Protection from non-alcoholic steatohepatitis and liver tumourigenesis in high fat-fed insulin receptor substrate-1-knockout mice despite insulin resistance
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Epidemiological studies have revealed that obesity and diabetes mellitus are independent risk factors for the development of non-alcoholic steatohepatitis (NASH) and hepatocellular carcinoma. However, the debate continues on whether insulin resistance as such is directly associated with NASH and liver tumourigenesis. Here, we investigated the incidence of NASH and liver tumourigenesis in Irs1−/− mice subjected to a long-term high-fat (HF) diet. Our hypothesis was that hepatic steatosis, rather than insulin resistance may be related to the pathophysiology of these conditions.
Mice (8 weeks old, C57Bl/6J) were given free access to standard chow (SC) or an HF diet. The development of NASH and liver tumourigenesis was evaluated after mice had been on the above-mentioned diets for 60 weeks. Similarly, Irs1−/− mice were also subjected to an HF diet for 60 weeks.
Long-term HF diet loading, which causes obesity and insulin resistance, was sufficient to induce NASH and liver tumourigenesis in the C57Bl/6J mice. Obesity and insulin resistance were reduced by switching mice from the HF diet to SC, which also protected these mice against the development of NASH and liver tumourigenesis. However, compared with wild-type mice fed the HF diet, Irs1−/− mice fed the HF diet were dramatically protected against NASH and liver tumourigenesis despite the presence of severe insulin resistance and marked postprandial hyperglycaemia.
IRS-1 inhibition might protect against HF diet-induced NASH and liver tumourigenesis, despite the presence of insulin resistance.
KeywordsHigh-fat diet Insulin receptor substrate-1 Insulin resistance Liver tumourigenesis Non-alcoholic steatohepatitis
Haematoxylin and eosin
HOMA of insulin resistance
Non-alcoholic fatty liver disease
The prevalence of obesity and diabetes has been increasing globally over the past 30 years [1, 2]. These diseases not only increase cardiovascular risk, but also cancer risk and mortality rates [3, 4]. Especially, hepatocellular carcinoma (HCC), the fifth most common cancer and the third leading cause of cancer death worldwide , accounts for the largest increase in cancer and mortality risk in individuals with obesity or diabetes [3, 4]. Certain cases of HCC may be associated with infection with hepatitis B or C virus, or chronic alcohol use. However, an increasing number of cases are associated with non-alcoholic fatty liver disease (NAFLD). NAFLD encompasses a clinicopathological spectrum of diseases ranging from isolated hepatic steatosis to non-alcoholic steatohepatitis (NASH), the more aggressive form of fatty liver disease, which may progress to cirrhosis and cirrhosis-related complications including HCC. The prevalence of NAFLD, including NASH, is also increasing in parallel with the growing obesity and diabetes epidemics .
Although the causal relationships between obesity or diabetes, and NASH or liver tumourigenesis have not yet been clearly elucidated, it is assumed that the insulin resistance associated with obesity and diabetes is involved in the development of hepatic steatosis and inflammation in the liver, which may progress to NASH and liver tumourigenesis . Indeed, long-term high-fat (HF) diet loading, which can induce obesity and insulin resistance, was sufficient to induce NASH and liver tumourigenesis in C57Bl/6J mice [6, 7, 8]. Thus, this experimental model supports the above-mentioned concept.
IRS-1 and -2 exhibit a high structural homology, are abundantly produced in the liver and are thought to be responsible for transmitting insulin signalling from the insulin receptor to intracellular effectors in the regulation of glucose and lipid homeostasis [9, 10]. Insulin receptor signalling can be almost exclusively mediated by IRS-1 and IRS-2 in the liver ; indeed, a dominant role of IRS-1 has been observed during nutrient excess . Moreover, HF diet-fed liver-specific Irs1−/− mice displayed severe insulin resistance, but not hepatic steatosis . Also, mice with acyl-CoA:diacylglycerol acyltransferase (DGAT)2 overabundance in the liver reportedly developed hepatic steatosis without abnormal plasma glucose and insulin levels , while liver-specific phosphoinositide 3-kinase (PI3K) p110α-knockout (Pik3ca−/−) mice fed an HF diet were protected against hepatic steatosis without ameliorating HF diet-induced glucose intolerance . Mice with genetic defects or targeted overexpression, such as hepatocyte-specific Pten-knockout mice and Pik3ca transgenic mice [14, 15], have been reported to be models of NAFLD and liver tumourigenesis, but might not reflect the natural aetiology of NASH and liver tumourigenesis in human participants. Rather, insulin sensitivity was improved in hepatocyte-specific Pten-knockout mice, compared with wild-type mice , and Pik3ca transgenic mice exhibited better glucose tolerance than wild-type mice . Therefore, liver steatosis can occur independently of insulin resistance.
