Deletion or inhibition of PTPRO mitigates diet-induced hepatic steatosis and inflammation in obesity

Chronic inflammation plays crucial roles in obesity-induced metabolic diseases. We herein demonstrated that mice lacking the protein tyrosine phosphatase receptor type O (PTPRO) exhibited the hyper-obese phenotype when fed a high-fat/high-sucrose diet. However, Ptpro-KO mice with hyperobesity showed the markedly small accumulation of ectopic fat in the liver, improvements in lipid and glucose homeostasis, and low-grade systemic inflammation associated with low macrophage activation. Expression of protein tyrosine phosphatase 1b (Ptp1b), an enzyme which is known to be implicated in metabolic disorders, was also suppressed in Ptpro-KO mice. The administration of AKB9778, a specific inhibitor of PTPRO, to highly obese ob/ob mice reproduced the phenotypes of Ptpro-KO mice along with the amelioration of inflammation. We revealed that an increase in the phosphorylation of Tyr(117) in vimentin, a component of intermediate filaments, by the inhibition of PTPRO promoted the growth of lipid droplets in adipocytes. The improvement in metabolic conditions with the attenuation of inflammation in Ptpro-KO mice was explained by the low activation of NFκb, a key transcription factor for inflammatory response, in adipose tissue. This is the first study to show that PTPRO is a promising target to ameliorate hepatic steatosis and metabolic disorders.

glucose metabolism in addition to a markedly larger adipose tissue mass than control 54 ob/ob mice (Kim et al., 2008). 55 Adipose tissue is well recognized as a significant depot of stromal cells, including 56 immune cells, which exert detrimental effects on overall health (Kahn et al., 2019; (TG), and cholesterol levels in the livers of ob/ob mice treated with vehicle or AKB9778. 126 Values are means ± SEM (n = 8 each). P values are based on the unpaired Student's t- 127 test. NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001.     In contrast to the livers of WT mice with obesity that turned yellowish due to fatty 145 liver, those of Ptpro-KO mice with hyper obesity showed a normal red appearance under 146 HFHSD at 24 weeks of age ( Figure 1C). The liver weight of Ptpro-KO mice was ~70% 147 that of WT mice (WT; 4.68 ± 0.18 g: KO; 3.14 ± 0.22 g: P < 0.0001) ( Figure 1D). HE  2.58 nM, respectively). 180 We intraperitoneally administered AKB9778 (10 mg/kg body weight) daily to 181 leptin-deficient ob/ob mice, a model of severe obesity under ND feeding, for 4 weeks 182 from 16 weeks of age. No significant differences were observed in body weight gains 183 between the AKB9778-treated group and vehicle group during this period. The final body 184 weight of the AKB9778-treated group (58.90 ± 0.88 g) was similar to that of the vehicle 185 group (58.64 ± 0.51 g) at 20 weeks of age. The visible appearance of the liver in the 186 vehicle group was obviously white, whereas that of the AKB9778-treated group 187 apparently improved at 20 weeks of age ( Figure 1G). Liver weight was significantly 188 lower in the AKB9778-treated group than in the vehicle group (AKB9778; 4.66 ± 0.08 g: 189 Vehicle; 5.14 ± 0.11 g: P = 0.0017) ( Figure 1H). HE and Bodipy FL stainings of liver 190 sections both indicated that the accumulation of lipids in the liver was significantly 191 reduced by the AKB9778 treatment ( Figure 1I). Quantitative biochemical analyses 192 11 also showed that total lipid, TG, and cholesterol levels in the liver were all significantly 193 lower in the AKB9778-treated group than in the control group ( Figure 1J). These 194 changes induced by the AKB9778 treatment were consistent with those observed in 195 Ptpro-KO mice. 196 We then compared the mRNA expression levels of inflammatory markers in the  The activities of alanine aminotransferase (ALT) and aspartate aminotransferase 208 (AST), markers of liver damage, were significantly lower in HFHSD-fed Ptpro-KO mice 209 than in HFHSD-fed WT mice (P = 0.0152 and P = 0.0045, respectively) ( Figure 2C).

