differential role of insulin/igf-1 receptor signaling in ...€¦ · cell reports article...

Article

Differential Role of Insulin
/IGF-1 Receptor Signalingin Muscle Growth and Glucose Homeostasis
Graphical Abstract

Highlights

d Insulin receptors (IRs) and IGF-1 receptors (IGF1Rs) are

required for muscle growth

d Deletion of muscle IRs/IGF1Rs is not sufficient to impair

glucose tolerance

d Loss of IRs/IGF1Rs in muscle increases Glut4 and glucose

uptake via decreased TBC1D1

d A dominant-negative IGF1R impairs glucose tolerance, even

without functional IRs/IGF1Rs

O’Neill et al., 2015, Cell Reports 11, 1220–1235May 26, 2015 ª2015 The Authorshttp://dx.doi.org/10.1016/j.celrep.2015.04.037

Authors

BrianT.O’Neill, HansP.M.M. Lauritzen, ...,

Laurie J. Goodyear, C. Ronald Kahn

[email protected]

In Brief

O’Neill et al. demonstrate insulin

receptors (IRs) and IGF-1 receptors

(IGF1Rs) are required for muscle growth

but not glucose tolerance. Muscle-

specific IR/IGF1R deletion decreases

TBC1D1, thereby increasing membrane-

localized glucose transporters and

glucose uptake. However,

overexpression of a dominant-negative

IGF1R induces glucose intolerance,

indicating that protein-protein

interactions with IRs/IGF1Rs can impair

glucose homeostasis.

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Cell Reports

Article

Differential Role of Insulin/IGF-1 ReceptorSignaling in Muscle Growth and Glucose HomeostasisBrian T. O’Neill,1 Hans P.M.M. Lauritzen,1 Michael F. Hirshman,1 Graham Smyth,1 Laurie J. Goodyear,1

and C. Ronald Kahn1,*1Section on Integrative Physiology and Metabolism, Joslin Diabetes Center, Harvard Medical School, Boston, MA 02215, USA

*Correspondence: [email protected]


This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

SUMMARY

Insulin and insulin-like growth factor 1 (IGF-1) arema-jor regulators of muscle protein and glucose homeo-stasis. To determine how these pathways interact,we generated mice with muscle-specific knockoutof IGF-1 receptor (IGF1R) and insulin receptor (IR).These MIGIRKO mice showed >60% decrease inmuscle mass. Despite a complete lack of insulin/IGF-1 signaling in muscle, MIGIRKO mice displayednormal glucose and insulin tolerance. Indeed,MIGIRKO mice showed fasting hypoglycemia andincreased basal glucose uptake. This was secondaryto decreased TBC1D1 resulting in increased Glut4and Glut1membrane localization. Interestingly, over-expression of a dominant-negative IGF1R in muscleinduced glucose intolerance in MIGIRKO animals.Thus, loss of insulin/IGF-1 signaling impairs musclegrowth, but not whole-body glucose tolerance dueto increasedmembrane localization of glucose trans-porters. Nonetheless, presence of a dominant-nega-tive receptor, even in the absence of functional IR/IGF1R, induces glucose intolerance, indicating thatinteractions between these receptors and other pro-teins in muscle can impair glucose homeostasis.

INTRODUCTION

Skeletal muscle insulin resistance is a prominent feature of type 2

diabetes that precedes and predicts the development of disease

in high-risk populations (Martin et al., 1992). In humans, up to 80%

of the glucose infused during a hyperinsulinemic euglycemic

clamp is disposed into muscle. However, genetic manipulation

of insulin signaling specifically in muscle of mice has shown little

effect on whole-body glucose metabolism. For example, genetic

deletion of the insulin receptor (IR) specifically in skeletal muscle

ofmice (MIRKO) did not cause dysglycemia or diabetes, although

it did result in hypertriglyceridemiaandmildobesity (Bruninget al.,

1998). On the other hand, overexpression of a kinase-deficient IR

in muscle of mice led to glucose intolerancewith increased circu-

lating insulin and triglyceride levels (Moller et al., 1996). Likewise,

mice that highly overexpress a dominant-negative, kinase-dead

insulin-like growth factor 1 (IGF-1) receptor (IGF1R) in muscle

1220 Cell Reports 11, 1220–1235, May 26, 2015 ª2015 The Authors

(MKR) develop severe glucose intolerance, insulin resistance,

and diabetes (Fernandez et al., 2001). Further study revealed

that expression of the MKR allele impairs both insulin and IGF-1

signaling in muscle due to hybrid receptor formation, suggesting

that the normal glucose tolerance in MIRKO mice might be due

to IGF1R compensating for loss of IR signaling in muscle.

Inaddition toglucoseuptakeandmetabolism, insulin and IGF-1

signaling affect muscle growth and protein turnover (Schiaffino

andMammucari, 2011; Meek et al., 1998). Both insulin and IGF-1

have been shown to stimulate muscle protein synthesis (Fulks

et al., 1975; Rommel et al., 2001) and inhibit protein degradation

via the ubiquitin-proteasomeand autophagy-lysosomepathways

(Mammucari et al., 2007; Sandri et al., 2004). Indeed, IGF-1 treat-

ment is sufficient to cause muscle hypertrophy via Akt activation

of mTOR and inhibition of GSK3b (Rommel et al., 2001). On the

other hand, deletion of IGF1R in muscle only modestly impairs

muscle growth, suggesting alternative pathways can induce

muscle growth, possibly via IRs (Mavalli et al., 2010).

At a molecular level, insulin and IGF-1 signal through highly

homologous tyrosine kinase receptors, which are virtually ubiq-

uitously expressed in mammals. IRs and IGF1Rs specifically

bind their respective ligands at physiological concentrations.

However, at high concentrations, each ligand can bind and

initiate signaling with the opposite receptor. Both IRs and

IGF1Rs then initiate intracellular signaling via similar cascades,

beginning with tyrosine phosphorylation of insulin receptor sub-

strates (IRSs), which leads to activation of the phosphatidylinosi-

tol 3-kinase (PI3K)/Akt pathway, as well as other downstream

signals (Taniguchi et al., 2006). In addition, IR and IGF1R interact

with Src homology and collagen domain protein (Shc) to activate

mitogen-activated protein kinase pathways. Signaling via these

two pathways leads to a broad range of cellular effects on

growth, proliferation, and metabolism.

In muscle, insulin-stimulated glucose uptake has been

extensively studied. IR-mediated activation of Akt leads to phos-

phorylation of AS160 and TBC1D1 to facilitate translocation of

vesicles containing the Glut4 glucose transporter to the plasma

membrane, where they fuse, leading to increased glucose

uptake into the cell (Klip, 2009). Not surprisingly, knockout of

Glut4 in muscle of mice leads to insulin resistance and hypergly-

cemia (Zisman et al., 2000). However, basal glucose uptake, i.e.,

that which occurs in the absence of insulin, has been ascribed to

other glucose transporters, such as Glut1, which have higher

constitutive association with the sarcolemma (Scheepers et al.,

2004; Marette et al., 1992; Wang et al., 1996). Exercise is also

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an important factor in glucose transport, inducing AMP-depen-

dent kinase (AMPK) activation and Glut4 vesicle translocation

via phosphorylation of AS160 and TBC1D1, which is indepen-

dent of insulin (Fujii et al., 2006; Koh et al., 2008).

