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Phosphoinositolglycan-Peptides from Yeast Potently Induce Metabolic Insulin Actions in Isolated Rat Adipocytes, Cardiomyocytes, and Diaphragms

Hoechst AG, Hoechst Marion Roussel (G.M., S.W., A.C.), Research Site Frankfurt, D-65926 Frankfurt am Main, Germany; and Laboratory of Molecular Cardiology (A.K., J.E.), Diabetes Research Institute, Auf’m Hennekamp 65, D-40225 Düsseldorf, Germany

Address all correspondence and requests for reprints to: Dr. Günter Müller, Hoechst AG, Hoechst-Marion-Roussel, Research Site Frankfurt, DG Metabolic Diseases, Building H825, D-65926 Frankfurt a.m., Germany.

	Abstract

Top Abstract Introduction Materials and Methods Results Discussion References

 
Polar headgroups of free glycosyl-phosphatidylinositol (GPI) lipids or protein-bound GPI membrane anchors have been shown to exhibit insulin-mimetic activity in different cell types. However, elucidation of the molecular mode of action of these phospho-inositolglycan (PIG) molecules has been hampered by 1) lack of knowledge of their exact structure; 2) variable action profiles; and 3) rather modest effects. In the present study, these problems were circumvented by preparation of PIG-peptides (PIG-P) in sufficient quantity by sequential proteolytic (V8 protease) and lipolytic (phosphatidylinositol-specific phospholipase C) cleavage of the GPI-anchored plasma membrane pro-tein, Gce1p, from the yeast Saccharomyces cerevisiae. The structure of the resulting PIG-P, NH₂-Tyr-Cys-Asn-ethanolamine-PO₄-6(Man1–2)Man1–2Man1–6Man1–4GlcNH₂1–6myo-inositol-1,2-cyclicPO₄, was revealed by amino acid analysis and Dionex exchange chromatography of fragments generated enzymatically or chemically from the neutral glycan core and is in accordance with the known consensus structures of yeast GPI anchors. PIG-P stimulated glucose transport and lipogenesis in normal, desensitized and receptor-depleted isolated rat adipocytes, increased glycerol-3-phosphate acyltransferase activity and translocation of the glucose transporter isoform 4, and inhibited isoproterenol-induced lipolysis and protein kinase A activation in adipocytes. Furthermore, PIG-P was found to stimulate glucose transport in isolated rat cardiomyocytes and glycogenesis and glycogen synthase in isolated rat diaphragms. The concentration-dependent effects of the PIG-P reached 70–90% of the maximal insulin activity with EC₅₀-values of 0.5–5 µM. Chemical or enzymic cleavages within the glycan or peptide portion of the PIG-P led to decrease or loss of activity. The data demonstrate that PIG-P exhibits a potent insulin-mimetic activity which covers a broad spectrum of metabolic insulin actions on glucose transport and metabolism.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
THE POSSIBILITY of existence of soluble mediators for metabolic insulin actions in fat and muscle cells has been raised in analogy to the regulation of glycogen storage and mobilization in the liver by ß-adrenergic hormones via the second messenger, cAMP (1). During the past 20 yr, some evidence has accumulated which seems to be compatible with this concept. 1) Treatment of plasma membranes from insulin-sensitive cells with phospholipases generated soluble small molecular mass substances exerting some insulin-mimetic activity. 2) Treatment of fat, liver, and muscle cells with insulin led to the release of small soluble and anionic substances, which in turn reproduced some of the metabolic actions of insulin when exposed to intact cells, e.g. increase of oxidative and nonoxidative glucose metabolism, inhibition of lipolysis (for reviews see Refs. 2–6).

Analytical and metabolic labeling studies indicated the presence of inositol, glucosamine, mannose, galactose, and phosphate in these soluble mediators. Chemical and enzymatic modifications have suggested that they consist of a core structure of phosphoinositol, glycosidically linked to nonacetylated glucosamine, which is itself coupled to additional monosaccharides of varied composition (for a recent review see 7 . Two different mediators were separated by Cheng and co-workers by sizing columns or by ion exchange resins (8). Upon chemical analysis, both mediators were shown to be phosphoinositolglycan (PIG) compounds, as previously reported, but they contained two different inositols and amino sugars.

With the first elucidation of the complete structure of a GPI lipid, the membrane anchor of the variant surface glycoprotein, VSG, from Trypanosoma brucei, structural similarity was recognized between the polar headgroup of a GPI membrane protein anchor consisting of a highly conserved glycan core with myo-inositol, nonacetylated glucosamine, and three mannose residues in characteristic glycosidic linkage (for a review, see 9 , and the PIG substances with insulin-mimetic activity identified so far. This concept gained additional attractiveness by a putative straightforward mode of generation of these molecules by a single lipolytic and/or double lipolytic/proteolytic processing from free GPI lipids and/or GPI membrane protein anchors of the plasma membrane, respectively, which would serve as inactive precursors (for reviews see Refs. 2, 7, 10, 11). In fact, it was known for many years that treatment of insulin-sensitive cells with certain proteases or phospholipases induces some metabolic effects that resemble insulin action to a limited degree. Compatible with these findings was the demonstration that insulin induces release of a set of GPI-anchored plasma membrane proteins from the surface of 3T3 adipocytes (12, 13), isolated rat adipocytes (14), BC₃H1-myocytes (15, 16) and hepatoma cells (17) and from membranes of skeletal muscle (18) via activation of a GPI-specific phospholipase and/or protease. Further evidence has accumulated for an involvement of regulated GPI metabolism in metabolic insulin signaling: 1) An antibody raised against a degalactosylated glycan fragment derived from the GPI membrane anchor of VSG from Trypanosoma brucei selectively inhibited some actions of insulin in intact BC₃H1 myocytes (19). 2) Mutant K562 erythroleukemia cells that exhibit insulin-stimulated receptor and IRS-1 tyrosine phosphorylation but are totally deficient in GPI synthesis were completely unresponsive to insulin regarding stimulation of glycogen synthesis (20). 3) PIG structures exhibit acute hypoglycemic activity in normal and streptozotocin diabetic rats (21, 22) and can be isolated from normal subjects and in reduced amounts from type II diabetic patients (23).

However, from the beginning the GPI mediator concept was faced with two major problems: 1) The exact structure of the putative GPI cleavage products remained unknown. 2) Systematic investigations on the range of metabolic actions covered by a given chemically defined molecule and on their molecular mechanism of action were lacking. Most studies were performed with impure and chemically heterogenous preparations preventing exact determinations of the concentrations required and were restricted to single or a few selected assays for metabolic insulin actions, only.

