Gordon C. Yaney

The Role of Long-Chain Fatty Acyl-CoA Esters in β-Cell Signal Transduction

Glucose-induced insulin secretion is associated with inhibition of free fatty acid (FFA) oxidation, increased esterification and complex lipid formation by pancreatic beta-cells. Abundant evidence favors a role for cytosolic long-chain acyl-CoA (LC-CoA), including the rapid rise in malonyl CoA, the inhibitory effect of hydroxycitrate or acetyl CoA carboxylase knockout, both of which prevent malonyl CoA formation, and the stimulatory effect of exogenous FFA. On the other hand, some evidence opposes the concept, including the fall in total LC-CoA levels in response to glucose, the stimulatory effect of LC-CoA on K(ATP) channels and the lack of inhibition of glucose-stimulated secretion either by overexpression of malonyl CoA decarboxylase, which markedly lowers malonyl CoA levels, or by triacsin C, which blocks FFA conversion to LC-CoA. Alternative explanations for these data are presented. A revised model of nutrient-stimulated secretion involving two arms of signal transduction that occur simultaneously is proposed. One arm depends on modulation of the K(ATP) channel evoked by changes in the ATP/ADP ratio. The other arm depends upon anaplerotic input into the tricarboxylic acid cycle, generation of excess citrate, and increases in cytosolic malonyl-CoA. Input from this arm is increased LC-CoA. Signaling through both arms would be required for normal secretion. LC-CoA esters and products formed from them are potent regulators of enzymes and channels. It is hypothesized that their elevations directly modulate the activity of enzymes, genes and various beta-cell functions or modify the acylation state of key proteins involved in regulation of ion channels and exocytosis.

Induction by glucose of genes coding for glycolytic enzymes in a pancreatic beta-cell line (INS-1).

Chronic elevation in glucose has pleiotropic effects on the pancreatic β-cell including a high rate of insulin secretion at low glucose, β-cell hypertrophy, and hyperplasia. These actions of glucose are expected to be associated with the modulation of the expression of a number of glucose-regulated genes that need to be identified. To further investigate the molecular mechanisms implicated in these adaptation processes to hyperglycemia, we have studied the regulation of genes encoding key glycolytic enzymes in the glucose-responsive β-cell line INS-1. Glucose (from 5 to 25 mM) induced phosphofructokinase-1 (PFK-1) isoform C, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (4-fold), and L-pyruvate kinase (L-PK) (7-fold) mRNAs. In contrast the expression level of the glucokinase (Gk) and 6-phosphofructo-2-kinase transcripts remained unchanged. Following a 3-day exposure to elevated glucose, a similar induction was observed at the protein level for PFK-1 (isoforms C, M, and L), GAPDH, and L-PK, whereas M-PK expression only increased slightly. The study of the mechanism of GAPDH induction indicated that glucose increased the transcriptional rate of the GAPDH gene but that both transcriptional and post transcriptional effects contributed to GAPDH mRNA accumulation. 2-Deoxyglucose did not mimic the inductive effect of glucose, suggesting that increased glucose metabolism is involved in GAPDH gene induction. These changes in glycolytic enzyme expression were associated with a 2-3-fold increase in insulin secretion at low (2-5 mM) glucose. The metabolic activity of the cells was also elevated, as indicated by the reduction of the artificial electron acceptor 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium. A marked deposition of glycogen, which was readily mobilized upon lowering of the ambient glucose, and increased DNA replication were also observed in cells exposed to elevated glucose. The results suggest that a coordinated induction of key glycolytic enzymes as well as massive glycogen deposition are implicated in the adaptation process of the β-cell to hyperglycemia to allow for chronically elevated glucose metabolism, which, in this particular fuel-sensitive cell, is linked to metabolic coupling factor production and cell activation.

Long-Chain Acyl CoA Regulation of Protein Kinase C and Fatty Acid Potentiation of Glucose-Stimulated Insulin Secretion in Clonalβ -Cells1

Pancreatic beta-cells contain protein kinase C (PKC) isoforms that may play a role in insulin secretion. Activity of PKC classes (cPKC, nPKC, aPKC) and their regulation by acyl-CoA derivatives was examined in extracts of clonal pancreatic beta-cells (HIT) by protein phosphorylation. PKC classes were distinguished based on their previously defined cofactor requirements. Down-regulation of PKC by phorbol esters was confirmed by Western blotting and resulted in the complete loss of cPKC activity, partial loss of nPKC activity and preservation of aPKC activity and glucose-stimulated insulin secretion. aPKC activity was potentiated 4- to 8-fold by the CoA esters of myristate, palmitate, and oleate with a half-maximal value of 3 microM. Both oleoyl- and myristol-CoA, but not palmitoyl-CoA, caused inhibition of nPKC activity. Oleoyl-CoA inhibited nPKC activity up to 75% with a half-maximal effect at 10 microM. This value was independent of the concentration of diacylglycerol used. The addition of exogenous oleate or palmitate potentiated glucose-stimulated insulin secretion 2-fold and was unaffected by PMA-induced down-regulation. Stimulation by glucose or glucose and oleate also increased the mass of PKC-zeta found in the particulate fraction. These data are consistent with increased cytosolic long-chain acylCoA-activating aPKC isoforms resulting in stimulation and/or potentiation of glucose-induced insulin secretion.

