The gut microbiota plays a key role in host metabolism. Toll-like receptor 5 (TLR5), a flagellin receptor, is required for gut microbiota homeostasis. Accordingly, TLR5-deficient (T5KO) mice are prone to develop microbiota-dependent metabolic syndrome. Here we observed that T5KO mice display elevated neutral lipids with a compositional increase of oleate [C18:1 (n9)] relative to wild-type littermates. Increased oleate contribution to hepatic lipids and liver SCD1 expression were both microbiota dependent. Analysis of short-chain fatty acids (SCFAs) and 13 C-acetate label incorporation revealed elevated SCFA in ceca and hepatic portal blood and increased liver de novo lipogenesis in T5KO mice. Dietary SCFAs further aggravated metabolic syndrome in T5KO mice. Deletion of hepatic SCD1 not only prevented hepatic neutral lipid oleate enrichment but also ameliorated metabolic syndrome in T5KO mice. Collectively, these results underscore the key role of the gut microbiota-liver axis in the pathogenesis of metabolic diseases.

In this study, we hypothesized that T5KO mice, which exhibit microbiota-dependent metabolic syndrome, may have altered hepatic lipogenesis that might promote an obese phenotype. Accordingly, we demonstrate that T5KO mice displayed elevated hepatic C18:1 in neutral lipids when compared to wild-type (WT) littermates. This difference was not seen in microbiota-ablated and germ-free (GF) T5KO mice. 1 H NMR metabolic profiling of cecal contents indicated decreased cecal oligosaccharides and elevated SCFA, suggesting profound changes in bacterial fermentation. Further, colonic SCFA receptors, hepatic lipogenic enzymes including SCD1, and pro-inflammatory genes were upregulated in T5KO mice. Using 13 C-acetate labeling, we showed that gut-derived SCFA reached to the liver and incorporated into both liver and plasma palmitate (C16:0) relatively to a greater extent in T5KO mice. Dietary SCFA further aggravated metabolic syndrome while hepatic deletion of SCD1 in T5KO mice prevented most of the indices of metabolic syndrome. Hence, the ability of T5KO mice to synthesize higher MUFA, probably as a “metabolic adaptation” to protect from lipotoxicity of SFA synthesized from SCFA but then potentiate metabolic syndrome. Even though numerous beneficial properties have been attributed to SCFA, their excess in conditions of innate immune deficiency coupled with bacterial overgrowth may increase susceptibility to metabolic diseases.

SCD1 negatively regulates inflammation possibly by controlling the homeostasis of MUFA and SFA. Specifically, inhibition of SCD1 exacerbates responses to exogenous pro-inflammatory challenges such as lipopolysaccharide (LPS), C. rodentium, and a chemical colitogen DSS (). Further, accumulation of SFA in Scd1KO mice promote inflammation via TLR4 signaling and promote atherosclerosis (), whereas loss of SCD1 attenuates adipose tissue inflammation (), suggesting that its activity in diverse tissues has distinct outcomes.

Microbiota exerts a substantial influence on host lipid metabolism (). One such pathway is the generation of short-chain fatty acids (SCFAs) such as acetate, butyrate, and propionate via anaerobic fermentation of dietary fiber. SCFAs not only serve as a major fuel (i.e., butyrate) for the colonocytes but also are absorbed via enterohepatic circulation and contribute to hepatic lipogenesis (). The major de novo synthesized lipotoxic saturated fatty acids (SFAs) in the liver are palmitic acid (C16:0) and stearic acid (C18:0), which are converted into the less lipotoxic monounsaturated fatty acids (MUFAs) palmitoleate (C16:1 n7) and oleate (C18:1 n9), a steatogenic agent. The addition of a double bond is catalyzed by hepatic microsomal, lipogenic rate-limiting enzyme stearoyl CoA desaturase-1 (SCD1), a key regulatory enzyme in the homeostasis of SFA and MUFA (). Despite oleate being a major MUFA from the diet, the expression of SCD1 is highly regulated by developmental, dietary, hormonal, and environmental factors. MUFA participates in the regulation of diverse processes, serve as better substrates for the synthesis of hepatic neutral lipids (triglycerides [TGs] and cholesterol esters [CEs]), and thus play a role in increasing tissue lipid load and may initiate insulin resistance (). Accordingly, SCD1 deficiency protects against high-fat-, high-carbohydrate-, and leptin-deficiency-induced obesity and hepatic steatosis (). Leptin-deficient mice exhibit increased SCD1 expression and associated with elevated levels of C16:1 and C18:1 in liver lipids.