To investigate to what extent insulin and/or IGF-1 signaling

pathways control glucose metabolism and protein homeostasis,

we have deleted IRs, IGF1Rs, or both in skeletal muscle using

genetic recombination. While mice with single-receptor dele-

tions show little or no change in glucose homeostasis or muscle

mass, mice with combined loss of IRs and IGF1Rs in muscle

(MIGIRKO) display dramatically decreased muscle mass and

fiber size. Nonetheless, MIGIRKO mice show normal glucose

and insulin tolerance and even have fasting hypoglycemia due

to enhanced basal glucose uptake into muscle secondary to

increased expression and translocation of glucose transporters.

Surprisingly, whenMIGIRKOmicewere crossed tomice carrying

a dominant-negative IGF1R, the resultant mice still developed

glucose intolerance and dyslipidemia. Thus, combined loss of

IRs and IGF1Rs in muscle dramatically impairs muscle growth,

but glucose tolerance is maintained by enhanced basal glucose

transport. The induction of glucose intolerance in these mice by

expression of a dominant-negative IGF1R indicates that the

dominant-negative receptor can interact with other proteins on

the cell to modify metabolic regulation.

RESULTS

Muscle-Specific Deletion of IRs and IGF1Rs DecreasesMuscle Growth and Leads to Early DemiseTo generate mice with skeletal-muscle-specific deletion of IRs

and IGF1Rs, we crossedmice that express the Cre recombinase

under the control of human skeletal muscle actin promoter

(ACTA1-Cre) with mice harboring floxed IR and IGF1R alleles.

Previous attempts at deleting IRs and IGF1Rs in muscle using

Cre under the muscle creatine kinase (MCK) promoter allowed

for expression in the heart, as well as skeletal muscle, and led

to death within 21 days from cardiac failure (Laustsen et al.,

2007). Cre expression in ACTA1-Cre mice is more restricted to

skeletal muscle (Miniou et al., 1999), allowing successful gener-

ation of mice with muscle specific deletion of IRs (M-IR�/�),IGF1Rs (M-IGF1R�/�), or both IGF1Rs and IRs (MIGIRKO) (Fig-

ure S1; Table S1). We have named mice that harbor IRlox/lox

alleles and the ACTA1-Cre transgene as M-IR�/� mice in order

to distinguish them from the MIRKO mouse, which was created

using MCK-Cre (Bruning et al., 1998). Genomic DNA isolated

from M-IR�/� and MIGIRKO muscle (which also contains

vascular cells, fibroblasts, and satellite cells/myoblasts) showed

a 50% recombination of IRlox (Insr locus) by qRT-PCR. IGF1Rlox

(Igf1r locus) was similarly recombined by 50% in M-IGF1R�/�

and MIGIRKO muscle without any change in liver DNA (Figures

S1A and S1B). M-IR�/� and MIGIRKO mice displayed a 90%

decrease of IR mRNA expression by qRT-PCR in muscle, while

M-IGF1R�/� and MIGIRKO mice showed a 50%–60% reduction

in IGF1R mRNA (Figure S1C). These changes in IR and IGF1R

mRNA expression correlated well with decreases in protein

levels corresponding to genotype (Figure 1A).

MIGIRKO mice showed an obvious growth phenotype with

decreased body weight as early as 3 weeks of age (Figures 1B

C

and1C).By7 to 10weeksof age,MIGIRKOmiceexhibited severe

muscle atrophy with spinal deformities and obvious kyphosis

(Figure 1B). These mice progressed to have breathing difficulties

and died between 15 and 25 weeks of age, most likely of respira-

tory failure (Figure 1D). By contrast, M-IR�/� orM-IGF1R�/�mice

had normal body weight and skeletal appearance and lived nor-

mally up to 52 weeks of age. By dual-energy X-ray absorptiome-

try (DEXA) scanning and assessment of tissueweight at sacrifice,

the decreased body weight inMIGIRKOmice could be attributed

almost entirely to a loss of muscle mass, with 59%–68% reduc-

tions in individual muscle weights and a 32% decreased in total

lean mass (Figures 1E–1G; Table S2). There was also a 9% loss

of lean mass and a decrease in muscle weights in M-IR�/�,whereasM-IGF1R�/� had normal leanmass andmuscleweights.

In addition, M-IGF1R�/� and MIGIRKO mice displayed a loss of

fat mass (Figure S1D; Table S2). The cause of this loss of fat

mass is unknown, but it was not due to recombination of IRs or

IGF1Rs in fat or other tissues (Figures S1F and S1G), suggesting

some form of communication between muscle and fat that is

dependent on IGF-1 action. None of the changes in body weight

andcompositionwere attributable todwarfismoranydecrease in

linear growth as assessed by femur length (Table S2).

Histologic analysis of tibialis anterior (TA) muscle using succi-

nate dehydrogenase (SDH) staining revealed marked atrophy or

lackof hypertrophyofMIGIRKOmuscle fibers (Figure1H). The to-

tal cross sectional area of the TA was mildly reduced in M-IR�/�

and markedly reduced in MIGIRKOmice (Figure S2). Quantifica-

tion of fiber number normalized to the area of the TAcross section

in square millimeters revealed that the decrease in muscle size in

these two strains was due to atrophy and not a loss of muscle

fibers (Figure 1I). Lastly, while glycolytic fiber number did not

change, M-IGF1R�/� and MIGIRKO mice showed increased

numbers of oxidative fibers (Figures 1J and S2C).

MIGIRKOMiceDemonstrate Normal Glucose and InsulinTolerance but Increased Basal Glucose Uptake inMuscle and Fasting HypoglycemiaGlucose levels were unchanged in randomly fed animals, but

after 16 hr of fasting, glucose levels were 35% lower in

MIGIRKO mice compared to controls (Figure 2A). This occurred

with no significant changes in either fasting or refed insulin

and triglyceride levels (Figures 2B and 2C). As expected, insu-

lin and IGF-1 signaling was abolished in skeletal muscle

from MIGIRKO animals injected with either 5 U of insulin

or 1 mg/kg IGF-1 via inferior vena cava (Figures 2D and S3A).