Phosphofructokinase Isozymes in Pancreatic Islets and Clonal β-Cells (INS-1)

Normal insulin secretion is oscillatory in vivo, and the oscillations are impaired in type II diabetes. We and others have shown oscillations in insulin secretion from isolated perifused islets stimulated with glucose, and in this study we show oscillations in insulin secretion from the glucose-sensitive clonal β-cell line INS-1. We have proposed that the oscillatory insulin secretion may be caused by spontaneous oscillations of glycolysis and the ATP:ADP ratio in the β-cell, analogous to those seen in glycolyzing muscle extracts. The mechanism of the latter involves autocatalytic activation of the key regulatory enzyme, phosphofructokinase (PFK), by its product fructose 1,6-bisphosphate (F16BP). However, of the three PFK subunit isoforms (M-[muscle], L-[liver], and C-type, predominant in fibroblasts), only M-type is activated by micromolar F16BP at near-physiological conditions. We therefore studied PFK isoforms in the β-cell. Western analysis of PFK subunits in isolated rat islets and INS-1 cells showed the presence of M-type, as well as C-type and perhaps lesser amounts of L-type. Kinetic studies of PFK activity in INS-1 cell extracts showed strong activation by micromolar concentrations of F16BP at near-physiological concentrations of ATP (several millimolar) and AMP and fructose 6-phosphate (micromolar), indicative of the M-type isoform. Activation by submicromolar concentrations of fructose 2,6-bisphosphate (F26BP) and potent inhibition by citrate were also observed. The F16BP-stimulatable activity was about one-half of the F26BP-stimulatable activity. These experiments demonstrate that μ-cells contain the M-type isoform of PFK with the requisite regulatory properties for generating glycolytic oscillations that may be the basis of oscillatory insulin secretion.

Ca2+, NAD(P)H and membrane potential changes in pancreatic β-cells by methyl succinate: comparison with glucose

The present study was undertaken to determine the main metabolic secretory signals generated by the mitochondrial substrate MeS (methyl succinate) compared with glucose in mouse and rat islets and to understand the differences. Glycolysis and mitochondrial metabolism both have key roles in the stimulation of insulin secretion by glucose. Both fuels elicited comparable oscillatory patterns of Ca2+ and changes in plasma and mitochondrial membrane potential in rat islet cells and clonal pancreatic beta-cells (INS-1). Saturation of the Ca2+ signal occurred between 5 and 6 mM MeS, while secretion reached its maximum at 15 mM, suggesting operation of a K(ATP)-channel-independent pathway. Additional responses to MeS and glucose included elevated NAD(P)H autofluorescence in INS-1 cells and islets and increases in assayed NADH and NADPH and the ATP/ADP ratio. Increased NADPH and ATP/ADP ratios occurred more rapidly with MeS, although similar levels were reached after 5 min of exposure to each fuel, whereas NADH increased more with MeS than with glucose. Reversal of MeS-induced cell depolarization by Methylene Blue completely inhibited MeS-stimulated secretion, whereas basal secretion and KCl-induced changes in these parameters were not affected. MeS had no effect on secretion or signals in the mouse islets, in contrast with glucose, possibly due to a lack of malic enzyme. The data are consistent with the common intermediates being pyruvate, cytosolic NADPH or both, and suggest that cytosolic NADPH production could account for the more rapid onset of MeS-induced secretion compared with glucose stimulation.

Glucagon-like peptide 1 and fatty acids amplify pulsatile insulin secretion from perifused rat islets

Glucose-induced insulin secretion from isolated, perifused rat islets is pulsatile with a period of about 5-10 min, similar to the insulin oscillations that are seen in healthy humans but which are impaired in Type II diabetes. We evaluated the pattern of enhancement by the potent incretin, glucagon-like peptide 1 (GLP-1). GLP-1 increased the amplitude of pulses and the magnitude of insulin secretion from the perifused islets, without affecting the average time interval between pulses. Forskolin and the phosphodiesterase inhibitor isobutylmethylxanthine had the same effect, suggesting that the effect was due to elevated cAMP levels. The possibility that cAMP might enhance the amplitude of pulses by reducing phosphofructo-2-kinase (PFK-2) activity was eliminated when the liver isoform of PFK-2 was shown to be absent from beta-cells. The possibility that cAMP enhanced pulsatile secretion, at least in part, by stimulating lipolysis was supported by the observations that added oleate had a similar effect on secretion, and that the incretin effect of GLP-1 was inhibited by the lipase inhibitor orlistat. These data show that the physiological incretin GLP-1 preserves and enhances normal pulsatile insulin secretion, which may be essential in proposed therapeutic uses of GLP-1 or its analogues.

