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Lipoapoptosis in Beta-cells of Obese Prediabeticfa/fa Rats

ROLE OF SERINE PALMITOYLTRANSFERASE OVEREXPRESSION*
Open AccessPublished:December 04, 1998DOI:https://doi.org/10.1074/jbc.273.49.32487
      We reported that the lipoapoptosis of beta-cells observed in fat-laden islets of obese fa/fa Zucker Diabetic Fatty (ZDF) rats results from overproduction of ceramide, an initiator of the apoptotic cascade and is induced by long-chain fatty acids (FA). Whereas the ceramide of cytokine-induced apoptosis may be derived from sphingomyelin hydrolysis, FA-induced ceramide overproduction seems to be derived from FA. We therefore semiquantified mRNA of serine palmitoyltransferase (SPT), which catalyzes the first step in ceramide synthesis. It was 2–3-fold higher in fa/fa islets than in +/+ controls. [3H]Ceramide formation from [3H]serine was 2.2–4.5-fold higher in fa/faislets. Triacsin-C, which blocks palmitoyl-CoA synthesis, andl-cycloserine, which blocks SPT activity, completely blocked [3H]ceramide formation from [3H]serine. Islets of fa/fa rats are unresponsive to the lipopenic action of leptin, which normally depletes fat and prevents FA up-regulation of SPT. To determine the role of leptin unresponsiveness in the SPT overexpression, we transferred wild type OB-Rb cDNA to their islets; now leptin completely blocked the exaggerated FA-induced increase of SPT mRNA while reducing the fat content. Beta-cell lipoapoptosis was partially prevented in vivo by treating prediabetic ZDF rats withl-cycloserine for 2 weeks. Ceramide content and DNA fragmentation both declined 40–50%. We conclude that lipoapoptosis of ZDF rats is mediated by enhanced ceramide synthesis from FA and that blockade by SPT inhibitors prevents lipoapoptosis.
      ZDF
      Zucker Diabetic Fatty
      FA
      fatty acid
      SPT
      serine palmitoyltransferase
      PBS
      phosphate-buffered saline
      TG
      triglyceride.
      Obesity has become the single most prevalent health problem in the United States and non-insulin-dependent diabetes mellitus its most common complication (
      • National Center for Health Statistics
      ). Yet the mechanism by which obesity can damage remotely situated tissues such as the pancreatic beta-cells has not been established. Recently however, considerable evidence has implicated fatty acid excess as the major factor in obesity-associated beta-cell dysfunction and damage in a rodent model of adipogenic non-insulin-dependent diabetes mellitus, the Zucker Diabetic Fatty (ZDF)1rat (
      • Lee Y.
      • Hirose H.
      • Ohneda M.
      • Johnson J.H.
      • McGarry J.D.
      • Unger R.H.
      ,
      • Lee Y.
      • Hirose H.
      • Zhou Y.-T.
      • Esser V.
      • McGarry J.D.
      • Unger R.H.
      ,
      • Shimabukuro M.
      • Zhou Y-T.
      • Levi M.
      • Unger R.H.
      ).
      Recent studies from this laboratory have demonstrated that long-chain fatty acids induce apoptosis of the beta-cells of obese ZDF rats (
      • Shimabukuro M.
      • Zhou Y-T.
      • Levi M.
      • Unger R.H.
      ). Furthermore, the evidence pointed to de novo ceramide synthesis as a major factor in the lipoapoptosis (
      • Shimabukuro M.
      • Zhou Y-T.
      • Levi M.
      • Unger R.H.
      ). Obese ZDF rats are homozygous for the fa mutation (fa/fa), a Gln-269 → Pro substitution in the leptin receptor (OB-R) (
      • Iida M.
      • Murakami T.
      • Ishida K.
      • Mizuno A.
      • Kuwajima M.
      • Shima K.
      ,
      • Phillips M.S.
      • Liu Q.
      • Hammond H.
      • Dugan V.
      • Hey P.
      • Caskey C.T.
      • Hess J.F.
      ). The lipid content of their islets is markedly elevated because of complete unresponsiveness of their fat-laden islets to the lipopenic action of leptin (
      • Shimabukuro M.
      • Koyama K.
      • Chen G.
      • Wang M-Y.
      • Trieu F.
      • Lee Y.
      • Newgard C.B.
      • Unger R.H.
      ). We theorized that the overproduction of ceramide was somehow linked to the overproduction of fat.
      Because each molecule of ceramide contains two molecules of long-chain fatty acids (FA), it seemed likely that fatty acids stimulated ceramide-mediated apoptosis by providing excess substrate for de novo ceramide synthesis rather than by causing sphingomyelin hydrolysis to ceramide. To test this possibility, we studied [3H]ceramide formation from [3H]palmitate and observed a marked increase in islets from fa/fa ZDF rats (
      • Shimabukuro M.
      • Zhou Y-T.
      • Levi M.
      • Unger R.H.
      ). This overproduction of ceramide was blocked with fumonisin-B1, an inhibitor of ceramide synthetase (
      • Wang E.
      • Norred W.P.
      • Bacon C.W.
      • Riley R.T.
      • Merrill Jr., A.H.
      ), which also blocked the apoptosis (
      • Shimabukuro M.
      • Zhou Y-T.
      • Levi M.
      • Unger R.H.
      ). Thus de novo synthesis of ceramide appeared to be important in fatty acid-induced apoptosis.
      However, the mechanism of de novo ceramide overproduction was not fully explained by the foregoing studies. Because the high triglyceride (TG) content of these islets provided an expanded source of palmitoyl-CoA for de novo ceramide formation (
      • Shimabukuro M.
      • Zhou Y-T.
      • Levi M.
      • Unger R.H.
      ), we considered an increase within the metabolic pathway of ceramide synthesis. The first step in the pathway involves serine palmitoyltransferase (SPT), the enzyme that catalyzes the condensation of palmitoyl-CoA and serine to form dehydrosphinganine, a precursor of sphingosine (
      • Weiss B.
      • Stoffel W.
      ,
      • Hanada K.
      • Hara T.
      • Nishijima M.
      • Kuge O.
      • Dickson R.C.
      • Nagiec M.M.
      ); sphingosine is then acylated to form ceramide (
      • Merrill Jr., A.H.
      • Jones D.D.
      ,
      • Obeid L.M.
      • Linardic C.M.
      • Karolak L.A.
      • Hannun Y.A.
      ). The following study was designed to determine whether there is an increase in SPT activity and, if so, whether fatty acid-induced ceramide-mediated apoptosis of beta-cells in islets of ZDF rats could be blocked by inhibiting SPT activity and if this prevents the partial destruction of beta-cells that accompanies this form of obesity.

