Ken Strynadka

Leptin activates hypothalamic acetyl-CoA carboxylase to inhibit food intake

Hypothalamic fatty acid metabolism has recently been implicated in the controls of food intake and energy homeostasis. We report that intracerebroventricular (ICV) injection of leptin, concomitant with inhibiting AMP-activated kinase (AMPK), activates acetyl-CoA carboxylase (ACC), the key regulatory enzyme in fatty acid biosynthesis, in the arcuate nucleus (Arc) and paraventricular nucleus (PVN) in the hypothalamus. Arc overexpression of constitutively active AMPK prevents the Arc ACC activation in response to ICV leptin, supporting the hypothesis that AMPK lies upstream of ACC in leptins Arc intracellular signaling pathway. Inhibiting hypothalamic ACC with 5-tetradecyloxy-2-furoic acid, a specific ACC inhibitor, blocks leptin-mediated decreases in food intake, body weight, and mRNA level of the orexigenic neuropeptide NPY. These results show that hypothalamic ACC activation makes an important contribution to leptins anorectic effects. Furthermore, we find that ICV leptin up-regulates the level of malonyl-CoA (the intermediate of fatty acid biosynthesis) specifically in the Arc and increases the level of palmitoyl-CoA (a major product of fatty acid biosynthesis) specifically in the PVN. The rises of both levels are blocked by 5-tetradecyloxy-2-furoic acid along with the blockade of leptin-mediated hypophagia. These data suggest malonyl-CoA as a downstream mediator of ACC in leptins signaling pathway in the Arc and imply that palmitoyl-CoA, instead of malonyl-CoA, could be an effector in relaying ACC signaling in the PVN. Together, these findings highlight site-specific impacts of hypothalamic ACC activation in leptins anorectic signaling cascade.

Peroxynitrite Is a Mediator of Cytokine-Induced Destruction of Human Pancreatic Islet β Cells

The proinflammatory cytokines, interleukin-1β (IL-1β), tumor necrosis factor α (TNFα), and interferon γ (IFNγ), are cytotoxic to pancreatic islet β cells, possibly by inducing nitric oxide and/or oxygen radical production in the β cells. Peroxynitrite, the reaction product of nitric oxide and the superoxide radical, is a strong oxidant and cytotoxic mediator; therefore, we hypothesized that peroxynitrite might be a mediator of cytokine-induced islet β-cell destruction. To test this hypothesis we incubated islets isolated from human pancreata with the cytokine combination of IL-1β, TNFα, and IFNγ. We found that these cytokines induced significant increases in nitrotyrosine, a marker of peroxynitrite, in islet β cells, and the increase in nitrotyrosine preceded islet-cell destruction. Peroxynitrite mimicked the effects of cytokines on nitrotyrosine formation and islet β-cell destruction. L-NG-monomethyl arginine, an inhibitor of nitric oxide synthase, prevented cytokine-induced nitric oxide production but not hydrogen peroxide production, nitrotyrosine formation, or islet β-cell destruction. In contrast, guanidinoethyldisulphide, an inhibitor of inducible nitric oxide synthase and scavenger of peroxynitrite, prevented cytokine-induced nitric oxide and hydrogen peroxide production, nitrotyrosine formation, and islet β-cell destruction. These results suggest that cytokine-induced peroxynitrite formation is dependent upon increased generation of superoxide (measured as hydrogen peroxide) and that peroxynitrite is a mediator of cytokine-induced destruction of human pancreatic islet β cells.

Mechanisms Underlying Resistance of Pancreatic Islets from ALR/Lt Mice to Cytokine-Induced Destruction

Nuclear and mitochondrial genomes combine in ALR/Lt mice to produce systemically elevated defenses against free radical damage, rendering these mice resistant to immune-mediated pancreatic islet destruction. We analyzed the mechanism whereby isolated islets from ALR mice resisted proinflammatory stress mediated by combined cytokines (IL-1β, TNF-α, and IFN-γ) in vitro. Such damage entails both superoxide and NO radical generation, as well as peroxynitrite, resulting from their combination. In contrast to islets from other mouse strains, ALR islets expressed constitutively higher glutathione reductase, glutathione peroxidase, and higher ratios of reduced to oxidized glutathione. Following incubation with combined cytokines, islets from control strains produced significantly higher levels of hydrogen peroxide and NO than islets from ALR mice. Nitrotyrosine was generated in NOD and C3H/HeJ islets but not by ALR islets. Western blot analysis showed that combined cytokines up-regulated the NF-κB inducible NO synthase in NOD-Rag and C3H/HeJ islets but not in ALR islets. This inability of cytokine-treated ALR islets to up-regulate inducible NO synthase and produce NO correlated both with reduced kinetics of IκB degradation and with markedly suppressed NF-κB p65 nuclear translocation. Hence, ALR/Lt islets resist cytokine-induced diabetogenic stress through enhanced dissipation and/or suppressed formation of reactive oxygen and nitrogen species, impaired IκB degradation, and blunted NF-κB activation. Nitrotyrosylation of β cell proteins may generate neoantigens; therefore, resistance of ALR islets to nitrotyrosine formation may, in part, explain why ALR mice are resistant to type 1 diabetes when reconstituted with a NOD immune system.

