Role of fatty acid elongase Elovl6 in the regulation of energy metabolism and pathophysiological significance in diabetes

Abstract

Type 2 diabetes mellitus (T2DM) is an expanding epidemic, closely linked to obesity. Peripheral insulin resistance and impaired insulin secretion remain the core defects in T2DM. Despite significant advances in unraveling the underlying these defects, many of the metabolic pathways and regulators involved in insulin resistance and β-cell dysfunction are not completely understood. This review proposes that manipulating the fatty acid (FA) composition by blocking ELOVL fatty acid elongase 6 (Elovl6) could protect against insulin resistance, impaired insulin secretion, and obesity-related disorders. The molecular mechanism of this new paradigm is also discussed. Elovl6 is a microsomal enzyme involved in the elongation of C16 saturated and monounsaturated FAs to form C18 FAs. We have reported that mice with Elovl6 deletion are protected against obesity-induced insulin resistance or β-cell failure when mated to leptin receptor-deficient db/db mice because the cellular FA composition is changed, even with concurrent obesity. Therefore, Elovl6 appears to be a crucial metabolic checkpoint, and limiting Elovl6 expression or activity could be a new therapeutic approach to treat T2DM.

Introduction

The increasing prevalence of obesity worldwide has become an alarming public health concern because of the associated rise in cases of obesity-associated diseases, including type 2 diabetes mellitus (T2DM) [1, 2]. Genetic and environmental factors contribute to the pathogenesis of T2DM, characterized by peripheral insulin resistance and insufficient compensation of insulin secretion by the pancreatic β-cells [3]. The accumulation of lipids, a phenomenon known as lipotoxicity, is a molecular link between obesity and glucose homeostasis dysregulation [4–6].

Most cellular lipid molecules include, as a major constituent, at least one fatty acid (FA). Most cells can synthesize FAs and the cellular FAs are diverse in carbon chain length and pattern of saturation/desaturation, which contributes to the variety of functions exhibited by the lipids [7]. Proper elongation and desaturation of FAs are essential for the maintenance of lipid homeostasis and disruption of these processes can have devastating consequences.

ELOVL fatty acid elongase 6 (Elovl6) is an endoplasmic reticulum enzyme that catalyzes the chain elongation of C12–16 saturated and monounsaturated FAs to form C18 FAs, such as stearate (C18:0), oleate (C18:1n-9), and vaccinate (C18:1n-7) (Fig. 1) [8, 9]. These long chain FAs with 16–18 carbons are the most abundant cellular FAs and are important components of triglycerides, cholesterol esters, phospholipids, and sphingolipids. Under normal dietary conditions, Elovl6 is expressed at a high level in the white adipose tissue, brown adipose tissue, liver, brain, adrenal gland, testis, and skin, where lipogenesis and steroidogenesis are active. Elovl6 mRNA levels in the liver and adipose tissue markedly elevate in a refed state after fasting through the sterol regulatory element-binding protein (SREBP)-1 dependent mechanism. Hepatic Elovl6 expression is also highly upregulated in genetically obese model such as ob/ob mice and db/db mice. Dietary polyunsaturated FAs cause a profound suppression of Elovl6 expression. Thus, this FA elongase is a member of the mammalian lipogenic enzymes regulated by SREBP-1 and plays an important role in de novo synthesis of long chain saturated and monounsaturated FAs with FA synthase (FAS) and stearoyl-CoA desaturase-1. Overall, Elovl6 affects the FA composition of membrane phospholipids, sphingolipids, triglycerides, and cholesterol esters, resulting in changes in membrane fluidity, lipid metabolism, and obesity.

Fig. 1.

Role of Elovl6 in mammalian fatty acid synthesis. ACL ATP citrate lyase, ACC acetyl-CoA carboxylase, FAS fatty acid synthase, SCD stearoyl-CoA desaturase

Role of Elovl6 in the development of obesity-induced insulin resistance

Studies on Elovl6^−/− mice showed that loss of Elovl6 function increased levels of palmitate (C16:0), palmitoleate (C16:1n − 7), and vaccinate (C18:1n-7), but reduced levels of stearate (C18:0) and oleate (C18:1n − 9), confirming that Elovl6 catalyzes the chain elongation of palmitate to stearate and the elongation of palmitoleate to vaccinate in vivo. Apart from dramatic alterations in FA composition, Elovl6^−/− mice do not differ from wild-type littermates regarding overall lipid concentration of the liver and plasma. On a chow diet, Elovl6^−/− mice appear grossly normal, although slightly leaner than their wild-type littermates. On a high-fat, high-sucrose (HFHS) diet, wild-type and Elovl6^−/− mice gain body weight and body fat at similar rates. In response to diet-induced obesity, wild-type mice exhibited insulin resistance. However, Elovl6^−/− mice are resistant to diet-induced insulin resistance, despite their being similar to wild-type mice regarding hepatosteatosis and obesity [10].