Differential modulation of L-type calcium channel subunits by oleate

Nonesterified fatty acids such as oleate and palmitate acutely potentiate insulin secretion from pancreatic islets in a glucose-dependent manner. In addition, recent studies show that fatty acids elevate intracellular free Ca(2+) and increase voltage-gated Ca(2+) current in mouse beta-cells, although the mechanisms involved are poorly understood. Here we utilized a heterologous system to express subunit-defined voltage-dependent L-type Ca(2+) channels (LTCC) and demonstrate that beta-cell calcium may increase in part from an interaction between fatty acid and specific calcium channel subunits. Distinct functional LTCC were assembled in both COS-7 and HEK-293 cells by expressing either one of the EYFP-tagged L-type alpha(1)-subunits (beta-cell Cav1.3 or lung Cav1.2) and ERFP-tagged islet beta-subunits (ibeta(2a) or ibeta(3)). In COS-7 cells, elevations in intracellular Ca(2+) mediated by LTCC were enhanced by an oleate-BSA complex. To extend these findings, Ca(2+) current was measured in LTCC-expressing HEK-293 cells that revealed an increase in peak Ca(2+) current within 2 min after addition of the oleate complex, with maximal potentiation occurring at voltages <0 mV. Both Cav1.3 and Cav1.2 were modulated by oleate, and the presence of different auxiliary beta-subunits resulted in differential augmentation. The potentiating effect of oleate on Cav1.2 was abolished by the pretreatment of cells with triacsin C, suggesting that long-chain CoA synthesis is necessary for Ca(2+) channel modulation. These results show for the first time that two L-type Ca(2+) channels expressed in beta-cells (Cav1.3 and Cav1.2) appear to be targeted by nonesterified fatty acids. This effect may account in part for the acute potentiation of glucose-dependent insulin secretion by fatty acids.

Mice Deficient in Phosphofructokinase‐M Have Greatly Decreased Fat Stores

Synthesis of triacylglycerol requires the glucose‐derived glycerol component, and glucose uptake has been viewed as the rate‐limiting step in glucose metabolism in adipocytes. Furthermore, adipose tissue contains all three isoforms of the glycolytic enzyme phosphofructokinase (PFK). We here report that mice deficient in the muscle isoform PFK‐M have greatly reduced fat stores. Mice with disrupted activity of the PFK‐M distal promoter were obtained from Lexicon Pharmaceuticals, developed from OmniBank OST#56064. Intra‐abdominal fat was measured by magnetic resonance imaging of the methylene proton signal. Lipogenesis from labeled glucose was measured in isolated adipocytes. Lipolysis (glycerol and free fatty acid release) was measured in perifused adipocytes. Intra‐abdominal fat in PFK‐M–deficient female mice (5–10 months old) was 17 ± 3% of that of wild‐type littermates (n = 4; P < 0.02). Epididymal fat weight in 15 animals (7–9.5 months) was 34 ± 4% of control littermate (P < 0.002), with 10–30% lower body weight. Basal and insulin‐stimulated lipogenesis in PFK‐M–deficient epididymal adipocytes was 40% of the rates in cells from heterozygous littermates (n = 3; P < 0.05). The rate of isoproterenol‐stimulated lipolysis in wild‐type adipocytes declined ∼10% after 1 h and 50% after 2 h; in PFK‐M–deficient cells it declined much more rapidly, 50% in 1 h and 90% in 2 h, and lipolytic oscillations appeared to be damped (n = 4). These results indicate an important role for PFK‐M in adipose metabolism. This may be related to the ability of this isoform to generate glycolytic oscillations, because such oscillations may enhance the production of the triacylglycerol precursor α‐glycerophosphate.

Modulation of Ca2+ release through ryanodine receptors in vascular smooth muscle by protein kinase Cα

The role of protein kinase C (PKC) in Ca2+ release through ryanodine receptors (RyRs) in the sarcoplasmic reticulum (SR) of vascular smooth muscle cells (SMCs) is not well understood. Caffeine was used to activate RyRs and the intracellular Ca2+ concentration ([Ca2+]i) was measured in both freshly isolated and cultured mouse aortic SMCs (ASMCs). Pre-activation of PKC with 1,2-dioctanoyl-sn-glycerol (DOG) prevented caffeine-induced [Ca2+]i transients. Application of the PKC inhibitor calphostin C caused [Ca2+]i transients which were not blocked by nifedipine or by removing extracellular Ca2+ but were abolished after inhibition of the SR Ca2+–ATPase with thapsigargin or after inhibition of RyRs with ryanodine. In addition, chelerythrine and GF109203X also elevated resting [Ca2+]i but no further [Ca2+]i increase was seen with subsequent application of caffeine. Selective inhibition of PKCα with safingol blocked caffeine-induced [Ca2+]i transients, but the PKCε inhibitory peptide V1-2 did not. In cells expressing a EGFP-tagged PKCα, caffeine-induced [Ca2+]i transients were associated with a rapid focal translocation near the cell periphery, while application of ionomycin and DOG caused translocation to the plasma membrane. Western blot showed that caffeine increased the relative amount of PKCα in the particulate fraction in a time-dependent manner. Co-immunoprecipitation of RyRs and PKCα indicated that they interact. In conclusion, our studies suggest that PKC activation can inhibit the gating activity of RyRs in the SR of ASMCs, and this regulation is most likely mediated by the Ca2+-dependent PKCα isoform.