De Novo Sphingolipid Biosynthesis Is Required for Adipocyte Survival and Metabolic Homeostasis

Abstract

Sphingolipids are a diverse class of essential cellular lipids that function as structural membrane components and as signaling molecules. Cells acquire sphingolipids by both de novo biosynthesis and recycling of exogenous sphingolipids. The individual importance of these pathways for the generation of essential sphingolipids in differentiated cells is not well understood. To investigate the requirement for de novo sphingolipid biosynthesis in adipocytes, a cell type with highly regulated lipid metabolism, we generated mice with an adipocyte-specific deletion of Sptlc1 Sptlc1 is an obligate subunit of serine palmitoyltransferase, the enzyme responsible for the first and rate-limiting step of de novo sphingolipid biosynthesis. These mice, which initially developed adipose tissue, exhibited a striking age-dependent loss of adipose tissue accompanied by evidence of adipocyte death, increased macrophage infiltration, and tissue fibrosis. Adipocyte differentiation was not affected by the Sptlc1 deletion. The mice also had elevated fasting blood glucose, fatty liver, and insulin resistance. Collectively, these data indicate that de novo sphingolipid biosynthesis is required for adipocyte cell viability and normal metabolic function and that reduced de novo sphingolipid biosynthesis within adipocytes is associated with adipocyte death, adipose tissue remodeling, and metabolic dysfunction.

Keywords: adipocyte; inflammation; lipodystrophy; serine palmitoyltransferase; sphingolipid.

FIGURE 1.

Generation of adipoSPTko mice. A, schematic of the sphingolipid metabolic pathway. The de novo biosynthesis portion is indicated by the green arrow. The uptake/recycling portion is indicated by the red arrow. B, schematic representation of the Sptlc1 targeting strategy. The structures of the WT Sptlc1 locus, the targeting vector, the Sptlc1 targeted allele (Sptlc1^fl(neo)), the Sptlc1^fl allele, and the Sptlc1 knock-out (KO) allele are shown. The locations of the 5′- and 3′-flanking probes are shown, along with the sizes of the HindIII and SpeI restriction digest fragments. C, Southern blotting analysis of HindIII- (left) and SpeI (right)-digested genomic DNA from embryonic stem cells hybridized with the indicated probe, showing correctly targeted clones (Targeted) and non-targeted clones (WT). D, relative mRNA expression, normalized to Gapdh mRNA expression, for Sptlc1 was determined by RT-qPCR in liver, BAT, and inguinal fat of 4–5-week-old Sptlc1^fl/fl control and adipoSPTko mice. Data represent means ± S.D. Student's t test, n = 9 for each genotype; *, p ≤ 0.05; **, p < 0.01.

FIGURE 2.

Sphingolipids in adipose tissue of adipoSPTko mice. Sphingolipid levels were determined by HPLC-tandem MS on lipid extracts from gonadal adipose tissue from 4-week-old (A and B) and 5-month-old (C and D) Sptlc1^fl/fl control and adipoSPTko mice. A and C, levels of individual ceramide species with different fatty acid chain lengths (Cer), dihydrosphingosine (dhSph), sphingosine (Sph), dihydroS1P, and S1P. Inset, total ceramide. B and D, levels of sphingomyelin (SM) species with different acyl-chain lengths. Inset, total sphingomyelin. Data represent means ± S.D. A and B, n = 7 for each genotype; C and D, n = 5 control and n = 7 adipoSPTko. Student's t test, *, p < 0.05; ** p < 0.01; *** p < 0.001.

FIGURE 3.

Metabolite levels in adipose tissue and serum of adipoSPTko mice. Triglycerides (A), phosphatidylcholine (PC) (B), and free fatty acid (FFA) (C) levels of gonadal adipose tissue extracts, normalized to DNA, were determined in 4-week-old mice, n = 3 mice per genotype. Free fatty acid (D) and glycerol (E) levels were determined in the serum from 3-month-old mice, n = 5 control, and n = 7 adipoSPTko. F, sphingomyelin levels were determined in serum (4-week-old mice, n = 6 each genotype). Data represent means ± S.D. Student's t test, ***, p < 0.001.

FIGURE 4.

