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. 2013 Mar;62(3):811-24.
doi: 10.2337/db11-1652. Epub 2012 Dec 3.

Proteasome dysfunction mediates obesity-induced endoplasmic reticulum stress and insulin resistance in the liver

Toshiki Otoda  1 , Toshinari Takamura, Hirofumi Misu, Tsuguhito Ota, Shigeo Murata, Hiroto Hayashi, Hiroaki Takayama, Akihiro Kikuchi, Takehiro Kanamori, Kosuke R Shima, Fei Lan, Takashi Takeda, Seiichiro Kurita, Kazuhide Ishikura, Yuki Kita, Kaito Iwayama, Ken-ichiro Kato, Masafumi Uno, Yumie Takeshita, Miyuki Yamamoto, Kunpei Tokuyama, Shoichi Iseki, Keiji Tanaka, Shuichi Kaneko
Affiliations

Proteasome dysfunction mediates obesity-induced endoplasmic reticulum stress and insulin resistance in the liver

Toshiki Otoda et al. Diabetes. 2013 Mar.

Abstract

Chronic endoplasmic reticulum (ER) stress is a major contributor to obesity-induced insulin resistance in the liver. However, the molecular link between obesity and ER stress remains to be identified. Proteasomes are important multicatalytic enzyme complexes that degrade misfolded and oxidized proteins. Here, we report that both mouse models of obesity and diabetes and proteasome activator (PA)28-null mice showed 30-40% reduction in proteasome activity and accumulation of polyubiquitinated proteins in the liver. PA28-null mice also showed hepatic steatosis, decreased hepatic insulin signaling, and increased hepatic glucose production. The link between proteasome dysfunction and hepatic insulin resistance involves ER stress leading to hyperactivation of c-Jun NH₂-terminal kinase in the liver. Administration of a chemical chaperone, phenylbutyric acid (PBA), partially rescued the phenotypes of PA28-null mice. To confirm part of the results obtained from in vivo experiments, we pretreated rat hepatoma-derived H4IIEC3 cells with bortezomib, a selective inhibitor of the 26S proteasome. Bortezomib causes ER stress and insulin resistance in vitro--responses that are partly blocked by PBA. Taken together, our data suggest that proteasome dysfunction mediates obesity-induced ER stress, leading to insulin resistance in the liver.