The biosynthesis of hepatic cholesterol esters and triglycerides is impaired in mice with a disruption of the gene for stearoyl-CoA desaturase 1.

Metabolic syndrome is a constellation of metabolic abnormalities associated with insulin resistance that leads to metabolic diseases such as obesity, type II diabetes, and hepatic and cardiovascular diseases. The etiology of metabolic syndrome is influenced by genetics and a number of environmental factors including gut microbiota (). Potential mechanisms by which the microbiota influences host metabolism include increased calorie extraction (), metabolic endotoxemia (), and generation of toxic metabolites (). Previously, we have demonstrated that mice lacking TLR5 (T5KO) are susceptible to spontaneous gut inflammation and metabolic syndrome and exhibit microbiotal dysbiosis (). Further, T5KO mice maintained on a high-fat diet (HFD) develop hepatic steatosis and insulitis. Specifically, the gut microbiota in T5KO mice induces low-grade, chronic, systemic inflammation resulting in desensitization of insulin receptors culminating in hyperphagia and metabolic syndrome ().

T5KO mice displayed substantial upregulation of hepatic SCD1 as well as increased presence of its product C18:1 in the hepatic lipid, suggesting its potential involvement in metabolic syndrome. Therefore, to investigate the role of hepatic SCD1, we generated liver-specific Scd1-deficient T5KO mice (T5KO-Scd1) and monitored for indices of metabolic syndrome in comparison to Scd1 floxed T5KO mice (T5KO-Scd1). Most notably, T5KO-Scd1mice exhibited reduced body weight and abdominal adiposity coupled with improved insulin resistance when compared to T5KO-Scd1mice ( Figures 7 A–7D). Even though serum insulin, TC, and TG levels were unaltered, hepatic TGs and CEs were reduced in T5KO-Scd1 Figures 7 E–7I). However, there was no change in hepatic FC ( Figure 7 J), indicating that non-hepatic SCD1 activity might be able to maintain serum lipids. As expected, liver total, TGs, and CE C18:1 level was substantially reduced ( Figures 7 K–7M) with a concomitant increase in total hepatic C18:0 and total C18:2 in T5KO-Scd1 Figures 7 N and 7O). Moreover hepatic palmitoleate (C16:1), was reduced in T5KO-Scd1mice (2.27 ± 0.07, T5KO-Scd1versus 1.96 ± 0.09, T5KO-Scd1). The hepatic transcripts of pro-inflammatory genes and the marker of insulin resistance Foxo1 were reduced in T5KO-Scd1mice ( Figure 7 P). Additionally, protein level of GLUT4, an insulin-responsive gene, was augmented in skeletal muscle of T5KO-Scd1mice ( Figure S7 A), suggesting that liver Scd1 deletion in T5KO mice is able to rescue the skeletal muscle insulin resistance. However, GLUT4 expression was unaltered in adipose tissue of T5KO-Scd1in comparison to T5KO-Scd1mice (data not shown). Moreover, expression of lipogenic genes remained unaltered in liver, adipose, and skeletal muscle of T5KO-Scd1mice ( Figures 7 P, S7 B, and S7C, respectively). Similarly, the expression of colonic SCFA transporters and pro-inflammatory genes along with colonic MPO activity, food intake, and gut bacterial load remained unaltered in T5KO-Scd1mice when compared to T5KO-Scd1mice ( Figures S7 D–S7G). As expected, C18:1 level was reduced in hepatic total and lipid fractions in WT-Scd1mice when compared to WT-Scd1. However, we did not observe any significant change in the metabolic-syndrome-related parameters between these groups ( Figures 7 A–7P). Altogether, our results suggest that loss of TLR5 results in a microbiota-generated increase in SCFAs. In turn, the increase in SCFA may drive hepatic SCD1-mediated lipogenesis, consequently promoting insulin resistance and inflammation in T5KO mice.

(A–G) (A) Immunoblot (upper panel) and bar graph representing the hepatic expression of SCD1 at protein and mRNA level, respectively. (B) Body weight, (C) percent fat pad, (D) insulin sensitivity test, (E) fasting serum insulin, (F) serum TC, and (G) serum TG. Hepatic lipids were analyzed in 5 hr fasted mice.