These problems may be circumvented by preparation of PIG molecules from a defined source available in sufficient amounts, i.e. by proteolytic/lipolytic digestion in vitro of a single purified GPI-anchored protein. This approach for obtaining chemically defined PIG-peptides (PIG-P), linked via a phosphoethanolamine bridge to one or a few amino acids derived from the carboxyl terminus of the protein moiety, was used in two previous studies. 1) A pronase fragment was prepared from the trypanosomal soluble (PI-specific phospholipase C-degraded) VSG, consisting of the PIG coupled to the carboxyl terminal amino acid of VSG, aspartate (24). This PIG-P specifically and concentration-dependently inhibited isoproterenol-induced lipolysis in isolated rat adipocytes, liver microsomal glucose-6-phosphatase and cytosolic fructose-1,6-bisphosphatase in a cell-free system as well as gluconeogenesis in isolated hepatocytes. However, high concentrations of the fragment (100–200 µM for maximal effects, EC₅₀-values of 62–130 µM) were required and their potency relative to that of insulin was not stated (24). 2) Digestion with PI-specific PLC of the proteinase K-fragment of GPI-anchored human erythrocyte acetylcholinesterase yielded a PIG linked to glycine (25). This PIG-P concentration-dependently antagonized the glucagon-stimulated activation of glycogen phosphorylase in intact rat hepatocytes. However, the cells used displayed limited insulin sensitivity, only, with insulin as well as the PIG-P (10 µM) reducing the glucagon response by 20–30% at maximum (25). In addition, both studies were restricted to the analysis of inhibitory effects of insulin and PIG-P on glucose and lipid metabolism.

Thus, it seemed useful to reevaluate the potential insulin-mimetic activity of PIG-P of known structure with respect to stimulatory effects of insulin on glucose and lipid metabolism. This was also justified by the fact that despite great efforts by numerous laboratories regulation of glucose transport, glycogen and lipid synthesis as well as of lipolysis by insulin via affecting the phosphorylation/dephosphorylation state of key metabolic enzymes remains largely unclear so far, leaving open a putative role of soluble PIG-P in mediating these processes (for a review, see 26 .

We decided to prepare chemically defined PIG-P from a GPI-anchored protein from the yeast Saccharomyces cerevisiae, the glycolipid-modified cAMP-binding ectoprotein, Gce1p, for the following reasons: 1) Albeit Gce1p is a minor plasma membrane protein of yeast (27, 28, 29), it can be isolated and purified in quantities sufficient for subsequent proteolytic and lipolytic cleavages in vitro. 2) The structure of the resulting PIG-P has been elucidated (M. Knauf, S. Petry, W. Frick, K. Sauber, manuscript in preparation). 3) The GPI anchor of Gce1p is susceptible to lipolytic/proteolytic processing in vivo during glucose-induced biosynthesis of the cell wall form of Gce1p (30, 31). PIG-P of similar or identical structure compared with the prepared ones should be generated in vivo, which may have signaling function. 4) A GPI-anchored protein homologous to Gce1p in yeast has been identified recently in cultured (13) and isolated adipocytes (32), which is lipolytically cleaved in response to insulin (13, 14). 4) GPI membrane protein anchors of yeast and higher eucaryotes possess identical glycan cores and phosphoethanolamine linkages to the carboxyl terminal amino acid of the protein moiety (33, 34). The only structural differences came from carbohydrate side chains branching off the common glycan cores. The presented work demonstrates that the chemically defined PIG-P obtained from yeast Gce1p exerts a broad spectrum of potent stimulatory effects on glucose and lipid metabolism in isolated rat adipocytes, cardiomyocytes, and diaphragms and offers a method for preparation of sufficient amounts of PIG molecules, which should facilitate studies on their molecular mechanism of action as well as on their potential use as antidiabetic drug.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
D-[3-³H]glucose (5–15 Ci/mmol), 2-deoxy-D-[2,6-³H]glucose (45 Ci/mmol), 3-O-[¹⁴C]methyl-D-glucose (54 mCi/mmol), L-[¹⁴C]glucose (55 mCi/mmol), and [³²P]ATP (5000 Ci/mmol) were obtained from Amersham-Buchler (Braunschweig, Germany); UDP-D-[U-¹⁴C]glucose (300 mCi/mmol) and [¹²⁵I]protein A (10 µCi/µg) were from DuPont-New England Nuclear (Bad Homburg, Germany); L-[2-³H]glycerol-3-phosphate (10 Ci/mmol) and U-[¹⁴C]glucose (20 mCi/mmol) were purchased from Biotrend (Köln, Germany); collagenase (Worthington CLSI, 190 U/mg) was provided by Biochrom (Berlin, Germany) for isolation of adipocytes and by Serva (Heidelberg, Germany) for isolation of cardiomyocytes; semisynthetic human insulin (I81 0182) and ß-amidotaurocholate were obtained from the pharmaceutical synthesis department of HMR, Hoechst AG (Frankfurt a.m., Germany); adenosine deaminase (bovine intestine), V8 protease (Staphylococcus aureus) and pronase were from Calbiochem (Bad Soden, Germany); -mannosidases (Aspergillus phoenicis, Jack bean) and PI-specific PLC (Bacillus cereus) were provided by Boehringer Mannheim (Mannheim, Germany); BioGel-P4 and ion exchange resins were from Bio-Rad Laboratories (Munich, Germany); cAMP Sepharose, octyl/phenyl Sepharose CL-4B, Sephacryl S-200, Mono Q and Sephadex LH-20 were bought from Pharmacia Biotech (Freiburg, Germany); Wistar rats were delivered from the animal breeding station of Hoechst AG (Kastengrund, Germany).