Insulin signaling was normal in M-IGF1R�/� and blunted in

M-IR�/� (Figure S3B). However, upon western blotting of mus-

cle extracts, MIGIRKO animals displayed an unexpected 3- to

10- fold increase in the protein levels of IRS-1, IRS-2, Akt1,

and Akt2. This dramatic increase was not seen at the mRNA

level, although mRNA for IRS-2 and Akt2 was increased in

MIGIRKO by 2.2- and 1.6-fold, respectively (Figure S3C). This

increase in protein levels was associated with a marked in-

crease in the amount of phosphorylated Akt in the basal state,

as well as an increase in downstream phosphorylation of

GSK3b, FoxO1, and FoxO3a (Figures 2D and S3D–S3G). We

hypothesized that other tyrosine kinases may constitutively

activate the IRS-PI3K-Akt pathway in the absence of IR and

ell Reports 11, 1220–1235, May 26, 2015 ª2015 The Authors 1221

A B

C D

E F G

H I J

Figure 1. Deletion of IRs and IGF1Rs in Muscle Dramatically Decreases Muscle Size and Survival

(A) Western blot of insulin receptor-b (IR-b) and IGF-1 receptor-b (IGF1R-b) was measured in quadriceps from mice with muscle-specific deletion of insulin

receptor (M-IR�/�), IGF-1 receptor (M-IGF1R�/�), or both IGF-1 receptor and insulin receptor (MIGIRKO).

(B) Representative profile and hindlimb dissection of control and MIGIRKO littermate mice.

(C) Body weight was measured weekly in control and MIGIRKO mice (n = 7–16).

(D) Survival curve of MIGIRKO mice compared to control, M-IR�/�, and M-IGF1R�/�, represented as ‘‘All other genotypes’’ (n = 14–20 per group).

(E) Body weight was measured at time of sacrifice of control, M-IR�/�, M-IGF1R�/�, and MIGIRKO mice (n = 5–8 knockout mice and pooled 20 controls).

(legend continued on next page)


IGF1R. Indeed, total EGFR levels are increased in MIGIRKO

muscle (Figure S3H).

Despite the lack of IRs and IGF1Rs in muscle, glucose toler-

ance and insulin tolerance in MIGIRKO were unchanged

compared to controls (Figures 2E and 2F). To determine the

fate of glucose in MIGIRKO animals, we performed an in vivo

glucose uptake assay under basal or insulin-stimulated condi-

tions as described in Experimental Procedures. Interestingly,

basal glucose uptake was increased in MIGIRKO muscles to

the level of insulin-stimulated control muscle, and it did not

increase further with insulin treatment (Figure 2G). In other tis-

sues, such as heart and brown adipose tissue (BAT), basal and

insulin-stimulated glucose uptake in MIGIRKO was similar to

controls, consistent with the absence of recombination of IRs

or IGFRs in these tissues (Figures S1F and S1G).

Deletion of IRs and IGF1Rs in Muscle ParadoxicallyIncreases Glucose Transporter Expression andMembrane LocalizationTo determine the relative contributions of IR and IGF1R signaling

to insulin-stimulated glucose uptake, we measured glucose

uptake in extensor digitorum longus (EDL) and soleus muscles

in vitro. Of note, insulin signaling in these two muscle groups

was similar to quadriceps (Figure S4A). While glucose uptake

in response to insulin in EDL from M-IR�/� was blunted, glucose

uptake in the soleus of M-IR�/� and in EDL or soleus of

M-IGF1R�/� was unchanged compared to controls (Figures 3A

and 3B). As found in vivo, in vitro basal glucose uptake was

increased in MIGIRKO muscle, and this was unresponsive to

insulin stimulation. This increase in basal glucose uptake in

MIGIRKO was associated with increased protein levels of the

glucose transporters Glut1 and Glut4 (Figures 3C and 3D). These

changes occurred with no changes in Glut1 expression at the

mRNA level and a decrease in Glut4 mRNA expression

(Figure 3E).

Since basal glucose uptake in vivo and in vitro was increased

in MIGIRKOmuscle and signaling downstream of Akt was signif-

icantly enhanced in the basal state, we hypothesized that Glut4

translocation was enhanced in fasted or unstimulated MIGIRKO

muscle. To evaluate Glut4 localization in vivo, we utilized a

method of intravital imagining of a Glut4-EGFP protein tran-

siently transfected into superficial muscle fibers of vastus latera-

lis utilizing a gene-gun approach (Lauritzen et al., 2002, 2006).

Glut4-EGFP remained in larger intracellular depots with minimal

surface localization in fibers from control mice, yet MIGIRKO

fibers displayed a diffuse pattern with dispersed GLUT4-EGFP

vesicle depots and increased surface localization (Figure 3F).

This pattern of diffuse fluorescence with increased surface local-

ization seen in MIGIRKO fibers is consistent with the pattern of

(F) Representative muscle dissection from control and MIGIRKO mice.

(G) Dissected muscle weights measured from control, M-IR�/�, M-IGF1R�/�, an(H) Representative cross section of TA muscle stained for SDH to demonstrate ox

M-IGF1R�/�, and MIGIRKO mice.

(I) Quantification of total number of muscle fibers normalized to cross sectional a

(J) Quantification of total oxidative and glycolytic fibers per TA section (n = 3–6 p

*p < 0.05, **p < 0.01 versus control (ANOVA). All mice were 11–15 weeks old. Qua

Gast, gastrocnemius; SDH, succinate dehydrogenase. Data are presented as m

C

Glut4-EGFP seen after stimulation with insulin (Figures S4C

and S4D) or muscle contractions (Lauritzen et al., 2006, 2010).

Muscle fractionation experiments confirmed increased levels

of Glut4 and Glut1 in plasma membrane isolates from MIGIRKO

muscle compared to controls (Figure 3G). Increased Glut4 trans-

location is consistent with our observation that signaling down-

stream of Akt was increased in muscle from fasted MIGIRKO

mice (Figure 2D). MIGIRKO muscles also displayed increased

phosphorylation of AMPK in the fed state (Figure 3H), indicating

activation of this pathway. AMPK phosphorylation remained

elevated evenwhen themicewere fastedwithmodest elevations

in p-ACC, but phosphorylation of neither protein changed in

response to insulin in either control or MIGIRKO mice (Fig-

ure S4B). Despite increased basal glucose uptake and Glut4

membrane localization, lactate levels were actually decreased

in MIGIRKO muscle and glycogen content was unchanged

in M-IR�/�, M-IGF1R�/�, and MIGIRKO muscle compared to

controls (Table S3).

Deletion of IRs and IGF1Rs in Muscle Leads toSuppression of TBC1D1, and Re-expression of TBC1D1Leads to Re-internalization of Glut4To gain insight into the mechanism for enhanced glucose

transporter translocation, we investigated the phosphorylation

status of AS160 and TBC1D1, both of which participate in

Glut4 translocation and glucose uptake in muscle. Consistent

with previous reports (Taylor et al., 2008), we found AS160

to be more abundant in oxidative soleus muscle and TBC1D1

more abundant in EDL, a more glycolytic muscle (Fig-

ure S4A). Phosphorylation of AS160 and TBC1D1 in response

to insulin showed no differences among control, M-IR�/�, andM-IGF1R�/�, but basal phosphorylation of AS160 was

increased in EDL and soleus from fasted MIGIRKO mice

(Figure S4A), consistent with increased Akt phosphorylation

and activation. Interestingly, while phosphorylation of the

160-kDa band using a phospho-Akt substrate antibody (PAS

160) and AS160T642 were increased, total levels of TBC1D1

were decreased compared to controls in EDL, soleus,

and quadriceps muscle (Figures 4A and S4A). qPCR analysis

revealed a significant �20% decrease in TBC1D1 mRNA from

M-IR�/� mice and a dramatic 72% decrease in MIGIRKO mus-

cle (Figure 4B). Conversely, AS160 mRNA levels were

increased 2.1- and 2.6-fold in M-IR�/� and MIGIRKO muscle,

respectively.

