Jessica C. Ralston

Individual Saturated and Monounsaturated Fatty Acids Trigger Distinct Transcriptional Networks in Differentiated 3T3-L1 Preadipocytes.

Background/Aims: Saturated fatty acids (SFA) are widely thought to induce inflammation in adipose tissue (AT), while monounsaturated fatty acids (MUFA) are purported to have the opposite effect; however, it is unclear if individual SFA and MUFA behave similarly. Our goal was to examine adipocyte transcriptional networks regulated by individual SFA (palmitic acid, PA; stearic acid, SA) and MUFA (palmitoleic acid, PMA; oleic acid, OA). Methods: Differentiated preadipocytes were treated with either 250 µM PA, SA, PMA, or OA for 48 h. Gene expression was analyzed using microarrays and real-time RT-PCR. Data were compared with those of a previous study reporting AT gene expression in humans following the consumption of SFA- or MUFA-enriched diets. Results: Individual fatty acid treatments had significant effects on adipocyte gene expression. Functional analyses revealed that PA induced the TLR signalling pathway, while PMA had the opposite effect. SA and OA had similar effects, with increases in key metabolic pathways including mTOR and PPAR signalling and a reduction in TLR signalling. Ccl5 was validated as a candidate gene that may mediate the differential inflammatory effects of SFA and MUFA in AT. Conclusions: Individual SFA and MUFA trigger distinct transcriptional responses in differentiated preadipocytes, with inflammatory and metabolic pathways particularly sensitive to these fatty acids.

Polyunsaturated fatty acid regulation of adipocyte FADS1 and FADS2 expression and function.

Polyunsaturated fatty acids (PUFAs) regulate fatty acid desaturase (FADS1, FADS2) expression in the liver; however, it is unknown whether PUFAs regulate FADS in adipocytes. This is important to study considering reports that link altered desaturase activity with adipose tissue PUFA profiles, body weight, and whole‐body glucose homeostasis. Therefore, the present study aimed to determine the direct effects of PUFAs on FADS expression in differentiated 3T3‐L1 adipocytes.

Inhibition of stearoyl-CoA desaturase-1 in differentiating 3T3-L1 preadipocytes upregulates elongase 6 and downregulates genes affecting triacylglycerol synthesis

Background:Stearoyl-CoA desaturase-1 (SCD1) is rate limiting for the conversion of saturated fatty acids palmitate (16:0) and stearate (18:0) to monounsaturated fatty acids palmitoleate (16:1n7) and oleate (18:1n9), respectively. Given that reduced SCD1 activity is associated with improved insulin sensitivity and decreased body weight, there is considerable interest to elucidate the role of this enzyme in adipocytes. During adipogenesis, SCD1 levels increase concomitantly with the accumulation of triacylglycerol (TG); however, the extent to which reduced SCD1 activity can influence TG synthesis and metabolic pathways in differentiating adipocytes remains relatively unexplored.Objective:The aim of this work was to delineate how reduced SCD1 activity affects gene expression, protein content and cellular fatty acids in differentiating murine preadipocytes.Methods:3T3-L1 preadipocytes were treated with an SCD1 inhibitor (10 nM) throughout differentiation. After 7 days, global gene expression, protein content and fatty acid profiles were examined using microarrays, western blotting and gas chromatography, respectively.Results:SCD1 inhibition increased the abundance of 16:0 and 18:0 (45% and 194%, respectively) and decreased 16:1n7 and 18:1n7 (61% and 35%, respectively) in differentiated preadipocytes. Interestingly, 18:1n9 levels increased by 61%. The augmented 18:0 suggested a possible increase in elongase activity. Elongase 6 (Elovl6) gene expression was increased 2.8-fold (P=0.04); however, changes were not detected for ELOVL6 protein content. Microarray analysis revealed that genes affecting TG synthesis were downregulated with SCD1 inhibition, which coincided with a 33% decrease in cellular TG content.Conclusion:These results provide new mechanistic insight into the role of SCD1 as a regulator of fatty acid profiles and TG synthesis in adipocytes, and reinforce that modulating SCD1 activity may help reduce the risk of obesity-related complications.