We next analyzed how SCFA supplementation impacted the liver lipids. Similar to data from the serum, T5KO mice fed SCFA showed an increase in liver TG and CE except liver FC ( Figures 6 I–6K) compared to vehicle-treated T5KO mice. Interestingly, dietary SCFA further increased liver SCD1 in T5KO mice ( Figure 6 L). Serum insulin remains unaltered in SCFA-fed T5KO mice ( Figure 6 E), suggesting that the increased hepatic SCD1 in these mice could be due to more SCFA availability as substrate. Further, dietary SCFA increased hepatic total C18:1, TG C18:1 and CE C18:1 in T5KO mice when compared vehicle given mice ( Figures 6 M–6O). Hepatic palmitoleate (C16:1) was also elevated in T5KO mice that received SCFA (2.36 ± 0.19, T5KO versus 2.91 ± 0.1, T5KO+SCFA). However, no change in adipose tissue fatty acid composition was observed in T5KO mice given SCFA (data not shown). Expression of hepatic inflammatory genes and lipogenic genes in liver ( Figure 6 P), adipose, and skeletal muscles ( Figures S6 A and S6B) were unaltered in SCFA-fed T5KO mice. Moreover, colonic expression of SCFA receptors/transporters and pro-inflammatory genes also remained unaltered, with the exception of Lcn2, which is reduced, in T5KO mice fed SCFAs ( Figure S6 C). Additionally, colonic MPO activity ( Figure S6 D) and gut bacterial load ( Figure S6 E) remained unchanged in T5KO mice fed with SCFAs.

Dietary SCFA can regulate gut inflammation (). SCFA produced in the cecum partake in hepatic lipogenesis in both mice and human (). Therefore, we next asked whether dietary SCFA would protect from low-grade inflammation or promote metabolic syndrome in T5KO mice. We administered a combination of acetate, butyrate, and propionate in drinking water for 21 days, as previously described (), and observed that T5KO mice on SCFA treatment exhibited further increase in body weight, fat pad, blood glucose, and insulin resistance ( Figures 6 A–6D) compared to vehicle-treated T5KO mice. In agreement with a recent report (), we observed a significant reduction in food intake in both WT and T5KO mice receiving dietary SCFAs ( Figure 6 F). Dietary SCFAs further elevated serum TC and TG in T5KO mice ( Figures 6 G and 6H). Interestingly, no significant differences were observed in the above parameters in WT mice with or without dietary SCFA ( Figures 6 A–6H) except reduced food intake. Taken together, these results suggest that SCFA participate in hepatic lipogenesis and contributing to insulin resistance in T5KO mice.

(I–P) (I) TG, (J) CE, (K) FC, (L) immunoblot represents hepatic SCD1, (M) C18:1 in total lipid, (N) C18:1 in CE, (O) C18:1 in TG, and (P) mRNA expression of hepatic lipogenic, inflammatory, and insulin resistance genes. Data are represented as mean ± SEM. ∗ p < 0.05.

Age-matched male T5KO mice and their WT littermates (16 week old, n = 6) received either a mixture of SCFA (67.5 mM sodium acetate, 40 mM sodium butyrate, and 25.9 mM sodium propionate) or equimolar sodium chloride in drinking water for 21 days.

Gut microbiota plays a key role in host energy homeostasis by generating SCFA via fermentation of dietary fiber, which serves as substrate for lipid biosynthesis (). The SCFAs are utilized by colonocytes or absorbed efficiently in the colon (). Therefore, we performed metabolic profiling of intestinal contents byH NMR. As shown in Figures 5 A–5E, cecal contents of T5KO mice displayed increased SCFA, specifically butyrate and propionate. Similar results were observed in calorie restricted T5KO mice (data not shown). Surprisingly, there was no significant difference observed in the major luminal SCFA acetate in the cecal contents of both strains. As expected, fecal metabolites byH NMR and fatty acid analysis by GC did not reveal any significant differences in WT and T5KO mice ( Figure S5 A; Table S2 ), indicating SCFA produced in ceca were absorbed in the colon. In support of this notion, we observed elevated colonic mRNA transcripts of SCFA receptors (G protein-coupled receptor (Gpr)41 and 43; Figure 5 F) and increased SCFA (propionate and butyrate) in the hepatic portal vein serum of T5KO mice ( Figure 5 G). However, no change in Gpr109A and Na-coupled transporter of SCFA Slc5a8 was observed between WT and T5KO mice ( Figures S5 B and S5C). These results suggest that T5KO mice, which have an increased microbial burden, utilize more oligosaccharides and generate higher levels of SCFA. To extend our demonstration how increased availability of SCFA to liver affects de novo lipogenesis (DNL), we fedC-acetate to WT and T5KO mice to measure label incorporation into liver and plasma lipids. The feeding achieved an overall enrichment of the C16:0-McLafferty ion(m/Z = 75) of 1.17% ± 0.07% above background. Under these conditions, the proportion of label appearing in C16:0 was increased in T5KO mice ( Figures 5 H and 5I), indicating SCFA contributes a greater fraction of TG in both the liver and plasma pools. This is apparent in serum TG of T5KO mice ( Figure 5 I), where increased serum TG-C16:0 results from an increased utilization of SCFA above that observed in WT. This indicates that the increased delivery of SCFA to hepatocytes combines with the observed increase in synthetic capacity ( Figure 2 ) to result in increased DNL in T5KO mice.