Adipose tissue in adipoSPTko mice. A and C, fat mass and lean mass weight as a percentage of total body weight, measured by EchoMRI, in 1- and 4-month-old Sptlc1^fl/fl control and adipoSPTko mice. B and D, weight of excised gonadal adipose tissue, inguinal adipose tissue, interscapular BAT, and liver in 1- and 4-month-old control and adipoSPTko mice. Data represent means ± S.E. Student's t test, 1-month-old (A): n = 13 control and n = 9 adipoSPTko; 1-month-old (B): n = 9 each genotype; 4-month-old (C and D): n = 7 control and n = 5 adipoSPTko; *, p < 0.05; **, p < 0.01; ***, p < 0.001. E, photographs of gonadal adipose tissue (top) and interscapular BAT (bottom) before excision from 4-month-old mice. F, photographs of gonadal adipose tissue (top) and interscapular BAT (bottom) after excision from 4-month-old mice. G, average daily food intake. 4-Week-old mice: n = 4 control and n = 5 adipoSPTko; 4-month-old mice: n = 5 control and n = 4 adipoSPTko.

FIGURE 5.

Adipocyte development in adipoSPTko mice. A, H&E-stained paraffin sections of gonadal adipose tissue from 1- and 5-month-old Sptlc1^fl/fl control and adipoSPTko mice. Scale bar, 100 μm. B, quantitation of adipocyte size in 5-month-old control and adipoSPTko mice from A. Data represent means ± S.E., n = 4. C–E, primary SVF cells isolated from gonadal adipose tissue of 3-month old control and adipoSPTko mice were differentiated under adipogenic conditions for 8 days. C, representative image of Oil Red O staining. Scale bar, 100 μm. D, quantification of lipid accumulation by Oil Red O recovery. Data represent means ± S.D., n = 3. E, relative mRNA levels for Sptlc1 and adipogenic genes (Pparγ, aP2, perilipin, and adiponectin) were determined by quantitative RT-PCR in differentiated SVF cell cultures. The control value was set to 1. Data represent means ± S.D., Student's t test, n = 3; **, p < 0.01. F, in vitro differentiation of adipocytes in charcoal-treated FBS. Primary SVF cells isolated from gonadal adipose tissue of 1-month-old control and adipoSPTko mice were differentiated under adipogenic conditions using charcoal-treated FBS for 8 days. Quantification of lipid accumulation was by Oil Red O recovery. Data represent means ± S.D., n = 3.

FIGURE 6.

Adipocyte death, tissue inflammation, and fibrosis in adipoSPTko mice. A, Mac-2 immunostaining in gonadal adipose tissue of 2- and 5-month-old Sptlc1^fl/fl control and adipoSPTko mice. Scale bar, 50 μm. B, perilipin immunostaining in gonadal adipose tissue of 2- and 5-month-old control and adipoSPTko mice. Asterisks denote degenerating perilipin-free adipocytes. Scale bar, 20 μm. C, paraffin sections of gonadal adipose tissue from 2- and 5-month-old control and adipoSPTko mice were stained with Sirius Red to visualize fibrotic accumulation of extracellular collagen. Scale bar, 100 μm. D, transmission electron micrographs of adipocytes from gonadal adipose tissue from 2-month-old control and adipoSPTko mice showing the presence of caveolae (arrows). Scale bar, 0.5 μm. E, quantitation of caveolae on adipocytes from 2-month-old control and adipoSPTko mice. Plasma membrane connected caveolae were counted on transmission electron micrographs images of adipocytes and normalized to plasma membrane surface area. Data represent means ± S.D., n = 3 for each genotype, 5–18 adipocytes per mouse. F, gene expression analysis of immune system and inflammatory genes in gonadal adipose tissue from 5-month-old control and adipoSPTko mice (n = 4). The heat map shows the raw signal values of genes that were significantly increased in adipoSPTko mice, using a cutoff of p < 0.05 and a fold-change of greater than 2 (29/84 genes).

FIGURE 7.

Metabolic profile of adipoSPTko mice. A, leptin, adiponectin, triglycerides, and cholesterol were measured in the serum of 4-month-old Sptlc1^fl/fl control and adipoSPTko mice. Data represent means ± S.D. Student's t test, n = 3; **, p < 0.01; ***, p < 0.001. B, frozen sections of liver from 5-month-old control and adipoSPTko mice were stained with Oil Red O. Scale bar, 100 μm. C and D, insulin tolerance and glucose tolerance tests (ITT and GTT, respectively) of 3-month-old control and adipoSPTko mice. Data represent means ± S.D. Student's t test, n = 3 for each genotype; *, p < 0.05. E, cold tolerance test (CTT) of 3-month-old control and adipoSPTko mice. Data represent means ± S.D., n = 4 for each genotype.