We hypothesized that the observed decrease of total TBC1D1

levels along with increased AS160 phosphorylation in MIGIRKO

muscle contributed to the re-localization of Glut4 to the

sarcolemma. To test this hypothesis directly, we transiently

re-expressed TBC1D1 in MIGIRKO muscle (Figure S4E) and

d MIGIRKO mice (n = 5–9 knockout mice and pooled 22 controls).

idative (purple) and glycolytic (gray/white) muscle fibers from control, M-IR�/�,

rea of TA sections in mm2 (n = 3–6 per group).

er group).

d, quadriceps; TA, tibialis anterior; EDL, extensor digitorum longus; Sol, soleus;

ean ± SEM. See also Figures S1 and S2 and Table S2.


A B C

D E

F

G

Figure 2. MIGIRKO Mice Display Normal Glucose Tolerance, Fasting Hypoglycemia, and Increased Basal Glucose Uptake into Muscle,

Despite Abolished Insulin Signaling in Muscle

(A) Blood glucose levels were measured in 8- to 10-week-old MIGIRKO and control mice fasted overnight or randomly fed (n = 9–10).

(B and C) Insulin (B) and triglyceride (C) levels from 8- to 10-week-old MIGIRKO and control mice fasted overnight or refed for 4 hr (n = 9–10).

(D) Insulin signaling was determined by western blot analysis in quadriceps muscle from 11- to 15-week-old MIGIRKO and control mice fasted overnight and

treated with saline or insulin intravenously.

(E and F) Intraperitoneal glucose tolerance test (GTT) (E) and insulin tolerance test (ITT) (F) were performed in 8- to 10-week-old MIGIRKO and control mice

(n = 9–10).

(G) In vivo 2-deoxyglucose uptake was performed as described in Experimental Procedures in control and MIGIRKO mice (n = 6–8 per group).

**p < 0.01 versus control (Student’s t test), #p < 0.05 versus control with same treatment, and yp < 0.05 versus basal of same genotype (ANOVA). Quad,

quadriceps; Gastroc, gastrocnemius; BAT, brown adipose tissue. Data are presented as mean ± SEM. See also Figure S3.


A

C

F G

H

D E

B

Figure 3. Deletion of Muscle IRs and IGF1Rs Paradoxically Increases Glucose Transporter Expression and Membrane Localization

(A and B) Ex vivo 2-deoxyglucose uptake wasmeasured in EDL (A) and soleus (B) from 8- to 10-week-old control, M-IR�/�, M-IGF1R�/�, andMIGIRKOmice (n = 5

knockouts and 12 pooled controls).

(C and D) Glut1 (C) and Glut4 (D) total protein measured by western blot in control, M-IR�/�, M-IGF1R�/�, and MIGIRKO quadriceps (n = 4).

(E) Glut1 and Glut4 mRNA levels were measured in quadriceps from control, M-IR�/�, M-IGF1R�/�, and MIGIRKO mice by qRT-PCR (n = 5–8).

(F) Glut4-EGFP was transfected into vastus lateralis muscle and visualized 5 days later as described in Experimental Procedures. Scale bar, 10 mm (n = 2).

(G) Glut1 and Glut4 levels in plasma membrane (PM) isolates from mixed hindlimb muscle (n = 3).

(H) Phosphorylation of AMPKT172 was measured in quadriceps (n = 4).

*p<0.05, **p<0.01versuscontrol; yp<0.05versusbasal of samegenotype (ANOVA).Dataarepresentedasmean±SEM.SeealsoFigureS4andTablesS1andS3.

Cell Reports 11, 1220–1235, May 26, 2015 ª2015 The Authors 1225

A B

C

D E

Figure 4. Deletion of IRs and IGF1Rs in Muscle Leads to Suppression of TBC1D1, and Re-expression of TBC1D1 Normalizes Glut4 Locali-

zation

(A) AS160 phospho- and total protein, phospho-Akt substrate 160-kDa band (PAS 160), and TBC1D1 total protein were measured by western blot in control and

MIGIRKO quadriceps (n = 4).

(B) TBC1D1 and AS160 mRNA levels were measured in TA muscle from control, M-IR�/�, M-IGF1R�/�, and MIGIRKO mice by qRT-PCR (n = 4–8).

(C) Glut4-EGFP was transfected into vastus lateralis muscle along with empty vector (EV) or with TBC1D1, and visualized 5 days later in the fed state. Scale

bar, 10 mm.



determined the localization of Glut4-EGFP using intravital

imaging of muscle fibers. Transient expression of Glut4-EGFP

with an empty vector (EV) in control muscle fibers again showed

large depots of Glut4, whereas a diffuse pattern with increased

membrane localization was seen in MIGIRKO fibers (Figure 4C).

Co-expression of TBC1D1 with Glut4-EGFP in MIGIRKO fibers

normalized the pattern of Glut4 localization back to large intra-

cellular depots. Quantification of the Glut4 depot area using

MetaMorph software was performed as described in Experi-

mental Procedures. Total area of GLUT4-EGFP vesicle depots

above 1 mm in size was lower in MIGIRKO animals compared

to controls, reflecting re-localization of the depots to t-tubules

and sarcolemma (Figures 4D and 4E). Re-expression of

TBC1D1 in MIGIRKO fibers significantly increased Glut4 depot

area compared to MIGIRKO + EV in intramyofibrillar compart-

ments (Figures 4D and 4E), where 90% of Glut4 vesicles reside

(Wang et al., 1996).

Increased Energy Expenditure in MIGIRKO Mice IsCorrelated with Increased Browning of SubcutaneousWhite Fat and Increased Glucose Uptake into FatMIGIRKO mice display fasting hypoglycemia and increased

basal muscle glucose uptake even in the fasted state, but

glucose tolerance and insulin tolerance were normal, indicating

that whole-body metabolic adaptations are likely to occur

when insulin signaling is abolished in skeletal muscle. To better

investigate these metabolic changes, we assessed metabolic

actions of MIGIRKO mice using the Comprehensive Laboratory

Animal Monitoring System (CLAMS). This revealed that

MIGIRKO mice ate �20% less food and drank less water per

mouse than their controls, but when normalized to lean body

mass, the water intake was unchanged and the food intake

was actually increased (Figure 5A). Likewise, oxygen con-

sumption and CO2 production normalized to lean body mass

were significantly increased in MIGIRKO mice during both

day and night cycles (Figure 5B), while respiratory exchange

ratio (RER) was unchanged (Figure 5C). This occurred despite

a significant decrease in activity of the MIGIRKO mice as

measured by number of times a horizontal axis was crossed

(Figure 5D).