Variation in the FADS1/2 gene cluster alters plasma n−6 PUFA and is weakly associated with hsCRP levels in healthy young adults

INTRODUCTION Past research has reported that single nucleotide polymorphisms (SNPs) in fatty acid desaturase 1 and 2 (FADS1/2) can influence plasma fatty acid (FA) profiles. Changes in FA profiles are known to influence inflammatory processes; therefore both FA and SNPs in FADS1/2 may affect inflammation. The goals of this study were to (i) examine the relationships between individual n-6 FA and estimates of FA desaturation with circulating high sensitivity C-reactive protein (hsCRP) levels, and (ii) determine whether SNPs in FADS1/2 are associated with changes in hsCRP. METHODS FA and hsCRP were measured in fasted plasma samples from 878 healthy young adults (20-29yrs). Circulating levels of plasma linoleic (LA), γ-linolenic (GLA), dihomo-γ-linolenic (DGLA) and arachidonic (AA) acids were measured by gas chromatography and used to calculate desaturase indices for FADS1/2. Nineteen SNPs in FADS1/2 were genotyped in all subjects and six (rs174579, rs174593, rs174626, rs526126, rs968567 and rs17831757) were further analyzed. RESULTS Significant inverse associations were found between LA and hsCRP (p=8.55×10(-9)) and the FADS1 desaturase index and hsCRP (p=4.41×10(-6)). A significant positive association was found between DGLA and hsCRP (p=9.10×10(-11)). Several SNPs were associated with circulating levels of individual FA and desaturase indices, with minor allele carriers having lower AA levels and reduced desaturase indices. A single SNP in FADS2 (rs526126) was weakly associated with hsCRP (p=0.05). CONCLUSIONS This study highlights the relationships between FA and hsCRP, and confirms that FA are strongly influenced by SNPs in FADS1/2. Furthermore, we found weak evidence that SNPs in FADS1/2 may influence hsCRP levels in young adults.

Vaccenic acid in serum triglycerides is associated with markers of insulin resistance in men.

Serum triglyceride levels are associated with metabolic disorders; however, it remains unclear whether the fatty acid (FA) composition of triglycerides is also changed. Although there were no differences in circulating triglyceride levels between normoglycaemic-normoinsulinaemic and hyperglycaemic-hyperinsulinaemic men, inspection of individual FA revealed that vaccenic acid was enriched with hyperglycaemia-hyperinsulinaemia. Moreover, vaccenic acid levels were positively correlated with insulin and HOMA-IR. This reinforces that examination of individual FA in the context of insulin resistance is warranted.

Ethnic- and sex-specific associations between plasma fatty acids and markers of insulin resistance in healthy young adults

BackgroundAlthough evidence indicates that fatty acids (FA) can affect insulin resistance (IR), not all FA contribute equally to the process. Indeed, monounsaturated FA (MUFA) and polyunsaturated FA (PUFA) are reported to reduce IR, whereas saturated FA (SFA) and trans FA appear to increase IR. However, it is not yet clear how individual FA are associated with markers of IR, and whether these relationships are influenced by ethnicity and/or sex. Therefore, the goal of this study was to examine the ethnic- and sex-specific relationships between plasma FA and markers of IR in a cohort of healthy young Caucasian, East Asian, and South Asian adults.MethodsGas chromatography was used to quantify fasting plasma FA from young Canadian adults (22.6 ± 0.1 yrs) of Caucasian (n = 461), East Asian (n = 362), or South Asian (n = 104) descent. Linear regression models were used to investigate associations between plasma FA and markers of IR (i.e. fasting insulin, glucose, and HOMA-IR) according to ethnicity and sex.ResultsNumerous significant associations (P < 0.05, adjusted for multiple testing) were identified between individual FA and markers of IR, with the majority identified in Caucasians. For SFA, positive associations were found between 14:0 and fasting insulin and HOMA-IR in Caucasian and East Asian populations, and 18:0 and fasting glucose in Caucasians only. Several positive associations were also found for specific MUFA (18:1t11 and 18:1t6-8 with HOMA-IR, and 18:1c9 with fasting glucose) and PUFA (18:2n6 with fasting glucose and 18:2c9t11 with HOMA-IR) in Caucasian adults only. Most of the aforementioned associations were stronger in males compared to females. Interestingly, no significant associations were found between FA and markers of IR in South Asian adults.ConclusionsWe report numerous associations between plasma FA and markers of IR in Caucasian and East Asian populations, but not in South Asian individuals. Furthermore, these associations appeared to be more robust in men. This demonstrates the importance of investigating associations between FA and markers of IR in an ethnic- and sex-specific manner in order to better understand the contribution of plasma FA to the development of IR and type-2 diabetes.