Lysoplasmenylethanolamine accumulation in ischemic/reperfused isolated fatty acid-perfused hearts.

Lysophospholipid accumulation has been implicated in the pathogenesis of irreversible injury during myocardial ischemia and reperfusion. Plasmalogens (phospholipids with a vinyl-ether bond in the sn-1 position) account for more than 50% of total myocardial sarcolemmal and sarcoplasmic reticulum phospholipids. Accumulation of plasmalogen choline and ethanolamine lysophospholipids (lysoplasmenylcholine and lysoplasmenylethanolamine) or the effects of exogenous fatty acids on lysoplasmalogen accumulation during ischemia and reperfusion have not been examined. Isolated working rat hearts perfused with buffer containing either 11 mM glucose or 11 mM glucose plus 1.2 mM palmitate were subjected to aerobic, ischemic, or ischemia/reperfusion protocols. Levels of lysoplasmenylcholine and lysoplasmenylethanolamine were quantified using a two-stage high-performance liquid chromatographic technique. In hearts perfused with glucose alone, no significant differences in levels of lysoplasmenylcholine or lysoplasmenylethanolamine were seen during ischemia or reperfusion. In fatty acid-perfused hearts, however, significant accumulation of lysoplasmenylethanolamine occurred during reperfusion but not during ischemia (723 +/- 112, 734 +/- 83, and 1,394 +/- 193 nmol/g dry wt for aerobic, ischemic, and ischemic/reperfused hearts, respectively; p less than 0.05 for ischemic/reperfused hearts versus aerobic or ischemic hearts). Lysoplasmenylcholine levels after ischemia and reperfusion did not differ significantly from aerobic values, regardless of whether fatty acids were present or absent from the perfusate. Aerobic and ischemic/reperfused rabbit hearts, in the presence of fatty acid, showed a similar profile in their lysoplasmalogen content. We conclude that differential lysoplasmenylethanolamine accumulation occurs during myocardial reperfusion when exogenous fatty acid concentrations are high. This may reflect the selective action of fatty acid intermediates on the metabolism of lysoplasmenylethanolamines.(ABSTRACT TRUNCATED AT 250 WORDS)

Photolabelling of the prostaglandin E2 receptor in cardiac sarcolemmal vesicles

A [3H]azidophenacyl ester of PGE2 ([3H]azido‐PGE2) was synthesized and used to photoaffinity label the protein component of the high affinity PGE2 binding site in cardiac sarcolemma membrane. Photolysis of the isolated cardiac sarcolemmal vesicles in the presence of [3H]azido‐PGE2 resulted in the covalent labelling of a protein component that migrated on sodium dodecyl sulfate‐polyacrylamide gels with an apparent molecular weight of 100 000. Incorporation of the [3H]azido‐PGE2 did not occur in the absence of photolysis. The photolabelling of the 100‐kDa protein by [3H]azido‐PGE2 was inhibited by excess unlabelled PGE2 and arido‐PGE2. Specific binding of [3H]azido‐PGE2 was displaced by excess unlabelled PGE2 or azido‐PGE2, but not PGF2α, 6‐keto‐PGF1α or PGD2. These results indicate that the 100‐kDa photoaffinity labelled [3H]azido‐PGE2 binding protein contains the binding site for PGE2 in isolated cardiac sarcolemma membranes.

Decreased Polyamine Content of Concanavalin A Stimulated Lymphocytes in Down's Syndrome Subjects

Summary: Increased polyamine content is associated with increased rates of cell growth. Several Downs syndrome (D.S.) tissues have been shown to have decreased growth rates. Studies were undertaken to determine if the polyamine content of stimulated D.S. lymphocytes was similar to that of stimulated normal cells. Lymphocytes were isolated and cultured in the presence of Concanavalin A for 4 or 5 days. Polyamines were then extracted and quantitated. After 4 days spermidine content for normal cells was 930.9 ± 127 and for D.S. cells 489.2 ± 113.1 nmoles/109 cells (P < 0.025). Spermine content of normal cells was 1152.8 ± 157.4 and for D.S. cells 533.9 ± 82.0 (P < 0.005). After 5 days in culture spermidine content of normal cells was 803.0 ± 75.9 and for D.S. cells 446.2 ± 76.5 nmoles/109 cells (P < 0.005). Spermine content was 1155.7 ± 121.9 for normal cells and 555.1 ± 68.4 nmoles/109 for D.S. cells. Decreased content of polyamines in D.S.-stimulated lymphocytes is most probably due to decreased rate of polyamine synthesis. Decreased content of polyamines in response to stimulation may be a factor in decreased growth rates and altered immune function seen in D.S. patients.Speculation: Polyamine content is associated with increased rates of DNA and cell replication. If decreased polyamine synthesis occurred in multiple tissues in Downs syndrome, it could result in a total decrease in cell number and small stature present in Downs syndrome patients.