In the present study, we investigated the effect of a long-term HF diet on the development of NASH and liver tumourigenesis using C57Bl/6J male mice. Next, we performed similar experiments in which the HF diet was switched to a standard chow (SC) diet to clarify the effect of improved insulin resistance on the development of these diseases. We also investigated the incidence of NASH and liver tumourigenesis in Irs1−/− mice subjected to a long-term HF diet. The hypothesis behind this part of the study was that hepatic steatosis, rather than insulin resistance, may be related to the pathophysiology of these conditions.
Mice (Irs1−/−) were generated as described elsewhere . We backcrossed these mice with C57Bl/6J mice more than nine times. Male littermates derived from the intercrosses were fed an SC diet until 8 weeks of age and then had free access to the SC diet or an HF diet. In the dietary switch experiment, 8-week-old C57Bl/6J male mice were subjected to the HF diet for 30 weeks and then switched to the SC diet for the next 30 weeks; these mice were then compared with mice from the same genetic background that had received the HF diet for the entire 60 weeks. The mice were housed under a 12 h light–dark cycle. The animals were maintained according to standard animal care procedures based on institutional guidelines. These experiments involving animals were approved by the local Ethics Committee of the Yokohama City University.
We used an SC diet (MF; Oriental Yeast, Tokyo, Japan) and an HF diet (High-Fat Diet 32; Clea Japan, Tokyo, Japan). The composition of each of these diets is shown in electronic supplementary material (ESM) Table 1. The fatty acid composition of the HF diet consisted of 22% (wt/wt) saturated fatty acid (12.6% palmitic acid, 7.5% stearic acid) and 77% (wt/wt) unsaturated fatty acid (64.3% oleic acid, 10.2% linoleic acid).
Measurement of biochemical variables
Blood glucose levels were measured using a portable glucose meter and Glutest Neo (Sanwa Chemical, Nagoya, Japan). Insulin levels were determined using an insulin ELISA kit (Morinaga, Yokohama, Japan). Plasma alanine aminotransferase (ALT) levels were assayed using an enzymatic method (Wako Pure Chemical, Osaka, Japan). The plasma levels of total adiponectin and leptin were measured using ELISAs (Otsuka Pharmaceutical, Tokyo, Japan, and Morinaga, respectively). The triacylglycerol content of the liver was determined as described elsewhere . HOMA of insulin resistance (HOMA-IR) was calculated by using the formula [fasting insulin (mU/l) × fasting plasma glucose (mmol/l)] / 22.5. When insulin is expressed in SI units as pmol/l, the constant changes to 156.26.
Glucose tolerance test
Mice were denied access to food for more than 16 h before the study and then orally loaded with glucose at 1.5 mg/g body weight. Blood samples were collected before, and at 15, 30, 60 and 120 min after glucose loading.
Insulin tolerance test
The insulin tolerance test was performed under non-fasting conditions. Insulin was injected intraperitoneally, and blood samples were collected before, and at 30, 60, 90 and 120 min after the injection.
Liver samples were immersion-fixed overnight in 10% formalin (vol./vol.) at 4°C. The tissues were then routinely processed for paraffin embedding, and 5 μm sections mounted on glass slides were processed for haematoxylin and eosin (H&E) staining. The presence of collagen, which can be used as an index of fibrosis in lesions, was examined using Masson trichrome-stained preparations.
Liver histology and scoring system
All the histopathological findings were scored by experienced pathologists, who were unaware of the genetic backgrounds and diets of the mice. The histological features were grouped into three broad categories: steatosis, inflammation and fibrosis. The scoring system used for the evaluation is detailed in ESM Table 2.
RNA preparation and real-time quantitative PCR
Total RNA was prepared from portions of the liver using a reagent (Isogen; NipponGene, Tokyo, Japan), according to the manufacturer's instructions, and these samples were used as the starting material for cDNA preparations. cDNA was synthesised using reagents (TaqMan Reverse Transcription; Applied Biosystems, Foster City, CA, USA), followed by TaqMan quantitative PCR (50°C for 2 min and 95°C for 10 min, followed by 40 cycles at 95°C for 15 s and at 60°C for 1 min), performed on a PCR instrument (ABI Prism 7500; Applied Biosystems) to amplify the following genes: Pparg, Srebp1c (also known as Srebf1), Fas, Scd1, Ppara, Cpt1 (also known as Cpt1a), Mcad (also known as Acadm), Tnfa (also known as Tnf), Mcp1 (also known as Ccl2), p22phox (also known as Cyba), gp91phox (also known as Cybb), p47phox (also known as Ncf1) and Irs2. The relative expression levels were then compared after normalisation to the expression of beta-actin.
