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. 2014 Mar 1;306(5):E529-42.
doi: 10.1152/ajpendo.00610.2012. Epub 2013 Dec 24.

Impact of insulin deprivation and treatment on sphingolipid distribution in different muscle subcellular compartments of streptozotocin-diabetic C57Bl/6 mice

Piotr Zabielski  1 , Agnieszka Blachnio-Zabielska, Ian R Lanza, Srinivas Gopala, S Manjunatha, Daniel R Jakaitis, Xuan-Mai Persson, Jaime Gransee, Katherine A Klaus, Jill M Schimke, Michael D Jensen, K Sreekumaran Nair
Affiliations

Impact of insulin deprivation and treatment on sphingolipid distribution in different muscle subcellular compartments of streptozotocin-diabetic C57Bl/6 mice

Piotr Zabielski et al. Am J Physiol Endocrinol Metab. .

Abstract

Insulin deprivation in type 1 diabetes (T1D) individuals increases lipolysis and plasma free fatty acids (FFA) concentration, which can stimulate synthesis of intramyocellular bioactive lipids such as ceramides (Cer) and long-chain fatty acid-CoAs (LCFa-CoAs). Ceramide was shown to decrease muscle insulin sensitivity, and at mitochondrial levels it stimulates reactive oxygen species production. Here, we show that insulin deprivation in streptozotocin diabetic C57BL/6 mice increases quadriceps muscle Cer content, which was correlated with a concomitant decrease in the body fat and increased plasma FFA, glycosylated hemoglobin level (%Hb A1c), and muscular LCFa-CoA content. The alternations were accompanied by an increase in protein expression in LCFa-CoA and Cer synthesis (FATP1/ACSVL5, CerS1, CerS5), a decrease in the expression of genes implicated in muscle insulin sensitivity (GLUT4, GYS1), and inhibition of insulin signaling cascade by Aktα and GYS3β phosphorylation under acute insulin stimulation. Both the content and composition of sarcoplasmic fraction sphingolipids were most affected by insulin deprivation, whereas mitochondrial fraction sphingolipids remained stable. The observed effects of insulin deprivation were reversed, except for content and composition of LCFa-CoA, CerS protein expression, GYS1 gene expression, and phosphorylation status of Akt and GYS3β when exogenous insulin was provided by subcutaneous insulin implants. Principal component analysis and Pearson's correlation analysis revealed close relationships between the features of the diabetic phenotype, the content of LCFa-CoAs and Cers containing C18-fatty acids in sarcoplasm, but not in mitochondria. Insulin replacement did not completely rescue the phenotype, especially regarding the content of LCFa-CoA, or proteins implicated in Cer synthesis and muscle insulin sensitivity. These persistent changes might contribute to muscle insulin resistance observed in T1D individuals.

Keywords: ceramide; long-chain fatty acid-coenzyme A; mitochondria; skeletal muscle; type 1 diabetes.

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Figures

Fig. 1.
Fig. 1.
Experimental design and time scale and exemplary blood glucose profile of control (C; n = 3) (solid line, ○), insulin-treated (weeks 1–5: combined D + I and D − I groups under insulin treatment, n = 6; week 6: D + I group only, n = 3) (dashed line, gray circles), and insulin-deprived animals (D − I; n = 3) (dotted line, ●). STZ, intraperitoneal injection of streptozotocin; VEH, intraperitoneal injection of vehicle. Values represent means ± SE. aP < 0.05 vs. C group by ANOVA.
Fig. 2.
Fig. 2.
Insulin deprivation affects both the content and composition of plasma free fatty acids (FFA; A) and skeletal muscle long-chain fatty acids-acyl-CoA esters (LCFa-CoA; B) in diabetic C57Bl/6J mice. Values are expressed in μmol/l of plasma for FFA (means ± SE; n = 8/group) or ng/mg of protein for LCFa-CoA (means ± SE; n = 13/group). aP < 0.05 vs. C group; bP < 0.05 vs. D + I group.
Fig. 3.
Fig. 3.
Insulin deprivation increases the content of ceramide in mouse skeletal muscle homogenates and sarcoplasmic fraction but not in mitochondria. Shown is the the impact of insulin deprivation (black bars) and insulin treatment (gray bars) on the content of individual ceramide (Cer) molecular species (left) and sphingoid bases (right) in mouse quadriceps muscle homogenates (A and B), sarcoplasmic fraction (C and D), and mitochondrial fraction (E and F). Values were normalized to protein content in appropriate fraction and represent the mean ng/mg of protein content ± SE; n = 13/group; aP < 0.05 vs. C group; bP < 0.05 vs. D + I group.
Fig. 4.
Fig. 4.
Insulin deprivation upregulates proteins of LCFa-CoA and Cer synthesis and affects the genes and proteins implicated in muscle insulin sensitivity. The impact of insulin deprivation and treatment on content of proteins of intramyocellular FFA uptake and LCFa-CoA synthesis (CD36 and FATP1/ACSVL5; A), sphingoid backbone formation and Cer synthesis [serine palmitoyltransferase (SPT), CerS1, and CerS5; B], genes implicated in muscle tissue insulin sensitivity [insulin receptor substrate-1 (IRS-1), glucose transporter type 4 (GLUT4), and glycogen synthase 1 (GYS1); C], and Akt-dependent insulin signaling (phosphorylation state of Akt and its downstream target GSK-3β; D) in mouse skeletal muscle. Values were normalized to vinculin expression for protein data and β2-microglobulin for mRNA data. Bars represent SE; n = 8/group (except for D; see materials and methods). aP < 0.05 vs. C group; bP < 0.05 vs. D + I group.
Fig. 5.
Fig. 5.
Scores scatter plot for 1st and 2nd components of principal component analysis: ●, individual D − I animals; gray circles, individual D + I animals; ○, individual C animals. Ovals represent clustering of animals from respective experimental groups. Arrows indicate directions of major variables that are responsible for between- and within-group differences. S-Cer, total sarcoplasmic ceramide; M-Cer, total mitochondrial ceramide; M-dhSPH, mitochondrial sphinganine.
Fig. 6.
Fig. 6.
Loadings scatterplot for first 2 components show clustering of the diabetic phenotype, muscle lipid-related variables, and representation of a diabetic group (D − I). A: scatterplot of all variables. The variables grouped in 3 major clusters (encircled) are described in results. B: for better visibility, the variables from clusters 1 and 2 are displayed. PC1, principal component 1 axis; PC2, principal component 2 axis.
Fig. 7.
Fig. 7.
Muscle content of LCFa-CoA is highly correlated with both muscle sarcoplasmic ceramide content and blood %Hb A1c value. Left: correlations between muscle total content of LCFa-CoA and total plasma FFA (A), total muscle ceramide (B), and muscle total sarcoplasmic ceramide (C). Right: relationship between blood %Hb A1c and the total content of muscle LCFa-CoA (D), muscle ceramide (E), and sarcoplamic ceramide (F). Linear regression scores (r2), Pearson r values, and their respective P values are shown next to the linear regression plots. ○, Nondiabetic C animals; gray circles, D + I animals; ●, D − I animals.
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