      DISCUSSION

      The results of this study provide several lines of new evidence that the high ceramide content observed in pancreatic islets of obesefa/fa ZDF rats, and implicated in beta-cell lipoapoptosis and adipogenic diabetes (
      • Shimabukuro M.
      • Zhou Y-T.
      • Levi M.
      • Unger R.H.
      ), represents newly synthesized ceramide rather than a product of sphingomyelin hydrolysis. First, expression of the enzyme SPT, the enzyme that catalyzes the condensation of serine and palmitoyl-CoA to form the ceramide precursor, dehydrosphinganine (
      • Wang E.
      • Norred W.P.
      • Bacon C.W.
      • Riley R.T.
      • Merrill Jr., A.H.
      ,
      • Weiss B.
      • Stoffel W.
      ,
      • Hanada K.
      • Hara T.
      • Nishijima M.
      • Kuge O.
      • Dickson R.C.
      • Nagiec M.M.
      ,
      • Merrill Jr., A.H.
      • Jones D.D.
      ,
      • Obeid L.M.
      • Linardic C.M.
      • Karolak L.A.
      • Hannun Y.A.
      ), was strikingly increased in the fa/fa islets with high ceramide content. Second, [3H]ceramide formation from [3H]serine, evidence of SPT enzyme activity, was increased in those islets, confirming our previous report of increased [3H]ceramide from [3H]palmitate (
      • Shimabukuro M.
      • Zhou Y-T.
      • Levi M.
      • Unger R.H.
      ). Third, the increased ceramide synthesis was inhibited by Triacsin-C blockade of fatty acyl-CoA synthetase, indicating that the excess ceramide formation is dependent on activation of long-chain fatty acids. Triacsin-C also blocked fatty acid-induced apoptosis (
      • Shimabukuro M.
      • Zhou Y-T.
      • Levi M.
      • Unger R.H.
      ), providing further evidence of their linkage. Finally, the competitive inhibitor of SPT, l-cycloserine, blocked the increase in [3H]ceramide, clear evidence that it was newly formed.De novo ceramide synthesis had not previously been identified as a source of ceramide in apoptosis; in cytokine-mediated autoimmune destruction of islets, for example, the ceramide is believed to be derived from sphingomyelin of membranes (
      • Sjoholm A.
      ,
      • Welsh N.
      ), although this has been questioned (
      • Kwon G.
      • Bohrer A.
      • Han X.
      • Corbett J.A.
      • Ma Z.
      • Gross R.W.
      • McDaniel M.L.
      • Turk J.
      ).
      It is noteworthy that in the [3H]serine experiments demonstrating the marked increase in [3H]ceramide formation in fa/fa ZDF islets, most of the label was recovered as sphingomyelin, phosphatidylcholine, and phosphatidylethanolamine, but these products were formed equally in fa/fa and +/+ islets. This strongly indicates that the serine → ceramide segment of sphingomyelin synthetic pathway is increased in fa/fa islets without any discernible post-ceramide differences (Fig. 7).
      Figure thumbnail gr7
      Figure 7A possible mechanism for increased ceramide synthesis in islets of fa/fa ZDF rats. SPT, serine palmitoyltransferase. Arrow widthindicates putative enzyme activity. Only SPT was actually quantified; the width of arrows is inferred from the metabolic studies of Fig. .
      The overexpression of SPT in the fa/fa islets appears to be secondary to the fa mutation, a Gln-269 → Pro substitution in the OB-R (
      • Iida M.
      • Murakami T.
      • Ishida K.