SCD1 mediates the influence of exogenous saturated and monounsaturated fatty acids in adipocytes: Effects on cellular stress, inflammatory markers and fatty acid elongation

Palmitate (PA), stearate (SA), palmitoleate (PMA) and oleate (OA) are among the most abundant fatty acids (FAs) in adipose tissue (AT). These FAs differentially regulate AT inflammation by altering adipocyte signalling pathways and the secretion of proinflammatory cytokines. Intracellular levels of these FAs are controlled, in part, by stearoyl-CoA desaturase 1 (SCD1). Therefore, SCD1 may have an important role mediating FA-regulation of adipocyte inflammation. Given this, we hypothesized that the influence of PA, SA, PMA and OA on inflammation and cellular stress, as well as FA metabolism, would be exacerbated with reduced SCD1 activity. Real-time RT-PCR, immunoassays, gas chromatography and western blotting were used to examine the expression and secretion of common inflammatory markers, as well as FA profiles and markers of cellular stress, in 3T3-L1 adipocytes. FA treatments differentially affected inflammatory markers and FA profiles in SCD1-inhibited adipocytes. Specifically, SA significantly increased the expression of Ccl5 (5.3-fold) and Mcp-1 (3.2-fold), and the secretion of IL-6 (17.8-fold) and MCP-1 (4.0-fold) in SCD1-inhibited adipocytes compared to controls. The proinflammatory effects observed with SA are particularly notable given that SCD1-inhibited adipocytes increased elongation of PA to SA, as determined using U-(13)C-PA. The effects of PA, PMA and OA were not as substantial as those of SA, although PA did significantly increase Ccl5 (2.7-fold) and Mcp-1 (1.2-fold) expression in SCD1-inhibited adipocytes. None of the FAs altered markers of cellular stress. Collectively, these results emphasize the differential effects of individual FAs and highlight how SCD1 influences their regulation of adipocyte inflammation.

SCD1 inhibition during 3T3-L1 adipocyte differentiation remodels triacylglycerol, diacylglycerol and phospholipid fatty acid composition

The conversion of saturated fatty acids (FAs) palmitate (16:0) and stearate (18:0) into monounsaturated FAs palmitoleate (16:1n-7) and oleate (18:1n-9) is catalyzed by stearoyl-CoA desaturase 1 (SCD1). These FAs represent the dominant constituents of adipocyte triacylglycerols (TAGs) and phospholipids (PLs). Given the critical role of SCD1 in lipid metabolism and the notable increase in its expression during adipogenesis, reductions in SCD1 activity have the potential to compromise the adipocytes ability to accumulate lipid. The current study used thin-layer and gas chromatography to examine the content and FA composition of TAGs, PLs, cholesteryl esters, diacylglycerols and free fatty acids in SCD1-inhibited differentiating 3T3-L1 adipocyte cells. SCD1 inhibition reduced total cellular PL and TAG content concurrent with the down-regulation of genes involved in TAG and PL biosynthesis; however, the relative amount of PL was unaltered. While total DAG levels were increased ~2.7-fold in SCD1-inhibited adipocytes, this did not induce JNK activation; however, phosphorylated (Ser473) AKT was significantly reduced. As expected, total SFA and MUFA content were increased (~1.3-fold) and decreased (~4.0-fold). Further, SCD1 inhibition caused a ~2.2-fold increase and a ~8.3-fold decrease in total cellular 18:0 and 16:1n-7 levels, respectively. Similar changes were also seen in other lipid fractions. The levels of other FAs, including polyunsaturated FAs, were also changed in SCD1-inhibited adipocytes. Together, these results add to the existing body of knowledge regarding SCD1 function in adipocytes and highlight its important role in regulating global adipocyte lipid composition.