Amelioration of whole-body insulin resistance in Elovl6^−/− mice can be attributed to the restoration of hepatic insulin sensitivity. Elovl6 deficiency restores suppressed Akt phosphorylation in the liver, but not in the skeletal muscle and white adipose tissue. Restoration of Akt phosphorylation is accompanied by increased total and phosphorylated insulin receptor substrate (IRS)-2 protein and Elovl6^{− / −} mice livers. Diacylglycerol (DAG) accumulation has been reported to be linked to increased protein kinase C epsilon (PKCɛ) activity and impaired insulin signaling [11]. In wild-type mice, hepatic DAG content and PKCɛ activity are significantly increased in the liver in response to an HFHS diet. However, the livers of Elovl6^−/− mice contain less DAG and show lower expression of PKC compared with wild-type mice. These results show that restoration of insulin signaling is mediated by recovering the IRS-2/Akt signaling pathway and suppressing the DAG/PKCɛ pathway in the livers of Elovl6^−/− mice. These findings suggested that the vital role of alterations in FA composition by Elovl6 deficiency extended beyond lipid accumulation and affected insulin sensitivity.

Role of Elovl6 in the development of T2DM

Studies show that Elovl6^−/− mice were protected against the development of obesity-induced insulin resistance, despite similar levels of hepatosteatosis and obesity between Elovl6^−/− and wild-type mice [10]. Therefore, Elovl6 inhibition could be a potential therapeutic approach in T2DM treatment. A critical question is whether inhibition of this elongase confers reduced susceptibility to T2DM. Thus, we investigated the effects of Elovl6 deletion in leptin receptor-deficient db/db mice by generating Elovl6-deficient db/db mice (db/db; Elovl6^−/−). Although obesity in db/db mice was not improved, Elovl6 deficiency significantly improved hyperglycemia and showed an adaptive increase in insulin in db/db mice [12]. These metabolic changes improved polydipsia, polyuria, and elevated HbA1c levels in db/db;Elovl6^−/− mice. Improved plasma glucose clearance and increased insulin secretion were observed in db/db; Elovl6^−/− mice by oral glucose tolerance tests. These results suggested that Elovl6 deficiency prevented T2DM progression by increasing insulin secretory capacity of pancreatic β-cells in db/db mice.

Elovl6 deficiency also improved impaired glucose-stimulated insulin secretion (GSIS) in db/db islets. The islets in db/db;Elovl6^−/− mice were markedly enlarged compared with those in db/db;Elovl6^+/+ mice with a significant increase in β-cell mass. Excessive lipid accumulation in pancreatic β-cells is associated with lipotoxicity and reduces insulin secretion [4, 5]. The triglyceride content of db/db;Elovl6^−/− islets was significantly lower than db/db;Elovl6^+/+ islets and almost similar to db/ + ;Elovl6^+/+ and db/ + ;Elovl6^−/− islets. Compared with db/db;Elovl6^+/+ islets, there was a marked reduction in oleate composition of db/db;Elovl6^−/− islets, similar to db/ + ;Elovl6^+/+ and db/ + ;Elovl6^−/− controls. Gene expression analysis of isolated islets revealed that Elovl6 deficiency suppressed the expression of genes involved in the islet inflammation and ER stress in db/db mice. These results suggested that the modulation of intracellular FA composition in β-cells by Elovl6 was associated with β-cell dysfunction and that Elovl6 deficiency could play a positive role in β-cell mass and function through reduction of oleate and triglyceride content and susceptibility to FA-induced inflammation and ER stress under diabetic conditions (Fig. 2).

Fig. 2.