Purification of Gce1p
Gce1p with uncleaved GPI anchor was purified from lactate-grown yeast cells (strain W303 1A, MATa, can^R1–100, ade2–1, his3–11, 15, leu2–3, 112, trp1–1, ura3–1) (27). Ten liters of yeast culture (3 x 10⁸ cells/ml) were combined with 50 ml suspension of yeast cells (5 x 10⁷ cells/ml) that had been metabolically labeled with myo-[³H]inositol (29) to allow monitoring of the purification of Gce1 protein and of the PIG-P derived from it. After conversion of the yeast cells into spheroplasts by digestion with Zymolyase 20,000, plasma membranes were prepared, purified by Ficoll gradient centrifugation (30), and solubilized in TEB [25 mM Tris/HCl, pH 7.4, 0.5 mM EDTA, 1 mM dithiothreitol (DTT), 20 mM KCl, 0.1 mM phenylmethylsulfonylfluoride (PMSF), soy bean trypsin inhibitor (10 µg/ml), 10 µM leupeptin, 1 mM iodoacetamide, 5% glycerol, 0.35% ß-amidotaurocholate (35)] at 2–3 mg protein/ml. A previous study has demonstrated that extraction of membranes with the detergent ß-amidotaurocholate results in a considerable enrichment of GPI-anchored proteins toward integral membrane proteins compared with other detergents (e.g. octyl glucoside) that are commonly used for solubilization of GPI-anchored proteins (35). After the initial solubilization of Gce1p (recovery 80–90% of total amount), the following purification steps were performed with TX-100 (see below). The solubilized plasma membranes were diluted with 10 volumes of ice-cold TX-114 (2%), 25 mM Tris/HCl (pH 7.4), 150 mM NaCl, the samples were subjected to phase separation. Gce1p contained in the detergent-enriched phase (two times reextracted) was purified by gel filtration chromatography on Sephadex S-300, affinity chromatography on N⁶-(2-aminoethyl)-cAMP Sepharose (elution with TEB containing 0.1 mM cAMP) and phenyl Sepharose chromatography (elution with the above buffer containing 0.75% TX-100 instead of ß-amidotaurocholate). Elution from the columns of [³H]inositol-labeled plasma membrane proteins and of a purified [¹⁴C]inositol-labeled Gce1p marker protein was followed in parallel by monitoring on line the ³H- and ¹⁴C-radioactivity. The partially purified Gce1p was precipitated with polyethylene glycol 4000 (final concentration 12%). Analysis of a small sample by SDS-PAGE and fluorography revealed the presence of a single radiolabeled band at 54 kDa, only, which comigrated with authentic photoaffinity-labeled Gce1p (for a see comparison 36 . This demonstrated that Gce1p has been prepared to radiochemical homogeneity and was the only protein in the preparation modified with a GPI anchor. According to binding of [³H]cAMP, Gce1p was enriched 200- to 500-fold (5–10 nmol cAMP bound per mg protein) compared with total yeast membranes.

Preparation and purification of the PIG-P
Digestion with V8 protease.
The precipitated and washed (two times with 0.8% polyethylene glycol 4000) Gce1p (400–500 nmol corresponding to 250,000 to 450,000 dpm) was dialyzed against water, reduced in volume (Speedvac concentrator) and solubilized in 3 ml of 0.1 M Hepes/KOH (pH 8.0), 20 mM CaCl₂, 0.5 mM DTT, 0.5% TX-100, 30 mg/ml V8 protease (Staphylococcus aureus). After incubation for 10 h at 50 C, a second aliquot of V8 protease and SDS (final concentration, 1%) was added and the digestion continued for 10 h. The digestion mixture was boiled (15 min), centrifuged (10,000 x g, 10 min) and applied to a Sephacryl S-200 column (1.5 x 90 cm) equilibrated in 20 mM sodium phosphate (pH 7.0), 250 mM KCl, 0.1% TX-100, 0.5 mM DTT. The elution of material during this and the following chromatographic runs was followed by measurement of ³H-radioactivity (Ramona Raytest on-line radioactivity monitor), UV absorption (A₂₂₀) and insulin-mimetic activity according to stimulation of lipogenesis for each fraction. The ³H-labeled anchor-containing GPI-peptide had an elution volume of 0.41 relative to the solvent marker K₂Cr₂O₇.

Gel filtration.
The peak fractions were pooled, concentrated to 300 µl, mixed with 1.2 ml of ice-cold formic acid, and applied to a water-cooled (4 C) Sephadex LH-60 column (1.5 x 65 cm) equilibrated in ethanol, 88% formic acid (3:1, by vol.). One-milliliter fractions were collected, immediately neutralized with NaOH, and measured for ³H-radioactivity. (A control experiment confirmed that the use of formic acid does not cause cleavage of inositol-1,2-cyclic phosphate, presumably due to the short running time [less than 10 min] and low temperature of the chromatographic procedure. Decyclization of inositol-1,2-cyclic phosphate as induced by treatment with HCl at high temperature [see below] could be followed as a shift in the elution profile during SAX anion exchange chromatography). The major radiolabeled digestion product had an apparent molecular mass of about 2 kDa relative to peptide standards. Fractions 83–100 were pooled, supplemented with NaCl (final concentration, 150 mM) and TX-114 (final concentration, 2%), incubated on ice (15 min) and centrifuged (10,000 x g, 10 min, 4 C). The supernatant was removed and warmed up to 37 C for 10 min to induce phase separation. After centrifugation (10,000 x g, 5 min, 30 C), the aqueous phase was removed. The remaining TX-114 phase containing about 85% of the ³H-radioactivity was washed two times by addition of the same volume of 100 mM Tris/HCl (pH 7.8), 1 mM EDTA, 0.5 mM DTT and a mix of protease inhibitors (see above).

Octyl Sepharose chromatography.
The sample was extracted with an equal volume of water-saturated 1-butanol. The butanol phase was acidified and adjusted to 5% 1-propanol, 100 mM ammonium acetate (pH 5.5). After heating (5 min, 100 C) and centrifugation (10,000 x g, 10 min), the supernatant was applied (2 ml/h) to a column (2.5 x 60 cm) of octyl Sepharose CL-4B equilibrated in 5% 1-propanol, 100 mM ammonium acetate (pH 5.5). The column was washed with 300 ml of equilibration buffer and eluted with a linear gradient of 5–100% propanol in water (10 ml/h) over 800 ml. Eight-milliliter fractions were collected and measured for radioactivity. ³H-radioactivity eluted between 34–38% 1-propanol (as determined by the refractive index). The peak fractions 71–79 were pooled, concentrated by evaporation, freeze-dried, and redissolved in 8 ml of 5% 1-propanol. The purification on octyl-Sepharose was repeated once and the eluted radiolabeled material was lyophilized.

Digestion with PI-specific PLC.
The sample was dissolved in 200 µl of 20 mM Tris/acetate (pH 7.4), 0.1% ß-amidotaurocholate, 1.5 U PI-specific PLC (Bacillus cereus) and incubated for 8 h at 25 C and following addition of further 5 U of PI-specific PLC for 16 h at 40 C under toluene atmosphere (head-over rotation). After addition of 0.3 ml water, the digest was extracted three times with 0.5 ml TX-100 (20%) to remove toluene. After phase separation, residual toluene was removed from the aqueous phase under a stream of N₂. The aqueous phase was subjected to TX-114 partitioning as described above. After phase separation the aqueous phase was passed through a 2.5-ml column of AG50X12(H⁺), and then eluted with 1 ml H₂O. The eluate was dried, dissolved in 100 mM ammonium formate (pH 2.8), and loaded onto a 5-ml Dowex 50W-X8 (100 mesh) cation exchange resin column, equilibrated in 100 mM ammonium formate. The [³H]inositol-containing PIG-P was eluted with the same solvent, lyophilized, dissolved in water and passed through a C18 Sep-Pak column.

