Transcriptional regulation of Acsl1 by CHREBP and NF-kappa B in macrophages during hyperglycemia and inflammation

Acyl-CoA synthetase 1 (ACSL1) is an enzyme that converts fatty acids to acyl-CoA-derivatives for lipid catabolism and lipid synthesis in general and can provide substrates for the production of mediators of inflammation in monocytes and macrophages. Acsl1 expression is increased by hyperglycemia and inflammatory stimuli in monocytes and macrophages, and promotes the pro-atherosclerotic effects of diabetes in mice. Yet, surprisingly little is known about the mechanisms underlying Acsl1 transcriptional regulation. Here we demonstrate that the glucose-sensing transcription factor, Carbohydrate Response Element Binding Protein (CHREBP), is a regulator of the expression of Acsl1 mRNA by high glucose in mouse bone marrow-derived macrophages (BMDMs). In addition, we show that inflammatory stimulation of BMDMs with lipopolysaccharide (LPS) increases Acsl1 mRNA via the transcription factor, NF-kappa B. LPS treatment also increases ACSL1 protein abundance and localization to membranes where it can exert its activity. Using an Acsl1 reporter gene containing the promoter and an upstream regulatory region, which has multiple conserved CHREBP and NF-kappa B (p65/RELA) binding sites, we found increased Acsl1 promoter activity upon CHREBP and p65/RELA expression. We also show that CHREBP and p65/RELA occupy the Acsl1 promoter in BMDMs. In primary human monocytes cultured in high glucose versus normal glucose, ACSL1 mRNA expression was elevated by high glucose and further enhanced by LPS treatment. Our findings demonstrate that CHREBP and NF-kappa B control Acsl1 expression under hyperglycemic and inflammatory conditions.


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CHREBP is a glucose responsive transcription factor that regulates metabolic genes, 220 including those involved in lipolysis and glycolysis [13][14][15]. An increase in intracellular glucose 221 levels relieves inhibition of CHREBP and promotes CHREBP translocation from the cytoplasm 222 into the nucleus, where it drives the expression of glucose responsive genes [16]. Elevated 223 glucose levels in diabetes is has been shown to increase CHREBP transcriptional activity in 224 liver and adipose tissue [17]. In addition, a ChIP-seq study for CHREBP from white adipose 225 tissue from the fasted to fed state showed CHREBP occupies multiple sites upstream of the 226 Acsl1 transcription start site [18], suggesting that a Acsl1 is a potential target of CHREBP.
227 Because the induction of Acsl1 by hyperglycemic is at the transcriptional level, and that 228 CHREBP occupies an upstream regulatory region of Acsl1 in adipose tissue, we hypothesized 229 that the induction of Acsl1 by hyperglycemia in macrophages is through CHREBP. To 230 investigate this we used both gain and loss of function approaches. Prior to embarking on these 231 experiments, we first determined the localization of CHREBP in BMDMs cultured under NG and 232 HG conditions. We observed an increase in CHREBP nuclear localization under HG compared 233 to NG conditions by cell fraction and immunofluorescence (S1_fig.pdf). This is consistent with 234 CHREBP being a potential transcriptional activator of Acsl1 under HG conditions.

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Next, we used a gain of function approach to determine whether overexpression of 236 CHREBP regulates Acsl1 promoter activity in a cell based reporter assay. We co-transfected 237 HEK293 cells with the same pACSL1-GLuc reporter as in Figure 1D, along with a CHREBP 238 expression construct, or an empty expression vector, under NG and HG conditions. Acsl1 239 promoter activity was higher in the cells expressing CHREBP in both NG and HG conditions 240 (Fig 2A). The basal promoter activity in cells with vector only was also higher in the HG 241 condition as compared to NG, and the Acsl1 promoter activity was further increased in cells 242 cultured in HG and overexpressing CHREBP ( Fig. 2A). This is consistent with CHREBP 243 inducing Acsl1 transcription in HG.

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To further examine the impact of CHREBP on glucose-dependent Acsl1 transcriptional 245 activation we turned to a loss of function approach. We evaluated Acsl1 expression in the 246 absence of CHREBP under NG and HG conditions using macrophages from Chrebp -/mice [15].
247 BMDMs from wild type littermate and Chrebp -/mice were differentiated under NG and HG 248 conditions, and Acsl1 mRNA expression was measured. Acsl1 expression was reduced in 249 Chrebp -/cells in both NG and HG conditions, with a greater reduction in Acsl1 expression in HG 250 from Chrebp -/cells compared to wild type controls (Fig. 2B). This result indicates that CHREBP 251 is required both for basal and glucose-induced expression of Acsl1.  274 Intriguingly, Acsl1 expression upon LPS treatment in HG-induced cells increased ~80-fold 275 relative to BMDMs not activated by LPS in NG. We also observed an increase ACSL1 protein 276 abundance and localization to membranes upon LPS treatment (S3_fig.pdf) as has been 277 described by others [4]. This suggests that both inflammatory and hyperglycemic signals 278 contribute to the regulation of Acsl1.

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We also compared the localization of CHREBP protein in unstimulated (M0) and LPS-280 treated inflammatory (M1) macrophages under NG and HG conditions. M1 macrophages show 281 increased nuclear CHREBP both in NG and HG conditions, with a slight increase in nuclear 282 CHREBP under HG conditions (Fig. 3B). This suggests that CHREBP is contributing to the 283 transcriptional regulation of ACSL1 during inflammation especially under HG conditions.

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To test the functional significance of NF-kappa B and CHREBP in establishing Acsl1 305 gene expression, we employed a cell based reporter assay using the pACSL1-GLuc reporter 306 gene containing the ~1.6 kB of upstream regulatory sequence. We transfected HEK293 cells 307 with pACSL1-GLuc, with expression vectors for CHREBP or RELA separately, or CHREBP and 308 RELA together, and measured the Acsl1 promoter activity. While there was a modest increase 309 in pACSL1-GLuc activity in cells transfected with CHREBP, there was no increase in reporter 310 activity with RELA alone (Fig. 4). Strikingly, co-expression of CHREBP and RELA showed a 311 synergistic increase Acsl1 promoter activity (Fig. 4). This suggests that CHREBP and NF-312 kappa B act together to increase Acsl1 transcriptional activity.

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Based on these findings, we propose that in macrophages under conditions of NG and 353 either no or low level inflammation the expression of Acsl1 is low and driven by a small pool of 354 active, nuclear CHREBP (Fig. 5A). Expression of Acsl1 is increased under conditions of HG by 355 virtue of an increase in the pool of nuclear CHREBP (Fig. 5B). We further posit that in 356 macrophages exposed to an acute inflammatory stimuli, such as by LPS, expression is of Acsl1 357 is increased by activation of NF-kappa B under both NG and HG, with even greater activation in 358 HG (Fig.5C). Whether NF-kappa B induction of Acsl1 also requires CHREBP remains an open 359 question, but is suggested by the low activity of the Acsl1 reporter with overexpressed RELA, 360 and that co-expression of both CHREBP and RELA synergistically activate the Acsl1 reporter. 369 Western blot of total cell lysates from BMDMs cultured in NG and HG using antibodies against 370 ACSL1 and β-actin as a loading control. C) Acsl1nascent RNA expression was determined by 371 qPCR using primers spanning the intron-exon junction relative to cyclophilin A1 and shown as 372 fold change between NG and HG. D) pACSL1-GLuc reporter was transfected in HEK 293 cells