Hepatic oxidative stress promotes insulin-STAT-5 signaling and obesity by inactivating protein tyrosine phosphatase N2

Abstract

Hepatic insulin resistance is a key contributor to the pathogenesis of obesity and type 2 diabetes (T2D). Paradoxically, the development of insulin resistance in the liver is not universal, but pathway selective, such that insulin fails to suppress gluconeogenesis but promotes lipogenesis, contributing to the hyperglycemia, steatosis, and hypertriglyceridemia that underpin the deteriorating glucose control and microvascular complications in T2D. The molecular basis for the pathway-specific insulin resistance remains unknown. Here we report that oxidative stress accompanying obesity inactivates protein-tyrosine phosphatases (PTPs) in the liver to activate select signaling pathways that exacerbate disease progression. In obese mice, hepatic PTPN2 (TCPTP) inactivation promoted lipogenesis and steatosis and insulin-STAT-5 signaling. The enhanced STAT-5 signaling increased hepatic IGF-1 production, which suppressed central growth hormone release and exacerbated the development of obesity and T2D. Our studies define a mechanism for the development of selective insulin resistance with wide-ranging implications for diseases characterized by oxidative stress.

Fig. 1. Oxidative stress and hepatic PTP oxidation

(a) 8 week-old male C57BL/6 mice were chow- or HFF for 24 weeks. Blood GSH and GSSG levels and GSH:GSSG ratios were determined. (b) Hepatocytes from 12-week chow- or HFF mice were either left untreated or serum-starved and stimulated with 100 nM insulin for 30 min and H₂O₂ production measured. (c, e-f) Livers from 24 week chow- or HFF mice were processed for an assessment of total PTP oxidation by (c) immunoblot analysis with the PTPox antibody, or (e) mass spectrometry determining relative PTP oxidation (PTP-SO₃H), or (f) processed for immunoblot analysis for the indicated PTPs. (d) Hepatocytes were processed immediately after isolation for an assessment of total PTP oxidation. In (c-d) arrows correspond to TCPTP. Results are means ± SEM for the indicated number of mice (a, e-f) or experimental repeats (b).

Fig. 2. Oxidative stress promotes insulin-induced STAT-5

(a) 8 week-old male C57BL/6 mice were chow- or HFF for 24 weeks, fasted, injected with PBS or insulin and livers processed for immunoblotting. (b) 20 week-old male C57BL/6 mice were subjected to hyperinsulinemic euglycemic clamps. Plasma GH levels were measured and livers processed for immunoblotting or quantitative real-time PCR to measure Igf-1. (c-g) Hepatocytes from chow-fed mice were treated with vehicle or 0.5 mM palmitate. (c, e-g) Hepatocytes were serum-starved, stimulated with 100 nM insulin and processed for immunoblotting. (d) H₂O₂ production was measured. Where indicated hepatocytes were pre-treated with vehicle, the JAK inhibitor CMP6 (10 µM), mitoTempol (10 µM) or SS31 (50 µM) for 2 h. In (f) hepatocytes were transfected with GFP control or Jak-2-specific siRNAs prior to palmitate treatment. (h) H₂O₂ production and (i) insulin (100 nM)-induced p-STAT-5 signaling in Gpx1+/+ and −/− hepatocytes. (j) 8 week-old male C57BL/6 mice were HFF for 12 weeks and treated with vehicle or SS31 for 10 days. Mice were fasted and injected with PBS or insulin (0.5 mU/g, 10 min) and livers extracted for immunoblotting. (k-l) Gpx1+/+ and −/− male mice were HFF for 24 weeks, fasted and livers processed for immunoblotting. Representative and quantified (means ± SEM) results are shown.