BioGel-P4 gel filtration.
The sample was chromatographed on BioGel-P4 (400 mesh) in H₂O containing 2 mM DTT. The column (1.5 cm x 1 m) was held at 55 C with a water jacket and eluted with H₂O containing 2 mM DTT (12 ml/h) in 1-ml fractions. Fractions 70–75 were pooled, dried, and dissolved in 25 mM Tris/HCl (pH 8.0). The column was calibrated in glucose units (Glu) using a glucose oligomer standard mix (prepared as described for Dionex chromatography).

SAX anion exchange chromatography.
The sample was loaded onto a SAX HPLC anion exchange column (0.5 x 5 cm) and eluted with 10 ml of 40 mM triethylamine-formate (pH 4.8), followed by a linear gradient of 60 ml of 40–400 mM triethylamine-formate in 0.5-ml fractions. Fractions 93–97 were pooled, dried, and dissolved in 50% methanol.

HPTLC analysis.
The sample was applied on Si-60 HPTLC plates that were developed in chloroform, methanol, 30% ammonium hydroxide, 1 M ammonium acetate, H₂O (180:140:9:8:23, by volume) and monitored for radioactivity using a Berthold radioactivity scanner. The major portion of the radiolabeled material remained at the origin. It was eluted from the scraped silica by sonication in 50% methanol, dried, dissolved in water, and applied onto another Si-60 HPTLC plate that was developed with 1-butanol, ethanol, ammonium hydroxide, H₂O (2:2:1:2, by volume). The plates were scanned for radioactivity. Fractions 21–26 (corresponding a distance of 21–26 mm from the origin) were scraped from the plate, eluted with methanol, dried, dissolved in H₂O containing 2 mM DTT and used for a second BioGel-P4 chromatography (see above). Fractions 70–75 were used for analysis by Dionex chromatography (after chemical and enzymic modification) and of insulin-mimetic activity.

Determination of the concentration
PIG-P were hydrolyzed (6 M HCl, 16 h, 110 C, in heat-sealed capillary tube). The amount of inorganic phosphate (2 mol/molecule) was determined. Tyrosine content (1 mol/molecule) was measured by amino acid analysis after derivatization with phenylisothiocyanate. Dried PIG-P was suspended in H₂O containing 2 mM DTT at a final concentration of 100 µM.

Chemical and enzymic modification of PIG-P
Nitrous acid deamination and sodium borohydride reduction.
The dried PIG-P (200,000 dpm) was dissolved in 50 µl of 0.1 M sodium acetate (pH 4.0), supplemented with 50 µl 0.5 M NaNO₂, and incubated for 3 h at 25 C. After addition of 20 µl 0.8 M boric acid, the mixture was adjusted to pH 10.5 by supplementation of 25–40 µl 2 M NaOH. The ("hot") reduction was initiated by addition of 40 µl NaB³H₄ (36 mM in 0.1 M NaOH) and after 2 h completed (cold reduction) by addition of 100 µl NaBH₄ and further incubation for 3 h. The samples were supplemented dropwise with 85 µl of acetic acid (50%) and then desalted by passage through 0.4 ml AG50X12(H⁺) (elution with water), dried, further purified to remove boric acid (rotary evaporation twice with 0.25 ml of acetic acid:methanol [5:95, by volume], twice with 0.25 ml methanol, twice with 50 µl toluene) and finally dissolved in water.

Dephosphorylation with hydrogen fluoride (HF).
The dried sample (deaminated and reduced; 200,000 dpm) was supplemented with 50 µl aqueous HF (48%), incubated for 48 h on ice and then transferred to 250–300 µl of frozen saturated LiOH, whereby the final pH has to be adjusted to 3–5. After vortexing, centrifugation and two washing cycles of the pellet fraction with 50 µl H₂O each, the combined supernatants were desalted (passage through 0.2 ml AG50X12(H⁺) over AG3X4(OH^-) over 0.1 ml QAE-Sephadex A25(OH^-), elution with 2.5 ml water), filtered (0.2 µm membrane) and dried (rotary evaporation).

Partial acetolysis.
The dried sample (100,000 dpm) was treated with 100 µl pyridine, acetic anhydride (1:1) for 30 min at 100 C, and again dried. After addition of 60 µl acetic anhydride, acetic acid, sulfuric acid (10:10:1) and incubation for 12 h at 37 C, the reaction was terminated by supplementation of 25 µl pyridine and 1 ml H₂O.

Decyclization.
Dried PIG-P was suspended in 100 mM HCl, incubated at 100 C for 15 min and subsequently immediately placed on ice. After neutralization with NaOH, the samples were dried and used for assay of insulin-mimetic activity. Decyclization was controlled by a shift in the elution profile during SAX anion exchange chromatography.

Digestion with -mannosidases from Aspergillus phoenicis and Jack bean.
A quantity of 0.05 mU or 1 U of enzyme, respectively, was incubated with substrate (50,000 dpm) in 25 µl 0.1 M sodium acetate (pH 5.0) for 20 h at 37 C. After heating to 95 C for 10 min the cleavage products were desalted, filtered and dried.

Pronase digestion.
Dried PIG-P was suspended in 100 mM NH₄HCO₃, 1 mM CaCl₂ and incubated at 37 C two times for 20 h with 1.4 mg pronase/ml each. After addition of 90% formic acid (25 µl/ml) and centrifugation (4000 x g, 5 min) the resulting supernatant was filtered (0.2 µm membrane), passed over a Dowex 50W-X8 column equilibrated with 100 mM ammonium formate (pH 2.8) and eluted with the same buffer. After neutralization, the samples were dried and used for assay of insulin-mimetic activity.

Dionex anion exchange chromatography
The sample was applied on a Dionex CarboPac PA-1 anion exchange HPLC equilibrated with 0.15 M NaOH/0.15 M NaOH, 0.25 M sodium acetate (95:5) at pH 13. The column was eluted with 0.15 M NaOH and 0.15 M NaOH, 0.25 M sodium acetate from 95:5 (0 min) to 80:20 (50 min) at 0.6 ml/min. The column was calibrated in Dionex units by inclusion of 5 µl of a glucose oligomer standard mix (20 mg/ml) prepared by partial acid hydrolysis (0.1 M HCl, 100 C, 5 h) to each run. The internal standards were detected using a pulsed amperiometric detector. The ³H-labeled fragments were followed by the Raytest Ramona on-line radioactivity monitor.

Mono Q anion exchange chromatography
The filtered (0.45 µm membrane) sample in 100 µl ionized water was injected into an HR5/5 Mono-Q column. The column was eluted at a flow rate of 1 ml/min with deionized water from 0–1 min, with a linear gradient to 50% of 350 mM ammonium acetate (pH 5.2) from 1–8 min and with a linear gradient to 100% of the same buffer from 8 to 25 min. A₂₂₀ was followed on-line.