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Figures

FIG. 1.
FIG. 1.
Proteasome dysfunction in mouse models of obesity. A: Gene expression and proteasome activity in the livers of patients with type 2 diabetes and obesity. Coordinate upregulation of genes involved in proteasome degradation pathways in the livers of type 2 diabetic patients with obesity compared with those without obesity. GenMAPP (Gene MicroArray Pathway Profiler [http://www.genmapp.org]) was used to annotate the pathway with expression ratios for the genes involved. The fold changes presented beside the gene names are for obese versus nonobese patients. Genes significantly upregulated in obesity (P < 0.05) are shown in red; genes analyzed whose expression was not significantly altered in obesity are shown in gray. B: Graphs showing associations between homeostasis model assessment of insulin resistance (HOMA-IR) and the mRNA expression of subunits of PA28: PA28α (PSME1), PA28β (PSME2), and PA28γ (PSME3). Data were normalized according to 18S mRNA level. C: Comparison of liver proteasome activity in 32-week-old C57BL6 mice fed STD and HFD for 28 weeks (from 4 to 32 weeks of age), in WT and db/db mice (20 weeks old), and in WT mice and ob/ob mice (20 weeks old). Proteasome activity was estimated by measuring chymotrypsin-like activity in liver tissues as described in research design and methods. Data represent means ± SE (n = 4 per group). *P < 0.05, **P < 0.01. D: Accumulation of polyubiquitinated proteins was assessed by Western blot in the livers isolated from C57BL6 mice fed the STD and the HFD, db/db mice, and ob/ob mice. GAPDH served as internal control.
FIG. 2.
FIG. 2.
PA28 KO mice show impaired proteasome function and accumulation of polyubiquitinated proteins in the liver. A: Mice were genotyped by PCR analysis as previously described (18,19). Genomic DNA extracted from mouse tails was analyzed by PCR. Primers used for genotyping were 5'-TTTCCTGTACGTGACTTCCATCCTGTTG-3' (primer 1), 5'-GGTCCACATACAATAAAGACATGGGCTG-3' (primer 2), and 5'-GATTGTGGTCCTCCTGCAACGCCTAAA-3' (primer 3). Primers 1 and 3 amplified 1.2-kb fragments from the wild-type PA28α allele. Primers 1 and 2 amplified 2.2-kb fragments from the mutant allele. Primers used for genotyping were 5'-CCGGGACAATAAGACACATCACTC-3' (primer 1), 5'-TTGTCCCTCCCTCCAGTTGTCTAA-3' (primer 2), and 5'-GATCCCCTCAGAAGAACTCGTCAA-3' (primer 3). Primers 2 and 3 amplified 0.9-kb fragments from the wild-type PA28γ allele. Primers 1 and 3 amplified 1.9-kb fragments from the mutant allele. B: RT-PCR analysis of total RNA prepared from liver of PA28 KO mice (n = 7). N.D., not determined. Transcripts of mouse PA28α, -β, and -γ were examined by quantitative RT-PCR. Data were normalized according to GAPDH mRNA level and presented as a value relative to that for WT mice. C: Western blot analysis of extracts prepared from the liver of PA28 KO mice. The blot was probed with the anti-PA28α, anti-PA28β, and anti-PA28γ antibodies. D: Body weights of WT and PA28 KO mice fed an STD or the HFD. ○, WT mice fed the STD (n = 5); ●, WT mice fed the HFD (n = 4); □, PA28 KO mice fed the STD (n = 4); ■, PA28 KO mice fed the HFD (n = 4). Data represent means ± SE. E: Comparison of liver proteasome activity in WT (n = 13, STD; n = 4, HFD) and PA28 KO (n = 11, STD; n = 4, HFD) mice. Data represent means ± SE. *P < 0.05. F: Proteasome activity was evaluated in the muscles isolated from 32-week-old WT mice (n = 5) and PA28 KO mice (n = 4). The activity was normalized to the total protein content. Data represent means ± SE. *P < 0.05. G: Western blot analyses of total ubiquitinated proteins in the livers of WT and KO mice. Livers were isolated from 32-week-old WT and PA28 KO mice fed the STD or the HFD for 28 weeks (from 4 to 32 weeks of age). Quantitation of ubiquitinated proteins levels is normalized to GAPDH and is represented as means ± SE (n = 3 per group). *P < 0.05.
FIG. 3.
FIG. 3.
PA28 KO mice show proteasome dysfunction, glucose intolerance, and attenuated insulin signaling in the liver. A: Blood glucose concentrations and serum insulin levels measured in an overnight fasting state at 20 weeks of age. WT (n = 9, STD; n = 5, HFD) and PA28 KO (n = 6, STD; n = 4, HFD) mice. Data represent means ± SE. *P < 0.05. B: Intraperitoneal glucose tolerance tests (IPGTT). C: Intraperitoneal insulin tolerance tests (IPITT). Blood glucose level measured during the intraperitoneal glucose tolerance tests and intraperitoneal insulin tolerance tests at 20 weeks of age. WT mice fed the STD, n = 5; WT mice fed the HFD, n = 4; PA28 KO mice fed the STD, n = 4; PA28 KO mice fed the HFD, n = 4. Data represent means ± SE. *P < 0.05. **P < 0.01. D: Insulin sensitivity was assayed by using a hyperinsulinemic-euglycemic clamp study in WT (n = 8) and PA28 KO (n = 10) mice. Left panel: Glucose infusion ratio. Middle panel: HGP before and after insulin clamp. Right panel: Glucose disposal rate. Data represent means ± SE. *P < 0.05. E: Equal amounts of protein in total lysates of liver and muscle were immunoblotted (IB) with anti–p-Akt (Ser473) and anti-Akt antibodies. p-Akt values of insulin-injected fasted mice were displayed relative to those of saline-injected mice (WT mice fed the STD, n = 7; PA28 KO mice fed the STD, n = 7). Data represent means ± SE. *P < 0.05. F: Expression of mRNAs encoding Irs2 in the livers of 12-week-old WT and PA28 KO mice analyzed by quantitative real-time RT-PCR. Expression values were normalized to 18S mRNA. Data represent means ± SE (n = 7 per group). *P < 0.05. Liver lysates from mice were immunoblotted with anti–IRS-2 antibody. G: Liver lysates from mice were immunoprecipitated (IP) using anti–IRS-1 antibody bound to protein A agarose. The immunoprecipitates were immunoblotted with anti–p–IRS-1 (Ser307) and p85. Representative results from five mice of each genotype are shown. Right panel: densitometry quantitation of IRS-1 to p85 signal ratio is shown. Data represent means ± SE. *P < 0.05.