[70] High-voltage electrophoresis and thin-layer chromatographic separation of vitamin B6 compounds

Publisher Summary This chapter describes the high-voltage electrophoresis and two dimensional, thin-layer chromatographic methods for the separation of vitamin B 6 compounds. To 30 g of cellulose powder, MN 300 is added in 160 ml of water. The slurry is mixed with a mechanical stirrer for 30 minutes, then poured into a Shannon Unoplan Thin-Layer Spreader set at 500 μm thickness. The slurry is sufficient to make three cellulose-coated 46 × 20 cm glass plates. The plates are air-dried for 24 hours before use. The separation of radiolabeled vitamin B 6 compounds in tissue extracts can be achieved with electrophoresis and two dimensional methods. An application of tissue extract without standard is applied to determine whether substances are present that would interfere with the migration of standards. To one of the spaces, 2μl of standard alone is applied. The plates are placed in the horizontal Savant electrophoresis apparatus and run in the dark as described for standard solutions.

Solubilization and purification of the prostaglandin E2 receptor from cardiac sarcolemma

A prostaglandin E2 (PGE2) receptor was solubilized and isolated from cardiac sarcolemma membranes. Its binding characteristics are almost identical to those of the membrane bound receptor. [3H]PGE2 binding to solubilized and membrane bound receptor was sensitive to elevated temperature and no binding was observed in the absence of NaCl. No significant effects of DTT, ATP, Mg2+, Ca2+ or of changes in buffer pH were observed on [3H]PGE2 binding to either solubilized or membrane-bound receptor. Unlabelled PGE1 displaced over 90% of [3H]PGE2 from the CHAPS-solubilized receptor. PGD2, PGI2, PGF2 alpha and 6-keto-PGF1 alpha were not effective in displacing [3H]PGE2 from the receptor. Scatchard analysis of [3H]PGE2 binding to CHAPS-solubilized receptor revealed the presence of two types of PGE2 binding sites with Kd of 0.33 +/- 0.05 nM and 3.00 +/- 0.27 nM and Bmax of 0.5 +/- 0.04 and 2.0 +/- 0.1 pmol/mg of protein. The functional PGE2 receptor was isolated from CHAPS-solubilized SL membrane using two independent methods: first by a WGA-Sepharose chromatography and second by sucrose gradient density centrifugation. Receptor isolated by these two methods bound [3H]PGE2. Unlabelled PGE1 and PGE2 displaced [3H]PGE2 from the purified receptor. Scatchard analysis of [3H]PGE2 binding to purified receptor revealed the presence of the two binding sites as observed for the membrane bound and CHAPS-solubilized receptor. SDS-polyacrylamide gel electrophoresis of the purified receptor fractions revealed the presence of a protein band of M(r) of approx. 100,000. This 100-kDa was photolabelled with [3H]azido-PGE2, a photoactive derivative of PGE2. We propose that this 100-kDa protein is a cardiac PGE2 receptor.

695 Serial whole blood B6 vitamer levels in breast, formula and parenterally fed premature infants

To assess adequacy of vitamin B6 intake in premature infants, serial blood samples were obtained for vit B6 determination. A 0.5ml sample was frozen, B6 vitamers extracted, separated and quantitated by a HPLC-fluorometric technique. The method measured pyridoxal phosphate (PLP), pyridoxamine phosphate (PMP), pyridoxal (PL), pyridoxine (PN) and pyridoxamine (PM). Multiple blood samples were obtained over a 2-8wk period. Most infants receiving breast milk had low levels of PLP but high PL resulted in total normal B6 levels but some had low total vitamin B6. Several infants receiving formula had marked elevations of PL and PLP -- PL 256ng/ml (Normal=2-8) and PLP 120ng/ml (Normal 6-15). The values decreased to normal with continued feeding. Five infants received total parenteral nutrition (TPN) using commercial solutions for varying causes for 2-6wk period. PL and PLP values increased. Infant I -- PL 941ng/ml whole blood; PLP 293ng/ml. Infant II PL 765ng/ml, PLP 85ng/ml. Infant III PL & PLP beyond measurement. PL & PLP remained high until TPN was terminated.Conclusion: 1) Breastfed infants may have low whole blood PLP levels. 2) Some premature infants receiving proprietary formula have very high blood levels of PL and PLP suggesting an immaturity of B6 degradation. 3) Premature infants receiving TNP develop very high levels of PL and PLP. Commonly used multivitamin solutions used in TPN have excessive amounts of B6 for premature infants.

Assessment of Vitamin B-6 Status in Infants and Children: Serial Pyridoxal Phosphate Levels in Premature Infants

At the 1976 Workshop on Human Vitamin B-6 Requirements, material was presented on which the present recommended allowances for vitamin B-6 are based. As an introduction, parts of that work will be presented, followed by work we have done over the past year on plasma pyridoxal phosphate levels in premature infants.