(H) Fractional enrichment of C16:0 using C16:0 as the primary end product of DNL and the fractional enrichment as an estimate of fractional synthesis.

(C–E) (C) OPLS-DA score plot (left) and correlation coefficient loading plot (right) showing the discrimination between 1 H NMR spectra of cecal contents from T5KO and WT mice, respectively (|r| cutoff value is 0.602, n = 10 for WT group and n = 11 T5KO mice group; C. R 2 X = 0.56, Q 2 = 0.58; CV-ANOVA: p = 1.89 × 10 −3 ). Keys: 1, n-butyrate; 2, propionate; 3, isoleucine; 4, leucine; 5, valine; 6, ethanol; 7, lactate; 8, alanine; 9, lysine; 10, acetate; 11, proline; 12, bile acids; 13, succinate; 14, pyruvate; 15, trimethylamine (TMA); 16, creatine; 17, α-ketoglutarate; 18, methanol; 19, oligosaccharides & amino acids; 20, α-glucose; 21,raffinose; 22, stachyose; 23, uracil; 24, taurine; 25, glycine; 26, uridine; 27, fumarate; 28, tyrosine; 29, choline; 30, methionine; 31, phenylalanine; 32, histidine; 33,urocanate; 34, adenine; 35,adenosine; 36, nicotinurate. Relative abundance of (D) SCFA (propionate and butyrate) and (E). oligosaccharides, glucose, raffinose, and stachyose in cecal content extracts from WT and T5KO mice.

In a similar line, we asked whether the cecal microbiota of T5KO mice is sufficient to modulate hepatic lipogenesis upon transplant to GF-WT recipients. Cecal microbiota transplantation influenced hepatic lipid MUFA levels in GF-WT mice that received either WT or T5KO cecal microbiota. As shown in Figures 4 J–4L, total lipid, TG, and CE C18:1 levels in GF-WT mice that received T5KO cecal microbiota were significantly higher than the WT cecal microbiota recipients. As observed in conventional mice, transplantation of cecal microbiota resulted in a greater than 50% increase in C18:1 level in GF-WT recipients. However, this effect was more profound in T5KO mice cecal recipients, suggesting that the T5KO microbiota is sufficient to induce hepatic neutral lipid C18:1enrichment ( Figures 4 J–4L).

These observations were further confirmed in GF-T5KO mice, and their WT littermates as GF-T5KO mice did not exhibit either symptoms of colitis or indices of metabolic syndrome ( Figures S4 A–S4E). Notably, hepatic TG, total C18:1 and C18:1 in TG and CE fractions in GF-T5KO mice were comparable to WT littermates ( Figures 4 E–4H). Interestingly, the levels of C18:1 (in both strains) was substantially reduced (∼50%) with concomitant reduction of hepatic SCD1 expression in microbiota-ablated and GF mice when compared to conventional mice ( Figure 4 I). These results suggest that the microbiota or its metabolites likely play a key role in de novo synthesis of hepatic MUFA.

TLR5 is predominantly expressed on gut epithelial cells and plays a key role in microbiota homeostasis (). Accordingly, T5KO mice exhibit 3-fold higher fecal bacterial load (data not shown). To demonstrate the role of the microbiota in liver lipogenesis, we eliminated ∼90% of bacteria via antibiotics. Microbiota ablation ameliorated the majority of symptoms of metabolic syndrome in T5KO mice and, moreover, normalized hepatic TG and C18:1 in total lipid and TG and CE fractions ( Figures 4 A–4D).

(J–L) (J) C18:1 in total lipid, (K) C18:1 in TG, and (L) C18:1 in CE. Data are represented as mean ± SEM. ∗ p < 0.05.

(E–I) (E) Liver TG, (F) C18:1 in total lipid, (G) C18:1 in TG, (H) C18:1 in CE, and (I) Immunoblot for hepatic SCD1 in conventional (conv), antibiotics-treated, and GF-T5KO mice and their WT littermates. Four-week-old male GF (Swiss Webster) WT mice (n = 5) were orally administered cecal microbiota from either WT or T5KO mice and housed in specific pathogen-free conditions. Hepatic C18:1 in lipid fractions was analyzed 6 weeks after transplantation.

