Protection from high fat diet-induced increase in ceramide in mice lacking plasminogen activator inhibitor 1

Abstract

Obesity increases the risk for metabolic and cardiovascular disease, and adipose tissue plays a central role in this process. Ceramide, the key intermediate of sphingolipid metabolism, also contributes to obesity-related disorders. We show that a high fat diet increased ceramide levels in the adipose tissues and plasma in C57BL/6J mice via a mechanism that involves an increase in gene expression of enzymes mediating ceramide generation through the de novo pathway (e.g. serine palmitoyltransferase) and via the hydrolysis of sphingomyelin (acid sphingomyelinase and neutral sphingomyelinase). Although the induction of total ceramide in response to the high fat diet was modest, dramatic increases were observed for C16, C18, and C18:1 ceramides. Next, we investigated the relationship of ceramide to plasminogen activator inhibitor-1 (PAI-1), the primary inhibitor of plasminogen activation and another key player in obesity. PAI-1 is consistently elevated in obesity and thought to contribute to increased artherothrombotic events and more recently to obesity-mediated insulin resistance. Interestingly, the changes in ceramide were attenuated in mice lacking PAI-1. Mechanistically, mice lacking PAI-1 were protected from diet-induced increase in serine palmitoyltransferase, acid sphingomyelinase, and neutral sphingomyelinase mRNA, providing a mechanistic link for decreased ceramide in PAI-1-/- mice. The decreases in plasma free fatty acids and adipose tumor necrosis factor-alpha in PAI-1-/- mice may have additionally contributed indirectly to improvements in ceramide profile in these mice. This study has identified a novel link between sphingolipid metabolism and PAI-1 and also suggests that ceramide may be an intermediary molecule linking elevated PAI-1 to insulin resistance.

Figures

FIGURE 1.

Weight gain and ceramide expression in plasma and adipose tissue of C57BL/6J mice in response to a HFD. Fat pad weight (A) and ceramide levels in plasma (total (B) and ceramide species (C)) and adipose tissues (total (D), ceramide species in epididymal fat (E), and ceramide species in subcutaneous fat (F)) were determined from C57BL/6J mice placed on a LFD or HFD for 16 weeks. For B and D, total ceramide is the sum of individual ceramide species. In all panels, data are means (n = 6) ± S.D. For B, plasma ceramide in LFD versus HFD, p < 0.01. For D, epididymal fat pad LFD versus HFD, p < 0.05; subcutaneous fat pad LFD versus HFD, p < 0.001.

FIGURE 2.

Adipose tissue expression of enzymes involved in ceramide generation and hydrolysis. SPTLC2 (A), SPTLC3 (B), ASMase (C), NSMase (D), SMase activity (E) and alkaline ceramidase (F) mRNA levels were determined by real time reverse transcription-PCR from epididymal and subcutaneous fat pads of C57BL/6J mice after 16 weeks on an LFD or HFD. E, SMase activity in adipose tissues of C57BL/6J mice after 16 weeks on a LFD or HFD was determined using the Amplex Red sphingomyelinase assay kit. In all panels, data are means (n = 6) ± S.D.

FIGURE 3.

Weight gain and fat accumulation in WT and PAI-1–/– mice after 16 weeks on a LFD or HFD. A, PAI-1 mRNA expression as determined by real time reverse transcription-PCR in epididymal and subcutaneous fat pads. Data are means (n = 6) ± S.D. For both epididymal and subcutaneous fat pads, HFD versus LFD, p < 0.01. B, body weight gain in WT and PAI-1–/– mice. Data are means (n = 8) ± S.D. WT versus PAI-1–/– mice on HFD, p < 0.01. C, abdominal subcutaneous fat pad weights of WT and PAI-1–/– mice. Data are means (n = 8) ± S.D. WT versus PAI-1–/– mice on HFD, p < 0.01. D, epididymal fat pad weights of WT and PAI-1–/– mice. Data are means (n = 8) ± S.D. WT versus PAI-1–/– mice on HFD, p < 0.05. E, hematoxylin and eosin-stained histological sections (×200) of epididymal and subcutaneous fat pads from WT and PAI-1–/– mice. Results are representative of n = 6 in each group. Upper panels, epididymal fat; lower panels, subcutaneous fat.

FIGURE 4.

Plasma and adipose ceramide levels in WT and PAI-1–/– mice after 16 weeks on a LFD or HFD. Ceramide levels in plasma (total (A) and ceramide species (B)) and adipose tissues (total (C), ceramide species in epididymal fat (D), and ceramide species in subcutaneous fat (E)) were determined from WT and PAI-1–/– mice. F, Lass1/CerS1 gene expression in epididymal adipose tissues of WT and PAI-1-deficient mice. For A and C, total ceramide is the sum of individual ceramide species, and data are means (n = 6) ± S.D. For A, LFD versus HFD in WT mice, p < 0.01; WT versus PAI-1–/– mice on HFD, p < 0.05. For C, epididymal fat pad LFD versus HFD in WT mice, p < 0.05; epididymal fat pad WT versus PAI-1–/– on HFD, p < 0.001; subcutaneous fat pad LFD versus HFD in WT mice, p < 0.001; subcutaneous fat pad WT versus PAI-1–/– on HFD, p < 0.001; subcutaneous fat pad WT versus PAI–/–mice on LFD, p < 0.01.

FIGURE 5.

Adipose tissue gene expression of enzymes involved in ceramide generation and hydrolysis in WT and PAI-1–/– mice. SPTLC2 (A), SPTLC3 (B), ASMase (C), NSMase (D), and alkaline ceramidase (E) mRNA levels were determined by real time reverse transcription-PCR from epididymal and subcutaneous fat pads of WT and PAI-1–/– mice after 16 weeks on a LFD or HFD. For all panels, data are means (n = 6) ± S.D.

FIGURE 6.

Plasma FFA and adipose TNF-α expression in WT and PAI-1–/– mice. Plasma free fatty acids (A) and adipose TNF-α mRNA expression (B) were measured in WT and PAI-1–/– mice after 16 weeks on an LFD or HFD. For all groups in A and B, data are means (n = 6) ± S.D. For TNF-α expression, epididymal fat, LFD versus HFD in WT mice, p < 0.001; epididymal fat pad, WT versus PAI-1–/– on HFD, p < 0.01. Subcutaneous fat, LFD versus HFD in WT mice, p < 0.05; WT versus PAI-1–/– on HFD, p < 0.05.

FIGURE 7.

Glucose homeostasis in WT and PAI-1–/– mice. Plasma glucose (A) and plasma insulin (B) were measured after 16 weeks on the high or low fat diet in WT and PAI-1–/– mice. Data are means (n = 8) ± S.D. Plasma glucose, LFD versus HFD in WT mice, p < 0.05; WT versus PAI-1–/– mice on HFD, p < 0.05. Plasma insulin, LFD versus HFD in WT mice, p < 0.05; LFD versus HFD in PAI-1–/– mice, p < 0.01; WT versus PAI-1–/– mice on HFD, p < 0.01; WT versus PAI-1–/– mice on LFD, p < 0.05. C, glucose tolerance test. Mice fasted for ∼6 h were injected with a bolus of glucose, and blood samples were obtained at the indicated time and analyzed for glucose levels. D, insulin tolerance test. Mice were injected with insulin (0.75 units/kg of body weight), blood samples were obtained at the indicated times, and glucose levels were monitored. Data in A–D are means (n = 6) ± S.D.