Presence of accessory abductor digiti minimi muscle in two cadavers.

During routine cadaveric dissection, accessory hypothenar muscles were incidentally discovered in two cadavers, both males, aged 86 and 92. Both muscles originated from the palmaris longus tendon in the distal portion of the forearm and were identified as accessory abductor digiti minimi (AADM) muscles, based on their association with abductor digiti minimi. While AADM is a common variant in the antebrachium, it is less typical for them to originate from the palmaris longus tendon. The presence of such an AADM could complicate surgical procedures requiring resection of the palmaris longus tendon. Moreover, the surrounding neurovasculature - namely the ulnar nerve as it passes through the ulnar canal between the pisiform and hook of the hamate - could be compressed by contractions of an AADM with such a proximal origin. This can manifest as ulnar neuropathies resulting in pain, weakness, or protracted flexion of the fourth and fifth digits (ulnar claw). Our description of these muscles adds to previous accounts of variation of the palmaris longus and abductor digiti minimi muscles while considering potential clinical implications.

Biochemical and immunological characterization of p190-calmodulin complex from vertebrate brain: a novel calmodulin-binding myosin.

We have recently identified a novel 190-kD calmodulin-binding protein (p190) associated with the actin-based cytoskeleton from mammalian brain (Larson, R. E., D. E. Pitta, and J. A. Ferro. 1988. Braz. J. Med. Biol. Res. 21:213-217; Larson, R. E., F. S. Espindola, and E. M. Espreafico. 1990. J. Neurochem. 54:1288-1294). These studies indicated that p190 is a phosphoprotein substrate for calmodulin-dependent kinase II and has calcium- and calmodulin-stimulated MgATPase activity. We now have biochemical and immunological evidence that this protein is a novel calmodulin-binding myosin whose properties include (a) Ca2+ dependent action activation of its Mg-ATPase activity, which seems to be mediated by Ca2+ binding directly to calmodulin(s) associated with p190 (maximal activation by actin requires the presence of Ca2+ and is further augmented by addition of exogenous calmodulin); (b) ATP-sensitive cross-linking of skeletal muscle F-actin, as demonstrated by the low-speed actin sedimentation assay; and (c) cross-reactivity with mAbs specific for epitopes in the head of brush border myosin I. We also show that p190 has properties distinct from conventional brain myosin II and brush border myosin I, including (a) separation of p190 from brain myosin II by gel filtration on a Sephacryl S-500 column; (b) lack by p190 of K(+)-stimulated EDTA ATPase activity characteristic of most myosins; (c) lack of immunological cross-reactivity of polyclonal antibodies which recognize p190 and brain myosin II, respectively; (d) lack of immunological recognition of p190 by mAbs against an epitope in the tail region of brush border myosin I; and (e) distinctive proteolytic susceptibility to calpain. A survey of rat tissues by immunoblotting indicated that p190 is expressed predominantly in the adult forebrain and cerebellum, and could be detected in embryos 11 d post coitus. Immunocytochemical studies showed p190 to be present in the perikarya and dendritic extensions of Purkinje cells of the cerebellum.

Primary structure and cellular localization of chicken brain myosin-V (p190), an unconventional myosin with calmodulin light chains.

Recent biochemical studies of p190, a calmodulin (CM)-binding protein purified from vertebrate brain, have demonstrated that this protein, purified as a complex with bound CM, shares a number of properties with myosins (Espindola, F. S., E. M. Espreafico, M. V. Coelho, A. R. Martins, F. R. C. Costa, M. S. Mooseker, and R. E. Larson. 1992. J. Cell Biol. 118:359-368). To determine whether or not p190 was a member of the myosin family of proteins, a set of overlapping cDNAs encoding the full-length protein sequence of chicken brain p190 was isolated and sequenced. Verification that the deduced primary structure was that of p190 was demonstrated through microsequence analysis of a cyanogen bromide peptide generated from chick brain p190. The deduced primary structure of chicken brain p190 revealed that this 1,830-amino acid (aa) 212,509-D) protein is a member of a novel structural class of unconventional myosins that includes the gene products encoded by the dilute locus of mouse and the MYO2 gene of Saccharomyces cerevisiae. We have named the p190-CM complex "myosin-V" based on the results of a detailed sequence comparison of the head domains of 29 myosin heavy chains (hc), which has revealed that this myosin, based on head structure, is the fifth of six distinct structural classes of myosin to be described thus far. Like the presumed products of the mouse dilute and yeast MYO2 genes, the head domain of chicken myosin-V hc (aa 1-764) is linked to a "neck" domain (aa 765-909) consisting of six tandem repeats of an approximately 23-aa "IQ-motif." All known myosins contain at least one such motif at their head-tail junctions; these IQ-motifs may function as calmodulin or light chain binding sites. The tail domain of chicken myosin-V consists of an initial 511 aa predicted to form several segments of coiled-coil alpha helix followed by a terminal 410-aa globular domain (aa, 1,421-1,830). Interestingly, a portion of the tail domain (aa, 1,094-1,830) shares 58% amino acid sequence identity with a 723-aa protein from mouse brain reported to be a glutamic acid decarboxylase. The neck region of chicken myosin-V, which contains the IQ-motifs, was demonstrated to contain the binding sites for CM by analyzing CM binding to bacterially expressed fusion proteins containing the head, neck, and tail domains. Immunolocalization of myosin-V in brain and in cultured cells revealed an unusual distribution for this myosin in both neurons and nonneuronal cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Potassium and angiotensin II increase the concentrations of phosphatidic acid, phosphatidylinositol, and polyphosphoinositides in rat adrenal capsules in vitro.