Results are expressed as means ± SEM (n). Differences between two groups were analysed for statistical significance using Student's t test. Individual comparisons among four groups were performed using an ANOVA followed by Fisher's protected least significant difference post-hoc test. Also, individual comparisons between two time points and two groups were performed using a two-way ANOVA. A value of p < 0.05 was considered statistically significant.
Effects of long-term HF diet on metabolic changes in C57Bl/6J mice
Effects of long-term HF diet on the risk of occurrence of NASH and liver tumourigenesis in C57Bl/6J mice
Switching from the HF to the SC diet: effects on metabolic changes in C57Bl/6J mice
Switching from the HF to the SC diet: effects on the incidence of NASH and liver tumourigenesis in C57Bl/6J mice
Effects of long-term HF diet on metabolic changes in Irs1−/− mice
Effects of long-term HF diet on the incidence of NASH and liver tumourigenesis in Irs1−/− mice
In the present study, we showed that long-term HF diet loading, which causes obesity and peripheral insulin resistance, was sufficient to induce NASH and liver tumourigenesis in C57Bl/6J mice, and that the reduction of obesity and peripheral insulin resistance by switching from the HF to an SC diet protected animals against the development of NASH and liver tumourigenesis. More importantly, the results of our study indicate that Irs1−/− mice fed the HF diet were dramatically protected against NASH and liver tumourigenesis, despite having severe insulin resistance and marked postprandial hyperglycaemia. These results are not consistent with the prevalent notion that the insulin resistance associated with obesity and diabetes is involved in the development of hepatic steatosis and inflammation in the liver, which may progress to NASH and liver tumourigenesis .
How can these results be explained? One explanation is the concept of selective or partial insulin resistance . Thus humans with insulin resistance caused by inherited mutations in the insulin receptor and mice with a liver-specific deletion of the insulin receptor have hyperglycaemia and hyperinsulinaemia, but are both protected against hepatic steatosis and hypertriacylglycerolaemia [23, 24]. This finding is consistent with the idea that not all signals are blunted in classical insulin-resistant states; instead, some signals are preserved, particularly those related to hepatic steatosis.
Insulin receptor signalling can be almost exclusively mediated by IRS-1 and IRS-2 in the liver, with IRS-2 mainly functioning during fasting and immediately after re-feeding, while IRS-1 functions primarily after re-feeding . In our results in wild-type mice, Irs2 levels were significantly decreased in the HF diet group, compared with those in the SC diet group, under fasting conditions, while levels of Irs1 in the HF diet group were similar to those in the SC diet group under re-feeding conditions (ESM Fig. 2). Insulin signalling might be decreased mainly under fasting conditions in the HF diet group, as the hyperinsulinaemia associated with an HF diet may suppress IRS-2 production . In this case, HF diet feeding might place the mice in a chronic postprandial state that preferentially inactivates IRS-2, with persistent IRS-1 signalling possibly promoting lipogenesis and leading to hepatic steatosis, since IRS-1 has been proposed to be the dominant regulator of expression of the hepatic genes controlling lipogenesis . In contrast, hepatic insulin signalling in Irs1−/− mice fed the HF diet was impaired, since IRS-1 was absent and IRS-2 signalling was suppressed by the hyperinsulinaemia associated with the HF diet. Thus, the pathophysiological features in Irs1−/− mice fed the HF diet might be similar to those in liver-specific Irs1/Irs2 double-knockout mice and in liver-specific insulin receptor knockout mice [11, 24]. A similar situation is seen with the liver-specific loss-of-function of the p110α subunit of PI3K  or of Akt . Since the IRS proteins lie between these steps , these previous studies using mouse models with genetic engineering of genes encoding the insulin receptor, IRS, PI3K and Akt are consistent with the phenotype of the Irs1−/− mice fed the HF diet in our study.
