INPP4B protects from metabolic syndrome and associated disorders

Figures

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  Fig. 1. Inpp4b−/− males accumulate more fat due to decreased activity, energy expenditure, and respiratory exchange ratio. a Expression of Inpp4b in mouse tissues. b Expression of Inpp4b in epididymal, retroperitoneal, mesenteric, inguinal, and brown adipose tissue of FVB males. c Body weights of LFD WT (N = 14), LFD Inpp4b−/− (N = 12), HFD WT (N = 9), and HFD Inpp4b−/− (N = 10) mice were recorded every week beginning on post-partum day 2. LFD WT, LFD Inpp4b−/−, HFD WT, and HFD Inpp4b−/− groups are represented by the blue line with squares, red line with up-pointing triangles, green line with down-pointing triangles, and purple line with circles, respectively (**p = 0.0048, ***p = 0.001). d–f Percent fat (**p = 0.0012) (d), and percent lean (**p = 0.0014) (e) were determined by NMR and the ratios of fat-to-lean mass (**p = 0.00015) (f) were calculated for WT (N = 6) or Inpp4b−/− (N = 10) mice. g, h Hourly food (**p = 0.0027) and water intake (***p = 0.00023) were recorded for the WT (N = 6) or Inpp4b−/− (N = 8) mice. i Mouse ambulatory activity was detected with XYZ beam array over 92 h in WT (N = 6) or Inpp4b−/− (N = 10) mice. Total distances were acquired by MetaScreen and processed…

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  Fig. 2. Comparison of the metabolic and inflammatory profiles of adipose tissues from WT and Inpp4b−/− males. a Protein lysates from eWAT were analyzed for FAS, HK2, CD68, and tubulin by western blot. b Quantification of FAS protein expression in (a). The FAS values were normalized to tubulin and are shown as fold change (N = 3 per group). c–g RNA was extracted from mouse eWAT of LFD WT (N = 12), LFD Inpp4b−/− (N = 11), HFD WT (N = 8), and HFD Inpp4b−/− (N = 9) mice. The expression levels of Lep/Adipoq (c), Il6 (d), Tnf (e), Adgre1 (f), and Cd68 (g) were assayed by qRT-PCR using 18S as an internal control. h Quantification of CD68 protein in (a). Average expression in LFD WT mice was designated as 1 (N = 3 per group). i–l Concentrations of leptin (i), insulin (j), C-peptide (k), Resistin (l) in serum of LFD WT (N = 11), LFD Inpp4b−/− (N = 11), HFD WT (N = 9), and HFD Inpp4b−/− (N = 9) mice (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Exact p values are provided in Supplementary Data 2).

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  Fig. 3. INPP4B protects mice from hyperglycemia and diabetes. a Oral glucose tolerance test of LFD WT (N = 7), LFD Inpp4b−/− (N = 7), HFD WT (N = 9), and HFD Inpp4b−/− (N = 6) mice following a 2 g/kg oral glucose challenge. # is used to indicate statistically significant difference between LFD groups and * is used to indicate statistically significant difference for HFD groups. LFD WT, LFD Inpp4b−/−, HFD WT, and HFD Inpp4b−/− groups are represented by blue line with squares, red line with up-pointing triangles, green line with down-pointing triangles, and purple line with circles, respectively. b, c Blood glucose was measured in the same group as (a) after 6 h of fasting (b) or non-fasting (c) LFD WT, LFD Inpp4b−/−, HFD WT and HFD Inpp4b−/− mice. d Blood insulin was measured by ELISA in the same groups as (a). e INPP4B expression in livers of subjects with or without type II diabetes (T2D) were exported from GSE15653. f Representative pancreata sections from LFD WT, LFD Inpp4b−/−, HFD WT, and HFD Inpp4b−/− mice were stained for insulin and counterstained with hematoxylin (*p < 0.05, ***p < 0.001, ****p < 0.0001. Exact p values are provided in Supplementary Data 2).

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  Fig. 4. INPP4B protects mice from liver steatosis. a Livers were dissected from LFD WT (N = 11), LFD Inpp4b−/− (N = 13), HFD WT (N = 9), and HFD Inpp4b−/− (N = 10) mice. Liver weights in 4 experimental groups (*p = 0.0422 for LFD groups and *p = 0.0374 for HFD groups). b Representative H&E staining of liver sections from designated experimental groups used for steatosis analysis. c Morphometric quantification of the unstained area on H&E stained liver sections (*p = 0.111, ***p = 0.0004). d Representative images of the Oil Red O/hematoxylin staining of frozen liver sections in designated groups. e Characteristic features of hepatocellular steatosis in livers of HFD and LFD Inpp4b−/− males. Examples of microvesicular (mic) and macrovesicular (mac) steatoses, Mallory bodies (closed arrowhead) and pyknotic nuclei (open arrowhead). f Number of individual features per 100 cells. g Total number of steatotic features per 100 cells in each treatment group (*p = 0.0118, **p = 0.0052). h INPP4B expression in Reads Per Kilobase of transcript, per Million mapped reads (RPKM) in normal liver tissue (N = 14), obese (N = 12 patients), and NAFLD (N = 15 patients) in GSE126848 dataset (exact p values are…