Isolation of ventricular cardiomyocytes
Ca²⁺-tolerant myocytes were isolated from male Wistar rats (260–320 g) by perfusion of the heart with collagenase as previously described (37, 38). The final cell suspension was washed three times with HEPES buffer (130 mM NaCl, 4.8 mM KCl, 1.2 mM KH₂PO₄, 25 mM HEPES, 5 mM glucose, 20 g/ml BSA, pH 7.4, equilibrated with oxygen) and incubated in silicone-treated Erlenmeyer flasks in a rotating waterbath shaker at 37 C. After 20 min, CaCl₂ and MgSO₄ (final concentration 1 mM) were added and incubation was continued until further use. Cell viability was checked by determination of the percentage of rod-shaped cells and averaged 90–95% under all incubation conditions.

Isolation of white adipocytes (according to Ref. 39 with the following modifications)
Male Wistar rats (140–160 g) were killed by cervical dislocation. Epididymal fat pads were placed in KRH (25 mM HEPES free acid, 25 mM HEPES sodium salt, 80 mM NaCl, 1 mM MgSO₄, 2 mM CaCl₂, 6 mM KCl, 1 mM sodium pyruvate, 0.5% BSA) and then washed with KRB (12 mM KH₂PO₄, 1.2 mM MgSO₄, 4.8 mM KCl, 25 mM NaHCO₃, 120 mM NaCl, 1.4 mM CaCl₂, 5 mM glucose and 2.5% (wt/vol) BSA, bubbled with 5% CO₂/95% O₂). Each pad was then cut into two to three pieces. Two pieces each were incubated with 1.5 ml of digestion buffer (10 mg collagenase in 9 ml KRB) for 15–30 min at 37 C in a shaking water bath. Released adipocytes were washed two times with KRH by flotation and then diluted to a final volume equal to 20 ml of KRH/g of fat (final titer about 2.5 x 10⁵ cells/ml).

Desensitization of adipocytes
Isolated rat adipocytes (10⁶ cells) were incubated in 20 ml of HEPES-buffered salt solution (40) containing either 5 mM glucose (normal cells) or 20 mM glucose, 16 mM glutamine, 10 nM insulin (desensitized cells) for 20 h at 37 C under gentle shaking. Thereafter, the cells were washed three times with HEPES-buffered salt solution lacking glucose, glutamine, and insulin, and then incubated in 2.5 ml of KRH (final titer about 3–4 x 10⁵ cells/ml) for 15 min at 37 C to allow deactivation of the adipocyte glucose transport system. For trypsin treatment, adipocytes in KRB (1.5 x 10⁵ cells/ml) were incubated with 40 µg/ml trypsin for 20 min at 37 C under gentle shaking. After addition of 0.4 ml bovine trypsin inhibitor (10 mg/ml) and incubation for 5 min at 20 C, the cells were washed two times and finally suspended in KRH (final titer see above).

Assays for insulin-mimetic activity
The rates for the basal and insulin-stimulated activities determined by the individual assays as outlined below are well within the ranges of published data and are given in the corresponding figure legends each.

Lipogenesis.
Lipogenesis was measured as incorporation of [³H]glucose into total toluene-extractable lipids according to Ref. 41 with the following modifications. Nine hundred microliters of adipocytes (3.5 x 10⁵ cells) in KRH containing 140 µM glucose were incubated with insulin or PIG-P in 10-ml scintillation vials for 20 min at 37 C. Lipogenesis was started by addition of 100 µl D-[3-³H]glucose (4 µCi/ml KRH). After incubation for 60 min at 37 C under an atmosphere of 5% CO₂/95% O₂ and gentle shaking, 10 ml toluene-based scintillation cocktail was added. The samples were vigorously mixed and counted for radioactivity. dpm values for radiolabeled lipids (as measured in the toluene phase) were corrected for a background value determined for an incubation mixture containing the same amount of [³H]glucose but lacking adipocytes.

Glycerol-3-phosphate acyltransferase.
GPAT was assayed as incorporation of [³H]glycerol-3-phosphate into butanol-extractable lipids according to Refs. 42, 43 with the following modifications. Two milliliters of adipocytes (6 x 10⁵ cells) were incubated with insulin or PIG-P for 20 min at 37 C in 10-ml scintillation vials under an atmosphere of 5% CO₂ and shaking (160 cycles/min). Washed cells were homogenized in 500 µl homogenization medium containing 25 mM Tris/HCl (pH 7.4), 250 mM sucrose, 2 mM EDTA, 2 mM DTT, 180 mM NaF, 1 mM NaVO₃, 1 mM benzamidine, 10 µg/ml aprotinin, 2 µg/ml leupeptin, 0.1 mM PMSF by 10 strokes in a Teflon-in-glass homogenisator. After centrifugation (1,000 x g, 5 min, 4 C) the infranatant below the fat cake was removed and centrifuged (14,000 x g, 20 min, 4 C). The supernatant was recentrifuged (150,000 x g, 60 min, 4 C) to obtain a crude microsomal fraction. The pellet was suspended in 25 mM Tris/HCl (pH 7.4), 250 mM sucrose, 0.5 mM EDTA, 1 mM DTT, 0.1 mM PMSF at 2 mg protein/ml and stored in liquid N₂ until determination of GPAT activity (43). The assay mixture contained 0.2 mM [³H]glycerol-3-phosphate (0.5 µCi), 150 µM palmitoyl-CoA and 100 µg microsomal protein in a total volume of 0.5 ml and was incubated for 3 min at 37 C.

Glucose transport in adipocytes.
Glucose transport was assayed as uptake of 2-deoxy-[³H]glucose according to Ref. 44 with the following modifications. One milliliter of adipocytes in KRH (3 x 10⁵ cells) were incubated with insulin or PIG-P for 20 min at 37 C. Initial rates of glucose transport were measured by adding 50 µl KRH containing 0.5 µCi of 2-deoxy-D-[³H]glucose (final substrate concentration, 0.1 mM) and after 5 min separating 100 µl-aliquots of the cells by centrifugation through 50 µl dinonylphtalate. Preincubation of the cells with 20 µM cytochalasin B was used to correct the values for simple diffusion and nonspecific trapping of the hexose.

Glucose transport in cardiomyocytes.
Transport experiments were performed at 37 C in HEPES buffer containing 1 mM MgCl₂, 1 mM CaCl₂. The reaction was started by pipetting a 50 µl aliquot of the cell suspension to 50 µl of HEPES buffer containing 3-O-[¹⁴C]methyl-D-glucose (final concentration 100 µM). Carrier-mediated glucose transport was then determined using a 10-sec assay period and L-[¹⁴C]glucose to correct for simple diffusion as described earlier (37, 45, 46).