We examined the effects of K+ and angiotensin II, the major regulators of aldosterone secretion, on phospholipid metabolism during incubation of glomerulosa-rich, adrenal capsules. Addition of increasing amounts of K+ and angiotensin II to the incubation media elicited progressive increases in corticosterone production and capsular concentrations of phosphatidic acid, phosphatidyl-inositol, and polyphosphoinositides. These effects are similar to those previously reported for ACTH in the whole adrenal cortex. A common mechanism, i.e., activation of the phosphatidate-polyphosphoinositide cycle, may be operative in the action of steroidogenic agents in their target tissues.

Subcellular localization of myosin-V in the B16 melanoma cells, a wild-type cell line for the dilute gene.

The discovery that the dilute gene encodes a class V myosin led to the hypothesis that this molecular motor is involved in melanosome transport and/or dendrite outgrowth in mammalian melanocytes. The present studies were undertaken to gain insight into the subcellular distribution of myosin-V in the melanoma cell line B16-F10, which is wild-type for the dilute gene. Immunofluorescence studies showed some degree of superimposed labeling of myosin-V with melanosomes that predominated at the cell periphery. A subcellular fraction highly enriched in melanosomes was also enriched in myosin-V based on Western blot analysis. Immunoelectron microscopy showed myosin-V labeling associated with melanosomes and other organelles. The stimulation of B16 cells with the alpha-melanocyte-stimulating hormone led to a significant increase in myosin-V expression. This is the first evidence that a cAMP signaling pathway might regulate the dilute gene expression. Immunofluorescence also showed an intense labeling of myosin-V independent of melanosomes that was observed within the dendrites and at the perinuclear region. Although the results presented herein are consistent with the hypothesis that myosin-V might act as a motor for melanosome translocation, they also suggest a broader cytoplasmic function for myosin-V, acting on other types of organelles or in cytoskeletal dynamics.

The molecular motor Myosin Va interacts with the cilia-centrosomal protein RPGRIP1L.

Myosin Va (MyoVa) is an actin-based molecular motor abundantly found at the centrosome. However, the role of MyoVa at this organelle has been elusive due to the lack of evidence on interacting partners or functional data. Herein, we combined yeast two-hybrid screen, biochemical studies and cellular assays to demonstrate that MyoVa interacts with RPGRIP1L, a cilia-centrosomal protein that controls ciliary signaling and positioning. MyoVa binds to the C2 domains of RPGRIP1L via residues located near or in the Rab11a-binding site, a conserved site in the globular tail domain (GTD) from class V myosins. According to proximity ligation assays, MyoVa and RPGRIP1L can interact near the cilium base in ciliated RPE cells. Furthermore, we showed that RPE cells expressing dominant-negative constructs of MyoVa are mostly unciliated, providing the first experimental evidence about a possible link between this molecular motor and cilia-related processes.

Effects of Ca2+ ionophore A23187 and Ca2+ deficiency on pancreatic phospholipids and amylase release in vitro.

The Ca2+ ionophore A23187 elicits a transient increase in pancreatic amylase release in vitro, and this is accompanied by a transient decrease in phosphatidyl inositol concentration. Effects of ionophore A23187 and carbachol on amylase release and phosphatidylinisitol breakdown are dependent on medium Ca2+. These results suggest that major secretagogue-induced, pancreatic phospholipid changes follow, rather than precede, changes in Ca2+ in the pancreas.

