Neuropeptide Y (NPY) and norepinephrine, found colocalized in sympathetic neurons innervating blood vessels, exert synergistic responses on vasoconstriction. To examine the signaling mechanisms involved, free of complications associated with mixed receptor populations, we have established a stable Chinese hamster ovary cell line expressing both Y1-NPY and alpha 1b-adrenergic receptors. Occupation of either receptor species, with 100 nM peptide YY (PYY) or 10 microM phenylephrine (PE), respectively, resulted in a rapid increase in the cytoplasmic free calcium concentration ([Ca2+]i) as assessed with Fura-2/AM. The rise due to PYY, but not that due to PE, was abolished by pretreatment with pertussis toxin. Both responses were largely maintained in the absence of extracellular Ca2+, but abolished by prior depletion of intracellular Ca2+ pools with either thapsigargin or 2,5-di-(t-butyl)-1,4-benzohydroquinone. Using cells prelabeled with myo-[3H]inositol, PE promoted a rapid (5 s) rise in inositol 1,4,5-trisphosphate (Ins(1,4,5)P3) as analyzed by anion-exchange high pressure liquid chromatography, whereas the response to PYY (first significant at > 15 s post-stimulation) was too slow to play a causative role in Ca2+ mobilization. Combination of PE and PYY resulted in increases in [Ca2+]i which were at best additive, whereas they promoted a clearly synergistic rise in Ins(1,4,5)P3 at both 15 and 60 s. Co-stimulation also resulted in a synergistic activation of both protein kinase C (PKC) and [3H]arachidonic acid release. In either instance PYY alone was without effect. The potentiation of arachidonic acid release was abolished by depletion of cellular PKC following chronic treatment with phorbol esters. It is suggested that the ability of PYY to mobilize Ca2+ in an Ins(1,4,5)P3-independent fashion minimizes the functional importance of the capacity to potentiate PE-stimulated Ins(1,4,5)P3 generation. Instead the major consequences of the synergistic activation of phospholipase C are mediated via PKC, the other route of the signaling pathway.

We have tested the hypothesis that changes in the levels and cellular location of protein kinase C (PKC) isozymes might be associated with the development of insulin resistance in skeletal muscles from the high-fat-fed rat. Lipid measurements showed that triglyceride and diacylglycerol, an activator of PKC, were elevated four- and twofold, respectively. PKC activity assays indicated that the proportion of membrane-associated calcium-independent PKC was also increased. As determined by immunoblotting, total (particulate plus cytosolic) PKC alpha, epsilon, and zeta levels were not different between control and fat-fed rats. However, the ratio of particulate to cytosolic PKC epsilon in red muscles from fat-fed rats was increased nearly sixfold, suggesting chronic activation. In contrast, the amount of cytosolic PKC theta was downregulated to 45% of control, while the ratio of particulate to cytosolic levels increased, suggesting a combination of chronic activation and downregulation. Interestingly, while insulin infusion in glucose-clamped rats increased the proportion of PKC theta in the particulate fraction of red muscle, this was potentiated by fat-feeding, suggesting that the translocation is a consequence of altered lipid flux rather than a proximal event in insulin signaling. PKC epsilon and theta measurements from individual rats correlated with triglyceride content of red gastrocnemius muscle; they did not correlate with plasma glucose, which was not elevated in fat-fed rats, suggesting that they were not simply a consequence of hyperglycemia. Our results suggest that these specific alterations in PKC epsilon and PKC theta might contribute to the link between increased lipid availability and muscle insulin resistance previously described using high-fat-fed rats.

We have recently shown that the reduction in insulin sensitivity of rats fed a high-fat diet is associated with the translocation of the novel protein kinase Cε(nPKCε) from cytosolic to particulate fractions in red skeletal muscle and also the downregulation of cytosolic nPKCθ. Here we have further investigated the link between insulin resistance and PKC by assessing the effects of the thiazolidinedione insulin-sensitizer BRL-49653 on PKC isoenzymes in muscle. BRL-49653 increased the recovery of nPKC isoenzymes in cytosolic fractions of red muscle from fat-fed rats, reducing their apparent activation and/or downregulation, whereas PKC in control rats was unaffected. Because BRL-49653 also improves insulin-stimulated glucose uptake in fat-fed rats and reduces muscle lipid storage, especially diglyceride content, these results strengthen the association between lipid availability, nPKC activation, and skeletal muscle insulin resistance and support the hypothesis that chronic activation of nPKC isoenzymes is involved in the generation of muscle insulin resistance in fat-fed rats.

The 30-residue human neuropeptide, galanin, was shown to bind to rat insulinoma RINm5F cells and to inhibit glyceraldehyde-stimulated insulin secretion from these cells in a manner quantitatively similar to that of porcine galanin. Neither human nor porcine galanin stimulated Ca2+ mobilization in cultured human small cell lung carcinoma cells. Sedimentation equilibrium analysis of human galanin showed that it was strictly monomeric in aqueous solution, indicating that the peptide interacts with its receptor(s) as a monomer. The monomeric nature of the peptide makes it especially suitable for structural studies using NMR. Nuclear Overhauser enhancement spectroscopy experiments performed on galanin dissolved in aqueous solution (150 mM KCl, pH 4) at both 33 and 3 degrees C indicate that certain regions of the peptide are capable of adopting detectable levels of short-range structure in rapid equilibrium with random coil. At 33 degrees C, the short-range structures include a nascent helix spanning residues 3-11 which incorporates a hydrophobic core from residues 6-11. Residues 14-18 and 22-30 display sequential NH-NH and C beta H-NH connectivities, indicating that these regions of the peptide adopt nonrandom conformations by significantly populating the alpha-region of conformational space. However, no medium-range dipolar connectivities indicative of nascent helix or turn conformations were observed. At 3 degrees C, almost all residues significantly populate the alpha-region of conformational space, and the nascent helix between residues 3 and 11, with its hydrophobic core, is retained.(ABSTRACT TRUNCATED AT 250 WORDS)