Fig. 3. Hepatic TCPTP deletion promotes obesity and insulin resistance

7 week-old lox/lox and LTKO mice were HFF for 12 weeks and (a, e) body weights, (b, f) gross morphology, (c, g) tissue weights, (d, h) body composition and (i) oxygen consumption, RER, energy expenditure and ambulatory activity assessed. (j) 7 week-old lox/+ and LTKO HET mice were HFF for 20 weeks and body weights and composition determined. (k) HFF mice were subjected to insulin tolerance tests and (l) fed and fasted blood glucose levels and plasma insulin levels determined. (m) Fasted HFF mice were injected with insulin and livers extracted for immunoblot analysis. Representative and quantified (means ± SEM) results are shown.

Fig. 4. LTKO mice exhibit steatosis and alterations in IGF-1 and GH

7 week-old male lox/lox and LTKO or lox/+ and LTKO HET mice were HFF for 12 and 20 weeks respectively. Livers were extracted from fasted mice and either (a, c) processed for histology, (b) lipid analyses, (c, d, g) immunoblotting, or (e, h) real time PCR. (f) Lipogenesis was assessed ex vivo in liver slices. (i) Hepatocytes from chow-fed C57BL/6 were transfected with GFP- or Ptpn2-specific siRNAs and after 48 h processed for real time PCR. (j) Lox/lox and LTKO mice were HFF for 12 weeks, fasted overnight and serum IGF-1 and GH levels determined. Results are means ± SEM.

Fig. 5. Hepatic TCPTP deletion exacerbates insulin-induced STAT-5 signaling

(a) 7 week-old male lox/lox and LTKO mice were HFF for 12 weeks. Livers from 4 h fasted mice were processed for immunoblotting. (b) Hepatocytes were serum-starved and stimulated with either 50 U/ml INF-γ, 100 U/ml IL-6 or 100 nM insulin and processed for immunoblotting. (c) 7 week-old male lox/+ and LTKO HET mice were HFF for 20 weeks. Livers from fasted mice were processed for immunoblotting. (d) Livers from 7 week-old fasted male lox/lox and LTKO mice were processed for immunoblotting. (e) Mice were HFF for 12 weeks, fasted and injected with PBS or insulin and livers processed for immunoblotting. Representative and quantified (means ± SEM) results are shown.

Fig. 6. Liver-specific STAT-5 heterozygosity corrects obesity but not steatosis

7 week-old male lox/lox, Ptpn2^lox/lox;STAT-5^lox/+ (lox/lox;STAT-5^lox/+), LTKO, and Alb-Cre;Ptpn2^lox/lox;STAT-5^lox/+ (LTKO;STAT-5^lox/+) mice were HFF for 12 weeks and (a) body and tissue weights, body composition and energy expenditure, determined, (b-c) fed and fasted blood glucose and plasma insulin measured and (d-f) livers extracted for (d) immunoblotting, (e) histology or (f) lipid analyses. Results shown are means ± SEM; significance (* LTKO and lox/lox, $ LTKO and lox/lox;STAT-5 ^lox/+, # LTKO and LTKO;STAT-5^lox/+) determined using a (a-c) one-way ANOVA or (f) a 2-tailed student’s t-test.

Fig. 7. JI-38 corrects the obesity in LTKO mice

(a-b) 7 week-old lox/lox, lox/lox;STAT-5 ^lox/+, LTKO, and LTKO;STAT-5^lox/+ mice were HFF for 12 weeks, fasted overnight and serum IGF-1 and GH levels (at 8 am) measured. (c-e) 7 week-old female lox/lox and LTKO mice were HFF for 24 days and administered vehicle or the synthetic GH releasing hormone agonist JI-38 (50 µg/kg in 100 µl) subcutaneously twice daily (8 am and 6 pm) for 20 days and body weights recorded. Serum GH levels (8 am; 30 min after JI-38) and body composition were determined. Results are means ± SEM; significance in a-b (^* LTKO and lox/lox, ^$ LTKO and lox/lox;STAT-5^lox/+, ^# LTKO and LTKO;STAT-5^lox/+) and c-d (^* LTKO and lox/lox, ^# LTKO + JI-38 and lox/lox) determined using one-way ANOVA.