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E4orf1 induction in adipose tissue promotes insulin-independent signaling in the adipocyte.

Kusminski CM, Gallardo-Montejano VI, Wang ZV, Hegde V, Bickel PE, Dhurandhar NV, Scherer PE - Mol Metab (2015)

Bottom Line: At the whole body level, this leads to reduced body-weight gain under a high fat diet challenge.Nevertheless, they are protected from diet-induced hepatic steatosis.The resulting systemic phenotype is complex, yet highlights the powerful nature of manipulating selective branches of the insulin signaling network within the adipocyte.

View Article: PubMed Central - PubMed

Affiliation: Touchstone Diabetes Center, Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, TX, USA.

ABSTRACT

Background/purpose: Type 2 diabetes remains a worldwide epidemic with major pathophysiological changes as a result of chronic insulin resistance. Insulin regulates numerous biochemical pathways related to carbohydrate and lipid metabolism.

Methods: We have generated a novel mouse model that allows us to constitutively activate, in an inducible fashion, the distal branch of the insulin signaling transduction pathway specifically in adipocytes.

Results: Using the adenoviral 36 E4orf1 protein, we chronically stimulate locally the Ras-ERK-MAPK signaling pathway. At the whole body level, this leads to reduced body-weight gain under a high fat diet challenge. Despite overlapping glucose tolerance curves, there is a reduced requirement for insulin action under these conditions. The mice further exhibit reduced circulating adiponectin levels that ultimately lead to impaired lipid clearance, and inflamed and fibrotic white adipose tissues. Nevertheless, they are protected from diet-induced hepatic steatosis. As we observe constitutively elevated p-Akt levels in the adipocytes, even under conditions of low insulin levels, this pinpoints enhanced Ras-ERK-MAPK signaling in transgenic adipocytes as a potential alternative route to bypass proximal insulin signaling events.

Conclusion: We conclude that E4orf1 expression in the adipocyte leads to enhanced baseline activation of the distal insulin signaling node, yet impaired insulin receptor stimulation in the presence of insulin, with important implications for the regulation of adiponectin secretion. The resulting systemic phenotype is complex, yet highlights the powerful nature of manipulating selective branches of the insulin signaling network within the adipocyte.

No MeSH data available.


