Circulating sphingolipid biomarkers in models of type 1 diabetes

Alterations in lipid metabolism may contribute to diabetic complications. Sphingolipids are essential components of cell membranes and have essential roles in homeostasis and in the initiation and progression of disease. However, the role of sphingolipids in type 1 diabetes remains largely unexplored. Therefore, we sought to quantify sphingolipid metabolites by LC-MS/MS from two animal models of type 1 diabetes (streptozotocin-induced diabetic rats and Ins2Akita diabetic mice) to identify putative therapeutic targets and biomarkers. The results reveal that sphingosine-1-phosphate (So1P) is elevated in both diabetic models in comparison to respective control animals. In addition, diabetic animals demonstrated reductions in plasma levels of omega-9 24:1 (nervonic acid)-containing ceramide, sphingomyelin, and cerebrosides. Reduction of 24:1-esterfied sphingolipids was also observed in liver and heart. Nutritional stress via a high-fat diet also reduced 24:1 content in the plasma and liver of mice, exacerbating the decrease in some cases where diabetes was also present. Subcutaneous insulin corrected both circulating So1P and 24:1 levels in the murine diabetic model. Thus, changes in circulating sphingolipids, as evidenced by an increase in bioactive So1P and a reduction in cardio- and neuro-protective omega-9 esterified sphingolipids, may serve as biomarkers for type 1 diabetes and represent novel therapeutic targets. Alterations in lipid metabolism may contribute to diabetic complications. Sphingolipids are essential components of cell membranes and have essential roles in homeostasis and in the initiation and progression of disease. However, the role of sphingolipids in type 1 diabetes remains largely unexplored. Therefore, we sought to quantify sphingolipid metabolites by LC-MS/MS from two animal models of type 1 diabetes (streptozotocin-induced diabetic rats and Ins2Akita diabetic mice) to identify putative therapeutic targets and biomarkers. The results reveal that sphingosine-1-phosphate (So1P) is elevated in both diabetic models in comparison to respective control animals. In addition, diabetic animals demonstrated reductions in plasma levels of omega-9 24:1 (nervonic acid)-containing ceramide, sphingomyelin, and cerebrosides. Reduction of 24:1-esterfied sphingolipids was also observed in liver and heart. Nutritional stress via a high-fat diet also reduced 24:1 content in the plasma and liver of mice, exacerbating the decrease in some cases where diabetes was also present. Subcutaneous insulin corrected both circulating So1P and 24:1 levels in the murine diabetic model. Thus, changes in circulating sphingolipids, as evidenced by an increase in bioactive So1P and a reduction in cardio- and neuro-protective omega-9 esterified sphingolipids, may serve as biomarkers for type 1 diabetes and represent novel therapeutic targets. Diabetes is a debilitating chronic disease that has no cure and can only be managed by pharmaceutical and/or nutritional interventions. Worldwide, the incidence of diabetes and diabetic complications is dramatically increasing, possibly due to a worldwide obesity epidemic. It is argued that although targeting obesity will radically decrease the incidence and complications of type 2 diabetes, no such general nutritional solution exists for patients with type 1 diabetes. Yet patients with type 1 diabetes who adhere to the American Diabetes Association diet guidelines and/or exhibit a low body mass index demonstrate significant reductions in complications. Despite the knowledge that altered lipid metabolism is a cardinal feature of both type 1 and 2 diabetes, the actual lipid species that contribute to complications such as diabetic nephropathy, retinopathy, neuropathy, and cardiovascular disease have not been comprehensively studied in type 1 models and may offer a targeted nutritional approach for these patients. Although sphingolipids comprise only a fraction of total lipids, a body of evidence