(A–D) (A) Liver TG, (B) C18:1 in total lipid, (C) C18:1 in TG, and (D) C18:1 in CE. Hepatic lipids were analyzed in 12-week-old males (age- and sex-matched) GF-T5KO mice and their WT littermates (n = 7).

Age matched male T5KO mice and their WT littermates (4 week old, n = 5) were treated with broad-spectrum antibiotics (ampicillin and neomycin) in drinking water for 8 weeks and analyzed for liver lipids.

Metabolic syndrome in T5KO mice is largely driven by hyperphagia; calorie restriction for 12 weeks normalized most of the indices of metabolic syndrome in T5KO mice except insulin resistance (). Analysis of liver lipids and fatty acid profiles of calorie-restricted WT and T5KO mice exhibited an increase in liver TG and liver total C18:1 mainly in TG and CE fractions ( Figures 3 M–3P) of T5KO mice in comparison to WT, suggesting that an increase in hepatic lipids might be contributing to insulin resistance. Further, the PUFA C18:2 and C20:4 levels showed a decreasing trend in the liver total lipids and TG and CE fractions as observed in ad libitum fed mice (data not shown).

Diets rich in saturated fat are a risk factor for insulin resistance, obesity, and hepatic steatosis. Accordingly, we demonstrated exacerbated metabolic syndrome and hepatic steatosis in T5KO mice fed HFD (). Because of the substantial reduction in plant-derived fiber in HFD, we envisioned that such diets might influence hepatic lipids. After 8 weeks on a HFD, T5KO mice displayed hyperglycemia and insulin resistance ( Figures 3 A–3C) with concomitant increases in serum TC and TG ( Figures 3 D and 3E). Moreover, elevated liver TG, total C18:1, and C18:1 in the TG fraction ( Figures 3 F–3H) were observed in comparison to HFD-fed WT mice. Interestingly, the HFD did not increase C18:1 in the CE fraction of liver lipids in HFD-fed T5KO mice ( Figure 3 I), as was observed in T5KO mice fed lab chow. Another major difference observed with the HFD was a reduction in total lipid C18:2 in T5KO mice ( Figure 3 J). A decreasing trend in C20:4 in the liver total lipid fraction of T5KO mice was observed in comparison to WT littermates ( Figure 3 K), suggesting altered delta-6 desaturase activity. Additionally, mRNA transcripts of hepatic Fas, Scd1, Hmgcr, Foxo1, and Lcn2 were elevated in HFD-fed T5KO mice ( Figure 3 L). There were no significant differences in the transcript levels of lipogenic genes in adipose and skeletal muscles of HFD-fed T5KO mice ( Figures S3 A and S3B). Colonic expression of SCFA receptors and pro-inflammatory genes except iNos were also comparable to WT mice ( Figure S3 C). Similar results were obtained with HFD-fed male T5KO mice (data not shown).

(M–P) (M) Liver TG, (N) C18:1 in total lipid, (O) C18:1 in CE, and (P) C18:1 in TG. Data are represented as mean ± SEM. ∗ p < 0.05.

(L) mRNA expression of hepatic lipogenic, inflammatory, and insulin resistance genes in HFD-fed WT and T5KO mice. Age-matched female T5KO mice and their WT littermates (n = 5) were subjected to calorie restriction for 12 weeks and hepatic TG and C18:1 were analyzed after 5 hr of fasting.

(A–E) Age-matched female T5KO mice and their WT littermates (12 week old, n = 5) maintained on a HFD for 8 weeks and 5 hr fasting serum and liver were analyzed for following parameters: (A) blood glucose, (B) insulin sensitivity test, (C) fasting serum insulin, (D) serum TC, and (E) serum TG.