Related in: MedlinePlus

E4orf1 induction in adipose tissue alters components of the insulin-signaling cascade in sWAT and liver tissues. (A) Representative Western blots of phospho (p)-Akt expression (∼60 kDa) in sWAT, BAT, gWAT and liver tissues from WT versus E4orf1-Tg mice prior to and post insulin injection (0, 5 and 15 min time-points) that were fed Dox-chow (600 mg/kg) for two-weeks. Mice were fasted overnight (∼14–16 h) prior to insulin injection (1 U/kg body-weight insulin i.p). (B) Western blots of p-Akt (upper panels) and total-Akt (∼60 kDa) (lower panels) in sWAT and liver tissues derived from WT mice versus E4orf1-Tg mice post Dox-chow (600 mg/kg) feeding. Bar graphs show p-Akt levels normalized to total-Akt levels in sWAT (left) and liver (right). (C) Western blots of the phospho-tyrosine (p-Tyr) insulin receptor β-subunit (Ins-Rec) (∼97 kDa) and p-Tyr insulin receptor substrate-1 (IRS-1) (∼170 kDa) in sWAT (upper panels) and liver (lower panels) tissues derived from Dox-chow (600 mg/kg) fed WT mice versus E4orf1-Tg mice prior and post insulin injection (5 and 15 min). The loading control β-actin is also provided. (D) Western blots of p-ERK (p-44/42 MAPK [ERK 1/2]) (Thr-202/Tyr-204) (∼42/44 kDa) and total-ERK expression levels in sWAT (upper panels) and liver (lower panels) tissues derived from Dox-chow (600 mg/kg) fed WT mice versus E4orf1-Tg mice prior and post insulin injection (5 and 15 min). (E) Western blots of p-Caveolin (Cav)-1 (p-Tyr14) (∼22 kDa) in sWAT (upper panel) and liver (lower panel) tissues derived from Dox-chow (600 mg/kg) fed WT mice versus E4orf1-Tg mice prior and post insulin injection (5 and 15 min). (F) Representative images of Glut4 immunofluorescence staining of sWAT from WT versus E4orf1-Tg mice post eight-weeks Dox-HFD feeding. Scale bar: 32 μm. (G) Real-time qPCR data showing the fold-changes (in comparison to WT) in gene expression levels of key components of the insulin signaling pathway (Ins-Rec, Irs-1/2, PI3K [p85/p110], Glut4 and Akt-1/2/3) in sWAT derived from WT mice versus E4orf1-Tg mice that underwent three-weeks of Dox-HFD (600 mg/kg) feeding. Mice were fasted for 3–4 h prior to tissue harvest (n = 9/group). (H) Real-time qPCR confirmation of the top significantly up-regulated genes (identified by RNA-Seq analyses) in E4orf1-Tg sWAT, when compared with WT sWAT, (n = 9 per group). (I) Real-time qPCR of the most significantly down-regulated genes (identified by RNA-Seq analyses) in E4orf1-Tg sWAT, when compared with WT sWAT, (n = 9 per group). (Student's t-test, *P < 0.05; **P < 0.01; ***P < 0.001).
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fig3: E4orf1 induction in adipose tissue alters components of the insulin-signaling cascade in sWAT and liver tissues. (A) Representative Western blots of phospho (p)-Akt expression (∼60 kDa) in sWAT, BAT, gWAT and liver tissues from WT versus E4orf1-Tg mice prior to and post insulin injection (0, 5 and 15 min time-points) that were fed Dox-chow (600 mg/kg) for two-weeks. Mice were fasted overnight (∼14–16 h) prior to insulin injection (1 U/kg body-weight insulin i.p). (B) Western blots of p-Akt (upper panels) and total-Akt (∼60 kDa) (lower panels) in sWAT and liver tissues derived from WT mice versus E4orf1-Tg mice post Dox-chow (600 mg/kg) feeding. Bar graphs show p-Akt levels normalized to total-Akt levels in sWAT (left) and liver (right). (C) Western blots of the phospho-tyrosine (p-Tyr) insulin receptor β-subunit (Ins-Rec) (∼97 kDa) and p-Tyr insulin receptor substrate-1 (IRS-1) (∼170 kDa) in sWAT (upper panels) and liver (lower panels) tissues derived from Dox-chow (600 mg/kg) fed WT mice versus E4orf1-Tg mice prior and post insulin injection (5 and 15 min). The loading control β-actin is also provided. (D) Western blots of p-ERK (p-44/42 MAPK [ERK 1/2]) (Thr-202/Tyr-204) (∼42/44 kDa) and total-ERK expression levels in sWAT (upper panels) and liver (lower panels) tissues derived from Dox-chow (600 mg/kg) fed WT mice versus E4orf1-Tg mice prior and post insulin injection (5 and 15 min). (E) Western blots of p-Caveolin (Cav)-1 (p-Tyr14) (∼22 kDa) in sWAT (upper panel) and liver (lower panel) tissues derived from Dox-chow (600 mg/kg) fed WT mice versus E4orf1-Tg mice prior and post insulin injection (5 and 15 min). (F) Representative images of Glut4 immunofluorescence staining of sWAT from WT versus E4orf1-Tg mice post eight-weeks Dox-HFD feeding. Scale bar: 32 μm. (G) Real-time qPCR data showing the fold-changes (in comparison to WT) in gene expression levels of key components of the insulin signaling pathway (Ins-Rec, Irs-1/2, PI3K [p85/p110], Glut4 and Akt-1/2/3) in sWAT derived from WT mice versus E4orf1-Tg mice that underwent three-weeks of Dox-HFD (600 mg/kg) feeding. Mice were fasted for 3–4 h prior to tissue harvest (n = 9/group). (H) Real-time qPCR confirmation of the top significantly up-regulated genes (identified by RNA-Seq analyses) in E4orf1-Tg sWAT, when compared with WT sWAT, (n = 9 per group). (I) Real-time qPCR of the most significantly down-regulated genes (identified by RNA-Seq analyses) in E4orf1-Tg sWAT, when compared with WT sWAT, (n = 9 per group). (Student's t-test, *P < 0.05; **P < 0.01; ***P < 0.001).