Glucose transporter isoform 4 (GLUT4) translocation.
Adipocytes (7 x 10⁶) were incubated in 100 ml of KRH containing 5.5 mM glucose with insulin or PIG-P for 20 min at 37 C under gentle shaking. Washed cells in KRH containing 2 mM KCN were suspended in 55 ml of 20 mM Tris/HCl (pH 7.4), 250 mM sucrose, 1 mM EDTA, 0.2 mM PMSF, 100 µM benzamidine, 20 µg/ml leupeptin, pepstatin, aprotinin, and antipain each, and homogenized 10-times in a Teflon-in-glass homogenizer. Plasma membranes and low density microsomes were prepared as described (47). GLUT4 was quantitatively immunoprecipitated with rabbit anti-GLUT4 antibodies raised against the carboxyl-terminal 16 amino acids of rat GLUT4 and protein A-Sepharose. The immunoprecipitated GLUT4 was separated by SDS-PAGE and its amount evaluated by immunoblotting with the same antibodies and [¹²⁵I]protein A followed by autoradiography of the dried gel (Kodak X-Omat AR). Quantitation was performed by phosphorimaging of the dried gel (Molecular Dynamics, Storm 840).

Glycogenesis.
Glycogenesis was assayed as incorporation of U-[¹⁴C]glucose into ethanol-precipitable glycogen. Intact hemidiaphragms with rib cage attached were dissected from male Wistar rats and incubated in DMEM (15 ml/hemidiaphragm) containing 10% FCS, 5.5 mM glucose, 1% BSA, 50 U penicillin/ml, 50 µg streptomycin sulfate/ml under constant bubbling of O₂:CO₂ (95:5) for 60 min at 37 C. Subsequently, the diaphragms were incubated with insulin or PIG-P in DMEM (15 ml/hemidiaphragm) for 30 min at 37 C in the presence of insulin or PIG-P. Washed hemidiaphragms were then incubated with 5 µCi [U-¹⁴C]glucose in 15 ml KRH containing 2 mM glucose for 20 min at 37 C in the presence of insulin or PIG-P. After blotting and cutting off the rib cage, the hemidiaphragms (80–100 mg wet weight) were washed, frozen in liquid N₂, and homogenized in 2 ml of 25 mM Tris-HCl (pH 7.4), 150 mM NaF, 5 mM EDTA, 0.1 mM PMSF in porcelain mortar on ice. One hundred microliters of the supernatant (200–500 µg protein) obtained by centrifugation (10,000 x g, 15 min, 4 C) was used for glycogen determination by precipitation of glycogen with ethanol (43).

Glycogen synthase (GS).
GS activity was assayed by measuring the incorporation of D-[¹⁴C]glucose from UDP-[¹⁴C]glucose into glycogen. Ten milliliters of adipocytes in KRB (about 3 x 10⁵ cells) were incubated with insulin or PIG-P in a shaking water bath under an atmosphere of 5% CO₂ for 30 min at 37 C. Washed cells were frozen in liquid N₂ and then homogenized in 0.5 ml of 10 mM N-[Tris(hydroxymethyl)methyl]glycine buffer (pH 7.5) containing 10 mM EDTA, 150 mM KF, 5 mM DTT by ten strokes in a Teflon-in-glass homogenizer on ice. The homogenate was centrifuged (5500 x g, 2 min, 10 C). The infranatant below the lipid layer was cleared from residual lipids by two additional centrifugations and served as source for GS. The reaction was started by adding 30 µl of the homogenate to 60 µl of a reaction mixture (prewarmed at 30 C) containing 33 mM Tris/HCl (pH 7.8), 0.2 mM UDP-[U-¹⁴C]glucose (4 µCi), 6.7 mg glycogen, 150 mM KF and 0.1 mM/10 mM glucose-6-P. After incubation for 20 min at 30 C, the reaction was terminated by addition of 2 ml of 66% ethanol, 10 mM LiBr (-20 C), rapid mixing and filtration over prewetted Whatman GF/C glass-fiber discs. The filters were washed five times with 5 ml of 66% ethanol each at 25 C, dried, and measured for radioactivity. Blank values determined by adding the homogenate to tubes containing the complete reaction mixture plus ice-cold ethanol were subtracted from the total values each. The fractional velocity was calculated as described (43). Measurements of GS activity in homogenates from adipocytes that had been incubated in the presence of 0.1 mM glucose instead of 5 mM glucose (the concentration routinely used) did not reveal significant differences with respect to both the activity ratio and the effect of PIG-P or insulin. Presumably, the dilution of the limited amount of glucose-6-phosphate, which accumulates during incubation with 5 mM extracellular glucose, during the subsequent preparation of the homogenate and the GS assay was high enough to prevent allosteric activation of GS. According to our experience, incubation of the isolated rat adipocytes in the presence of 5 mM glucose has a positive impact on their viability, in general, and insulin sensitivity (glucose transport, glycogen synthesis), in particular.

Lipolysis.
Lipolysis was assayed as release of glycerol. One milliliter of adipocyte suspension in KRB (3 x 10⁵ cells) was incubated with insulin or PIG-P for 10 min at 37 C under an atmosphere of 5% CO₂. After addition of 1 µM isoproterenol and 1 unit of adenosine deaminase, the incubation was continued for 60 min. Following addition of 200 µl of extraction medium containing 120 mM Hepes/KOH (pH 7.4), 6 mM isobutylmethylxanthine, 60 mM EDTA, 180 mM NaF, the cells were homogenized by 10 strokes of a Teflon-in-glass homogenizer. The extract was centrifuged (10,000 x g, 15 min, 4 C). The infranatant below the fat cake was stored in liquid N₂ and used for glycerol determination (43).

Protein kinase A.
PKA was assayed by incorporation of ³²P from [³²P]ATP into histone H1. Twenty microliters of the cleared adipocyte extract (see "lipolysis") was added to 80 µl of a reaction mixture containing 20 mM 3-(N-morpholino)propanesulfonic acid (pH 7.2), 4 mM DTT, 12.5 mM MgCl₂, 0.2 mM PMSF, 1 mM isobutylmethylxanthine, 50 µM [³²P]ATP (2.5 µCi) with or without 1 µM cAMP. After incubation for 10 min at 30 C, the reaction mixture was supplemented with 1 ml of ice-cold 20% trichloroacetic acid, 10 mM ATP, 5 mM NaPP_i, 100 mM NaF. The precipitates were centrifuged (12,000 x g, 5 min, 4 C) and then dissolved in 100 µl of 1 N NaOH. After addition of 1 ml of 20% trichloroacetic acid, 5 mM NaPP_i, 10 mM ATP, the precipitates were decanted onto Whatman GF/C glass fiber filters under vacuum prewetted with washing solution (20% trichloroacetic acid, 5 mM NaPP_i). After washing (five times with 3 ml each of washing solution), the filters were dried and counted for radioactivity. Blank values determined by inclusion of 0.6 mg/ml PKA inhibitor from bovine heart into the reaction mixture were subtracted from the total values each. The PKA activity ratio indicating the portion of PKA active at the time-point of homogenization compared with total enzyme content was expressed as the ratio between phosphorylation of histone H1 with and without cAMP.

