RNA-binding protein HuD reduces triglyceride production in pancreatic β cells by enhancing the expression of insulin-induced gene 1

Abstract

Although triglyceride (TG) accumulation in the pancreas leads to β-cell dysfunction and raises the chance to develop metabolic disorders such as type 2 diabetes (T2DM), the molecular mechanisms whereby intracellular TG levels are regulated in pancreatic β cells have not been fully elucidated. Here, we present evidence that the RNA-binding protein HuD regulates TG production in pancreatic β cells. Mouse insulinoma βTC6 cells stably expressing a small hairpin RNA targeting HuD (shHuD) (βTC6-shHuD) contained higher TG levels compared to control cells. Moreover, downregulation of HuD resulted in a decrease in insulin-induced gene 1 (INSIG1) levels but not in the levels of sterol regulatory element-binding protein 1c (SREBP1c), a key transcription factor for lipid production. We identified Insig1 mRNA as a direct target of HuD by using ribonucleoprotein immunoprecipitation (RIP) and biotin pulldown analyses. By associating with the 3'-untranslated region (3'UTR) of Insig1 mRNA, HuD promoted INSIG1 translation; accordingly, HuD downregulation reduced while ectopic HuD expression increased INSIG1 levels. We further observed that HuD downregulation facilitated the nuclear localization of SREBP1c, thereby increasing the transcriptional activity of SREBP1c and the expression of target genes involved in lipogenesis; likewise, we observed lower INSIG1 levels in the pancreatic islets of HuD-null mice. Taken together, our results indicate that HuD functions as a novel repressor of lipid synthesis in pancreatic β cells.

Keywords: HuD; INSIG1; RNA-binding protein; SREBP1c; Triglyceride.

Fig. 1.

Downregulation of HuD accumulates triglyceride in pancreatic β cells. (a) Cell lysates from pancreatic βTC6 cells expressing pshHuD or control plasmid (βTC6-shHuD or βTC6-shCtrl) were prepared and the levels of HuD and housekeeping control protein β-actin levels were assessed by Western blot analysis. (b) Cells were fixed and incubated with 0.2% Oil-Red-O solution for 30 min and lipid accumulation was observed using a Zeiss Axioimager M1 microscope. Quantification of the Oil-Red-O-stained area of the lipid droplet in βTC6 cells was analyzed using ImageJ software. (c) The relative triglyceride content in cells was determined at 0, 3, and 6 days. (d) In cells processed as described in (a), the levels of SREBP1c, INSIG1, and β-actin were determined by Western blot analysis. Images in (a), (b), and (d) are representative from three independent experiments. Data in (c) represent the means ± SEM from three independent experiments; *, p < 0.05, **, p < 0.01.

Fig. 2.

HuD regulates INSIG1 expression by interacting with the Insig1 3′UTR. (a) βTC6 cell lysates was subjected to RIP followed by RT-qPCR analysis to measure the enriched Insig1 mRNA in anti-HuD IP compared control IgG IP. Gapdh mRNA was used for normalization. (b) Top, schematic of the Insig1 mRNA depicting the 5′UTR, coding region, and 3′UTR as well as the biotinylated transcripts (5U, CR, 3U-1, 3U-2, and 3U-3) that were synthesized for biotin pulldown analysis. Bottom, after incubation of each biotinylated transcript with βTC6 cell lysate, the interactions between the biotinylated transcripts and HuD were analyzed by Western blot analysis using anti-HuD antibody. The numbers indicate the mean ± SEM from three independent experiments. (c) Schematic of reporter plasmids pEGFP (control) and pEGFP + Insig1 3U-3. (d) Forty-eight hours after transfection of HuD-direct siRNA (siHuD) or overexpression of HuD using a plasmid (pHuD) along with appropriate controls (siCtrl and pcDNA, respectively), together with each reporter plasmid, the levels of GFP, HuD, and loading control β-actin were assessed by Western blot analysis. Data in (a) represent mean ± SEM from three independent experiments; *, p < 0.05, **, p < 0.01. Images in (b) and (d) are representative from three independent experiments.

Fig. 3.