Mentions: To assess AT insulin signaling in the presence of E4orf1, mice underwent two weeks of Dox-chow feeding (600 mg/kg) and insulin-stimulated phospho-Akt (p-Akt) levels were examined. Interestingly, we observed a significant increase in baseline, non-insulin-stimulated p-Akt expression in E4orf1-Tg sWAT compared to WT sWAT (Figure 3A), while total-Akt expression levels remained unchanged (Supplementary Figure 2). A similar pattern was also observed in E4orf1-Tg BAT, the fat-depot that achieves the highest level of E4orf1 protein induction. Such an increase in p-Akt by E4orf1 under baseline conditions suggests that E4orf1 may activate the insulin-signaling cascade to enhance p-Akt independently of insulin. Upon insulin-stimulation (5, 10 and 15 min post insulin injection), conversely, markedly reduced levels of p-Akt expression are evident in E4orf1-Tg sWAT (Figure 3A). While E4orf1 impairs insulin-mediated effects, it raises the question how E4orf1 has the ability to trigger the insulin-signaling cascade in the absence of insulin; this is extremely important, particularly in the context of diabetic conditions with conventional insulin resistance. Quantitatively, in the basal un-stimulated state, we further substantiated the significant increase in p-Akt expression in E4orf1-Tg sWAT, by normalizing to total-Akt levels (Figure 3B). A marked reduction in p-Akt expression was further apparent in E4orf1-Tg livers (Figure 3B), suggesting some degree of hepatic insulin resistance as a secondary consequence of the dysfunctional WAT in E4orf1-Tg mice. Upon examination of other targets of the insulin-signaling pathway, we observed a marked reduction in insulin-stimulated p-tyrosine (p-Tyr) (insulin receptor β-subunit and IRS-1) expression levels in E4orf1-Tg sWAT and liver tissues (Figure 3C). Similarly upon Dox-chow feeding, assessing p-ERK, a downstream target of the Ras-mediated pathway, we noticed it to be markedly lower in insulin-stimulated E4orf1-Tg sWAT and liver tissues (Figure 3D). Lower baseline and insulin-stimulated levels of caveolin-1 (Cav-1) p-Tyr 14 were also apparent in sWAT and liver tissues from E4orf1-Tg mice (Figure 3E). Finally, we examined glucose transporter levels in sWAT by immunofluorescence staining, which revealed more prominent Glut4 staining in E4orf1-Tg sWAT, when compared with WT sWAT (Figure 3F).


E4orf1 induction in adipose tissue promotes insulin-independent signaling in the adipocyte.

Kusminski CM, Gallardo-Montejano VI, Wang ZV, Hegde V, Bickel PE, Dhurandhar NV, Scherer PE - Mol Metab (2015)