Miscellaneous procedures
Published methods were used for protein determination (43), generation of the trifluoroacetic acid (TFA)-fragment from the PIG-P (48), immunoprecipitation and immunoblotting of GLUT4 (47) and immunoprecipitation/immunodepletion of the PIG-P with anti-CRD antibodies (28, 30).

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Preparation, purification, and characterization of the PIG-P
A GPI anchor fragment was prepared from Gce1p, which had been partially purified by gel filtration, cAMP-affinity, and phenyl Sepharose chromatographies. In this preparation, Gce1p was the only GPI-anchored protein present because Gce1p from yeast cells, which had been metabolically labeled with myo-[³H]inositol and added as a tracer to the large-scale culture of unlabeled cells, represented the only radiolabeled band observed after SDS-PAGE and fluorography (data not shown). The unlabeled and ³H-labeled Gce1p was completely digested with V8 protease from Staphyloccocus aureus to cleave the protein moiety near its carboxyl terminus linked to the anchor. The resulting GPI anchor peptide eluted during gel filtration in the presence of detergent with an apparent molecular mass of about 2 kDa (fractions 83–100; Fig. 1A). It was then subjected to hydrophobic interaction chromatography on octyl-Sepharose (Fig. 1B). The radiolabeled GPI anchor peptide eluted as a sharp peak between 34–38% 1-propanol (fractions 71–79). It was subsequently treated with exogenous PI-specific PLC from Bacillus cereus to remove diacylglycerol from the anchor. After recovery from the aqueous phase of TX-114 partitioning, the [³H]inositol-labeled, polar PIG-P eluted during BioGel-P4 chromatography at a volume corresponding to 8–8.5 glucose units as the major peak (fractions 70–75). It was further purified by anion-exchange chromatography on a SAX HPLC column (Fig. 1D). PIG-P (fractions 93–97) constituted the only peak material with respect to both radiolabel and A₂₂₀ and was finally subjected to two successive HPTLC runs. The material migrating as the single radioactive and UV-absorptive spot during the second HPTLC plate (Fig. 1E) was recovered (fractions 21–26). It was analyzed by BioGel-P4 chromatography (Fig. 1F) and shown to be radiochemically and spectroscopically pure. The elution behavior (fractions 70–75) from the column, calibrated with a glucose oligomer standard mix, with a retention time of 8.5 glucose units corresponding to about 1.5 to 1.6 kDa is compatible with the apparent molecular mass of a polar fragment derived from Gce1p by digestion with pronase and bacterial PI-specific PLC (29).

View larger version (39K): [in this window] [in a new window]   Figure 1. Preparation and purification of the PIG-P. PIG-P was prepared from partially purified Gce1p. Purification steps were monitored by following myo-[3H]inositol-labeled tracer Gce1p and absorbance at 220 nm. A, Gel filtration on Sephadex LH-60 of V8-protease-digested Gce1p. B, Hydrophobic interaction chromatography on octyl Sepharose CL-4B of combined fractions 83–100 from step A after TX-114 partitioning. C, Combined fractions 71–79 from step B were rechromatographed on octyl Sepharose CL-4B, digested with bacterial PI-specific PLC and subjected to TX-114 partitioning. The hydrophilic material was passed sequentially over anion and cation exchange columns and then analyzed by BioGel-P4 chromatography. D, Anion exchange chromatography on SAX HPLC of combined fractions 70–75 from step C. E, HPTLC purification of combined fractions 93–97 from step D by two successive runs on Si-60 HPTLC plates with different solvent systems (only the second run is shown). F, Gel filtration on BioGel-P4 of fractions 21–26 from step E. Open circles, myo-[3H]inositol radioactivity; filled circles, A220 recording.

View larger version (22K): [in this window] [in a new window]   Figure 2. Evaluation of the efficiency of the purification procedure for PIG-P by Mono Q anion exchange chromatography. Equivalent volumes of combined peak fractions obtained by the purifications steps C0, D, E and F (see Table 1) were subjected to chromatography on a Mono Q column. The elution was followed by measurement of A220. The retention times of the major peaks are given. The arrows indicate the peak containing PIG-P (see Results).

Structural analysis of the material from step F revealed the presence of components, only, typically for the polar head group of yeast GPI anchors and of the carboxyl terminal tripeptide of Gce1p, Tyr-Cys-Asn (determined by sequence determination using manual Edman degradation) that are covalently linked (data not shown, manuscript in preparation). Determination of free amino groups in the intact PIG-P demonstrated the presence of two such groups per mol of myo-inositol, one belonging to the nonacetylated glucosamine, the other one to the tyrosine residue. Other amino acids (determined after total acid hydrolysis by the ninhydrin method) were not found in the PIG-P preparation. This provides strong evidence that the PIG-P is derived from a single GPI-anchored protein, Gce1p, and demonstrates its chemical homogeneity. Furthermore, the GPI anchor of Gce1p appears to be very closely related to the trypanosomal or vertebrate GPI anchors because, after cleavage by PI-specific PLC, it is recognized by antibodies against the cross-reacting determinant of soluble VSG from Trypanosoma brucei. This suggests a structural arrangement of the glycan core constituents of Gce1p in a fashion similar or identical to that of VSG.

