SPTLC3 regulates plasma membrane sphingolipid composition to facilitate hepatic gluconeogenesis

Abstract

SPTLC3, an inducible subunit of the serine palmitoyltransferase (SPT) complex, causes production of alternative sphingoid bases, including a 16-carbon dihydrosphingosine, whose biological function is only beginning to emerge. High-fat feeding induced SPTLC3 in the liver, prompting us to produce a liver-specific knockout mouse line. Following high-fat feeding, knockout mice showed decreased fasting blood glucose, and knockout primary hepatocytes showed suppressed glucose production, a core function of hepatocytes. Stable isotope tracing revealed suppression of the gluconeogenic pathway, finding that SPTLC3 was required to maintain expression of key gluconeogenic genes via adenylate cyclase/cyclic AMP (cAMP)/cAMP response element binding protein (CREB) signaling. Additionally, by employing a combination of a recently developed lipidomics methodology, exogenous C14/C16 fatty acid treatment, and in situ adenylate cyclase activity, we implicated a functional interaction between sphingomyelin with a d16 backbone and adenylate cyclase at the plasma membrane. This work pinpoints a specific sphingolipid-protein functional interaction with broad implications for understanding sphingolipid signaling and metabolic disease.

Keywords: CP: Metabolism; MAFLD; SPT; SPTLC3; adenylate cylcase; ceramide; cyclic-AMP; gluconeogenesis; metabolic disease; sphingolipid; sphingomyelin.

Figure 1.. Male SPT3-hKO and Alb-Cre controls were placed on an HFD for 16 weeks and assessed for metabolic phenotype.

(A) SPTLC3 expression in liver tissue homogenate of mice following high-fat diet (HFD) feeding versus low-glycemic control diet (CD) feeding. Expression was quantified by ΔΔcq method with a reference gene panel consisting of β-actin, Tbp1, and Hmbs1. (B) Representative images of H&E staining of liver tissue. Scale bars: 200 μm. (C) Quantification of macrosteatosis observed in histology images. (D) Liver weight. (E) TGs in liver homogenate. (F) Liver glycogen following HFD feeding. (G) Blood glucose following a 6 h fast. (H) ipGTT. (I) Area under the curve for the GTT. (J) Serum insulin. All graphical data are represented as mean ± SEM. The p values were calculated by Student’s t test. *p < 0.05, **p < 0.01, ****p < 0.001; n = 5 biological replicates.

Figure 2.. Evaluation of gluconeogenesis in primary mouse hepatocytes isolated from SPT3-hKO and Alb-Cre mice.

(A) Glucose levels in medium produced by Alb-Cre and SPT3-hKO primary hepatocytes after 6 h. *p < 0.05 by t test. n = 5 biological replicates. (B) Expression by real-time qPCR of gluconeogenic genes following treatment with 100 nM glucagon (GC) for 3 h. Expression was measured by ΔΔcq method with a reference gene panel consisting of β-actin, Hmbs1, and Tbp1. Significance was determined by ANOVA. **p < 0.01, *p < 0.05. The dashed line indicates p < 0.01 for control versus GC for both Alb-Cre and SPT3-hKO. n = 3 biological replicates. (C) Western blot showing expression of PCK1 protein with GC treatment. This blot is representative of triplicate measurements. (D) Western blot showing P-CREB (Ser-133) phosphorylation in response to a GC dose of 0, 5, 10, 25, 50, or 100 nM. This blot is representative of triplicate measurements. (E) Stable isotopic labeling of metabolites feeding gluconeogenesis via the TCA cycle. Hepatocytes isolated from Alb-Cre and SPT3-hKO mice were treated with 4 mM U¹³C-glutamine for 6 h in DMEM without glucose, glutamine, or phenol red. Universally labeled target metabolites were targeted for analysis to assess the first pass through the TCA cycle. Cellular metabolite levels are expressed relative to total protein. The p values were calculated by t test. *p < 0.05, n = 6 biological replicates. (F) Expression of amino acid transporters in primary mouse hepatocytes by ΔΔcq method with a reference gene panel consisting of β-actin, Hmbs1, and Tbp1. Significance was determined by t test. **p < 0.01, n = 3 biological replicates. All graphical data are represented as mean ± SEM.