E4orf1 induction in adipose tissue alters components of the insulin-signaling cascade in sWAT and liver tissues. (A) Representative Western blots of phospho (p)-Akt expression (∼60 kDa) in sWAT, BAT, gWAT and liver tissues from WT versus E4orf1-Tg mice prior to and post insulin injection (0, 5 and 15 min time-points) that were fed Dox-chow (600 mg/kg) for two-weeks. Mice were fasted overnight (∼14–16 h) prior to insulin injection (1 U/kg body-weight insulin i.p). (B) Western blots of p-Akt (upper panels) and total-Akt (∼60 kDa) (lower panels) in sWAT and liver tissues derived from WT mice versus E4orf1-Tg mice post Dox-chow (600 mg/kg) feeding. Bar graphs show p-Akt levels normalized to total-Akt levels in sWAT (left) and liver (right). (C) Western blots of the phospho-tyrosine (p-Tyr) insulin receptor β-subunit (Ins-Rec) (∼97 kDa) and p-Tyr insulin receptor substrate-1 (IRS-1) (∼170 kDa) in sWAT (upper panels) and liver (lower panels) tissues derived from Dox-chow (600 mg/kg) fed WT mice versus E4orf1-Tg mice prior and post insulin injection (5 and 15 min). The loading control β-actin is also provided. (D) Western blots of p-ERK (p-44/42 MAPK [ERK 1/2]) (Thr-202/Tyr-204) (∼42/44 kDa) and total-ERK expression levels in sWAT (upper panels) and liver (lower panels) tissues derived from Dox-chow (600 mg/kg) fed WT mice versus E4orf1-Tg mice prior and post insulin injection (5 and 15 min). (E) Western blots of p-Caveolin (Cav)-1 (p-Tyr14) (∼22 kDa) in sWAT (upper panel) and liver (lower panel) tissues derived from Dox-chow (600 mg/kg) fed WT mice versus E4orf1-Tg mice prior and post insulin injection (5 and 15 min). (F) Representative images of Glut4 immunofluorescence staining of sWAT from WT versus E4orf1-Tg mice post eight-weeks Dox-HFD feeding. Scale bar: 32 μm. (G) Real-time qPCR data showing the fold-changes (in comparison to WT) in gene expression levels of key components of the insulin signaling pathway (Ins-Rec, Irs-1/2, PI3K [p85/p110], Glut4 and Akt-1/2/3) in sWAT derived from WT mice versus E4orf1-Tg mice that underwent three-weeks of Dox-HFD (600 mg/kg) feeding. Mice were fasted for 3–4 h prior to tissue harvest (n = 9/group). (H) Real-time qPCR confirmation of the top significantly up-regulated genes (identified by RNA-Seq analyses) in E4orf1-Tg sWAT, when compared with WT sWAT, (n = 9 per group). (I) Real-time qPCR of the most significantly down-regulated genes (identified by RNA-Seq analyses) in E4orf1-Tg sWAT, when compared with WT sWAT, (n = 9 per group). (Student's t-test, *P < 0.05; **P < 0.01; ***P < 0.001).
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fig3: E4orf1 induction in adipose tissue alters components of the insulin-signaling cascade in sWAT and liver tissues. (A) Representative Western blots of phospho (p)-Akt expression (∼60 kDa) in sWAT, BAT, gWAT and liver tissues from WT versus E4orf1-Tg mice prior to and post insulin injection (0, 5 and 15 min time-points) that were fed Dox-chow (600 mg/kg) for two-weeks. Mice were fasted overnight (∼14–16 h) prior to insulin injection (1 U/kg body-weight insulin i.p). (B) Western blots of p-Akt (upper panels) and total-Akt (∼60 kDa) (lower panels) in sWAT and liver tissues derived from WT mice versus E4orf1-Tg mice post Dox-chow (600 mg/kg) feeding. Bar graphs show p-Akt levels normalized to total-Akt levels in sWAT (left) and liver (right). (C) Western blots of the phospho-tyrosine (p-Tyr) insulin receptor β-subunit (Ins-Rec) (∼97 kDa) and p-Tyr insulin receptor substrate-1 (IRS-1) (∼170 kDa) in sWAT (upper panels) and liver (lower panels) tissues derived from Dox-chow (600 mg/kg) fed WT mice versus E4orf1-Tg mice prior and post insulin injection (5 and 15 min). The loading control β-actin is also provided. (D) Western blots of p-ERK (p-44/42 MAPK [ERK 1/2]) (Thr-202/Tyr-204) (∼42/44 kDa) and total-ERK expression levels in sWAT (upper panels) and liver (lower panels) tissues derived from Dox-chow (600 mg/kg) fed WT mice versus E4orf1-Tg mice prior and post insulin injection (5 and 15 min). (E) Western blots of p-Caveolin (Cav)-1 (p-Tyr14) (∼22 kDa) in sWAT (upper panel) and liver (lower panel) tissues derived from Dox-chow (600 mg/kg) fed WT mice versus E4orf1-Tg mice prior and post insulin injection (5 and 15 min). (F) Representative images of Glut4 immunofluorescence staining of sWAT from WT versus E4orf1-Tg mice post eight-weeks Dox-HFD feeding. Scale bar: 32 μm. (G) Real-time qPCR data showing the fold-changes (in comparison to WT) in gene expression levels of key components of the insulin signaling pathway (Ins-Rec, Irs-1/2, PI3K [p85/p110], Glut4 and Akt-1/2/3) in sWAT derived from WT mice versus E4orf1-Tg mice that underwent three-weeks of Dox-HFD (600 mg/kg) feeding. Mice were fasted for 3–4 h prior to tissue harvest (n = 9/group). (H) Real-time qPCR confirmation of the top significantly up-regulated genes (identified by RNA-Seq analyses) in E4orf1-Tg sWAT, when compared with WT sWAT, (n = 9 per group). (I) Real-time qPCR of the most significantly down-regulated genes (identified by RNA-Seq analyses) in E4orf1-Tg sWAT, when compared with WT sWAT, (n = 9 per group). (Student's t-test, *P < 0.05; **P < 0.01; ***P < 0.001).
Mentions: To assess AT insulin signaling in the presence of E4orf1, mice underwent two weeks of Dox-chow feeding (600 mg/kg) and insulin-stimulated phospho-Akt (p-Akt) levels were examined. Interestingly, we observed a significant increase in baseline, non-insulin-stimulated p-Akt expression in E4orf1-Tg sWAT compared to WT sWAT (Figure 3A), while total-Akt expression levels remained unchanged (Supplementary Figure 2). A similar pattern was also observed in E4orf1-Tg BAT, the fat-depot that achieves the highest level of E4orf1 protein induction. Such an increase in p-Akt by E4orf1 under baseline conditions suggests that E4orf1 may activate the insulin-signaling cascade to enhance p-Akt independently of insulin. Upon insulin-stimulation (5, 10 and 15 min post insulin injection), conversely, markedly reduced levels of p-Akt expression are evident in E4orf1-Tg sWAT (Figure 3A). While E4orf1 impairs insulin-mediated effects, it raises the question how E4orf1 has the ability to trigger the insulin-signaling cascade in the absence of insulin; this is extremely important, particularly in the context of diabetic conditions with conventional insulin resistance. Quantitatively, in the basal un-stimulated state, we further substantiated the significant increase in p-Akt expression in E4orf1-Tg sWAT, by normalizing to total-Akt levels (Figure 3B). A marked reduction in p-Akt expression was further apparent in E4orf1-Tg livers (Figure 3B), suggesting some degree of hepatic insulin resistance as a secondary consequence of the dysfunctional WAT in E4orf1-Tg mice. Upon examination of other targets of the insulin-signaling pathway, we observed a marked reduction in insulin-stimulated p-tyrosine (p-Tyr) (insulin receptor β-subunit and IRS-1) expression levels in E4orf1-Tg sWAT and liver tissues (Figure 3C). Similarly upon Dox-chow feeding, assessing p-ERK, a downstream target of the Ras-mediated pathway, we noticed it to be markedly lower in insulin-stimulated E4orf1-Tg sWAT and liver tissues (Figure 3D). Lower baseline and insulin-stimulated levels of caveolin-1 (Cav-1) p-Tyr 14 were also apparent in sWAT and liver tissues from E4orf1-Tg mice (Figure 3E). Finally, we examined glucose transporter levels in sWAT by immunofluorescence staining, which revealed more prominent Glut4 staining in E4orf1-Tg sWAT, when compared with WT sWAT (Figure 3F).