At sacrifice, subcutaneous white adipose tissue (sWAT) from

the inguinal region of MIGIRKO mice was noted to be more

brown in color than in normal mice, and H&E staining of

sWAT revealed large patches of adipocytes with multiloculated

lipid droplets and abundant capillaries indicating browning

of the white fat in MIGIRKO mice (Figure 5E). Consistent with

browning, mRNA expression of BAT markers such as Ucp1,

Dio2, and Elovl3 were increased by 3- to 5-fold in sWAT of

MIGIRKO mice, but not in epididymal white adipose tissue

(eWAT) (Figure 5F), basal glucose uptake was increased into

sWAT, and insulin-stimulated glucose uptake was increased

in both sWAT and eWAT (Figure 5G). Recent studies have

(D and E) Quantification of average area of all Glut4 depots >1 mm from control an

with Glut4-EGFP + TBC1D1 (n = 2 control and n = 3 MIGIRKO mice per group w

*p < 0.05, **p < 0.01 versus control (ANOVA); xp < 0.05 versus MIGIRKO + EV (S

Table S1.

C

implicated a circulating protein called irisin, which is derived

from FNDC5 and secreted from muscle, in browning of WAT

(Bostrom et al., 2012); however, levels of Fndc5 mRNA were

decreased in quadriceps muscle from MIGIRKO (Table S4).

FGF21 is another circulating hormone that can lead to brown-

ing of sWAT and has recently been implicated in metabolic

adaptations to autophagy inhibition in muscle (Kim et al.,

2013), as well as other stresses such as ER stress. Interest-

ingly, Fgf21 mRNA levels in quadriceps were modestly

increased, especially in the fasted state (Figure 5H), with in-

creases in mRNA levels of macrophage markers, but not ER

stress markers (Table S4). However, when we tested circulating

levels of FGF21, these were not changed in randomly fed

MIGIRKO mice (Figure 5I).

Deletion of Insulin and IGF-1 Receptors in MuscleDoes Not Predispose Mice to Diabetes, Even aftera High-Fat DietTo determine if deletion of IRs, IGF1Rs, or both in muscle

would predispose mice to metabolic derangements or dia-

betes, all genotypes were challenged with a high-fat diet

(HFD). MIGIRKO mice were 25% smaller when dietary chal-

lenge was initiated, but mice of each genotype gained a similar

percent of weight (15%) on an HFD after 8 weeks compared to

mice on a chow diet (CD) (Figure 6A). In control mice, metabolic

derangements were present as early as 4 weeks on an HFD as

indicated by increased insulin levels and increased serum tri-

glycerides (Figure 6B). Interestingly, although serum triglycer-

ides were equally elevated in MIGIRKO mice as in controls on

an HFD, insulin levels did not increase in MIGIRKO animals

on an HFD.

Glucose tolerance tests again revealed fasting hypoglycemia

in MIGIRKO compared to controls on the same diet (Figure 6C).

However, both control and MIGIRKO mice on an HFD exhibited

impaired glucose tolerance, as measured by increased area

under the curve (AUC), with no differences between genotypes

on the same diet (Figure 6D). M-IR�/� mice did show modest

impairment of glucose tolerance on CD, but similar to MIGIRKO

animals, M-IR�/� and M-IGF1R�/� mice became glucose intol-

erant with increased AUC on an HFD, and no differences were

observed when compared to IRlox/lox or IGF1Rlox/lox controls on

the same diet (Figures S5B and S5C). Insulin tolerance tests at

a dose of 1.0 mU/g body weight also remained similar between

control and MIGIRKO mice, regardless of diet (Figure 6E-6F).

Finally CLAMS analysis of both control and MIGIRKO animals

on an HFD revealed increases in VO2 and VCO2 compared to

CD mice, with increases in both VO2 and VCO2 in MIGIRKO

compared to controls, regardless of the diet (Figures 6G and

6H). Both control and MIGIRKO mice showed the expected

suppression of RER on an HFD indicating increased fat utiliza-

tion, with no differences between genotypes on the same diet

(Figure 6I).

d MIGIRKO mice transfected with Glut4-EGFP + EV and MIGIRKO transfected

ith three to seven fibers each).

tudent’s t test). Data are presented as mean ± SEM. See also Figure S4 and


A B C

D

E F

G H I

Figure 5. Increased Energy Expenditure in MIGIRKO Mice Is Associated with Browning of Subcutaneous Fat

(A) Daily food and water intake were measured in control and MIGIRKO animals and normalized per mouse or per milligram of lean body weight (LBW) (n = 9–10).

(B) Oxygen consumption (VO2) and carbon dioxide production (VCO2) per kg of LBW were measured using CLAMS metabolic cages (n = 9–10).

(C) Respiratory exchange ratio (RER) was measured in control and MIGIRKO mice (n = 9–10).

(D) Activity was measured as the number of times an animal crossed a horizontal laser (n = 9–10).

(E) H&E staining was performed on inguinal subcutaneous white adipose tissue (sWAT) from control and MIGIRGO animals.

(F) Markers of brown adipose tissue (BAT) were measured by qRT-PCR in sWAT and epididymal WAT (eWAT); (n = 6).

(G) In vivo 2-deoxyglucose uptake under basal or insulin-stimulated conditions was measured in sWAT and eWAT from control and MIGIRKO mice (n = 6–8).



Overexpression of a Dominant-Negative IGF1R inMuscle of MIGIRKO Mice Leads to MetabolicDerangements, Despite the Absence of IRs and IGF1RsPrevious studies have shown that mice overexpressing a domi-

nant-negative, kinase-inactive IGF1R in muscle (MKR) develop

overt diabetes, presumably through inhibition of endogenous

IR and IGF1R function (Fernandez et al., 2001). To further explore

this hypothesis, we expressed the mutant IGF1R in MIGIRKO

muscle by crossing MKR and MIGIRKO mice (MKR-MIGIRKO).

As has been previously reported (Fernandez et al., 2002), we

observed that MKR mice have reduced body weight compared

to controls (Figure 7A). Unlike MKR mice on an FVB background

(Fernandez et al., 2001), MKR mice on this mixed genetic back-

ground exhibited no differences in glucose levels upon fasting or

refeeding (Figures 7B and 7C); however, they did develop signif-

icant glucose intolerance when compared to controls (Figures

7D and 7E). Expression of the MKR allele in MKR-MIGIRKO

mice did not further reduce the body weight of MIGIRKO mice

(Figure 7A), nor did it affect the development of mild hypoglyce-

mia upon fasting when compared to control and MKRmice (Fig-

ure 7B). However, upon refeeding for only 4 hr, MKR-MIGIRKO

mice displayed an exaggerated rebound in glucose levels.