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Consistently, the AKB9778-treated group of ob/ob mice also showed significantly lower 211 ALT and AST activities than the vehicle group (P = 0.0457 and P = 0.0308, respectively) 212 ( Figure 2D). Therefore, the liver phenotypes induced by obesity were significantly 213 suppressed by the Ptpro deficiency and AKB9778 treatment, suggesting a crucial role for 214 PTPRO activity in the induction of hepatic steatosis and inflammation in obesity.    Values are means ± SEM (n = 8 each). P values are based on the unpaired Student's t- 230 test. NS, not significant; *P < 0.05, **P < 0.01, ***P < 0.001. The Ptpro deficiency and AKB9778 treatment induce the expansion of adipose tissue 241 In contrast to the liver, the weights of adipose tissues, including epididymal adipose tissue, 242 were ~45% higher on average in Ptpro-KO mice than in WT mice at 24 weeks of age 243 under HFHSD (P = 0.0006) ( Figure 3A and Table1); the weights of individual adipose 244 tissues in Ptpro-KO mice (epididymal, 2.96 ± 0.19 g; mesenteric 1.32 ± 0.03 g; 245 retroperitoneal, 2.52 ± 0.06 g; subcutaneous, 7.76 ± 0.21 g; n = 8) were markedly higher 246 than those in WT mice (epididymal, 2.06 ± 0.09 g; mesenteric, 0.96 ± 0.03 g; 247 retroperitoneal, 1.74 ± 0.07 g; subcutaneous, 5.09 ± 0.21 g; n = 8). The ratio of the fat 248 mass to the body weight of each part was significantly higher in Ptpro-KO mice than in 249 WT mice (Table 1). In paraffin sections of epididymal adipose tissue, Ptpro-KO mice 250 had a higher number of larger adipocytes than WT mice; the average diameter of 251 adipocytes in Ptpro-KO mice was ~1.3-fold larger than those in WT mice (P < 0.0001) 252 ( Figure 3B). Consistently, the mass of all adipose tissues increased by ~20% on average 253 in ob/ob mice treated with AKB9778 ( Figure 3C and Table 2           higher in Ptpro-KO mice than in WT mice (P < 0.0001 and P = 0.0159, respectively).

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On the other hand, no significant differences were observed in the expression levels of  tissue was smaller in AKB9778-treated ob/ob mice (P = 0.0106) ( Figure 5D). 427 We also examined the activation of the transcriptional factor NFκb, which is  To elucidate the molecular mechanism involving PTPRO in adipose tissue expansion, we 607 examined differences in the phosphorylation of tyrosine (Tyr) in cellular proteins in 3T3-608 L1 adipose cells upon the AKB9778 treatment. By using the two-dimensional 609 electrophoresis of Tyr-phosphorylated proteins, we found that a protein spot with ~53 610 kDa was markedly increased by the AKB9778 treatment ( Figure 8D). This protein was   In PTPRO-deficient mice, inflammation in adipose tissue (Figure 4) and the liver 783 46 (Figure 2) was markedly low and the metabolic state remained healthy (Figures 2, 3), 784 despite the significant expansion of adipose tissue (Figure 1). In more details, the 785 accumulation of ectopic fat in the liver was markedly lower than in control mice ( Figure   786 1) and the expression of pro-inflammatory factors was significantly suppressed (Figures   787   2, 4), while on the other hand, lipid and glucose homeostasis (Figures 1, 10)  insulin sensitivity (Figures 4, 10).

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PTPRO activity is assumed to be involved in the metabolic function of tissues Ptpro-KO and AKB9778-treated ob/ob mice (Figures 6 and 7). Mild fibrosis may also 877 be one of the mechanisms underlying the enlargement of adipocytes associated with 878 improved metabolism in these mice.   Immunostaining was performed as previously described (Shintani et al., 2017).

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Images were acquired with a BZ-X810 fluorescence microscope (Keyence Corporation,

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Osaka, Japan). Stained areas were quantified in 4 non-overlapping fields randomly