Effects of dibutyryl cyclic AMP and theophylline on rat pancreatic phospholipids in vitro. Ca2+-sensitive decrease in phosphatidylinositol and cycloheximide-sensitive increase in phosphatidic acid.

We evaluated the effects of dibutyryl cyclic AMP and theophylline on rat pancreatic phospholipid metabolism in vitro. Dibutyryl cyclic AMP decreased mean phosphatidylinositol concentration by 30%, increased phosphatidic acid and phosphatidylglycerol concentrations by 90 and 25%, respectively, and increased [32P]phosphate incorporation into phosphatidic acid and phosphatidylinositol several-fold. Theophylline provoked similar changes in phosphatidic acid and phosphatidylinositol concentrations, and both stimulatory agents enhanced amylase and insulin secretion. Effects of dibutyryl cyclic AMP on amylase secretion and phospholipid levels were dependent on Ca2+. Cycloheximide blocked induced increases in phosphatidic acid, but did not diminish phosphatidylinositol breakdown or amylase secretion. Contrary to previous postulations, the present findings suggest: (a) cyclic AMP provokes large-scale phosphatidylinositol breakdown in the pancreas; (b) this phosphatidylinositol breakdown is dependent on Ca2+; and (c) phosphatidylinositol breakdown may contribute to exocytosis. In addition, it appears that a labile protein is required for synthesis of phosphatidic acid from 1,2-diacylglycerol and ATP.

Phosphatidylinositol hydrolysis and phosphatidylinositol 4',5-diphosphate hydrolysis are separable responses during secretagogue action in the rat pancreas.

Rat pancreatic fragments and acinar preparations were incubated in vitro to characterize further the changes in phosphoinositide metabolism that occur during secretagogue action. Two distinct responses were discernible. The first response, most notably involving a decrease in phosphatidylinositol content, was (a) observed at lower carbachol concentrations in dose-response studies, (b) inhibited by incubation in Ca2+-free media containing 1 mM EGTA, (c) associated with increases in inositol monophosphate production, and (d) provoked by all tissue secretagogues (carbachol, cholecystokinin, secretin, insulin, dibutyryl cAMP and the ionophore A23187), regardless of whether their mechanism of action primarily involved Ca2+ mobilization or cAMP generation. This decrease in phosphatidylinositol content was at least partly due to phospholipase C (and/or D) activation, as evidenced by the increase in inositol monophosphate. The second response, most notably involving markedly increased incorporation of 32PO4 into phosphatidic acid and phosphatidylinositol, was (a) observed at higher carbachol concentrations, (b) not influenced by incubation in Ca2+-free media containing 1 mM EGTA, and (c) associated with increases in inositol triphosphate production. This 32PO4 turnover response was probably largely the result of phospholipase C-mediated hydrolysis of phosphatidylinositol 4',5'-diphosphate, which, as shown previously, also occurs at higher carbachol concentrations and is insensitive to comparable EGTA-induced Ca2+ deficiency. This phosphatidylinositol 4',5'-diphosphate hydrolysis response was only observed in the action of agents (carbachol and cholecystokinin) which mobilize Ca2+ via activation of cell surface receptors. The present results indicate that phosphatidylinositol and phosphatidylinositol 4',5'-diphosphate hydrolysis are truly separable responses to secretagogues acting in the rat pancreas. Furthermore, phosphatidylinositol 4',5'-diphosphate, rather than phosphatidylinositol hydrolysis is more likely to be associated with receptor activation and Ca2+ mobilization.

Interaction of insulin and prostacyclin production in the rat.

