high glucose (HG, 27.8 mmol/L) or low glucose (LG, 6.9 mmol/L) medium. The effects of ketones were also examined in acinar cells isolated from rats fed purified high fat (HF, 67% of energy from fat) or low fat (LF, 11% of energy from fat) diet. Cellular lipase was significantly higher in cells from HF-fed rats at both 24 and 48 h (264% and 145% of LF values, respectively; P less than 0.0001). beta-Hydroxybutyrate significantly increased (P less than 0.04) lipase activity in LF cells at 48 h but did not affect lipase activity in HF cells. These studies suggest that ketones may be involved in the regulation of pancreatic lipase in rats fed a LF diet, but their role is complex and interactive with dietary carbohydrate and fat.

Release of lipoprotein lipase from fat cells in vitro

Release of lipoprotein lipase from rat fat cells incubated at 20°C in medium with albumin, but without glucose proceeded at a constant rate for 30 min. The initial rate of release was increased when serum was present in the medium. Maximal stimulation (100–300%) was produced with 3.8% serum. The maximal increment in release caused by serum was always greater than that produced by heparin and when both were added release was greater than it was with either one alone. The active component(s) of serum, nondialyzable and stable for 30 min at 56°C, was present in sera from humans and rats in the fed or fasted state. Glucose plus insulin (but neither alone) enhanced the rate of lipase release in the presence of serum but not in its absence. The half-life of the lipase in basal medium at 20° C was 90 min. Heparin decreased this to about 50 min and serum markedly prolonged it whether or not heparin was present. Lipoprotein lipase activity in cells and fractions thereof was assayed in extracts of acetone powders. After centrifugation of fat cell homogenates at 600 × g for 15 min, only 50–60% of the activity was recovered in the supernatant. After centrifugation at 100 000 × g for 60 min, the supernatant contained about 10% of the total activity and the sediment 40%. In some experiments, most of the rest was recovered in the floating fat fraction. Total lipoprotein lipase activity of cells plus medium increased steadily during incubation of fat cells for 1 h at 30° C. The major increment occurred in the cells and activity in the medium was always less than 15% of the total. Our observations are consistent with the view that activation may be an important determinant of fat cell lipoprotein lipase activity as well as an integral part of the release process.