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. 2019 Feb 27;10(1):947.
doi: 10.1038/s41467-019-08591-6.

Hepatic Sdf2l1 controls feeding-induced ER stress and regulates metabolism

Takayoshi Sasako  1   2   3   4   5 , Mitsuru Ohsugi  1 , Naoto Kubota  1   2   6 , Shinsuke Itoh  1   7 , Yukiko Okazaki  1   3 , Ai Terai  1   3 , Tetsuya Kubota  1   8   9 , Satoshi Yamashita  10 , Kunio Nakatsukasa  11   12 , Takumi Kamura  11 , Kaito Iwayama  13 , Kumpei Tokuyama  13 , Hiroshi Kiyonari  14   15 , Yasuhide Furuta  14   15 , Junji Shibahara  16 , Masashi Fukayama  16 , Kenichiro Enooku  17 , Kazuya Okushin  17 , Takeya Tsutsumi  18 , Ryosuke Tateishi  17 , Kazuyuki Tobe  19 , Hiroshi Asahara  10 , Kazuhiko Koike  17 , Takashi Kadowaki  20   21   22   23 , Kohjiro Ueki  24   25   26
Affiliations

Hepatic Sdf2l1 controls feeding-induced ER stress and regulates metabolism

Takayoshi Sasako et al. Nat Commun. .

Abstract

Dynamic metabolic changes occur in the liver during the transition between fasting and feeding. Here we show that transient ER stress responses in the liver following feeding terminated by Sdf2l1 are essential for normal glucose and lipid homeostasis. Sdf2l1 regulates ERAD through interaction with a trafficking protein, TMED10. Suppression of Sdf2l1 expression in the liver results in insulin resistance and increases triglyceride content with sustained ER stress. In obese and diabetic mice, Sdf2l1 is downregulated due to decreased levels of nuclear XBP-1s, whereas restoration of Sdf2l1 expression ameliorates glucose intolerance and fatty liver with decreased ER stress. In diabetic patients, insufficient induction of Sdf2l1 correlates with progression of insulin resistance and steatohepatitis. Therefore, failure to build an ER stress response in the liver may be a causal factor in obesity-related diabetes and nonalcoholic steatohepatitis, for which Sdf2l1 could serve as a therapeutic target and sensitive biomarker.