Excessive hepatic lipogenesis may lead to liver inflammation and contribute to hepatic insulin resistance. Therefore, we next sought to determine whether increased lipid deposition associated with altered hepatic gene expression. Notably, the hepatic mRNA transcripts of lipogenic genes (sterol regulatory element binding protein 1c [Srebp1c], acetyl-CoA carboxylase [Acc], fatty acid synthase [Fas], Scd1, 3-hydroxy-3-methyl-glutaryl-CoA [HMG-CoA] reductase [Hmgcr], and HMG-CoA synthase [Hmgcs]) and enzymes responsible for the esterification of MUFA to cholesterol and TG via acyl CoA: cholesterol acyl transferase 2 (Acat2) and diacylglycerol acyl transferase 1 and 2 (Dgat1 and 2) were elevated in T5KO mice ( Figures 2 A–2D and 2F–2J ), including SCD1 at the protein level ( Figure 2 E). Interestingly, mRNA transcripts of key inflammatory cytokines Tnfα, Lcn2, and chemokine (C-X-C motif) ligand 1 (Kc) were augmented in T5KO mice ( Figures 2 K–2M). Further, the T5KO mice also showed increased expression of genes associated with insulin resistance such as Forkhead box protein O1 (Foxo1), peroxisome proliferator-activated receptor gamma coactivator 1 alpha (Pgc1α) transcription coactivator, and gluconeogenic enzyme phosphoenolpyruvate carboxykinase (Pepck) ( Figures 2 N–2P). Collectively, these results suggest that the elevated C18:1 in T5KO mice is a result of increased upregulation of a spectrum of lipogenic genes associated with markers of inflammation and insulin resistance. In accordance with unaltered fatty acid composition of adipose tissue in WT and T5KO mice, we did not observe any significant differences in the transcripts of above lipogenic genes in adipose tissue and skeletal muscles of these mice ( Figures S1 J and S1K).

Hepatic expression of lipogenic enzymes, inflammatory, and insulin resistance marker genes were analyzed in male T5KO mice and their WT littermates (20 week old, n = 5).

Given that both diet and gut microbiota composition change drastically at the suckling-to-weaning transition, we next asked whether the elevated C18:1 in hepatic lipids of T5KO mice is present beginning from birth or occurs after weaning. At the age of 3 weeks, mice were weaned on chow diet, and liver total C18:1 was measured at 3, 5, 8, and 12 weeks of age. At the time of weaning, both WT and T5KO mice displayed similar levels of C18:1 in liver, which increased within 2 weeks of weaning in both WT and T5KO mice ( Figure 1 Q). However, the percent increase in C18:1 in T5KO mice was significantly higher than in WT littermates. In addition to C18:1, liver total TG levels increased with age in T5KO mice (data not shown). Thus, the elevated C18:1 in the liver is an acquired characteristic after weaning perhaps due to the dietary switch from suckling to lab chow. In contrast to leptin-deficient mice, the elevated C18:1 was not seen in either serum or adipose tissue, while fecal lipids of T5KO mice are comparable to WT littermates ( Table S2 ).

Obesity in leptin-deficient mice is associated with elevated tissue MUFA levels and is SCD1 dependent (). Similar to leptin-deficient mice, T5KO mice exhibited elevated plasma insulin, an anabolic hormone known to upregulate SCD1 (). We therefore examined whether obese T5KO mice displayed similar MUFA-enriched lipids. Liver lipid analysis in T5KO mice showed an increase (25.0%) in C18:1 in the total liver lipids ( Figure 1 K), mainly attributed to increased C18:1 in the CE (16.0%) and TG (9.0%) fractions ( Figures 1 L and 1M). The C18:1 level in PL fraction was comparable between WT and T5KO mice ( Figure 1 N). Interestingly, palmitoleate (C16:1), a minor product of SCD1, was comparable in the total lipid (although with large variation), TG, CE, and PL fractions of T5KO mice and WT littermates ( Figures S1 F–S1I). Notably, mRNA transcript of lipogenic genes in adipose and skeletal muscle remained unaltered in T5KO mice ( Figures S1 J and S1K). In general, female mice tend to have higher levels of MUFA in the liver when compared to males (), but we did not observe such sex based differences (data not shown). In the total liver lipids, a decreasing trend in essential fatty acids (EFAs) linoleic acid (C18:2 n6) and arachidonic acid (C20:4 n6) was observed but not significant (data not shown). Similarly, in the liver CE, but not the TG fraction, C18:2 levels were decreased in T5KO mice relative to WT littermates (data not shown). T5KO mice with elevated hepatic lipids displayed insulin resistance, as measured by phosphorylation of AKT, an insulin-responsive protein, and whole-body insulin sensitivity test ( Figures 1 O and 1P). Similar to male, female T5KO mice also exhibited metabolic syndrome with hepatic lipid oleate enrichment ( Figures S2 A–S2J).