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  Fig. 5. INPP4B regulates metabolic and PPAR pathways in mouse liver. a RNAs were extracted from livers of HFD WT (N = 4) and HFD Inpp4b−/− (N = 4) mice and submitted for RNA sequencing. The changes in gene expression were compared between HFD WT and HFD Inpp4b−/− mice. b KEGG pathway functional enrichment analysis was done using DAVID functional annotation module or GSEA. The vertical axis represents the KEGG pathway terms significantly enriched by the loss of INPP4B in mouse liver. The horizontal axis shows the logarithmic scale of the p value. c–i RNA from LFD WT (N = 10), LFD Inpp4b−/− (N = 12), HFD WT (N = 8), and HFD Inpp4b−/− (N = 10) mice were analyzed for Pparg (c), Cd36 (d), Mogat1 (e), Vldlr (f), Cyp2b9 (g), Cyp2b13 (h), and Hao2 (i) by qRT-PCR using 18S as an internal control. j Correlation between INPP4B expression and PPARG expression in 289 human liver samples (GSE24335) (**p < 0.01, ***p < 0.001, ****p < 0.0001. Exact p values are provided in the Source data).

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  Fig. 6. INPP4B regulates lipogenesis and AKT and PKC signaling pathways in mouse liver. a RNA from Fig. 4(c–i) were analyzed for Srebf1 expression by qRT-PCR using 18S as control. b Proteins were extracted from livers of LFD WT, LFD Inpp4b−/−, HFD WT, and HFD Inpp4b−/− mice and used to compare levels of unprocessed SREBP1, processed SREBP1, and tubulin by western blotting. c Quantification of processed SREBP1 levels were presented as an average of three independent samples. The protein levels were normalized to tubulin and were shown in fold change from LFD WT. d Protein lysates from (b) were analyzed for pAKT, total AKT, pPKCβII, pPKCζ, pPKCε, and tubulin using western blotting. e, f Quantification of pAKT and pPKCβII protein from (d) (N = 3). g–i The Inpp4b−/− animals were treated with vehicle DMSO (N = 4), 6 mg/kg of AZD5363 (N = 2) or BIM-I (N = 4) for 16 h. Livers were dissected and used for protein and RNA purification. One-way ANOVA (Dunnett’s multiple comparison test) was used. (g) Fifty µg of protein extracted from livers of Inpp4b−/− animals treated with vehicle, BIM-I, or AZD5363 were used to compare levels of unprocessed and processed forms of SREBP1 and tubulin. (h)…

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  Fig. 7. Loss of Inpp4b leads to the development of PIN and inflammation in obese males. a H&E staining of prostates from LFD WT, LFD Inpp4b−/−, HFD WT, and HFD Inpp4b−/− males. b Representative tissue sections from LFD WT, LFD Inpp4b−/−, HFD WT, and HFD Inpp4b−/− prostates were stained for α-SMA and counterstained with hematoxylin. Anterior prostate (AP), dorsolateral prostate (DLP), and ventral prostate (VP) were dissected from LFD WT (N = 8), LFD Inpp4b−/− (N = 8), HFD WT (N = 9), and HFD Inpp4b−/− (N = 9) mice. c–j RNA was extracted and the expression levels of Pbsn (c), Apof (d), Nkx3.1 (e), Msmb (f), Igfbp3 (g), Il6 (h), Tnf (i), and Il1b (j) were compared by qRT-PCR using 18S as an internal control. k Protein concentrations of IL-6 in mouse DLPs were measured using IL-6 ELISA (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Exact p values are provided in the Source data).

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  Fig. 8. Physiological functions of INPP4B in liver, WAT, and prostate. The loss of INPP4B activates SREBP1, stimulates hepatic expression of Pparg, causing liver steatosis, insulin resistance, de novo lipogenesis (DNL), and an increased rate of gluconeogenesis. When consuming a HFD, the Inpp4b−/− males developed type 2 diabetes, inflammation, and macrophage (MØ) infiltration of WAT, DNL, and an increased leptin/adiponectin ratio, all of which were shown to stimulate the development of PIN. Consumption of a HFD promoted macrophage infiltration in the prostates of both WT and Inpp4b−/− mice; however, increased production of proinflammatory cytokines was only evident in dorsolateral lobes of prostates of Inpp4b−/− mice.