Inhibition of Foxo1 signaling rescues the metabolic deficits caused by mTORC2 inhibition - PowerPoint Presentation

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Inhibition of Foxo1 signaling rescues the metabolic deficits caused by mTORC2 inhibition Reji Babygirija1,2,3, Michaela Murphy1,2, Shelly Shonsella1,2, Sabrina Dumas1,2, Victoria Flores1,2, Cara Green1,2, Heidi Pak1,2 and Dudley W. Lamming1,2,31Department of Medicine, University of Wisconsin-Madison, 2William S. Middleton Memorial Veterans Hospital, 3Department of Nutritional Sciences, University of Wisconsin-Madison, Madison WI, and3Graduate Program in Cellular and Molecular Biology, University of Wisconsin-Madison, Madison, WI, USA

IntroductionRapamycin, an inhibitor of mechanistic target of rapamycin complex 1 (mTORC1) is an immunosuppressant and anticancer drug which has previously been shown to extend life span of model organisms including mice.We have previously demonstrated that impaired glucose homeostasis, which is notably one of the most severe negative effects of chronic administration of rapamycin results from the inhibition of mTOR complex 2 (Lamming et al. 2012) while its beneficial effects on aging is mediated via mTORC1 inhibition.Hepatic deletion of Rictor, which is one of the proteins in mTORC2 complex can reproduce the metabolic deficits caused by rapamycin.One major question that needs to be answered is how can we block the side effects of rapamycin? Can the inhibition of any of the downstream targets in PI3-AKT-FOXO rescue these metabolic defects?Forkhead

boxO (FOXO) transcription factors, Foxo1 are important targets of insulin activity within cells, required to maintain glucose homeostasis especially during fasted state by promoting gluconeogenesis.Foxo1 distribution in many of the insulin-responsive tissues as well as its regulation by phosphorylation of AKT which gets stimulated by insulin-

IgF

signaling pathway allows Foxo1 to mediate metabolic functions.

Foxo1 can be the primary effector of many of the metabolic phenotypes observed either in mice lacking hepatic

Rictor

or as a result of treatment with rapamycin and therefore

we hypothesized that genetic depletion of Foxo1 can have a protective effect in rescuing the metabolic defects caused by mTORC2 inhibition.

MethodologyAlb-Cre RictorLoxP/LoxPAlb-

Cre FoxoLoxP

/

LoxP

Alb-

Cre

Rictor

LoxP/

LoxP

Foxo

LoxP/LoxP

RictorLoxP/LoxP

Foxo1LoxP/LoxP

RictorLoxP/LoxP and Foxo1LoxP/LoxP

Metabolic phenotyping

Glucose Tolerance Tests (GTT)

Pyruvate Tolerance Tests (PTT)

A-L) Glucose Tolerance Tests (GTT) and AUC analysis of both males and females :Male Rictor knockout (KO) mice were glucose intolerant following GTT. However female mice (C-D) didn’t show any significant differences in glucose levels compared to their wild type littermates. mice ** = p < 0.01. Hepatic deletion of Foxo1 resulted in a decrease in blood glucose levels in both male and female mice (A-D) and male mice lacking both Foxo1 and Rictor exhibited a better glycemic control compared with their wild type littermates. ** = p < 0.01. RKO-Rictor knockout, RWT-Rictor wild type, FKO-Foxo1 knockout,FWT-Foxo1 wild type, RFKO-Rictor Foxo1 double knockout, RFWT-Rictor Foxo wild type, AUC-Area under the Curve.

Hepatic deficiency of Foxo1 protects mice from developing glucose intolerance

A

B

C

D

E

F

G

H

I

J

K

L

**

**

**

**

Downregulation of hepatic gluconeogenesis following Foxo1 deletion

A

B

C

D

E

F

G

H

I

J

K

L

A-L)

Pyruvate Tolerance Tests (PTT) and AUC analysis of both males and females

. Foxo1 has been shown to regulate hepatic gluconeogenesis, we examined this using pyruvate tolerance test in all the phenotypes. After the pyruvate injection, blood glucose was decreased in Foxo

1 knock out (KO) as well as the double knock out mice compared with their littermates.

** = p < 0.01. RKO-

Rictor

knockout, RWT-

Rictor

wild type, FKO-

Foxo

1 knockout,FWT-Foxo1 wild type, RFKO-

Rictor

Foxo1 double knockout, RFWT-

RictorFoxo

wild type, AUC-Area under the Curve

**

**

Conclusions and Future PerspectivesHepatic deletion of Foxo1 resulted in a decrease in blood glucose levels in both male and female mice.Inhibiting Foxo1 signaling rescued the impaired glucose tolerance caused by deletion of rictor, with a stronger effect seen in males than females. These preliminary results suggests that blocking Foxo1 signaling can rescue metabolic effects caused by mTORC2 inhibitionOverall our results could potentially open new treatment options for diabetes as well as other metabolic syndromes thereby promoting health aging and an increase in overall survival rate.This is an ongoing study and future studies focus on further differentiating the metabolic phenotyping in these mice and perform a series of glucose, insulin, and pyruvate tolerance tests, and a glucose-stimulated insulin secretion assay in both sexes.

Acknowledgements

We would like to thank

all the members of the Lamming lab for their assistance and insight, and the

Merrins

,

Kimple

, and Davis labs for their continual support. The Lamming Lab is supported in part by the NIH/National Institute on Aging (AG056771, AG061635 and AG062328 to D.W.L.), by the U.S. Department of Veterans Affairs (I01-BX004031), and by funding from the University of Wisconsin-Madison School of Medicine and Public Health and Department of Medicine to D.W.L. This work was supported using facilities and resources from the William S. Middleton Memorial Veterans Hospital. The content is solely the responsibility of the authors and does not necessarily represent the official views of the NIH. This work does not represent the views of the Department of Veterans Affairs or the United States Government.