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Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
ER stress induced by feeding in the liver and regulation of Sdf2l1 expression. a–d C57BL/6J mice in an ad libitum-fed state were fasted for 24 h and then refed for 6 h, and ER stress markers were analyzed by a RT-PCR (n = 4), b Western blotting of total lysates (n = 3), c Western blotting of nuclear extracts (XBP-1s and nATF6 were detected at 55 and 50 kDa, respectively) (n = 4), and d Western blotting of microsomal fractions (n = 4). e–g Sdf2l1 promoter assays in Fao cells by transfecting luciferase (Luc) plasmids, with tunicamycin treatment, assessed with one-way ANOVA (n = 3), e using deletion mutants of upstream sequences, f using partial or total deletion mutants of the 11 bases of interest, and g using reporter plasmids, with knocking down of Xbp1 and/or Atf6. h ChIP assay, using antibodies recognizing XBP-1 or ATF6, for analysis of binding with the Sdf2l1 promoter in the liver, in a 24-h fasted state and a 3-h refed state (n = 3). The relative expression levels were normalized by the upstream region (−3.5 kb) and then by control IgG (rabbit or mouse IgG, respectively). Values of the data are expressed as mean ± SEM. *P < 0.05, **P < 0.01. Unpaired 2-tailed t-test was used for assessment
Fig. 2
Fig. 2
Functions of Sdf2l1 in vitro. a–c, f Cultured cells were infected with Ad-InsC96Y, whose lysates were immunoprecipitated for Western blotting: a, b Sdf2l1-floxed MEF cells, with a co-infection of Ad-Cre to knock out Sdf2l1, and with b further co-infection of Ad-Sdf2l1-FLAG to restore the expression; c NIH/3T3 cells, with either Sdf2l1 or Hspa5 knocked down, or with treatment with thapsigargin for 6 h; f primary hepatocytes, with Sdf2l1 or Tmed10 knocked down. In a, the lanes were run on the same gel but were noncontiguous. Multi: multiubiquitinated insulin, Mono: monoubiquitinated insulin. d RT-PCR to analyze ER stress marker gene expression in NIH/3T3 cells with Sdf2l1 and/or Hspa5 knocked down, treated with tunicamycin treatment (n = 3). e Wild-type mice were administered with Ad-Sdf2l1-FLAG intravenously (2.0 × 107 PFU/g body weight [BW]), and refed for 6 h after fasting for 24 h, whose microsomal fractions were immunoprecipitated for Western blotting. g RT-PCR to analyze ER stress marker gene expression in primary hepatocytes, with ER-resident molecule(s) knocked down, treated with 100 nM insulin for 2 h after serum starvation for 16 h (n = 4). Values of the data are expressed as mean ± SEM. *P < 0.05, **P < 0.01. One-way ANOVA was used for assessment
Fig. 3
Fig. 3
Functions of Sdf2l1 in vivo. Wild-type mice were administered with Ad-Sdf2l1 RNAi intravenously (2.0 × 107 PFU/g BW) to knock down the gene in the liver. a–c ER stress and insulin signaling in the liver (n = 4–5), analyzed by a RT-PCR (n = 4–5), b Western blotting of microsomal fractions (n = 4), and c Western blotting of total lysates (n = 4). d–f Effects of knocking down on glucose metabolism (n = 9–11): d ad libitum-fed plasma glucose, e plasma glucose in insulin tolerance test (ITT), after intraperitoneal injection of human regular insulin (0.75 U/kg BW), f plasma glucose and insulin levels in an oral glucose tolerance test (OGTT), after oral administration of glucose (0.75 g/kg BW), following 16 h of fasting. g–i Results of hyperinsulinemic-euglycemic clamp studies after 3 h of fasting (2.5 mU/kg/min, n = 6–7), in wild-type mice with Sdf2l1 knocked down: g glucose infusion rate (GIR), endogenous glucose production (EGP), and rate of glucose disappearance (Rd); h, i the liver samples after the clamp studies were analyzed by h Western blotting and i RT-PCR. j, k Effects of knocking down on lipid metabolism, analyzed with j triglyceride contents quantification (n = 4–5), and k Oil Red O staining. Values of the data are expressed as mean ± SEM. *P < 0.05, **P < 0.01. Unpaired 2-tailed t-test was used for assessment. Scale bars: 100 μm
Fig. 4
Fig. 4
Generation and phenotypes of the knock out model of Sdf2l1. Sdf2l1-floxed mice were administered with Ad-Cre (3.0 × 107 PFU/g BW), to generate a liver-specific Sdf2l1 knockout model in adults. a Detection of deleted exon 2 of the Sdf2l1 gene by PCR. b, d–i Metabolic phenotypes analyzed 3–6 weeks after the adenovirus administration (n = 8–9): b body weight, d ad libitum-fed plasma glucose, e plasma glucose in insulin tolerance test (ITT), after intraperitoneal injection of human regular insulin (0.75 U/kg BW), f plasma glucose levels in an pyruvate tolerance test, after intraperitoneal injection of pyruvate (1.5 g/kg BW), g plasma glucose levels in an oral glucose tolerance test (OGTT), after oral administration of glucose (0.75 g/kg BW), following 16 h of fasting. c ER stress in the liver (n = 3–6), analyzed by RT-PCR. h, i Effects on lipid metabolism, analyzed with h triglyceride contents quantification (n = 3–6), and i Oil Red O staining. Values of the data are expressed as mean ± SEM. *P < 0.05, **P < 0.01. Unpaired 2-tailed t-test was used for assessment. Scale bars: 100 μm
Fig. 5
Fig. 5
ER stress responses in obesity and diabetes. Both db/db mice and m/m mice were fasted for 24 h and refed for 6 h. a–c, e ER stress markers were analyzed by a Western blotting of total lysates (n = 4), b RT-PCR (n = 3–6), c Western blotting of nuclear extracts (n = 3–7), and e Western blotting of microsomal fractions (n = 3–4). d ChIP assay, using antibodies recognizing XBP-1 or ATF6, for analysis of binding of transcription factors with the Sdf2l1 promoter in the liver, in a 1-h refed state after 24 h of fasting (n = 3). The relative expression levels were normalized by the upstream region (−3.5 kb) and then by control IgG (rabbit or mouse IgG, respectively). Values of the data are expressed as mean ± SEM. *P < 0.05, **P < 0.01. Unpaired 2-tailed t-test was used for assessment
Fig. 6
Fig. 6
Restoration of Sdf2l1 expression in an obesity and diabetes model. a–h db/db mice were administered with Ad-Sdf2l1 intravenously (2.5 × 107 PFU/g BW), for restoration of the gene expression in the liver. a–c ER stress and insulin signaling in the liver, analyzed by a RT-PCR (n = 3–5), b Western blotting of microsomal fractions (n = 3–5), and c Western blotting of total lysates (n = 3–4). d–f Effects of the restoration on glucose metabolism (n = 9–10), including d plasma glucose levels in an ad libitum-fed state, e plasma glucose levels in OGTT, as well as insulin levels, after oral administration of glucose (0.75 g/kg BW), following 24 h of fasting. f Results of hyperinsulinemic-euglycemic clamp studies (12.5 mU/min/kg, n = 6–7), after 3 h of fasting. g, h Effect of the restoration on lipid metabolism, analyzed with g triglyceride contents quantification (n = 8–10), and h Oil Red O staining. i, j Phenotypes of db/db mice co-administered with Ad-Sdf2l1 and Ad-BiP-HA, assessed with one-way ANOVA: i plasma glucose in an ad libitum-fed state (n = 4–6), and j the area under the curve (AUC) of plasma glucose in ITT, after intraperitoneal injection of human regular insulin (1.5 U/kg BW) (n = 6–8). Values of the data are expressed as mean ± SEM. *P < 0.05, **P < 0.01. a–g Unpaired 2-tailed t-test was used, and i, j one-way ANOVA was used for assessment. Scale bars: 100 μm
Fig. 7
Fig. 7
ER stress responses in human subjects. Expression of ER stress marker genes and the ratios between them, in liver biopsy samples of human male subjects with suspected nonalcoholic fatty liver disease (NAFLD), analyzed by RT-PCR. a Schematic description of factors affecting expression of marker genes and ratios. b Correlation with glucose tolerance (n = 64). c Comparison between diabetic (n = 25) and matched nondiabetic subjects (n = 25). d–h Correlation with progression of comorbid diseases in diabetic (n = 25) and matched nondiabetic subjects (n = 25), including d–f insulin resistance, and g, h staging of NASH. Data are shown in scatter dot plot with the median and interquartile range. *P < 0.05, **P < 0.01, T tertile. b, g, h Spearman’s rank correlation was used, and c, e, f the Mann–Whitney U test was used for assessment
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