The PIG-P obtained after the final purification step F were analyzed by Dionex anion exchange chromatography. For this, they were deaminated by nitrous acid treatment and then reduced with radiolabeled sodium borohydride for replacement of (radiolabeled) inositol-1,2-cyclic phosphate with radiolabeled 2,5-anhydromanitol (AHM). The material was then subjected to aqueous HF dephosphorylation for removal of the terminal phosphoethanolamine moiety. Subsequently, three different chemical or enzymic treatments were performed to specifically cleave off mannose residues before analysis of the size of the degradation fragments radiolabeled at the terminal AHM by Dionex chromatography (Fig. 3). The radiolabeled HF-fragment was eluted at 3.2 Dionex units corresponding to the neutral glycan core with a single carbohydrate side chain branching off, Man1–2Man1–2Man1–6Man1–4AHM (Fig. 3A). It thus constitutes the glycan portion of 80% of the whole spectrum of GPI anchors made by yeast cells (34). The lack of terminal phosphoethanolamine allowed access of mannose-specific exoglycosidases to this structure. -mannosidase (Aspergillus phoenicis) specific for -D-Man(1, 2)D-Man glycosidic bonds released a radiolabeled fragment from the deaminated, reduced and dephosphorylated PIG-P eluting at 2.2 Dionex units compatible with the structure, Man1–6Man1–4AHM (Fig. 3B). -mannosidase (Jack bean), specific for any terminal, nonsubstituted, -D-Man, generated a radiolabeled fragment from the neutral glycan core eluting at 1.0 Dionex units corresponding to a structure completely devoid of the mannose residues, i.e. AHM (Fig. 3C). Finally, partial acetolysis, which is relatively specific for Man()1–6Man glycosidic bonds (but also cleaves Man()1–4AHM to a minor degree), produced a radiolabeled structure from the neutral glycan core eluting at 1.1 Dionex units consistent with Man1–4AHM as well as a small amount of free radiolabeled AHM (Fig. 3D). Taken together, the size analysis of the exoglycosidase digestion and partial acetolysis products of the deaminated, reduced and dephosphorylated PIG-P by Dionex anion exchange chromatography is in full agreement with a conserved glycan core structure of the headgroup of GPI anchors common for protozoa and mammals, Man1–2Man1–6Man1–4GlcN1–6inositol-1-phosphate, which is modified with a single Man1–2 branch at the distal mannose residue typical for the 80%-component of total yeast GPI anchors (34). In addition, this structure was confirmed by 1) NMR spectroscopy of the complete as well as deaminated/dephosphorylated PIG-P (Knauf, M., unpublished results); 2) GC/MS analysis of the chemically or enzymatically treated PIG-P (Petry, S., unpublished results); and 3) GC analysis of the complete GPI anchor peptide generated by pronase digestion of Gce1p (29). In the latter study, the purified GPI anchor peptide was dephosphorylated with HF, the oligosaccharides hydrolyzed with trifluoroacetic acid, the sugars reduced with NaBH₄ and then acetylated, and the final products separated and identified by GC and amino acid analysis. Using these methods, the stoichiometry of the individual components was determined as follows: ethanolamine: Man: GlcN: inositol = 1.2: 3.9: 1: 1.6.

View larger version (20K): [in this window] [in a new window]   Figure 3. Analysis of PIG-P by Dionex chromatography. [3H]inositol-labeled PIG-P was subjected to nitrous acid deamination and subsequent reduction with [3H]NaBD4. The resulting [3H]AHM-labeled cleavage product was treated with HF and then analyzed by Dionex anion exchange chromatography either directly (A; 50,000 dpm applied) or after digestion with -mannosidase from Aspergillus phoenicis (B; 50,000 dpm applied) or Jack bean (C; 30,000 dpm applied) or after partial acetolysis (D; 25,000 dpm applied). Number of Dionex units as indicated above the arrows were derived from the elution behavior of a glucose oligomer standard mix run in parallel with the radiolabeled samples.

View larger version (20K): [in this window] [in a new window]   Figure 4. Effect of PIG-P on lipogenesis in normal, desensitized and trypsin-treated adipocytes. Normal (circles), desensitized (squares), and trypsin-treated (triangles) adipocytes were incubated with increasing concentrations of PIG-P (upper panel) or insulin (lower panel) for 20 min before start of lipogenesis assay. The stimulation factor was calculated as ratio between the stimulated and basal state. Each point represents the mean of six different adipocyte preparations with incubations and lipogenesis assays performed in quadruplicate. A stimulation factor of 20 corresponds to 10.5 nmol glucose/3 x 105 cells · 1 h incorporated into acylglycerols.

View larger version (23K): [in this window] [in a new window]   Figure 5. Effect of PIG-P on GPAT activity in adipocytes. Isolated rat adipocytes were incubated with increasing concentrations of PIG-P (upper panel) or insulin (lower panel) for 20 min. Microsomes were prepared and assayed for GPAT activity. Each point represents the mean ± SD of four different adipocyte preparations with incubations and GPAT assays performed in triplicate. 8000 dpm corresponds to 0.7 nmol glycerol-3-phosphate/100 µg protein · 3 min incorporated into acylglycerols.

View larger version (23K): [in this window] [in a new window]   Figure 6. Effect of PIG-P on glucose transport in adipocytes. Normal (closed circles) and desensitized (open circles) rat adipocytes were incubated with increasing concentrations of PIG-P (upper panel) or insulin (lower panel) for 20 min before start of glucose transport assay. Each point represents the mean ± SD of five different adipocyte preparations with incubations and glucose transport assays performed in quadruplicate. 5000 dpm corresponds to 0.44 nmol deoxyglucose/3 x 104 cells · 5 min transported.

View larger version (24K): [in this window] [in a new window]   Figure 7. Effect of PIG-P on GLUT4 translocation in adipocytes. Isolated rat adipocytes were incubated with increasing concentrations of PIG-P or insulin for 20 min. Plasma membranes (PM) and low density microsomes (LDM) were prepared and assayed for the amount of GLUT4 by immunoprecipitation and subsequent immunoblotting with anti-GLUT4 antibodies as described in Materials and Methods. A, Autoradiogram of a typical experiment. For quantitative evaluation of the stimulation of GLUT4 translocation (B, upper section: PIG-P, lower section: insulin), the amount of GLUT4 in the plasma membrane of basal cells was set at 1 arb. unit. Each point represents the mean ± SD of three different adipocyte preparations with incubations and GLUT4 determinations performed in triplicate.

View larger version (26K): [in this window] [in a new window]   Figure 8. Inactivation of PIG-P. Isolated rat adipocytes were incubated with 10 µM PIG-P, which had been treated with nitrous acid, aqueous HF, mild acid, pronase, and TFA or had been depleted with anti-CRD antibodies or had been left untreated for 15 min before start of lipogenesis (open bars) or glucose transport assay (filled bars). Identical amounts of PIG-P were used for the individual pretreatments. Each bar represents the mean ± SD of three different preparations with incubations and assays performed in quadruplicate.

View larger version (40K): [in this window] [in a new window]   Figure 9. Effect of PIG-P on 3-O-methylglucose transport in ventricular cardiomyocytes. Freshly isolated cardiomyocytes were incubated for 15 min at 37 C in the absence or presence of insulin (3.5 x 10-7 M), PIG-P (2 µM) or a combination of both agents. The transport of 3-O-methylglucose was then determined over a 10-sec assay period, as detailed in Materials and Methods. Data are mean values ± SEM of three to four separate experiments. *, Significantly different from PIG-P alone (P < 0.05); **, not significantly different from insulin alone (P > 0.05).