has now identified dysfunctional sphingolipid metabolism and generation of specific sphingolipid metabolites as contributors to diabetic complications (1Fox T.E. Kester M. Therapeutic strategies for diabetes and complications: a role for sphingolipids?.Adv. Exp. Med. Biol. 2010; 688: 206-216Crossref PubMed Scopus (27) Google Scholar). Most of the evidence pointing to a role of sphingolipids in diabetes has utilized models of type 2 diabetes. Treatment of Zucker fa/fa rats and diet-induced obese mice with the de novo sphingolipid inhibitor, myriocin, improved glucose tolerence and insulin sensitivity (2Holland W.L. Brozinick J.T. Wang L.P. Hawkins E.D. Sargent K.M. Liu Y. Narra K. Hoehn K.L. Knotts T.A. Siesky A. Inhibition of ceramide synthesis ameliorates glucocorticoid-, saturated-fat-, and obesity-induced insulin resistance.Cell Metab. 2007; 5: 167-179Abstract Full Text Full Text PDF PubMed Scopus (883) Google Scholar). Similarly, treatment strategies that inhibit glucosylceramide synthase have proven effective in diabetic animal models. Treatment of obese (ob/ob) mice with the selective glucosylceramide synthase inhibitor, N-(5-Adamantane-1-yl-methoxy-pentyl)-deoxynojirimycin, lowered blood glucose levels, improved oral glucose tolerance, reduced hemoglobin A1c, and improved insulin sensitivity in muscle and liver (3Aerts J.M. Ottenhoff R. Powlson A.S. Grefhorst A. van Eijk M. Dubbelhuis P.F. Aten J. Kuipers F. Serlie M.J. Wennekes T. Pharmacological inhibition of glucosylceramide synthase enhances insulin sensitivity.Diabetes. 2007; 56: 1341-1349Crossref PubMed Scopus (250) Google Scholar). Similar beneficial metabolic effects were observed in high-fat-fed mice and Zucker rats (3Aerts J.M. Ottenhoff R. Powlson A.S. Grefhorst A. van Eijk M. Dubbelhuis P.F. Aten J. Kuipers F. Serlie M.J. Wennekes T. Pharmacological inhibition of glucosylceramide synthase enhances insulin sensitivity.Diabetes. 2007; 56: 1341-1349Crossref PubMed Scopus (250) Google Scholar). Diminished blood glucose, improved glucose tolerance, and reduced A1c levels were also observed with a structurally distinct glucosylceramide synthase inhibitor, Genz-123346, in Zucker diabetic fatty rats and diet-induced obese mice. This inhibitor also limited the loss of pancreatic β-cell function, suggesting that sphingolipid metabolism might be affected in type 1 diabetes (4Zhao H. Przybylska M. Wu I.H. Zhang J. Siegel C. Komarnitsky S. Yew N.S. Cheng S.H. Inhibiting glycosphingolipid synthesis improves glycemic control and insulin sensitivity in animal models of type 2 diabetes.Diabetes. 2007; 56: 1210-1218Crossref PubMed Scopus (193) Google Scholar). Although these studies have demonstrated therapeutic efficacy in modulating sphingolipid metabolism, the actual diabetes-induced changes in sphingolipids remain poorly characterized. Herein, we analyzed the circulating sphingolipids in two distinct models of type 1 diabetes, the strepzotocotin (STZ)-induced diabetic rat and the Ins2Akita diabetic mouse. We also determined the sphingolipid profile of Ins2Akita mice in the presence or absence of nutritional “stress” (high-fat diet) and the effects of subcutaneous-implanted delivery of insulin to correct dysfunctional sphingolipid metabolism in the murine model. All lipid standards were obtained from Avanti Polar Lipids (Alabaster, AL). All solvents were HPLC grade or higher and obtained from Thermo Fisher Scientific (Waltham, MA) or Honeywell Burdick and Jackson (Morristown, NJ). Male Ins2Akita mice and wild-type littermates (C57BL/6J background) were housed under controlled temperature and a 12 h light/dark cycle with free access to food (Teklad 2019) and water (5Hong E.G. Jung D.Y. Ko H.J. Zhang Z. Ma Z. Jun J.Y. Kim J.H. Sumner A.D. Vary T.C. Gardner T.W. Nonobese, insulin-deficient Ins2Akita mice develop type 2 diabetes phenotypes including insulin resistance and cardiac remodeling.Am. J. Physiol. Endocrinol. Metab. 