Interestingly, expression of the dominant-negative IGF1R in

MKR-MIGIRKO mice also resulted in development of glucose

intolerance in MIGIRKO mice, similar to that observed when

the MKR transgene was expressed in control mice (Figures 7D

and 7E). The impaired glucose tolerance in MKR and MKR-

MIGIRKO mice was associated with elevated circulating triglyc-

erides, similar to that observed in HFD-fed animals (Figure S6A),

but with no significant change in insulin levels (Figure S6B).

Surprisingly, MKR-MIGIRKO show decreased Glut1 levels

compared to MIGIRKO (Figure 7C), but the total level was similar

to that observed in MKR. By contrast, MKR-MIGIRKO muscle

shows markedly increased levels of Glut4 protein compared to

control andMKRmice, which were similar to what was observed

in MIGIRKO muscle.

We determined in vivo glucose uptake during an intravenous

glucose tolerance test (IV GTT) to seewhich tissuesmay account

for the changes in glucose tolerance in MKR-MIGIRKO mice.

Glucose values during the IV GTT again demonstrated mild but

significant glucose intolerance in MKR compared to controls

and glucose intolerance in MKR-MIGIRKO compared to

MIGIRKOs (Figure S6C). Glucose uptake into quadriceps and

gastrocnemius muscle during IV GTT was unchanged in MKR

mice compared to controls (Figure 7F). MIGIRKO and MKR-

MIGIRKO mice showed increased glucose uptake in skeletal

muscle compared to control mice with no changes between

MIGIRKO and MKR-MIGIRKO muscle. Interestingly, glucose

uptake into heart was significantly decreased in MKR and

MKR-MIGIRKO mice compared to controls (Figure 7F), whereas

no change was observed in other insulin-sensitive tissues (Fig-

ure S6D). As previously observed for other genes on the MCK

(H) Fgf21 mRNA levels from quadriceps of MIGIRKO and control mice either ran

(I) Serum FGF21 levels in randomly fed control and MIGIRKO mice (n = 5).

*p < 0.05, **p < 0.01 versus control (Student’s t test); #p < 0.05 versus control with

presented as mean ± SEM. See also Table S1 and S4.

C

promoter (Bruning et al., 1998), expression of the MKR allele

was very high in both skeletal muscle and heart as shown by

IGF1R western blot analysis (Figure 7G). Total levels of Akt iso-

forms, as well as phosphorylation of Akt, in MKR-MIGIRKO

skeletal muscle were the same as MIGIRKO, but these levels

were elevated compared to that observed in control and MKR

skeletal muscle and did not respond to insulin or IGF-1 treatment

(Figures 7G and S7). However, insulin signaling in heart as

measured by phosphorylation of Akt, GSK3b, and FoxO isoforms

was unchanged (Figure 7G), indicating that changes in Akt

signaling in MKR and MKR-MIGIRKO hearts are unlikely to

account for the impaired glucose uptake when compared to

controls.

DISCUSSION

Skeletal muscle insulin resistance is an important component in

the pathogenesis of type 2 diabetes and metabolic syndrome,

and it may occur years prior to onset of disease (Martin et al.,

1992). However, while deletion of IRs in skeletal muscle of

MIRKO mice causes some features of the metabolic syndrome,

it alone does not cause diabetes or hyperglycemia (Bruning

et al., 1998). Two potential explanations for this discrepancy

are the possibility that there is residual insulin signaling in muscle

via the IGF1R or that exercise-induced glucose uptake compen-

sates for this insulin resistance and maintains glucose uptake.

Consistent with the first hypothesis, Fernandez et al. (Fernandez

et al., 2001) have reported that MKR mice, which overexpress a

dominant-negative form of the human IGF1R in muscle, develop

diabetes at a young age, suggesting that this receptor can

hybridize with IRs and IGF1Rs to block insulin and IGF-1

signaling and induce hyperglycemia.

To test the first hypothesis directly, in the present study, we

specifically deleted IRs and IGF1Rs in muscle to create the

MIGIRKO mouse. Indeed, we find that IRs and IGF1Rs compen-

sate for each other to maintain muscle growth, such that when

both are deleted, the mice display no insulin or IGF-1 signaling

in skeletal muscle and have a marked decrease in muscle

mass and fiber size. Despite this, these mice display normal

whole-body glucose tolerance, indicating that, in mice, neither

of these receptors alone or in combination is required in muscle

to maintain normal glucose tolerance. Furthermore, loss of IRs

and IGF1Rs in muscle does not lead to diabetes, even when

themice are challengedwith an HFD. That is not to say that these

MIGIRKO mice do not display perturbations in muscle glucose

metabolism. To the contrary, MIGIRKOmice show fasting hypo-

glycemia, which is mediated by increased glucose transporter

protein levels and translocation leading to increased basal

glucose uptake in muscle. Furthermore, energy expenditure

was increased and associated with increased glucose up-

take in BAT, WAT, and increased markers of browning of

sWAT, possibly contributing to the normal glucose tolerance in

domly fed or fasted for 16 hr were measured by qRT-PCR ( n = 4 per group).

same treatment, yp < 0.05 versus basal of same genotype (ANOVA). Data are


A B

C D

E F

G H I

Figure 6. MIGIRKO Mice Are Not Predisposed to Diabetes, Even after High-Fat Diet Feeding

(A) Body weights of control and MIGIRKO mice were measured weekly while on a chow diet (CD) or high-fat diet (HFD) beginning at 6 weeks of age (n = 4–8).

(B) Serum insulin levels from randomly fed mice and triglycerides from overnight fasted control and MIGIRKO mice on a CD or HFD for 4 weeks (n = 4–8).

(C and D) Intraperitoneal glucose tolerance test (GTT) (C) was performed and area under the curve (AUC) (D) was calculated for MIGIRKO and control mice on a

CD or HFD for 9 weeks (n = 3–9).

(E and F) Intraperitoneal insulin tolerance test (ITT) (E) was performed and area above the curve (AAC) (F) was calculated for mice on a CD or HFD for 8 weeks

(n = 3–9).

(G–I) VO2 (G), VCO2 (H), and RER (I) were measured in animals on a CD or HFD during both light and dark cycles using CLAMS metabolic cages (n = 3–9).

*p < 0.05 versus control with same diet, yp < 0.05 versus CD of same genotype (Student’s t test). Data are presented as mean ± SEM. See also Figure S5.

MIGIRKO mice. Surprisingly, despite the lack of effect of IR/

IGF1R knockout in muscle on whole-body glucose tolerance,

expression of the dominant-negative IGF1R in muscle does

lead to glucose intolerance and some of the metabolic derange-


ments associated with metabolic syndrome. Thus, insulin

signaling via the IGF1R in muscle is not a compensatory mech-

anism by which glucose tolerance is maintained when the IR is

deleted in muscle.

