Irs2 and Irs4 synergize in non-LepRb neurons to control energy balance and glucose

homeostasis

Mariana Sadagurski, X. Charlie Dong, Martin G. Myers Jr., and Morris F. White

Presented by: Alden ParkerBS 860

2/23/15

Journal/Article Info

• Molecular Metabolism – “reporting breakthroughs from all stages of the discovery and development of novel and improved personalized medicines for obesity, diabetes, and associated diseases.” (

http://www.molecularmetabolism.com/about_us.php)

- First Issue: December 2012, open access, published by Elsevier

- 1 issue 2012, 4 issues 2013, 9 issues 2014, monthly since June 2014, ~20 primary articles/issue

- Impact Factor? Unable to find one because the Journal is so new

- This article published online Oct. 2013, in print Feb. 2014, cited 5 times to date

http://en.1globaltranslators.com/people-like-german-language/http://www.molmetab.com/

Author Info• Marianna Sadagurski

– 2006 Ph.D from Tel Aviv University, Presently at University of Michigan Geriatrics– Assistant Research Scientist in Internal Medicine, Div. of Metab. – Fellowship At Harvard Medical School Children’s Hospital 2007 – 2012– Specializes in metabolism and aging– 11 publications, cited ~220 times

• Morris F. White– B.A. & Ph. D University of Michigan– Assistant Professor of Biochemistry, Harvard University– Researcher at Children’s Hospital in Boston– First to ID Insulin Receptor Substrate (Irs) family of proteins– Specializes in insulin – like signaling and progression of metabolic disease– 94 publications, cited ~47,000 times– Eli Lilly award for Excellence in Scientific Achievement from ADA 1999

http://www.hms.harvard.edu/dms/bbs/fac/WhiteMorris.php

http://medicine.umich.edu/dept/dgpm/marianna-sadagurski-phd

Glucose Sensing• Glucose – sensitive neurons in hypothalamus

• Assess energy status of the body distribute resources to provide energy for each system without compromising other systems (brain vs. body, Routh et al., 2014)

• Other tissues: GLUT transporters, GCK in hepatic/pancreatic cells, taste receptors (Efayan et al., 2015)

(Efayan et al., 2015)

Glucose Homeostasis – Major Hormonal Players

• Insulin – peptide hormone, β-cells in pancreas, promotes cellular absorption of glucose from blood muscle and fat fat storage. Also inhibits glucose production in liver

• Glucagon – peptide hormone, α-cells in pancreas, promotes glycogenolysis↑glucose in liverplasma glucose levels↑.

• Other: Thyroid Hormones (↑ energy production/glucose oxidation)

• Contributors - Leptin (↓ appetite), Ghrelin (↑ appetite)

• Feedback system to maintain stable level– 80 – 110 mg/dL during fasting, <140mg/dL after meal– ADA recommends 70 – 130 mg/dL fasting, <180mg/dL after meal as “safe range” for

diabetics

Glucose Homeostasis - Overview

(Herman & Kahn, 2006)

Δ glucose Δ ligand binding in a variety of receptors in neurons and other tissues

Glucose Homeostasis - Overview

https://14chohir.files.wordpress.com/2013/10/01-05_negfeedback_1.jpg

Diabetes

• High blood sugar over a long duration. - Type 1: insulin-dependent

• Usually genetic, loss of β cells in pancreas, usually destroyed via autoimmunity

• Irregular/unpredictable hyperglycemia, ketosis, impaired response to hypoglycemia, infection, etc.

- Type 2: insulin-independent• Obesity, genetics, lifestyle, stress, lack of exercise, etc.• Insulin resistance, reduced insulin secretion?, defect in IR?

Irs – Insulin receptor substrates• Group of proteins that are targets for IR and IGF1R tyrosine kinases

• Cytoplasmic adapter proteins – scaffolds to build signaling complexes (Mardilovich et al., 2009)

• Distinct functions based on sequence differences (structure differences), as well as divergent expression levels and locations.

– Irs1: widely expressed, embryonic/postnatal growth, insulin sensitivity• KO of Irs1 insulin resistance, ↑ insulin, β cell growth

– Irs2: widely expressed, β cell growth/survival, CNS/hypthalamus nutrient sensing, endothelial cell function, sensitivity to neurodegeneration, act in part on LepRb-expressing neurons in hypothalamus• KO of Irs2 diabetes from insulin resistance and β cell death

– KO of both Irs1 and Irs2 early death

– Irs4: mainly in hypothalamus in healthy mammals, function????• KO of Irs4 very little effect on energy balance or glucose metabolism

Research Questions• What is the physiological function of Irs4? Is there a functional role for it in LepRb-expressing neurons (where

Irs2 acts) or does it act elsewhere? Is there a synergy between Irs2 and Irs4 in metabolic sensing and glucose homeostasis?

Objectives: • Aim 1: Generate KO mice, Confirm the phenotypic effects of KOs. Confirm effects by visualization of behavior (energy

expenditure/consumption), physical characterization and by molecular techniques

• Aim 2: Determine if effects are due to changes in hypothalamic neuropeptide expression that resulted from KO (i.e if they’re leptin – dependent) or not.