HuD promotes translation of INSIG1 mRNA. (a) Forty-eight hours after transfection of βTC6 cells with either of HuD-direct siRNA (siHuD) or overexpression plasmid (pHuD) along with appropriate controls (siCtrl and pcDNA, respectively), the levels of Insig1 mRNA were measured by RT-qPCR and normalized to Gapdh mRNA. (b) Lysates prepared from βTC6 cells transfected as described in (a) were used to assess the level of INSIG1, HuD, and loading control β-actin by Western blot analysis. (c) Lysates prepared from βTC6 cells transfected as described in (a) were fractionated through sucrose gradients (left), and the relative distribution of Insig1 mRNA and Gapdh mRNA in each of 12 fractions was studied by RT-qPCR analysis (right). 40S, small ribosome subunits; 60S, large ribosome subunits; 80S, monosome; polysomes, polyribosomes. Polysome profiles are representative of two different fractionation analyses. (d) Nascent EGFP production was assessed by a brief (20-min-long) incubation with L-[³⁵S]methionine and L-[³⁵S]cysteine after transfection of HuD siRNA or HuD plasmid along with appropriate controls. Lysates were subjected to IP using anti-EGFP or anti-GAPDH antibodies, and the incorporation of radiolabeled amino acids into newly synthesized EGFP proteins was assessed by SDS-PAGE and visualized using a PharoseFX Plus. The data are representative of three independent experiments and the numbers indicate fold changes in EGFP levels. Numbers indicate the mean ± SEM from three independent experiments. *, p < 0.05; **, p < 0.01.

Fig. 4.

Downregulation of HuD enhances the transcriptional activity of SREBP1c in pancreatic β cells. (a) Forty-eight hours after transfection with siCtrl or siHuD, βTC6 cells were incubated with or without 0.4 mM of palmitate for 72 h, and the nuclear fractions were prepared using digitonin. The levels of nSREBP1c, HuD, lamin B, GAPDH, and loading control β-actin were assessed by Western blot analysis. (b) RNAs were prepared from cells as described in (a) and the relative expression of Fasn and Gpat mRNAs was determined by RT-qPCR analysis and normalized to the levels of Gapdh mRNA. (c) Schematic of reporter plasmids; control vector (pGL3-promoter-Luc) and luciferase vector containing triplicate SRE binding domains (pGL3-SREx3-Luc). (d) 100 ng of pGL3-SREx3-Luc or control plasmid was transfected to βTC6-shHuD or βTC6-shCtrl cells and both cell populations were incubated with or without 0.4 mM palmitate for 15 h. The relative luciferase activity was determined by measuring luminescent signal and normalized to each concentration of proteins. Data represent the means ± SEM from three independent experiments. *, p < 0.05; **, p < 0.01.

Fig. 5.

INSIG1 level was downregulated in HuD-null mice. (a and b) Pancreatic sections from WT (HuD+/+) and HuD-null (HuD−/−) mice were stained with anti-INSIG1 antibody. The intensity of INSIG1-positive areas in twenty islets from three pairs of mice was quantified using ImageJ. Data represent the means ± SEM. *, p < 0.05.

Fig. 6.

HuD expression was downregulated in the pancreas of db/db mice. (a and b) The relative levels of HuD mRNA and HuD protein in the pancreases of WT and db/db mice was analyzed by RT-qPCR and Western blot analysis. (b) Relative HuD and INSIG1 protein levels in the islet of WT or db/db mice (n = 3) was analyzed by immunohistochemistry using HuD and INSIG1 antibody. The intensity of HuD and INSIG1 was quantified using ImageJ. Data represent the means ± SEM. *, p < 0.05; **, p < 0.01. (d) Schematic diagram of INSIG1 regulation by HuD in pancreatic β-cells. HuD positively regulates INSIG1 expression at the post-transcription level by binding to the 3′UTR of Insig1 mRNA and enhancing its translation, thereby suppressing transcriptional activity of nSREBP1 (left). Downregulation of HuD results in the transcriptional activation of lipid biosynthetic genes such as Fasn and Gpat by nSREBP1; glucotoxic and lipotoxic conditions can promote the action of nSREBP1 by suppressing HuD function and thus lowering INSIG1 levels.