2007; 293: E1687-E1696Crossref PubMed Scopus (11) Google Scholar). The Akita allele is a spontaneous, dominantly inherited mutation in one of the two mouse insulin genes. The Ins2Akita mutation arose on the C57BL/6 background and upon transfer to The Jackson Laboratory (JAX) was bred to C57BL/6J. We imported the line from JAX and have continuously maintained the line by breeding to C57BL/6J stock. Ins2Akita/+ male mice are bred to Ins2+/+ female stock mice to generate the experimental male littermates, Ins2Akita/+(Ins2Akita) and Ins2+/+ (wild-type). All mice were housed under controlled temperature and a 12 h light/dark cycle with free access to food (Teklad 2019) and water. Ins2Akita mice demonstrate increased food intake, increased energy expenditure (despite being less active), and a significant reduction in respiratory exchange ratio (5Hong E.G. Jung D.Y. Ko H.J. Zhang Z. Ma Z. Jun J.Y. Kim J.H. Sumner A.D. Vary T.C. Gardner T.W. Nonobese, insulin-deficient Ins2Akita mice develop type 2 diabetes phenotypes including insulin resistance and cardiac remodeling.Am. J. Physiol. Endocrinol. Metab. 2007; 293: E1687-E1696Crossref PubMed Scopus (11) Google Scholar). For some studies (see Table 1 for details), animals at about 12 weeks of age (∼8 weeks hyperglycemia), were switched to a high-fat diet (Teklad 93075) for 6 weeks. Whole body fat and lean mass were noninvasively measured in awake Ins2Akita and wild-type mice at 14 weeks of age using 1H-MRS (Echo Medical Systems, Houston, TX). Some Ins2Akita diabetic mice received continuous insulin treatment via subcutaneous implant of LinBit pellets (LinShin, Canada, Inc.) 6 weeks prior to harvest following the manufacturer's instructions (3–4 pellets/animal determined by animal weight). Blood glucose concentrations and percent glycosylated hemoglobin were measured from drops of blood acquired by nicking the tail using a Lifescan glucose meter and Siemens DCA Analyzer, respectively. Cartridges for the quantitative measurement of hemoglobin A1c are based on specific monoclonal antibody agglutination.TABLE 1Animal informationStudyGroupnWeight(g)Blood Glucose (mg/dl)HbA1C(%)Body Fat(g)Figs. 1A, 2A–CControl (4wk)8422.8 ± 8.498.8 ± 3.6Diabetic (STZ - 4wk)7277.8 ± 16.2420.9 ± 19.9Figs. 1B, 2D–F, 3Wild-type (14wk)1028.5 ± 0.5194 ± 6.93.9 ± 0.2Ins2 (14wk)725.1 ± 0.6Above limit3.2 ± 0.1Wild-type + High Fat1133 ± 1.1212 ± 15.310.2 ± 1.0Ins2 + High Fat1025.6 ± 0.4512 ± 23.24.5 ± 0.1Figs. 4, 6Wild-type (16 wk)728.4 ± 1.5223 ± 16.94.4 ± 0.09Ins2 (16 wk)722.3 ± 0.6382 ± 50.613.3 ± 0.26Ins2 + insulin (16wk)725.2 ± 0.5Above limit6.7 ± 0.31Fig. 5Control (4wk)7391.1 ± 14.7108.6 ± 4.3Diabetic (STZ-4wk)7303.6 ± 9.6409.3 ± 17.6Fig. 7Wild-type (14wk)1027.1 ± 0.4168 ± 6.24.0 ± 0.2Ins2 (14wk)924.9 ± 0.7533 ± 23.23.0 ± 0.1Wild-type + High Fat1035.4 ± 1.35161 ± 4.111.8 ± 0.9Ins2 + High Fat1025.7 ± 0.2452 ± 22.04.4 ± 0.2 Open table in a new tab Male Sprague-Dawley rats (Charles River Laboratories, Wilmington, MA) were fasted overnight and given a single intraperitoneal injection of STZ (65 mg/kg; Sigma-Aldrich, St. Louis, MO) freshly dissolved in 10 mmol/l sodium citrate buffer (pH 4.5) (6Fox T.E. Han X. Kelly S. Merrill 2nd, A.H. Martin R.E. Anderson R.E. Gardner T.W. Kester M. Diabetes alters sphingolipid metabolism in the retina: a potential mechanism of cell death in diabetic retinopathy.Diabetes. 2006; 55: 3573-3580Crossref PubMed Scopus (71) Google Scholar). Diabetes was confirmed 6 days later by blood glucose > 250 mg/dl (Lifescan, Milpitas, CA). Age-matched control and diabetic rats were monitored regularly by weight and blood glucose tests. Rats were group-housed in solid plastic-bottom cages with bedding as well as ad libitum food (Teklad Global 18% protein rodent diet) and water under a normal 12 h light/dark schedule. Animals were maintained by the Juvenile Diabetes Research Foundation Animal Core Facility at Pennsylvania State University in accordance with the institutional animal care and use committee guidelines. The average weight and blood glucose level of each animal was recorded on the day they were euthanized and are indicated in Table 1. All procedures were approved by the Pennsylvania State University Institutional Animal Care and Use Committee. Sphingolipids were analyzed by LC/ESI-MS/MS based on the method described by Merrill et al. (7Merrill Jr, A.H. Sullards M.C. Allegood J.C. Kelly S. Wang E. Sphingolipidomics: high-throughput, structure-specific, and quantitative analysis of sphingolipids by liquid chromatography tandem mass spectrometry.Methods. 