• Aim 3: Confirm the synergism (or lack thereof) between Irs2 and Irs4 in glucose homeostasis, as well as the location of their concerted action, should it exist.

Hypotheses – Irs2 and Irs4 have a synergistic effect, complementing each other in metabolic sensing and glucose homeostasis. Irs4’s effects are

leptin-dependent (i.e. they occur in the same neurons as Irs2’s effects).

Methodology – KO mice and controls• Irs4-/Y KO mice (remove Irs4 everywhere)

• bIrs2-/-, Irs4-/Y double KO mice (remove Irs2 in neurons, Irs4 everywhere)

• LeprΔIrs2 (Irs2 KO in LepRb neurons, specifically)

• Positive Control – bIrs2-/- (Irs2 knocked out in neurons, diabetic phenotype)

• Negative Control – Irs2L/L (Irs2 coding sequence surrounded by LoxP sites, no pathological phenotype, baseline control)

(Sadagurski et al., 2012)

Methodology – KO generation• Irs4 gene ID’d from genomic DNA library from ES cells ↘• Ligation into pPNT vector 5’ to neo cassette, flanked by restriction sites based on Irs-4

regions 5’ and 3’ to the deleted coding region. ↘• Electroporation into ES cells, Irs4+/- neomycin selection + Ganciclovir

↘• Injection into C57BL/6 blastocysts chimeric ♂ ↘• chimeric ♂ + (WT) C57BL/6 = WT♀ +♂ Irs4 +/- ♀

↘• WT +♂ Irs4 +/- = ♀ Irs4-/Y ♂ + Irs4 +/- ♀

↘• Irs4-/Y ♂ + Irs4 +/- = ♀ Irs4-/Y ♂ + Irs4 -/- ♀

• Other KOs were similarly generated (Sadagurski et al., 2012)

http://www.addgene.org/vector-database/3825/

• Dual-Energy X-ray absorptiometry (fat content determination)

• Glucose meter (post – glucose injection/insulin injection)

• ELISA for insulin and leptin detection

• Food intake over 48 hours

Methodology – Metabolic Analyses

Methodology – Histology/Morphology

• Hematoxylin/Eosin stain

• Adipose morphology with ImageJ

• Pancreatic Islet area determination software

Methodology – Energy Expenditure• Physical activity (motion sensing) and energy

expenditure (heat production) monitored for 72 hours

RT-PCR on downstream Leptin molecules

• RNA extractioncDNAcustom primers for: – Nueropeptide Y, NPY (↑fat growth)

– Agouti-related protein, AgRP (↑ appetite, ↓ metabolism/energy expenditure)

– Pro-opiomelanocortin, Pomc (precursor to α-Melanocyte Stimulating Hormone, ↓ appetite)

Results – KO checks

Irs4-/Y KO is restricted to the CNS, in practice, since Irs4 expression is essentially restricted to ventral hypothalamus

Results: growth, Figure 1

Results: growth, Figure 1

Results: adipose/hepatocyte profiles, Figure 2

Results – Appetite, Energy, Figure 3

Results –Adipose Morphology, Figure 3

Results – RT-PCR on hypothalamic NPs, Figure 4

Results – Glucose Homeostasis & Tolerance, Insulin Resistance Figure 5

ADA recommends 70 – 130 mg/dL fasting, <180mg/dL after meal as “safe range” for diabetics

(HOMA2-IR = IR quantification & β cell function,Matthews et al., 1985)

Results – β cell islet morphology, Figure 6

Results – LepRb Dependence?, Figure 7

Discussion/Conclusions

• Irs4 KO only affected growth, appetite, and morphology in the absence of Irs2 Irs2 and Irs4 work in concerted action to regulate adiposity, glucose intolerance, insulin action, and hepatic lipid content. (energy balance and glucose homeostasis)

• Double KO had a more of an effect than either single KO

• Irs4 acts on neurons devoid of LepRb, so Irs2 acts on these neurons, too

• Irs4 does not change hypothalamus neuropeptide expression(i.e. leptin-regulated), but does function to regulate insulin sensitivity and glucose homeostasis

Critiques/ Future directions• Statistics: T-test only, could have done ANOVA since they have 3-4 groups of mice in

every assay, would have reduced error. low n for most figures, <10. Should have measured more KOs to potentially reduce error bars

• Figure 4, big error bars on 4C control, double KO all sections

• Why use Pomc, the precursor? Why not just use α-Melanocyte Stimulating Hormone?

• Figure 6, should have used 3.5 months for Irs4 KO, too, just as reference

• Editing flaws: In abstract! (Irs4-null mice …ARE...barely distinguishable, abstract = 181 words), When referencing 6A (page 59, top right), should have said “double KO mice at 3.5 months displayed 3-fold greater β-cell mass”

• What happens when Irs2 and Irs4 are overexpressed or co - overexpressed?

Future Directions

• Overexpression

• ID particular Irs2/Irs4 – expressing cells reveal their independent function in glucose homeostasis and energy balance

• Interaction of Irs1 and Irs4, or of 1, 2, & 4?

Questions?

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