Mechanistically, this study defines a critical role for IRs/

IGF1Rs in muscle to suppress basal glucose transport, espe-

cially in the fasted state (low insulin). Normally, muscle utilizes

glucose for energy production primarily in the fed state and tran-

sitions to fatty acids as a primary fuel source upon fasting. Upon

refeeding, the muscle rapidly switches back to glucose

utilization in a paradigm termed metabolic flexibility (Storlien

et al., 2004). One component of muscle metabolic flexibility is

the activation of glucose transport by insulin, which occurs via

enhanced Glut4 translocation. Somewhat unexpectedly, we

find that the total absence of IR and IGF1R signaling leads to a

paradoxical increase in Glut1 and Glut4 proteins and increased

localization of these to the plasma membrane, even in the fasted

state. Thus, the MIGIRKOmouse develops mild fasting hypogly-

cemia, rather than hyperglycemia.

Several mechanisms contribute to the increased basal

glucose uptake in muscle following loss of IRs and IGF1Rs. First,

deletion of IRs/IGF1Rs in muscle leads to decreased levels of

TBC1D1. TBC1D1 is a Rab-GAP protein expressed primarily

in glycolytic muscle and is homologous to the Rab-GAP AS160

(also known as TBC1D4) (Taylor et al., 2008). Both AS160 and

TBC1D1 are inhibited by Akt- or AMPK-mediated phosphoryla-

tion to promote GLUT4 translocation to the plasma membrane

(Taylor et al., 2008). We observe that re-expression of TBC1D1

in MIGIRKO muscle in vivo is able to reverse the abnormal

Glut4 localization. These data are further supported by in vitro

studies, which show that silencing of TBC1D1 in L6 myotubes

or adipocytes results in increased basal Glut4 and Glut1 translo-

cation, respectively (Zhou et al., 2008; Ishikura and Klip, 2008).

Interestingly, germline deletion of TBC1D1 results in decreased

glucose uptake in muscle, which was consistently associated

with decreased Glut4 levels (Dokas et al., 2013; Szekeres

et al., 2012). In MIGIRKO mice, we find decreased levels of

TBC1D1 protein but increased levels of Glut1 and Glut4, which

are related to decreased protein turnover, as mRNA levels

were unchanged or decreased. In addition, Akt and AMPK are

chronically activated in MIGIRKO muscle, which was unex-

pected and may relate to energy stress, changes in protein turn-

over, or unmasking of a feedback loop. While little is known

about the control of TBC1D1 expression, our data indicate that

IR/IGF1R signaling plays an important role in the regulation of

TBC1D1 and AS160 levels in muscle.

It has been known for some time that IGF-1 treatment can

induce muscle hypertrophy via the Akt-mTOR pathways, yet

previous studies have indicated that deletion of IGF1Rs alone

in muscle only modestly changes myocyte size and morphology

(Schiaffino and Mammucari, 2011; Mavalli et al., 2010). The

present study indicates that signaling via either IRs or IGF1Rs

is sufficient to maintain muscle mass. This indicates that phys-

iologic levels of insulin or IGF-1 ligand are sufficient to promote

proteins synthesis and suppress protein degradation as long as

either the IR or the IGF1R are present. Recent work has identi-

fied FoxO transcription factors, which are known targets for IR/

IGF1R signaling, as critical mediators of muscle protein degra-

dation and atrophy (Mammucari et al., 2007; Sandri et al.,

2004).

The browning of the sWAT in MIGIRKO mice also contributes

to the metabolic phenotype. Previous reports confirm that

C

changes in the autophagy pathway in muscle can lead to in-

creases energy expenditure via FGF21 induced browning of

WAT (Kim et al., 2013). Although circulating FGF21 levels were

not increased, local FGF21 production by the muscle remains

a possible mechanism for the browning of sWAT observed in

these mice. These mice were raised at room temperature

(25�C), which is not thermo-neutral, and the reduced body size

may contribute to increased need for thermogenic capacity.

Our work also identifies a distinction between lack of IR/IGF1R

signaling and insulin resistance in which the receptors are

present but activation by ligand is decreased. Thus, while com-

bined deletion of muscle IRs and IGF1Rs does not alter glucose

or insulin tolerance in mice, expression of a dominant-negative

IGF1R in skeletal muscle (and heart) in both control and

MIGIRKO mice can produce mild glucose intolerance and lipid

abnormalities. This is similar to previous observations demon-

strating that MKR mice develop diabetes associated with dysli-

pidemia, hepatic steatosis, and insulin resistance (Kim et al.,

2003; Vaitheesvaran et al., 2010), but in our study, this occurs

even in mice lacking normal insulin and IGF1Rs in muscle.

We speculate that at least two possibilities contribute to this

phenomenon. First, the dominant-negative IGF1R may bind to

a receptor other than IR/IGF1R, such as Met (Fafalios et al.,

2011), or to one or more downstream signaling proteins, such

as IRS-1 and Shc, to transmit a signal to the myocyte that

actively perturbs lipid homeostasis and interrupts tissue cross-

talk, whereas deletion of the receptors does not transmit such

a signal. Second, cardiac insulin/IGF-1 resistance, when com-

bined with skeletal muscle resistance, may contribute to more

glucose intolerance and lipid abnormalities than seen with skel-

etal muscle insulin resistance alone.

Our lab has previously shown that deletion of IRs/IGF1Rs in

preadipocytes protects them from apoptosis and reintroduction

of a non-functional IR transmitted a signal that conferred sus-

ceptibility to apoptosis (Boucher et al., 2010). This suggests

that the unoccupied insulin and IGF1Rs can generate a signal

that is different from that normally mediated by the occupied

receptor. The current study likewise indicates that deletion of

IRs or IGF1Rs is fundamentally different from loss of insulin or

IGF-1 signaling in which the receptors are present but the ligand

or ligands are missing. Further work will be needed to fully char-

acterize the nature of the signals coming from unoccupied insulin

and IGF1Rs and the specific IR/IGF1R receptor-protein interac-

tions that contribute to metabolic disease.

In summary, our study demonstrates that IR or IGF1R signaling

is critical for normal muscle growth but that deletion of both re-

ceptors in muscle does not lead to impaired glucose tolerance

due to underlying feedback loops, which maintain a high level

of glucose uptake, even in the fasted/unstimulated state. In addi-

tion, deletion of IRs and IGF1Rs inmuscle is unable to induce dia-

betesorworsenmetabolic parameters inmicechallengedwith an

HFD.On theother hand, lossof IRs and IGF1Rs inmuscle leads to

increased basal glucose uptake due to increases in levels of

Glut1 and Glut4 transporters, chronic activation of Akt and

AMPK signaling, and a loss of TBC1D1 expression. Finally, we

find that the presence of a non-functional IGF1R in muscle of an-

imals lacking IRs and IGF1Rs can induce glucose intolerance and

metabolic derangements, indicating a novel mechanism of


A

D

F G

E

B C

Figure 7. Overexpression of a Dominant-Negative, Kinase-Inactive IGF1R in Muscle of MIGIRKO Mice Induces Glucose Intolerance and

Impaired Glucose Uptake in Heart

(A) Body weight was measured in 8- to 10-week-old control mice, mice with overexpression of a kinase inactive IGF1R in muscle (MKR), MIGIRKO mice, and

MKR-MIGIRKO mice (n = 3–7).