Proposed mechanisms by which Elovl6 modulates β-cell lipotoxicity during progression to T2DM. Obesity and its associated metabolic changes including glucose intolerance, insulin resistance, and increased FFA delivery lead to palmitate- and oleate-induced β-cell dysfunction, inflammation, ER stress, and apoptosis in pancreatic islets. The loss of Elovl6 function inhibits the elongation of palmitate and synthesis of oleate, resulting in protection against impaired insulin secretion, inflammation, terminal UPR, and β-cell apoptosis in pancreatic islets, maintenance of β-cell compensation, and prevention of T2D

This study has showed the beneficial effect of Elovl6 loss in db/db mice. The results, combined with our previous work that showed Elovl6 deficiency improved obesity-induced peripheral insulin resistance, suggest that limiting Elovl6 expression or activity in individuals during early diabetes or in those with metabolic syndrome might be beneficial T2DM prevention and treatment.

Liver-specific deletion of Elovl6 enhances insulin sensitivity through inhibiting C18:0-ceramide production in the liver

We generated liver-specific Elovl6 knockout (LKO) mice to investigate the role of Elovl6 in the hepatic control of lipid metabolism and energy homeostasis [13]. To determine whether liver-specific Elovl6 deletion produces the same metabolic phenotype as that of Elovl6^−/− mice, Flox and LKO mice were fed an HFHS diet. In contrast to Elovl6^−/− mice, liver-specific Elovl6 deletion did not improve mice's insulin resistance on an HFHS diet. This result shows that hepatocyte-specific Elovl6 deficiency does not ameliorate insulin resistance induced by an HFHS diet.

We next fed Flox and LKO mice a high sucrose diet (HSD) to induce Elovl6 activity and de novo lipogenesis, which revealed several phenotypic differences. The HSD induced a similar hepatosteatosis in both genotypes compared to the chow diet. The Elovl6 deletion exacerbated the changes in hepatic FA composition induced by HSD feeding. Specifically, HSD feeding of LKO mice significantly lowered the stearate and oleate content but increased the palmitate, palmitoleate, and vaccinate content, relative to Flox mice. HSD-fed LKO mice had significantly lower plasma glucose and insulin levels compared to HSD-fed Flox controls. During insulin tolerance tests (ITTs), HSD-fed LKO mice showed lower plasma glucose concentrations than HSD-fed Flox mice. Akt's insulin-induced phosphorylation was much higher in the livers of HSD-fed LKO mice than HSD-fed Flox mice. These results suggest that LKO mice are more insulin sensitive than Flox mice when consuming an HSD by enhancing the hepatic insulin signaling via Akt.

Livers from Flox and LKO mice fed an HSD were profiled using microarray analysis to identify candidate genes associated with the enhancement in hepatic insulin sensitivity in HSD-fed LKO mice. Pathway analysis revealed highly significant downregulation of pathways involved in lipid metabolism, including patatin-like phospholipase domain containing 3 (Pnpla3 that encodes a membrane-bound protein with a predominant triacylglycerol lipase activity [14, 15]. To determine if the Pnpla3 reduction contributes to the higher insulin sensitivity in HSD-fed LKO mice, Flox and LKO mice that were consuming an HSD were injected with a recombinant adenovirus encoding mouse Pnpla3 (Ad-Pnpla3) or green fluorescent protein (Ad-GFP). The restoration of hepatic Pnpla3 protein expression in HSD-fed LKO mice remarkably reversed both the increase in insulin sensitivity and the increase in the insulin-stimulated phosphorylation of hepatic Akt. These results suggest that Elovl6- and Pnpla3-dependent lipid metabolic pathways are crucial in regulating hepatic insulin signaling under lipogenic conditions.

We hypothesized that Elovl6 and Pnpla3 cooperate in regulating hepatic insulin sensitivity by changing the acyl-chain composition of specific lipid(s) that modulate insulin action. To test this hypothesis, we performed lipidomics on liver samples from HSD-fed Flox and LKO mice injected with either Ad-GFP or Ad-Pnpla3. Elovl6-related changes occurred in a variety of lipids, including ceramides, cholesterol esters, FFAs, phospholipids, lysophospholipids, sphingomyelins, and triacylglycerol. Consistent with Pnpla3′s physiological role, its expression altered the distribution of triacylglycerol, free FA species, ceramide, and sphingomyelin species. Among these, ceramide(d18:1/18:0) exhibited changes corresponding to the effects of manipulating Elovl6 and Pnpla3; the concentration of ceramide(d18:1/18:0) was lower in LKO liver than in Flox liver and was increased by the restoration of Pnpla3 expression in LKO mice.