Hepatic BSCL2 (Seipin) Deficiency Disrupts Lipid Droplet Homeostasis and Increases Lipid Metabolism via SCD1 Activity

Berardinelli-Seip congenital lipodystrophy (BSCL) is an autosomal recessive disorder. The more severe form, designated BSCL2, arises due to mutations in the BSCL2 gene. Patients with BSCL2, as well as Bscl2−/− mice, have a near total absence of body fat, an organomegaly, and develop metabolic disorders including insulin resistance and hepatic steatosis. The function of the Seipin (BSCL2) protein remains poorly understood. Several lines of evidence have indicated that Seipin may have distinct functions in adipose versus non-adipose cells. Here we present evidence that BSCL2/Bscl2 plays a role in lipid droplet (LD) biogenesis and homeostasis in primary and cultured hepatocytes. Our results show that decreasing BSCL2/Bscl2 expression in hepatocytes increases the number and size of LD, as well as the expression of genes implicated in their formation and stability. We also show that knocking down SCD1 expression reverses the phenotype associated with Seipin deficiency. Interestingly, BSCL2 knockdown induces SCD1 expression and activity, potentially leading to increased basal phosphorylation of proteins involved in the insulin signaling cascade, as well as further increasing fatty acid uptake and de novo lipogenesis. In conclusion, our results suggest that a hepatic BSCL2/Bscl2 deficiency induces the increase and expansion of LD, potentially via increased SCD1 activity.

North American ginseng influences adipocyte–macrophage crosstalk regulation of inflammatory gene expression

Background Adipocyte–macrophage communication plays a critical role regulating white adipose tissue (WAT) inflammatory gene expression. Because WAT inflammation contributes to the development of metabolic diseases, there is significant interest in understanding how exogenous compounds regulate the adipocyte–macrophage crosstalk. An aqueous (AQ) extract of North American (NA) ginseng (Panax quinquefolius) was previously shown to have strong inflammo-regulatory properties in adipocytes. This study examined whether different ginseng extracts influence adipocyte–macrophage crosstalk, as well as WAT inflammatory gene expression. Methods The effects of AQ and ethanol (EtOH) ginseng extracts (5 μg/mL) on adipocyte and macrophage inflammatory gene expression were studied in 3T3-L1 and RAW264.7 cells, respectively, using real-time reverse transcription polymerase chain reaction. Adipose tissue organ culture was also used to examine the effects of ginseng extracts on epididymal WAT (EWAT) and inguinal subcutaneous WAT (SWAT) inflammatory gene expression. Results The AQ extract caused significant increases in the expression of common inflammatory genes (e.g., Mcp1, Ccl5, Tnf-α, Nos2) in both cell types. Culturing adipocytes in media from macrophages treated with the AQ extract, and vice versa, also induced inflammatory gene expression. Adipocyte Ppar-γ expression was reduced with the AQ extract. The AQ extract strongly induced inflammatory gene expression in EWAT, but not in SWAT. The EtOH extract had no effect on inflammatory gene expression in either both cell types or WAT. Conclusion These findings provide important new insights into the inflammo-regulatory role of NA ginseng in WAT.