2005; 36: 207-224Crossref PubMed Scopus (456) Google Scholar) with some modifications (8Wijesinghe D.S. Allegood J.C. Gentile L.B. Fox T.E. Kester M. Chalfant C.E. Use of high performance liquid chromatography-electrospray ionization-tandem mass spectrometry for the analysis of ceramide-1-phosphate levels.J. Lipid Res. 2010; 51: 641-651Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). These modifications ensure that the samples are properly neutralized to prevent breakdown of sphingomyelin and lysosphingomyelin into other lipids if dried down under alkaline conditions (8Wijesinghe D.S. Allegood J.C. Gentile L.B. Fox T.E. Kester M. Chalfant C.E. Use of high performance liquid chromatography-electrospray ionization-tandem mass spectrometry for the analysis of ceramide-1-phosphate levels.J. Lipid Res. 2010; 51: 641-651Abstract Full Text Full Text PDF PubMed Scopus (64) Google Scholar). Lipids from heparinized plasma liver and were and on an HPLC were as described (7Merrill Jr, A.H. Sullards M.C. Allegood J.C. Kelly S. Wang E. Sphingolipidomics: high-throughput, structure-specific, and quantitative analysis of sphingolipids by liquid chromatography tandem mass spectrometry.Methods. 2005; 36: 207-224Crossref PubMed Scopus (456) Google with on a maintained at for of the and The was analyzed with an mass with a The for the sphingolipid were with that of the All are based on mass and are as for plasma and protein for and liver lipids were for fatty analysis from plasma as described R.E. Anderson R.E. of the lipid of membranes from 2005; PubMed Scopus Google Scholar). lipid were to by of in and at for of and were as were on and fatty were with dried under and dissolved in The fatty were determined by of each at with the ratio to using a and in a Wilmington, and an The temperature was to at for 1 increased to at and at for 10 at The temperature was to The were and on were identified by with were were using with if to the of sphingolipids as contributors to the of disease of sphingolipid Med. 2007; Full Text Full Text PDF PubMed Scopus Google we sought to a of diabetes-induced changes of sphingolipids in models of type 1 diabetes. We the sphingolipid profile from lipids from the plasma of the a diabetic and the Ins2Akita a of diabetes. Ins2Akita mice a mutation in the insulin 2 a that the protein in results in pancreatic β-cell death and of diabetes weeks of M. T. T. A. novel to on in C57BL/6 PubMed Scopus Google Scholar). we the sphingosine-1-phosphate and levels of So1P were observed in both rats and Ins2Akita mice to the was as were the metabolic and The fatty sphingolipid species of and sphingomyelin were also in the we observed that the and fatty were elevated in the diabetic rat not in the Ins2Akita mouse It is if is due to a of the STZ or due to in the models. For rats are and Ins2Akita have higher insulin of the fatty of both models a significant decrease in the 24:1 fatty (nervonic in the diabetic rat and in the Ins2Akita model. This decrease was also observed in the sphingomyelin Ins2Akita and Ins2Akita of the 24:1 were no other fatty changes in the sphingomyelin or of It be that the levels of in the plasma are to other the effects of nutritional wild-type and Ins2Akita mice were a high-fat diet for 10 weeks. This high-fat diet has been utilized in the as a for diet-induced obesity in C57BL/6J mice T. J.C. of diet on the of J. Physiol. Endocrinol. Metab. PubMed Scopus Google Kim H.J. A. R. T. Liu J. of diet-induced in fatty and insulin 2005; PubMed Scopus Google M. M. Jr, is with in PubMed Scopus Google H. J. A. in fat a role in the of insulin PubMed Scopus Google