Fig 2: Increased energy expenditure in IEX-1 KO mice on high fat diet.Indirect calorimetry assessment of for 3 consecutive days showed no significant difference in (A) oxygen consumption (VO2) and (C) heat production between IEX-1 KO and WT mice when on ND. However, (B) VO2 and (D) heat production both significantly increased in KO mice when fed a HFD for 7–8 weeks as compared to WT littermates on same diet. (E) No difference in respiratory exchange ratio (RER) between two strains of mice was observed when on HFD. Mean analysis of 3 days measurements shown in (A–D) indicated significantly greater (F) VO2 and (G) heat production in IEX-1 KO mice (fill) than WT littermates (empty) on HFD. The values presented in (A–G) are normalized by lean body mass. (H) Analysis for lean and fat mass in mice fed with HFD or ND for 8 weeks revealed that IEX-1 deficiency resists the gain in fat mass after HFD without affecting lean mass. n = 4–5. **p < 0.01, ***p < 0.001 as indicated. N – night; D – Day.

Fig 3: Null mutation of IEX-1 resists the development of HFD-induced obesity and hyperinsulinemia.(A) The absolute and (B) percent change in body weight of IEX-1−/− (KO) and their WT littermates fed with HFD or ND over 20 weeks, illustrating installation of obesity in WT but not in IEX-1−/− mice (n = 7). (C) Shown in inset one KO and WT mouse each representing respective group of mice fed with HFD for 20 weeks. Measurements of (D) total daily food intake, (E) rectal temperature, and fasted (F) blood glucose, and (G) plasma insulin in KO (fill) and WT (empty) mice, demonstrate increased food consumption and rectal temperature but reduced glucose and insulin levels in KO mice on HFD as compared to WT control (n = 4–7). Fasted plasma (H) cholesterol and (I) non-esterified fatty acids (NEFA) are also lower in KO than WT mice. ELISA assay in plasma obtained from mice described in (A) revealed lower levels of circulating TNF-α whereas higher levels of IL-10 in KO than in WT mice after HFD feeding for 20 weeks (n = 4–6). *p < 0.05, **p < 0.01, ***p < 0.001 WT-HFD vs KO-HFD; and †p < 0.05, ††p < 0.01 KO-HFD vs KO-ND in (A,B) or as indicated.

Fig 4: Increased glucose clearance, reduced adipocytes enlargement and hepatic steatosis in IEX-1 deficiency.(A) Intraperitoneal glucose tolerance (IGTT) and (B) insulin tolerance (ITT) tests demonstrate that HFD feeding for 5 or 20 weeks impaired glucose clearance and installed insulin resistance in WT mice (empty). These effects were markedly inhibited in IEX-1 KO mice (fill), indicating improved glucose metabolism in IEX-1 deficiency (n = 7). (C) ELISA assay for plasma insulin concentration shows a robust increase in circulating insulin levels in WT mice on HFD in response to glucose administration during IGTT, an effect that was abrogated in KO mice (n = 4). Quantification of adipocytes diameter on eWAT sections shown in (F) indicated (D,E) diameter distribution shifting toward smaller sizes in KO compared with WT mice. (F) Histological analysis by H&E staining of eWAT (left panel) and liver (right panel) displayed increase in adipocytes area and liver fat accumulation (hepatic steatosis) in WT mice after 20 weeks on HFD. These effects were markedly inhibited in IEX-1 KO mice. Shown in (F) is one representative result each of 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 versus corresponding KO value or as indicated. Bars in F = 100 μM.

Fig 5: IEX-1 deficiency induces browning of white adipose tissues.(A) Immunoblotting of the whole cell lysate of indicated tissues obtained from mice fed with HFD or ND for 8 weeks demonstrated no difference in UCP1 protein levels in scWAT, eWAT, and BAT between two IEX-1 KO and WT mice when on ND. HFD feeding markedly suppressed UCP1 levels in scWAT and eWAT but not in BAT in WT mice. This effect was completely inhibited in KO mice, giving rise to greater levels of UCP1 in KO than in WT mice. α-tubulin was used as a loading control for UCP1. Each lane represents sample from individual animal (B) qRT-PCR analysis demonstrated no major impact of IEX-1 deficiency on thermogenic genes expression in BAT of mice on either diet described under (A). (C) Immunohistochemistry staining of scWAT (upper panel) and eWAT (lower panels) sections of these mice further showed increased UCP1 activity (purple) in KO mice on HFD as compared to WT control. (D) Hematoxylin and eosin (H&E) staining of the scWAT (upper panels) and eWAT (lower panels) sections displayed the appearance of multilocular adipocytes (insets) in KO but not in WT mice on HFD. qRT-PCR analysis revealed increased expression of thermogenic genes as indicated in both scWAT (E) and eWAT (F) of IEX-1 KO mice on HFD as compared to WT littermates (n = 4–6). Shown in (A,C and D) are representative immunoblots and images of 3 independent experiments. *p < 0.05, **p < 0.01, ***p < 0.001 as indicated. Bar in C and D = 50 and 100 μM, respectively.