Figure 3.. Evaluation of GC-driven cyclic AMP signaling in primary hepatocytes isolated from SPT3-hKO and Alb-Cre mice.

(A) cAMP levels measured by ELISA following treatment of primary mouse hepatocytes with 100 nM GC. Significance was determined by t test. **p < 0.01, ****p < 0.0001. n = 3 biological replicates. (B) Expression by real-time qPCR of key gluconeogenic genes following treatment with cell-permeable cAMP chlorophenylthiol-cAMP (pCPT-cAMP). Expression was measured by ΔΔcq method with a reference gene panel consisting of β-actin, Hmbs1, and Tbp1. The dashed line indicates the level of significance between treated and control samples within each genotype. Significance was determined by ANOVA. *p < 0.05, ****p < 0.0001, n = 3 biological replicates. (C) Successful isolation of PM from whole liver was validated by western blot of sodium-potassium ATPase (Na⁺, K⁺ ATPase) in lysate (Lys), supernatant (Sup), and plasma membrane (PM) fractions. Replicate low- and high-exposure images capture the Lys and PM fraction bands, respectively. This blot is representative of the 5 replicate PM extractions used to generate AC activity data shown in (E). (D) In vitro AC activity was determined by measuring cAMP produced by partially purified PM extracts from whole liver. Positive control (PC) samples consisted of pooled PM extracts treated with an AC activator (24 μM forskolin). NC samples were prepared with PM extracts and no ATP substrate. Sample blanks (SBs) contained no PM extract. ****p < 0.0001; significance was determined by t test; n = 3 technical replicates. (E) AC activity of partially purified PM fractions from isolated livers of 5 individual HFD-fed mice. *p < 0.05; significance was determined by t test; n = 5 biological replicates. (F) In situ AC activity on hepatocytes isolated from Alb-Cre and SPT3-hKO littermate controls. Cells were pretreated for 24 h with 250 μM palmitate (PA), myristate (MA), or vehicle (0.25% DMSO in 2% fatty acid free BSA). Fatty acids were removed and replaced with 100 nM GC plus a phosphodiesterase/ATPase inhibitor cocktail. After a 30 min of pre-treatment with GC/inhibitors, 5 mM isotopically labeled ATP was added for an additional 30 min, and the reaction was quenched with 80% methanol. The isotopic cAMP product was quantified by LC-MS/MS, and specific activity was calculated using total protein. Significance was determined by individual t tests. **p < 0.01, n = 3 biological replicates. All graphical data are represented as mean ± SEM.

Figure 4.. Lipidomics analysis revealing sphingoid base composition of PM sphingomyelin pools in primary hepatocytes isolated from SPT3-hKO and Alb-Cre mice.

(A) Structures of ceramides and sphingomyelin, highlighting the range of reported side chains incorporated into the d16 or d18 sphingoid backbone. Current methods for quantification of sphingomyelins do not distinguish between side chain versus backbone. (B) Schematic of the MS/MS transition used to identify sphingomyelin peaks, illustrating the limitations of standard methods for determining backbone and side-chain composition. All illustrations were produced with BioRender. (C) Schematic of bacterial sphingomyelinase (bSMase) post treatment, which allows the identification of the backbone and side-chain structures of PM sphingomyelin in primary mouse hepatocytes. (D) Total sphingomyelin levels determined by LC-MS/MS on Alb-Cre and SPT3-hKO primary mouse hepatocytes with control or bSMase post treatment. Total cellular SM is shown in Alb-Cre and SPT3-hKO without post treatment. Alb-Cre+bSMase and SPT3-hKO+bSMase reveal the amount of SM after PM SM has been converted to ceramide. The difference between the two groups reveals the initial level of PM SM. Individual SM species are labeled by total carbons and double bonds. (E) D16.1-based ceramide levels determined by LC-MS/MS on Alb-Cre and SPT3-hKO primary mouse hepatocytes with control or bSMase post treatment. The p values were determined by ANOVA. **p < 0.05, ****p < 0.0001. n = 5 biological replicates. All data are represented as mean ± SEM.