Glycosylphosphatidylinositol-specific phospholipase D immunoreactivity is present in islet amyloid in type 2 diabetes.

Numerous apolipoproteins associate with amyloid plaques. A minor high-density lipoprotein-associated protein, glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD), has recently been described by the authors and others. Since GPI-PLD is synthesized by, and secreted from, pancreatic islet beta cells, the present study examined the hypothesis that GPI-PLD associates with islet amyloid. GPI-PLD immunoreactivity was examined in pancreatic tissues from type 2 diabetic and non-diabetic humans. GPI-PLD binding to heparan sulphate proteoglycan was determined in the absence or presence of heparan sulphate or heparin. Fibril formation from human islet amyloid polypeptide was determined in the absence or presence of GPI-PLD. In non-diabetics, GPI-PLD immunoreactivity was present and co-localized with insulin, as opposed to co-localizing with amyloid in diabetics. No immunoreactivity for apolipoprotein A-I was present in islet cells or islet amyloid. Heparan sulphate proteoglycan, which is commonly present in most amyloid, bound GPI-PLD in vitro. GPI-PLD inhibited the formation of amyloid fibrils from synthetic islet amyloid polypeptide in vitro. GPI-PLD is therefore present in islet amyloid and appears to derive from local production from islets. This localization likely derives from interaction between GPI-PLD and heparan sulphate proteoglycan. Since GPI-PLD also inhibited islet amyloid polypeptide fibril formation in vitro, it is concluded that GPI-PLD may play a role in islet amyloid formation in type 2 diabetes.

Glucose and insulin regulate glycosylphosphatidylinositol-specific phospholipase D expression in islet beta cells.

Insulin resistance is associated with a compensatory islet hyperactivity to sustain adequate insulin biosynthesis and secretion to maintain near euglycemia. Both glucose and insulin are involved in regulating proteins required for insulin synthesis and secretion within the islet and islet hypertrophy. We have determined that glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is present within the secretory granules of islet beta cells. To determine if GPI-PLD is regulated in islet beta cells, we examined the effect of glucose and insulin on GPI-PLD expression in rat islets and murine insulinoma cell lines. Glucose (16.7 mmol/L) increased cellular GPI-PLD activity and mRNA levels 2- to 7-fold in isolated rat islets and betaTC3 and betaTC6-F7 cells. Insulin (10(-7) mol/L) also increased GPI-PLD mRNA levels in rat islets and betaTC6-F7 cells 2- to 4-fold commensurate with an increase in GPI-PLD biosynthesis. To determine if islet GPI-PLD expression is increased in vivo under conditions of islet hyperactivity, we compared GPI-PLD mRNA levels in islets and liver from ob/ob mice and their lean littermates. Islet GPI-PLD mRNA was increased 5-fold while liver mRNA and serum GPI-PLD levels were reduced 30% in ob/ob mice compared with lean littermate controls. These results suggest that glucose and insulin regulate GPI-PLD mRNA levels in isolated islets and beta-cell lines. These regulators may also account for the increased expression of GPI-PLD mRNA in islets from ob/ob mice, a model of insulin resistance and islet hyperactivity.

Increased expression of GPI-specific phospholipase D in mouse models of type 1 diabetes.

Glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD) is a high-density lipoprotein-associated protein. However, the tissue source(s) for circulating GPI-PLD and whether serum levels are regulated are unknown. Because the diabetic state alters lipoprotein metabolism, and liver and pancreatic islets are possible sources of GPI-PLD, we hypothesized that GPI-PLD levels would be altered in diabetes. GPI-PLD serum activity and liver mRNA were examined in two mouse models of type 1 diabetes, a nonobese diabetic (NOD) mouse model and low-dose streptozotocin-induced diabetes in CD-1 mice. With the onset of hyperglycemia (2- to 5-fold increase over nondiabetic levels), GPI-PLD serum activity and liver mRNA increased 2- to 4-fold in both models. Conversely, islet expression of GPI-PLD was absent as determined by immunofluorescence. Insulin may regulate GPI-PLD expression, because insulin treatment of diabetic NOD mice corrected the hyperglycemia along with reducing serum GPI-PLD activity and liver mRNA. Our data demonstrate that serum GPI-PLD levels are altered in the diabetic state and are consistent with liver as a contributor to circulating GPI-PLD.

Midportion antibodies stimulate glycosylphosphatidylinositol-specific phospholipase D activity.