Scholar). Ins2Akita mice on of fat with wild-type mice, on average Ins2Akita mice were to a high-fat diet-induced increase in body with type 1 diabetes. the high-fat Ins2Akita diabetic as well as wild-type mice significant in for ceramide sphingomyelin and In the decrease in 24:1 sphingomyelin and was with the high-fat These demonstrate that both a high-fat diet and diabetes in significant of We determined insulin these selective in So1P and by diabetes. Ins2Akita mice were given subcutaneous insulin for 6 weeks. This insulin treatment blood glucose levels and hemoglobin A1c levels in Ins2Akita mice analyzed the sphingolipid content from the plasma of these animals. the diabetic animals a significant increase in So1P levels and with insulin a of So1P to We also that insulin treatment in diabetic animals 24:1 levels to that of for ceramide and sphingomyelin not 24:1 fatty content of esterified sphingolipids from the we analyzed the fatty profile of the total lipids in the plasma of the diabetic analysis of total fatty we observed 24:1 the of 24:1 in free or is low with other fatty in the total lipid the of 24:1 on sphingolipid is higher This that is to sphingolipids We also that metabolic to omega-9 and are in in to in and fatty of of not to these demonstrate that diabetes results in the selective decrease in omega-9 fatty both elevated So1P levels and have been with cardiovascular complications disease with J. PubMed Scopus Google E. K. J. Y. K. fatty and and J. 2005; PubMed Scopus Google sphingolipid species were also from of wild-type and Ins2Akita diabetic mice. have that circulating So1P is of disease disease with J. PubMed Scopus Google Scholar). In addition, has been that a on and may have a on cardiovascular E. K. J. Y. K. fatty and and J. 2005; PubMed Scopus Google Scholar). a of the sphingolipids in the as with the However, we observed a significant reduction in and sphingomyelin not in other fatty In these we also the of insulin by of a subcutaneous to we have observed in the insulin was to decrease in We also So1P levels in the wild-type and diabetic Ins2Akita in the a significant increase in So1P levels was observed in diabetic although not reduced So1P levels in of animals. sphingolipids, and were not altered by diabetes not These demonstrate that the has a to diabetes as the sphingolipids can be in the the liver as a in altered sphingolipids in We observed diabetes-induced changes in the liver of the Ins2Akita in the and were observed a decrease in 24:1 was as in the This reduction in is not specific to diabetes and a high-fat diet reduced the content of the sphingomyelin and of We a of a high-fat diet on 24:1 in diabetic animals. It be that content is less other esterified that reduced 24:1 sphingolipids in may be a of altered liver sphingolipid We also So1P levels in the liver of the Ins2Akita diabetic model. we not changes in liver So1P in diabetic animals The of diabetes-induced So1P changes a of elevated Although insulin and are the for diabetes and to the metabolites that contribute to a are poorly We to sphingolipid metabolites due to evidence that of of lipids can have therapeutic in models of type 2 diabetes (2Holland W.L. Brozinick J.T. Wang L.P. Hawkins E.D. Sargent K.M. Liu Y. Narra K. Hoehn K.L. Knotts T.A. Siesky A. Inhibition of ceramide synthesis ameliorates glucocorticoid-, saturated-fat-, and obesity-induced insulin resistance.Cell Metab. 2007; 5: 167-179Abstract Full Text Full Text PDF PubMed Scopus (883) Google J.M. Ottenhoff R. Powlson A.S. Grefhorst A. van Eijk M. Dubbelhuis P.F. Aten J. Kuipers F. Serlie M.J. Wennekes T. Pharmacological inhibition of glucosylceramide synthase enhances insulin sensitivity.Diabetes. 2007; 56: 1341-1349Crossref PubMed Scopus (250) Google H. Przybylska M. Wu I.H. Zhang J. Siegel C. Komarnitsky S. Yew N.S. Cheng S.H. Inhibiting glycosphingolipid synthesis improves glycemic control and insulin sensitivity in animal models of type 2 diabetes.Diabetes. 