T5KO mice exhibit hallmark features of metabolic syndrome including hyperlipidemia. To investigate the extent to which serum hyperlipidemia is reflected in the liver, we analyzed liver TGs, CEs, free cholesterol (FC), phospholipids (PLs), insulin sensitivity, and signaling in 20-week-old T5KO mice and their WT littermates. As observed in other facilities (Emory and Georgia State University), T5KO mice housed at PSU displayed higher body weights, abdominal adiposity, hyperglycemia ( Figures 1 A–1C) with associated splenomegaly ( Figure S1 A), and elevated colonic expression of pro-inflammatory genes (Lipocalin 2 (Lcn2), iNos, Pro-Il-1β, and Kc) ( Figures S1 B–S1E), indicating a low-grade chronic inflammatory state. Further, T5KO mice were hyperinsulinemic and hyperlipidemic ( Figures 1 D–1F). T5KO mice displayed elevated hepatic neutral lipids TG and CE compared to WT littermates ( Figures 1 G and 1H). There was no significant difference in the liver FC, but a significant decrease in PL levels in T5KO mice was observed ( Figures 1 I and 1J, respectively). A similar trend of elevated lipid parameters was observed in the female T5KO mice (data not shown).

(Q) Liver total lipid oleate (%) levels were quantified in male T5KO and their WT littermates (n = 6) at different age 3, 5, 8, and 12 weeks. Data are represented as mean ± SEM. ∗ p < 0.05.

(K–N) Fatty acid composition of hepatic lipid fractions were analyzed by gas chromatography and represented as (%) C18:1 (K) C18:1 in total lipid, (L) C18:1 in CE, (M) C18:1 in TG and (N) C18:1 in PL.

(A–F) Age-matched T5KO male mice and their WT littermates (20 week old, n = 10 to 11) were monitored for (A) body weight, (B) fat pad, (C) 15 hr fasting blood glucose, (D) fasting serum insulin, (E) serum total cholesterol (TC), and (F) serum TGs.

Discussion

Gut microbiota is known to play a key role in the host energy homeostasis and is thus considered an environmental risk factor for metabolic diseases. Accordingly, mice deficient in TLR5 develop an altered microbiota that promotes metabolic syndrome. Herein, we report a potential molecular mechanism involving elevated C18:1 hepatic neutral lipids underlying the development of metabolic syndrome in T5KO mice. Such elevated hepatic C18:1 levels likely result, at least in part, from augmented SCD1 expression. We further observed that hepatic enrichment of oleate is microbiota dependent.

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Gewirtz A.T. Metabolic syndrome and altered gut microbiota in mice lacking Toll-like receptor 5. 1H NMR. In this study, using a metabolomic approach, we found that T5KO mice with increased microbiotal load generated substantial levels of SCFA in the cecum. This is transferred to the liver and contributes to increased hepatic DNL. Notably, the relative contribution of acetate to lipogenesis was more in T5KO mice. It is likely that the gut-derived SCFA drive increased lipogenesis by provision of greater amounts of substrate for the elevated lipid synthetic machinery in T5KO mice. The cecum is a transient reservoir for digested food and therefore contains the majority of complex dietary fiber that undergoes anaerobic bacterial fermentation that generates acetate, propionate, and butyrate in molar ratio of 60:20:20 (). Recent studies demonstrate a direct link of obesogenic dysbiosis (i.e., altered Firmicutes/Bacteroidetes ratio with metabolic syndrome in both mice and humans) (). We observed altered species primarily in Firmicutes and Bacteroidetes phyla in T5KO mice when compared to their WT littermates, but relative abundance of these phyla was unaltered (), suggesting that elevated gut microbiota along with altered bacterial species might contribute to increased cecal SCFA generation in T5KO mice. These SCFA not only serve as a fuel for intestinal epithelia (specifically butyrate) (), but also contribute additional sources of energy to the host (). A recent study using stable isotopes of SCFA demonstrated that acetate and butyrate are lipogenic whereas propionate is gluconeogenic () in mice. Specifically, this study showed that cecal acetate and butyrate contribute to synthesis of hepatic palmitate and cholesterol. In a similar vein,H NMR-based metabolic profiling of cecal content showed considerably elevated levels of butyrate and propionate accompanied with a decreasing trend of oligosaccharides in T5KO mice. Increased expression of colonic SCFA receptors, particularly Gpr41 and Gpr43, affords efficient absorption of these SCFAs and promoting the inflammatory response via MAP kinase signaling in T5KO mice (). Acetate levels in cecal contents of T5KO mice did not differ significantly in comparison to WT, suggesting its rapid conversion into butyrate by the gut bacteria (). Previously, we showed no difference in either energy content in feces (bomb calorimetry) or SCFA levels in the cecal contents of T5KO mice and their WT littermates via GC-MS (). The discrepancy could be due to differences in collection of samples from previous studies, including (i) age of the mice, (ii) ad libitum fed mice, (iii) use of wet rather than dry stools, or (iv) differences in processing of samples for analysis withH NMR. In this study, using a metabolomic approach, we found that T5KO mice with increased microbiotal load generated substantial levels of SCFA in the cecum. This is transferred to the liver and contributes to increased hepatic DNL. Notably, the relative contribution of acetate to lipogenesis was more in T5KO mice. It is likely that the gut-derived SCFA drive increased lipogenesis by provision of greater amounts of substrate for the elevated lipid synthetic machinery in T5KO mice.