The effects of hyperinsulinemia on production of prostacyclin (PGI2), a potent inhibitor of platelet aggregation, was studied in vitro in the rat. Aortic rings following 1-h preincubation at 37 degrees C and 1 h at 4 degrees C were incubated with and without purified porcine insulin in Krebs' glucose at 37 degrees C for 5 min and immediately percent inhibition of platelet aggregation was determined. PGI2 production in ng/mg aorta was estimated from a curve using PGI2 standard. The mean PGI2 production was significantly decreased in those rings incubated with insulin in concentration of 2500, 500, and 250 microunits/ml. Likewise, incubation of rings at 22 degrees C for 30 min resulted in at least ten times less PGI2 with insulin. Since PGI2 appears to exert its antiaggregatory effect through cyclic AMP, theophylline was added to the incubation medium resulting in potentiation of inhibition of aggregation which was decreased to control levels when insulin was also added to the medium. Pancreatic slices yielded no significant change in insulin obtained when incubated with and without 5 ng of PGI2 in 2 ml of 2.8 mM glucose, but in a high glucose medium (28 mM) the PGI2-treated slices yielded significantly less insulin. Since PGI2 may play a role in the formation of atherosclerotic plaques, these results suggest a possible deleterious effect of elevated insulin levels in type II and in insulin-treated type I diabetics with regard to macrovascular disease. Suppression of insulin production in presence of PGI2 in high glucose medium resembles the action of other prostaglandins and suggests it may inhibit insulin secretion after a glucose load.

Insulin and its secretagogues activate Ca2+-dependent phosphatidylinositol breakdown and amylase secretion in rat pancreas in vitro.

We studied the effects of insulin and insulin secretagogues on pancreatic phospholipids and the release of amylase from pancreatic fragments incubated in vitro. Insulin provoked rapid, dose-related increases in amylase release, and these were accompanied by decreases in phosphatidylinositol and increases in phosphatidic acid. Glucose and other insulin secretagogues elicited similar changes in amylase release and pancreatic phospholipids; epinephrine blocked these effects of glucose, presumably via inhibition of glucose-induced insulin secretion. Effects of insulin and glucose on phospholipids and amylase release were dependent on Ca2+ in the incubation medium. These findings suggest that, in the pancreas, insulin enhances Ca2+-dependent phosphatidylinositol breakdown, which in turn influences amylase secretion. The effects of insulin on phospholipids may also explain certain other actions of insulin.

The mechanism of action of insulin on phospholipid metabolism in rat adipose tissue. Requirement for protein synthesis and a carbohydrate source, and relationship to activation of pyruvate dehydrogenase.

We studied certain metabolic requirements for insulin-induced increases in phospholipids, and the relationship of phospholipid changes to the insulin-induced activation of pyruvate dehydrogenase, in rat adipocytes and fat pads in vitro. Increases in the contents of phosphatidylinositol and phosphatidylserine mass were maximal in rat fat pads within 10 min of incubation with insulin, and preceded or accompanied measurable increases in pyruvate dehydrogenase activity. In dose-response studies, the contents of these phospholipids and pyruvate dehydrogenase activity increased in parallel in response to increasing concentrations of insulin. Cycloheximide and puromycin inhibited insulin-induced increases in the mass of both of these phospholipids, as well as (in confirmation of previous reports) pyruvate dehydrogenase activity. Effects of insulin on phospholipid metabolism and pyruvate dehydrogenase were found to require an exogenous carbohydrate source, and fructose was nearly as effective as glucose in this regard. Insulin-induced increases in phosphatidylinositol and phosphatidylserine were demonstrated in the mitochondrial fraction, which is also the subcellular locus of pyruvate dehydrogenase. The present findings suggest that there is a relationship between insulin-induced increases in phospholipids and pyruvate dehydrogenase activity, but the nature of this relationship remains to be defined.

The human androgen receptor: complementary deoxyribonucleic acid cloning, sequence analysis and gene expression in prostate.

Androgenic hormones mediate their effects on male sex differentiation and development through a high affinity receptor protein. We report here cloning of the complete coding sequence of the human androgen receptor (hAR). By sequence homology hAR is a member of the nuclear receptor family, with closest sequence identity to the progesterone, mineralocorticoid, and glucocorticoid receptors. Regions of highest homology include the DNA-binding domain and a small region within the hydrophobic ligand-binding domain. Comparison of the deduced 919 amino acid sequence of hAR (98,999 mol wt) to the 902 amino acid sequence of rat AR (98,227 mol wt) reveals identical sequences in the DNA- and hormone-binding domains, with an overall homology of 85%. In human prostate, the major androgen receptor mRNA species is 10 kilobases while a less abundant mRNA is approximately 7 kilobases. Rabbit polyclonal antibodies were raised against a synthetic peptide from the N-terminal region of hAR. Immunocytochemical analysis of human prostate tissue demonstrated that AR is localized predominantly in nuclei of glandular epithelial cells.

A novel SDS-stable dimer of a heterogeneous nuclear ribonucleoprotein at presynaptic terminals of squid neurons.