2007; 56: 1210-1218Crossref PubMed Scopus (193) Google Scholar). Despite evidence of targeting sphingolipids as a for type 2 diabetes, the actual changes to sphingolipid metabolites in type 1 diabetes have The changes we observed in two distinct models of type 1 diabetes an of So1P levels and a selective decrease in sphingolipids in circulating in So1P levels have for diabetes, in cardiovascular disease. Research that the and from the diabetic rat have increased was in animals given an insulin A. C. of the by high glucose the of Res. 2005; PubMed Scopus Google Scholar). are with results the of is elevated in the diabetic heart. and So1P levels, to to and to A. C. of the by high glucose the of Res. 2005; PubMed Scopus Google Scholar). This high glucose to is by of a 1 A. C. of the by high glucose the of Res. 2005; PubMed Scopus Google in diabetes-induced cardiovascular disease. in diabetic mice, 1 via injection of reduced blood glucose, to reduced and fatty increased and cardiac Wang Wang H. Y. J. Zhang J. 1 in insulin and glucose metabolism and homeostasis in diabetic 2007; PubMed Scopus Google Scholar). So1P also of the So1P in the type 1 diabetic mouse S. K. in type 1 diabetic mice of the Res. 2006; PubMed Scopus Google Scholar). of circulating So1P in samples that So1P is of disease other including diabetes, lipid and disease with J. PubMed Scopus Google Scholar). The of the roles of So1P may have including and targets. These roles may have effects on where So1P is in a to the is in in in models Z. Zhang T. J.H. J. J.H. and in J. Physiol. Physiol. 2007; PubMed Scopus Google Scholar). The also has been in and results in in both mice and J. E. C. S. J. and and Biol. Full Text Full Text PDF PubMed Scopus Google M. R. J. J. C. C. J. cell and cardiovascular effects of in are via distinct Exp. PubMed Scopus Google K. R. T. S. A. of a novel in PubMed Google Scholar). This the and of So1P and targets has not been in diabetic models and a of of type 1 diabetic was a decrease in esterified in sphingolipid and have significant for diabetic complications. It has been that a on and may have a on metabolic E. K. J. Y. K. fatty and and J. 2005; PubMed Scopus Google Scholar). reveal that a high-fat diet also and that nutritional stress can decrease diabetes-induced reductions of In addition, a reduction of in diabetic cardiac was also The role of omega-9 fatty such as in remains poorly It is or with or the mechanism for content remains It is that we observed in omega-9 species in the diabetic plasma from the total lipid these are not in sphingolipids for Diabetes has been demonstrated to fatty and fatty the and for the synthesis of omega-9 and fatty and to can be by diabetes or a high-fat diet Y. J. E. J.M. of fatty and in diabetes and Lipid Res. 2006; Full Text Full Text PDF PubMed Scopus Google the reduction observed in the diabetic In to cardiovascular may also diabetic Diminished has been observed in such as and K. R. and PubMed Scopus Google Scholar). also in in diabetes J. in diabetes PubMed Scopus Google and of in diabetic patients the complications of metabolism in can to diabetes. For although the So1P levels increased in the and the diabetic liver not demonstrate a We also have studies elevated glucosylceramide at the of ceramide in the (6Fox T.E. Han X. Kelly S. Merrill 2nd, A.H. Martin R.E. Anderson R.E. Gardner T.W. Kester M. Diabetes alters sphingolipid metabolism in the retina: a potential mechanism of cell death in diabetic retinopathy.Diabetes. 2006; 55: 3573-3580Crossref PubMed Scopus (71) Google and have a in the R. K. N.S. role for glycosphingolipid in the of diabetes PubMed Scopus Google Scholar). not in the Therefore, a of sphingolipid metabolism is in to altered content in with diabetic complications. studies have identified novel diabetes-induced in circulating sphingolipids, as evidenced by an increase in the So1P and a reduction in cardio- and neuro-protective esterified These changes diabetes-induced such as and including cardiovascular disease and sphingosine-1-phosphate

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