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Brown L. Stearoyl-CoA desaturase: a vital checkpoint in the development and progression of obesity. Several studies have demonstrated that SFA C16:0 and C18:0, dietary or end products of DNL, are lipotoxic (). Increased SCFAs serve as substrates for SFA synthesis, which are converted into less lipotoxic MUFA via SCD1 () and play a role in the development of obesity (). Notably, hepatic SCD1 level is substantially reduced in GF and microbiota-ablated WT and T5KO mice, indicating that liver SCD1 is partly regulated via gut microbial metabolites. Accordingly, our results indicate that dietary SCFA aggravates the metabolic syndrome phenotype in T5KO mice probably via overproduction of SFA from SCFA.

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Ntambi J.M. Loss of Stearoyl-CoA desaturase-1 attenuates adipocyte inflammation: effects of adipocyte-derived oleate. The conversion of SFA into MUFA is known to prevent negative feedback inhibition of fatty acids (palmitate) on ACC (). The blockade of SFA removal as MUFA in Scd1KO mice underlies its lean phenotype. Although T5KO mice exhibited elevated C18:1-enriched hepatic neutral lipids, those lipid species were not enriched with palmitoleate (C16:1 n7), a minor product of SCD1. It is interesting to note that C16:1 has been shown to act as a lipokine, strongly induces muscle insulin action, and suppresses hepatic lipogenesis in mice (). In addition, adipocyte-derived oleate (18:1 n9), but not palmitoleate (16:1 n7), mediates inflammation in macrophages and adhesion responses in endothelial cells ().

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Bäckhed F. Microbial modulation of energy availability in the colon regulates intestinal transit. Deletion of SCD1 or supplementing leptin to Ob/Ob mice ameliorates obesity (). Interestingly, T5KO mice exhibited upregulation of a spectrum of hepatic lipogenic enzymes, including SCD1, even though they are hyperleptinemic (), suggesting they may be in a state of leptin resistance. Elevated hepatic SCD1 in T5KO mice may be driven by increase in (i) stability of this protein, (ii) insulin level, (iii) precursor (e.g., SCFA) availability (), (iv) low-grade hepatic inflammation, and (v) Srebp1c, a transcription factor known to stimulate lipogenic genes including Scd1 in hepatocytes. Accordingly, we demonstrated that deletion of hepatic Scd1 prevented accumulation of C18:1 and improved metabolic syndrome in T5KO mice. Taken together these results reinforce the concept that the microbiota-generated SCFA contribute, at least in part, to hepatic lipogenesis in T5KO mice. MUFA generated via SCD1 activity, but not dietary MUFA, function as endogenous fatty acid amide hydrolase (FAAH) inhibitor and thereby increases hepatic arachidonoylethanolamide (AEA), which induces insulin resistance by activating liver endocannabinoid/CB1 receptor (). Endogenous MUFA, synthesized by SCD1, but not dietary MUFA is required for hepatic TG synthesis, as oleate-enriched diet failed to restore hepatic TG level in global Scd1-deficient mice (). These observations support the notion that there is a difference in the metabolic fate of endogenously produced versus ingested metabolites. HFD aggravated metabolic syndrome and caused insulitis and hepatic steatosis in T5KO mice. Surprisingly, HFD-fed T5KO mice displayed modest increase in C18:1; this could reflect a reduced supply of SCFA as the HFD lacks plant-derived complex carbohydrates. However, the HFD used in this study is rich in palmitate (26%) and oleate (44%), which together may be enough to maintain C18:1 level on a higher side in T5KO mice. A recent study bydemonstrated that total SCFA in cecal contents is greatly reduced (>50%) in HFD-fed mice compared to chow-fed mice. Although there was a substantial reduction in EFA C18:2 and C20:4 in PL fractions of HFD-fed T5KO mice, they were far from EFA deficiency, as indicated by no detectable levels of eicosatrienoic acid (C20:3 n9), a surrogate marker of EFA deficiency.