The presence of mRNAs in synaptic terminals and their regulated translation are important factors in neuronal communication and plasticity. Heterogeneous nuclear ribonucleoprotein (hnRNP) complexes are involved in the translocation, stability, and subcellular localization of mRNA and the regulation of its translation. Defects in these processes and mutations in components of the hnRNP complexes have been related to the formation of cytoplasmic inclusion bodies and neurodegenerative diseases. Despite much data on mRNA localization and evidence for protein synthesis, as well as the presence of translation machinery, in axons and presynaptic terminals, the identity of RNA-binding proteins involved in RNA transport and function in presynaptic regions is lacking. We previously characterized a strongly basic RNA-binding protein (p65), member of the hnRNPA/B subfamily, in squid presynaptic terminals. Intriguingly, in sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), p65 migrated as a 65-kDa protein, whereas members of the hnRNPA/B family typically have molecular masses ranging from 35 to 42kDa. In this report we present further biochemical and molecular characterization that shows endogenous p65 to be an SDS-stable dimer composed of ∼37-kDa hnRNPA/B-like subunits. We cloned and expressed a recombinant protein corresponding to squid hnRNPA/B-like protein and showed its propensity to aggregate and form SDS-stable dimers in vitro. Our data suggest that this unique hnRNPA/B-like protein co-localizes with synaptic vesicle protein 2 and RNA-binding protein ELAV and thus may serve as a link between local mRNA processing and presynaptic function and regulation.

Adrenocorticotropin and adenosine 3',5'-monophosphate stimulate de novo synthesis of adrenal phosphatidic acid by a cycloheximide-sensitive, CA++-dependent mechanism.

We tested further our postulate that enhanced de novo synthesis of phosphatidic acid is responsible for ACTH- and cAMP-induced increases in adrenal phospholipids in the phosphatidate polyphosphoinositide pathway. During incubation of adrenal sections or cells in vitro, ACTH and cAMP increased the concentrations of and incorporation of [3H]glycerol and [14C]palmitate into phosphatidylcholine and phosphatidylethanolamine, two major phospholipids which are derived from phosphatidic acid, but are extrinsic to the inositide pathway. Thus, it is unlikely that ACTH and cAMP increase inositide phospholipids at the expense of other phospholipids. Similar to previously reported effects on phosphatidic acid and inositide phospholipids, cycloheximide blocked the effects of ACTH and cAMP on phosphatidylcholine and phosphatidylethanolamine. In addition, Ca++ was required for these effects, as well as for cAMP-induced increases in phosphatidic acid, inositide phospholipids, and steroidogenesis. Our findings strongly suggest that ACTH, via cAMP, stimulates de novo phosphatidate synthesis by a cycloheximide-sensitive, Ca++-dependent process, and this stimulation causes a rapid generalized increase in adrenal phospholipids. Moreover, the increased incorporation of labeled glycerol and palmitate into phospholipids suggests that ACTH and cAMP may stimulate the glycerol-3'-PO4 acyltransferase reaction. This stimulatory effect may play a central role in the steroidogenic and trophic actions of ACTH and cAMP.

Rapid effects of angiotensin-II on polyphosphoinositide metabolism in the rat adrenal glomerulosa.

Angiotensin-II (A-II) provoked a rapid decrease in 32p in triphosphoinositide (TPI) in 32p-prelabeled rat adrenal glomerulosa cells. This effect (presumably reflecting TPI hydrolysis) of A-II was nearly maximal at 5 sec of incubation and appeared to precede increases in labeling of phosphatidic acid and phosphatidylinositol. Other aldosterone-stimulating agents (ACTH, K+ and serotonin) did not provoke this effect. Since this effect appeared to be independent of Ca++, it is possible that TPI hydrolysis may be important for Ca++ mobilization during A-II action in glomerulosa tissue.

Kinetic aspects of cycloheximide-induced reversal of adrenocorticotropin effects on steroidogenesis and phospholipid metabolism in rat adrenal sections in vitro.