FIG. 4.
FIG. 4.
PA28 KO mice show hepatic SREBP-1c activation and steatosis. A: Oil-red O staining of lipid droplets in the livers of 12-week-old WT and PA28 KO mice. The highlighted region of the upper panel is shown at a higher magnification in the lower panel. Scale bars, 100 μm. B: Triglyceride contents in the livers of WT and PA28 KO mice. Data are expressed as milligrams per gram of liver tissue. Data represent means ± SE (n = 4–5 per group). *P < 0.05. C: Expression of mRNAs encoding Srebf1 and Acc1 in the livers of 12-week-old WT and PA28 KO mice analyzed by quantitative real-time RT-PCR. Expression values were normalized to GAPDH mRNA. Data represent means ± SE (n = 7–9 per group). D: Whole cell, membrane, and nuclear fractions in liver extracts were subjected to SDS-PAGE and blotted using an anti–SREBP-1 antibody. GAPDH, pan-cadherin, and lamin A/C served as internal controls. Quantitation of SREBP-1 68-kDa protein levels is normalized to GAPDH and is represented as means ± SE. *P < 0.05.
FIG. 5.
FIG. 5.
Deletion of PA28 genes causes ER stress in the liver. A: Hematoxylin-eosin–stained liver sections from PA28 KO and WT mice. Scale bar, 200 μm. B: Electron microscopic analyses of the ER in livers from WT and PA28 KO mice. The highlighted region of the upper panel is shown at a higher magnification in the lower panel. Scale bars: 10 μm (upper panel) and 1 μm (lower panel). C: Western blotting for the ER stress–associated markers GRP78, CHOP, p-PERK, p-eIF2α, and p-IRE1α in the livers of 12-week-old male WT and PA28 KO mice. Upper right panel: Each expression level was quantified (n = 3 per group). Data represent means ± SE. *P < 0.05. Lower right panel: Western blotting for nuclear spliced form of XBP-1s protein amounts in the livers of 12-week-old male WT (n = 5) and PA28 KO (n = 5) mice. Lamin A/C served as internal control. Data represent means ± SE. *P < 0.05. D: Expression of mRNAs encoding CHOP and XBP-1s in the livers of WT (□) and PA28 KO (■) mice. Expression values were normalized to Actb mRNA. Data represent means ± SE (n = 4–7 per group). *P < 0.05. E: Western blotting for p-JNK, p–c-Jun, and total JNK in the livers of WT and PA28 KO mice.
FIG. 6.
FIG. 6.
Effects of a chemical chaperone, PBA, administration on proteasome dysfunction–induced ER stress and insulin resistance in PA28 KO mice. A–C: WT and PA28 KO mice were administered mixed PBA (4 mg/mL) through drinking water for 3 weeks. A: Electron microscopic analyses of the ER in livers from WT and PA28 KO mice administered orally with or without PBA. The highlighted region of the left panel (magnification ×5,000) is shown at a higher magnification in the right panel (magnification ×20,000). Scale bars: 10 μm (left panel) and 1 μm (right panel). B: IRE1α phosphorylation and CHOP levels in the livers of WT and PA28 KO mice. C: WT and PA28 KO mice fed STD were starved overnight and injected with insulin (10 IU/kg i.p.). Equal amounts of protein in total lysates of liver and muscle were immunoblotted with anti–p-Akt (Ser473) and anti-Akt antibodies. p-Akt values of insulin-injected fasted mice values were displayed relative to those of saline-injected mice. Data represent means ± SE (n = 3 per group). *P < 0.05. t-, total.
FIG. 7.
FIG. 7.
Proteasome dysfunction upregulates FoxO1 protein amounts and gluconeogenic gene expression in the liver of PA28 KO mice. A: Total liver extracts and nuclear fractions from the livers of WT and PA28 KO mice were analyzed by Western blotting for phosphorylated and total FoxO1. GAPDH served as internal control. Data represent means ± SE (n = 4–5 per group). *P < 0.05. Nuclear fractions from in the livers of WT and PA28 KO mice were analyzed by Western blotting for total FoxO1. Lamin A/C GAPDH served as internal control. Data represent means ± SE (n = 5 per group). *P < 0.05. B and C: Relative mRNA levels of FoxO1, G6pc, Pck1, Igfbp1, and Ppargc1a in the liver of WT and PA28 KO mice were analyzed by RT-PCR. Data were normalized according to GAPDH levels. Data represent means ± SE (n = 7 per group). *P < 0.05.
FIG. 8.
FIG. 8.
The proteasome inhibitor bortezomib (BZ) induces ER stress and insulin resistance in H4IIEC3 cells. A and B: H4IIEC3 cells were treated with the indicated concentrations of bortezomib (in DMEM supplemented with 10% FBS) for 24 h. After washing, cells were serum starved for 16 h and then treated with insulin (1 nmol/L) or phosphate-buffered saline for 15 min. Cells were solubilized, and equal amounts of proteins were analyzed by Western blotting using BiP-, p-IRE1α–, CHOP-, p-JNK–, total JNK (t-JNK)-, p-Akt–, and total Akt (t-Akt)-specific antibodies. C and D: H4IIEC3 cells were pretreated or not for 24 h with 2 mmol/L PBA and then treated with the indicated concentrations of bortezomib (in DMEM supplemented with 10% FBS) for 24 h. Cells were washed, serum starved for 16 h, and treated with insulin (1 nmol/L) or phosphate-buffered saline for 15 min. Cells were solubilized, and equal amounts of proteins were analyzed by Western blotting using BiP-, CHOP-, p-JNK–, total JNK–, p-Akt–, and total Akt–specific antibodies. Blots of p-Akt were quantitated densitometrically and expressed as ratios to total Akt (n = 4 for each condition). Relative density is mean ± SE fold increase over control. *P = 0.05 vs. treatment with insulin alone.
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