Importantly, the mice in the present study, unlike liver-specific knockout mice, had impaired IRS-1 functions in all their tissues. It thus remains unclear whether the protection against NASH and liver tumourigenesis is due to a global loss of insulin signalling or a liver-specific loss. Unfortunately, the current data do not answer this question. However, we assumed that the protection might be due to a liver-autonomous effect, since liver-specific Irs1−/− mice fed an HF diet, but not liver-specific Irs2−/− mice fed an HF diet were reportedly protected from hepatic steatosis  and the steatotic host microenvironment probably sets the stage for tumour development, even during the initially reversible and treatable stages of fatty liver disease . Therefore, this hypothesis should be further examined using liver-specific Irs1−/− and Irs2−/− mice  in the future.
Another question that remains unclear is whether the contribution or compensation of IRS-2 signalling plays a role in the above-mentioned protective effect. Our data show that Irs2 expression in Irs1−/− mice on the HF diet was significantly higher than in wild-type mice fed the HF diet. However, levels were significantly lower than those in wild-type mice fed the SC diet. Moreover, the basal and insulin-stimulated phosphorylation of Akt in Irs1−/− mice on the HF diet was lower than in wild-type mice fed the HF diet (data not shown). These results suggest that IRS-2 may not fully compensate for the loss of IRS-1 in Irs1−/− mice on an HF diet. We therefore assumed that the contribution of residual IRS-2 signalling was limited to the livers of Irs1−/− mice on the HF diet.
What is the relevance of the present results to the clinical management of humans with diabetes? Therapeutic targeting of IRS-1 may not be advisable, since IRS1 is an insulin resistance gene in humans  and our results suggest that blocking IRS-1-mediated signalling exacerbated glucose tolerance, even though it was able to protect against NASH and liver tumourigenesis. Epidemiological evidence suggests that people with diabetes have significantly higher risks of many forms of cancer . Recently, a meta-analysis of several studies showed that liver cancer is more common in patients with diabetes . Johnson et al commented that the accumulation of experimental and epidemiological evidence is more consistent with the hyperinsulinaemia hypothesis and less so with the hyperglycaemia hypothesis  with regard to the increased risk of cancer in patients with diabetes. Here, we showed that the reduction of obesity and hyperinsulinaemia by switching from an HF to an SC diet protected mice against the development of NASH and liver tumourigenesis without changing blood glucose levels. These results support the hyperinsulinaemia hypothesis. How can our results for Irs1−/− mice fed an HF diet be explained? We assumed that the protection against NASH and tumour development in Irs1−/− mice fed the HF diet was caused by the downregulation of IRS-1-mediated insulin action in the liver, despite systemic hyperinsulinaemia. As described above, the hyperinsulinaemia associated with an HF diet suppresses IRS-2 production, and persistent IRS-1 signalling promotes lipogenesis and hepatic steatosis. Thus, when mice were fed the HF diet, the wild-type mice developed hepatic steatosis, but the Irs1−/− mice were protected against the development of NASH and liver tumourigenesis, despite the presence of hyperinsulinaemia. Thus, these results for Irs1−/− mice fed an HF diet are consistent with the above-mentioned hyperinsulinaemia hypothesis. Therefore, the prevention of hyperinsulinaemia using glucose-lowering agents such as metformin and thiazolidinedione could not only protect against diabetes, but also against liver tumourigenesis. The effects of metformin on NASH and liver tumourigenesis in this mouse model are now under investigation.
In conclusion, long-term HF diet loading was sufficient to induce NASH and liver tumourigenesis in C57Bl/6J mice. Switching from an HF to an SC diet reduced obesity and insulin resistance, and protected against the development of NASH and liver tumourigenesis in the same mice. Moreover, Irs1−/− mice fed an HF diet were dramatically protected against NASH and liver tumourigenesis, suggesting that IRS-1 inhibition might protect against HF diet-induced NASH and liver tumourigenesis, despite the presence of insulin resistance.
We thank M. Kaji and E. Sakamoto (Department of Endocrinology and Metabolism, Graduate School of Medicine, Yokohama City University, Yokohama, Japan) for their excellent technical assistance and animal care.
This work was supported in part by: Grants-in-Aid for Scientific Research (B) 19390251 and (B) 21390282 from the Ministry of Education, Culture, Sports, Science and Technology (MEXT) of Japan; a Medical Award from the Japan Medical Association; a Grant-in-Aid from the Uehara Memorial Foundation; a Grant-in-Aid from the Daiichi–Sankyo Foundation of Life Science; and a Grant-in-Aid from the Naito Foundation (to Y. Terauchi).
All the authors conceived and designed the study, and participated in the analysis and interpretation of the data. AN drafted the manuscript, and all the other authors revised it critically for intellectual content. All the authors approved the final version of the paper.
Duality of interest
The authors declare that there is no duality of interest associated with this manuscript.
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