Bottom Line: At the whole body level, this leads to reduced body-weight gain under a high fat diet challenge.Nevertheless, they are protected from diet-induced hepatic steatosis.The resulting systemic phenotype is complex, yet highlights the powerful nature of manipulating selective branches of the insulin signaling network within the adipocyte.

View Article: PubMed Central - PubMed

Affiliation: Touchstone Diabetes Center, Department of Internal Medicine, The University of Texas Southwestern Medical Center, Dallas, TX, USA.

ABSTRACT

Background/purpose: Type 2 diabetes remains a worldwide epidemic with major pathophysiological changes as a result of chronic insulin resistance. Insulin regulates numerous biochemical pathways related to carbohydrate and lipid metabolism.

Methods: We have generated a novel mouse model that allows us to constitutively activate, in an inducible fashion, the distal branch of the insulin signaling transduction pathway specifically in adipocytes.

Results: Using the adenoviral 36 E4orf1 protein, we chronically stimulate locally the Ras-ERK-MAPK signaling pathway. At the whole body level, this leads to reduced body-weight gain under a high fat diet challenge. Despite overlapping glucose tolerance curves, there is a reduced requirement for insulin action under these conditions. The mice further exhibit reduced circulating adiponectin levels that ultimately lead to impaired lipid clearance, and inflamed and fibrotic white adipose tissues. Nevertheless, they are protected from diet-induced hepatic steatosis. As we observe constitutively elevated p-Akt levels in the adipocytes, even under conditions of low insulin levels, this pinpoints enhanced Ras-ERK-MAPK signaling in transgenic adipocytes as a potential alternative route to bypass proximal insulin signaling events.

Conclusion: We conclude that E4orf1 expression in the adipocyte leads to enhanced baseline activation of the distal insulin signaling node, yet impaired insulin receptor stimulation in the presence of insulin, with important implications for the regulation of adiponectin secretion. The resulting systemic phenotype is complex, yet highlights the powerful nature of manipulating selective branches of the insulin signaling network within the adipocyte.

No MeSH data available.


Related in: MedlinePlus