      • Mizuno A.
      • Kuwajima M.
      • Shima K.
      ,
      • Phillips M.S.
      • Liu Q.
      • Hammond H.
      • Dugan V.
      • Hey P.
      • Caskey C.T.
      • Hess J.F.
      ). One consequence of this mutation is total loss of the lipopenic action of leptin on islets (
      • Shimabukuro M.
      • Koyama K.
      • Chen G.
      • Wang M-Y.
      • Trieu F.
      • Lee Y.
      • Newgard C.B.
      • Unger R.H.
      ,
      • Zhou Y-T.
      • Shimabukuro M.
      • Koyama K.
      • Lee Y.
      • Wang M-Y.
      • Trieu F.
      • Unger R.H.
      ). In its presence, lipogenic enzymes are overexpressed (
      • Lee Y.
      • Hirose H.
      • Zhou Y.-T.
      • Esser V.
      • McGarry J.D.
      • Unger R.H.
      ) and enzymes of fatty acid oxidation are underexpressed (
      • Zhou Y-T.
      • Shimabukuro M.
      • Koyama K.
      • Lee Y.
      • Wang M-Y.
      • Trieu F.
      • Unger R.H.
      ), resulting in striking increase in islet lipid content exceeding more than 50 times that of normal (
      • Lee Y.
      • Hirose H.
      • Ohneda M.
      • Johnson J.H.
      • McGarry J.D.
      • Unger R.H.
      ,
      • Lee Y.
      • Hirose H.
      • Zhou Y.-T.
      • Esser V.
      • McGarry J.D.
      • Unger R.H.
      ). Based on the foregoing, it seemed reasonable to consider as a mechanism for SPT overexpression either a loss of direct leptin-induced inhibition or a lipid-mediated up-regulatory effect. Because loss of leptin activity is so tightly coupled to lipid accumulation (
      • Shimabukuro M.
      • Koyama K.
      • Chen G.
      • Wang M-Y.
      • Trieu F.
      • Lee Y.
      • Newgard C.B.
      • Unger R.H.
      ), we could not exclude the former possibility and focused instead on the role of lipids. Fatty acids up-regulated SPT mRNA in both normal+/+ and obese fa/fa ZDF rat islets, but the baseline level of SPT expression and the fatty acid-induced up-regulation was far greater in the fa/fa islets. This correlated well with the much higher base-line TG content and the fatty acid-induced increase in TG content in fa/fa islets.
      Evidence consistent with leptin-mediated regulation of islet lipid was also obtained by overexpressing the wild-type OB-Rb or, as a control, β-galactosidase in the fa/fa islets. This conferred leptin responsiveness to the former islets, whereas leptin had no effect in the β-gal controls. In the OB-Rb-overexpressing islets, 20 ng/ml leptin reduced islet TG content to normal and completely blocked fatty acid-induced up-regulation of SPT mRNA.
      Finally, we tested the possibility that SPT blockade might protect the fat-laden islets of prediabetic ZDF rats from ceramide-mediated lipoapoptosis of beta-cells (
      • Shimabukuro M.
      • Zhou Y-T.
      • Levi M.
      • Unger R.H.
      ), which may be a factor in their diabetes. Prediabetic ZDF rats were treated withl-cycloserine for 2 weeks, and ceramide content and DNA fragmentation were measured. The results suggest that agent was partially effective, reducing [3H]ceramide formation from [3H]serine and DNA fragmentation by ∼50%.

      ACKNOWLEDGEMENTS

      We thank Tagan Ferguson and Kay McCorkle for excellent technical work and Tess Perico for secretarial assistance.

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