We examined the rate of cycloheximide-induced reversal of ACTH effects on steroidogenesis and phospholipid metabolism in adrenal sections in vitro. In the absence of cycloheximide, ACTH treatment elicited sustained increases in steroidogenesis (approximately 3- to 5-fold) and adrenal concentrations (approximately 2-fold) of phosphatidic acid, phosphatidylinositol, and polyphosphoinositides. These increases in phospholipids were not dependent on steroidogenesis, since they were also apparent in aminoglutethimide-blocked adrenal sections. The addition of cycloheximide 30 min after ACTH treatment reversed the stimulatory effects of ACTH on steroidogenesis and phospholipids, and the rates of decay for both processes were identical, viz 28 min. This relatively slow turnover in adrenal sections in vitro contrasts with a much more rapid turnover observed previously in vivo and presently in dispersed adrenal cells in vitro; thus, it appears that an artefact of the in vitro adrenal section system stabilizes the putative labile protein. More importantly, the identical rates of decay for steroidogenesis and phospholipids suggest that the same labile protein is required for both phospholipid metabolism and steroidogenesis. These findings are in keeping with our postulate that the labile protein is required for phospholipid metabolism, which, in turn, is required for steroidogenesis.

A23187 inhibits adrenal protein synthesis and the effects of adrenocorticotropin (ACTH) on steroidogenesis and phospholipid metabolism in rat adrenal cells in vitro: further evidence implicating phospholipids in the steroidogenic action of ACTH.

We examined the effects of ACTH and the Ca++ ionophore, A23187, on steroidogenesis and phospholipid metabolism during incubation of dispersed rat adrenal cells. Increasing doses of ACTH elicited nearly parallel increases in corticosterone production and adrenal inositide (mono- and di-) concentrations. As reported previously by other investigators in Y1 cells, A23187 inhibited ACTH- and cAMP-stimulated, but not basal or pregnenolone-stimulated, corticosterone production. A23187 also inhibited ACTH-induced increases in phosphatidic acid, phosphatidylinositol, and diphosphoinositide, and this was attended by inhibition of [3H]leucine incorporation into protein. These findings support our previous contentions that: 1) a labile protein is required for ACTH-induced increases in adrenal phospholipids in the phosphatidate-polyphosphoinositide-polyglycerophospholipid pathway; and 2) these phospholipids are involved in the steroidogenic action of ACTH.

Comparison of effects of adrenocorticotropin and Lys-gamma 3-melanocyte-stimulating hormone on steroidogenesis, adenosine 3',5'-monophosphate production, and phospholipid metabolism in rat adrenal fasciculata-reticularis cells in vitro.

Synthetic bovine Lys-gamma 3MSH was found to potentiate the steroidogenic action of ACTH during incubation of rat adrenal fasciculata-reticularis cells in vitro. On the other hand, Lys-gamma 3MSH did not increase basal levels of or ACTH-induced (submaximal) increases in cellular concentrations of cAMP or phosphatidylinositol. Thus, Lys-gamma 3MSH does not appear to simply increase the overall action of ACTH, and moreover, it appears to potentiate steroidogenesis by a mechanism that is considerably different from that employed by ACTH. Observance of an effect of ACTH and failure to observe an effect of gamma 3MSH on adrenal phosphatidylinositol are in keeping with our previous postulation that phospholipids in the phosphatidate-inositide cycle play an important role in promoting cholesterol side-chain cleavage, since ACTH, but not gamma 3MSH, reportedly increases cholesterol side-chain cleavage. In addition, we also presently observed that Lys-gamma 3MSH can markedly increase corticosterone production in the face of a fixed, relatively small, submaximal, ACTH-induced increase in phosphatidylinositol. Thus, if gamma 3MSH enhances steroidogenesis by increasing free cholesterol availability, the latter may require another factor to initiate steroidogenesis, but is, nevertheless, an important determinant of the rate of steroidogenesis.

Rapid glucose-dependent increases in phosphatidic acid and phosphoinositides in rat pancreatic islets.

Glucose effects on islet phospholipids were examined during direct incubation or after 3 days of 32P prelabeling in primary culture. In both cases, glucose increased the 32P content of phosphatidic acid (PA), phosphatidylinositol (PI), and polyphosphoinositides (PPI). Glucose-induced increases in PA, PI, and PPI in the culture-prelabeling experiments were evident within 1 min, dose related, and reflective of increases in phospholipid mass, which was confirmed in direct incubations by measurement of PI phosphorus. Thus, in addition to increasing PI-PPI hydrolysis, glucose increases de novo phospholipid synthesis in pancreatic islets. The latter may result from enhanced glycolysis and substrate availability for PA-PI-PPI synthesis, since glyceraldehyde and pyruvic acid also increased PI levels. Our findings raise the possibility that increases in PA, PI, and PPI synthesis could serve as a mechanism to enhance the generation of intracellular mediators, which are purported to regulate insulin secretion.