Limited information is known regarding the regulation, structural features, and functional domains of glycosylphosphatidylinositol-specific phospholipase D (GPI-PLD, EC 3. 1.4.50). Previous studies demonstrated that trypsin cleavage of GPI-PLD at or near Arg325 and/or Arg589 in bovine serum GPI-PLD was associated with an increase in enzymatic activity. Since the Arg325 is predicted to be in a region between the catalytic domain and predicted beta-propeller structure in the C-terminal portion of GPI-PLD (T. A. Springer, Proc. Natl. Acad. Sci. USA 94, 65-72, 1997), we hypothesized that this connecting region is important for catalytic activity. Trypsin cleavage of human serum GPI-PLD, which has an Arg325 but lacks the Arg589 present in bovine serum GPI-PLD, also increased GPI-PLD activity. Peptide-specific antibodies to residues 275-296 (anti-GPI-PLD(275)) and a monoclonal antibody, 191, with an epitope encompassing Arg325, also stimulated GPI-PLD activity. Pretreating human GPI-PLD with trypsin demonstrated that anti-GPI-PLD(275) only stimulated the activity of intact GPI-PLD. These results suggest that trypsin activation and anti-GPI-PPLD(275) may have similar effects on GPI-PLD. Consistent with this is the observation that both manipulations decreased the affinity of GPI-PLD for mixed micelle substrates. These results indicate that the midportion region of GPI-PLD is important in regulating enzymatic activity.

Phosphoproteins regulated by the interaction of high-density lipoprotein with human skin fibroblasts.

Interaction of HDL with cells activates protein kinase C (PKC), a process that may be important in stimulating efflux of excess cellular cholesterol. Here we report that HDL treatment of cholesterol-loaded fibroblasts increases 32P labeling of three acidic phosphoproteins. These phosphoproteins, called pp80, pp27, and pp18 based on apparent M(r) in kD, were also phosphorylated by acute treatment of cells with phorbol myristate acetate, suggesting that they are regulated in response to PKC activation. The HDL-stimulated phosphorylation of pp80 and pp18 was significant after only 30 seconds and was sustained for at least 30 and 120 minutes, respectively, while increased phosphorylation of pp27 was transient, reaching a maximum at 10 minutes. Both pp27 and pp18 were phosphorylated on serine/threonine residues, whereas pp80 was phosphorylated on serine/threonine and tyrosine residues. Immunoprecipitation studies suggested that pp80 is the myristoylated alanine-rich C kinase substrate protein, but the identities of pp27 and pp18 are unknown. HDL and trypsin-digested HDL stimulated phosphorylation of pp80 and pp27, while purified apoA-I, apoA-II, or apoE had no stimulatory effects, indicating that the active component in HDL was trypsin resistant and unlikely to be an apolipoprotein. Conversely, HDL, apoA-I, apoA-II, and apoE all stimulated pp18 phosphorylation, while trypsin-digested HDL had less effect, consistent with pp18's being responsive to HDL apolipoproteins. Treatment of cholesterol-depleted cells with apoA-I also stimulated phosphorylation of pp18, but only transiently. These results suggest that HDL interaction with cells activates diverse PKC-mediated pathways that target different phosphoproteins. Of these three phosphoproteins, only pp18 has a phosphorylation response consistent with its being involved in apolipoprotein-mediated lipid transport.

High density lipoproteins stimulate mitogen-activated protein kinases in human skin fibroblasts.

Protein kinase C (PKC) seems to play an important role in many of HDL effects on cells, including removal of excess cholesterol. HDL removes cholesterol by at least two mechanisms. One mechanism involves desorption/diffusion of cholesterol from the plasma membrane onto the acceptor particle, whereas the second is mediated by apolipoproteins and may involve intracellular translocation of cholesterol to the plasma membrane for subsequent efflux. In this report, we examined the possibility that mitogen-activated protein (MAP) kinase is one of the downstream events from HDL activation of PKC. Using a gel kinase assay with myelin basic protein incorporated into the gel, HDL (50 micrograms protein/mL) stimulated multiple kinases of 42, 50, 52, 58, and 60 kDa. The 42-kDa protein kinase, corresponding to the unresolved MAP kinases ERK1 and ERK2 based on immunoblotting, was activated over 2-fold by HDL. HDL activated all identified kinases in a concentration- and time-dependent manner, which became maximal within 5 to 10 minutes and remained activated for at least 60 minutes. HDL activation of MAP kinase seems to be partially mediated by PKC, because down-regulation of PKC and known PKC inhibitors inhibited the HDL effect by 40 to 50%. Free apolipoproteins A-I (10 micrograms/mL) and A-II (10 micrograms/mL) had no significant effect on MAP kinase activation. Moreover, modifying HDL with trypsin or tetranitromethane, which abolishes apolipoprotein-mediated cholesterol efflux, had no effect on HDL activation of MAP kinase. These results suggest that HDL activates MAP kinase via multiple signal transduction pathways that are likely involved in an HDL effect unrelated to apolipoprotein-mediated cholesterol translocation and efflux.