Ceramides have been implicated in the lipid-induced inhibition of insulin sensitivity by activating protein phosphatase 2A (PP2A) and PKCζ [16, 17]. HSD feeding significantly increased PP2A activity in the liver of Flox mice but not LKO mice. The restoration of hepatic Pnpla3 expression in HSD-fed LKO mice significantly increased protein phosphatase 2A (PP2A) activity to a level comparable to HSD-fed Flox mice. Ceramide activates PP2A activity in part via direct binding of Inhibitor 2 of PP2A (I2PP2A/SET oncogene), which prevents the interaction between PP2A and I2PP2A [18]. Notably, I2PP2A preferentially binds C18-ceramides over C14–C16 ceramides [18, 19]. We hypothesized that if the major effect of Elovl6 deficiency on hepatic insulin signaling is to reduce hepatic ceramide(d18:1/18:0) production, this effect would be altered by altering I2PP2A–PP2A binding. To test this hypothesis, HepG2 cells transfected with HA-tagged PP2A catalytic subunit alpha isoform (PPP2CA) and FLAG-tagged I2PP2A were treated with ceramide(d18:1/16:0), ceramide(d18:1/18:0), or ceramide(d18:1/20:0). Ceramide(d18:1/16:0) did not change the PPP2CA–I2PP2A interaction but ceramide(d18:1/18:0) and ceramide(d18:1/20:0) significantly did. These results suggest that the higher hepatic insulin sensitivity in HSD-fed LKO mice may be mediated through the suppression of C18:0-ceramide production, resulting in a reduction in ceramide-induced PP2A activity in the liver and lower PP2A-dependent downregulation of insulin signaling in an I2PP2A-dependent manner, implying a disinhibition of insulin signaling (Fig. 3).

Fig. 3.

Role of the Elovl6/Pnpla3/CerS pathway regulating hepatic insulin signaling. Excess consumption of carbohydrate and hyperinsulinemia activate de novo lipogenesis and upregulate the expression of Elovl6 and Pnpla3 in the liver. Activation of Elovl6 increases the amount of C18:0, enhancing C18:0-ceramide production through formation of an enzyme complex with ceramide synthase (CerS) on the endoplasmic reticulum (ER). Pnpla3 acts on the surface of lipid droplets (LDs), releases FAs from triacylglycerols (TAGs), and modifies ceramide FA composition by forming an enzyme complex with CerS at the ER-LD interface. Both Elovl6 and Pnpla3 are linked to the generation of C18:0-ceramide, which activates protein phosphatase 2A (PP2A) by disrupting the interactions with its endogenous inhibitor I2PP2A. As a consequence, PP2A inhibits insulin signaling by impairing Akt activation

Liver-specific deletion of Elovl6 enhances insulin sensitivity in mice by reducing the generation of ceramides involved in the endogenous activation of PP2A. Our observations linking Elovl6 and ceramides could provide insights into the pathophysiology of insulin resistance and suggest that inhibition of Elovl6 may represent a therapeutic target for the amelioration of insulin resistance.

Conclusion

Resolution of insulin resistance and impaired insulin secretion in obese mice is usually accompanied by loss of fat or body weight. As outlined in this review, global and liver-specific Elovl6^−/− mice are unique in that their insulin resistance and/or impaired insulin secretion is improved without amelioration of obesity or hepatosteatosis. These results highlight the importance of cellular and tissue FA composition in insulin sensitivity and lipotoxicity, especially the ratio of C16–18 FAs, which is controlled by Elovl6 activity. These findings reveal that Elovl6 is an important metabolic control point in insulin sensitivity and lipotoxicity. Importantly, studies in humans have shown that genetic variations in the ELOVL6 gene are associated with risk of T2DM [20–22]. Therefore, Elovl6 can be recommended as a promising therapeutic target for the prevention and treatment of diabetes and other obesity-related metabolic diseases.

Distinct effects of different long chain FAs depending upon their extent of desaturation have been observed. Our current studies suggest that the length of FAs is also important in energy metabolism and insulin sensitivity. Our observations could lead to new therapeutic strategies for diabetes and obesity-related diseases that target elongase enzymes. Therefore, a better understanding of the function and/or regulation of the enzymes that control the quality of lipids may help the development of potential therapeutic approaches for this disease.

Compliance with ethical standards

Conflict of interest

The author declares that he has no conflict of interest.

Human or animal rights

This article does not contain any studies with human or animal subjects performed by any of the authors.