(B) Blood glucose was measured in 8- to 10-week-old control, MKR, MIGIRKO, and MKR-MIGIRKO mice after an overnight fast and after 4 hr of refeeding

(n = 3–7).

(C) Glut1 and Glut4 were measured in quadriceps (n = 8–10).

(D and E) Intraperitoneal glucose tolerance test (GTT) (D) was performed and area under the curve (AUC) (E) was calculated for 7- to 15-week-old mice (n = 4–7).



altered signaling by this receptor mutant. These data add a new

layer of understanding, such that the metabolic changes that

occur in insulin-resistant statesmay be a consequence of signals

transmitted from a poorly functional IR or IGF1R in muscle.

Further investigation of the protein interactions of IRs and IGF1Rs

in the insulin-resistant statewill potentially providenew targets for

the treatment of type 2 diabetes and its complications.

EXPERIMENTAL PROCEDURES

Animal Care and Use

Animal studies were performed according to protocols approved by the Insti-

tutional Animal Care and Use Committee (IACUC). Male mice were used for all

studies other than MKR-MIGIRKO studies, which used both males and fe-

males. Muscle IR knockout (M-IR�/�), muscle IGF1R knockout (M-IGF1R�/�),and combined muscle IR/IGF1R knockout (MIGIRKO) mice were each gener-

ated by crossing mice carrying the Cre recombinase driven by a skeletal

muscle actin promoter, ACTA1-Cre (Jackson Laboratory, stock number

006149), with mice carrying both floxed insulin and IGF1R receptor alleles

(Boucher et al., 2012), i.e., IRlox/loxIGF1Rlox/lox, then maintained as separate

colonies. Since no differences were observed among the IRlox/lox, IGF1Rlox/lox,

IRlox/+IGF1Rlox/lox, and IRlox/loxIGF1Rlox/lox mice, the results on controls were

pooled. MKR transgenic mice, which have themurine MCK promoter directing

expression of the human IGF1R gene containing the K1003R mutation, were

purchased from Jackson Laboratory (stock number 016618) and have been

previously described (Fernandez et al., 2001). See the Supplemental Experi-

mental Procedures for more information.

In Vivo Glucose Uptake

Glucose uptake into tissue was measured by intravenous injection with either

saline or 1 mU/g insulin in combination with 0.33 mCi [14C]2-deoxyglucose/g

administered via the retro-orbital sinus. After 45 min, [14C] levels in blood

and tissue were determined. For full details, see the Supplemental Experi-

mental Procedures.

Ex Vivo Muscle Glucose Uptake

Glucose uptake was measured in EDL and soleus strips as previously

described (Hayashi et al., 1998). Briefly, mice were fasted starting at 22:00

and muscle harvested the next day between 10:00 and 13:00. EDL and

isolated soleus strips were incubated with resting tension in the basal state

or stimulated with 5 mU/ml of insulin for 40 min with the addition of [3H]-2-de-

oxyglucose for the last 10 min.

Plasmid Transfection and Intravital Microscopy

The construction of GLUT4-EGFP (Lauritzen et al., 2002, 2008) and TBC1D1

(An et al., 2010) have been described previously. Mice were transfected using

the Helios gene gun system (Bio-Rad) as previously described (Lauritzen

et al., 2002; Lauritzen, 2010; Lauritzen and Schertzer, 2010). Briefly, mice

were anaesthetized with 90 mg/kg pentobarbital; the skin was opened to

expose the vastus lateralis to the bombardment of DNA/gold particles using

the gene gun. Five days after transfection, vastus lateralis in random-fed mice

were imaged. Quantification of Glut4-EGFP vesicular depots above 1 mm in

size and measurement of average GLUT4-EGFP area were generated using

MetaMorph software. See the Supplemental Experimental Procedures for

more information.

Physiological and Analytical Measurements

CLAMS (Columbus Instruments) and DEXA measurements were performed

at the Joslin Diabetes Research Center (DRC) core. Glucose tolerance tests

(F) In vivo 2-deoxyglucose uptake was performed during an IV GTT in control,

(G) Western blots for IGF1R expression and insulin signaling were performed on

after insulin injection via IVC.

*p < 0.05, **p < 0.01 versus control (ANOVA), #p < 0.05 versus MIGIRKO (Stude

C

and insulin tolerance tests were performed as previously described (Bruning

et al., 1998). Insulin levels were measured using a mouse insulin ELISA kit

(Crystal Chem), triglycerides were measured using a triglyceride assay kit

(Abnova), and FGF21 serum levels were measured with a mouse/rat ELISA

kit (R&D Systems, catalog number MF2100). In vivo insulin and IGF-1

signaling was performed in anesthetized, overnight-fasted mice by injecting

either 5 U of regular insulin or 1 mg/kg of human IGF-1 (Sigma-Aldrich) via

inferior vena cava (IVC). Then, 10–15 min later, tissues were harvested and

snap frozen in liquid nitrogen. Lactate levels were measured in gastrocne-

mius at the Mayo Clinic Metabolomics Resource Core using time-of-flight

mass spectrometry.

Statistical Analyses

All data are presented as mean ± SEM. Student’s t test was performed for

comparison of two groups, and ANOVA was performed for comparison of

three or more groups to determine significance.

SUPPLEMENTAL INFORMATION

Supplemental Information includes Supplemental Experimental Procedures,

seven figures, and four tables and can be found with this article online at

http://dx.doi.org/10.1016/j.celrep.2015.04.037.

AUTHOR CONTRIBUTIONS

B.T.O. designed the study, researched data, and wrote the manuscript.

H.P.M.M.L. performed intravital microscopy experiments and helped write

the manuscript. M.F.H. researched data and helped prepare the manuscript.

L.J.G. provided reagents and helped design experiments. C.R.K. designed

the study and helped write the manuscript.

ACKNOWLEDGMENTS

This work was supported by NIH grants R01 DK-031036 (to C.R.K.) and

R01AR42238 (to L.J.G.). B.T.O. was funded by a K08 training award from

the NIDDK of the NIH (K08DK100543), Mayo Clinic Metabolomics Resource

Core grant U24DK100469 from the NIDDK, which originates from the NIH

Director’s Common Fund, and Mayo Clinic CTSA grant UL1 TR000135 from

NCATS of the NIH. The Joslin Diabetes Center DRC core facility was used

for part of this work (P30 DK36836).

Received: December 2, 2014

Revised: March 11, 2015

Accepted: April 17, 2015

Published: May 14, 2015

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Cell Reports

Supplemental Information

Differential Role of Insulin/IGF-1 Receptor

Signaling in Muscle Growth and Glucose Homeostasis

Brian T. O’Neill, Hans P.M.M. Lauritzen, Michael F. Hirshman, Graham Smyth, Laurie J.

Goodyear, and C. Ronald Kahn

Figure S1. Relates to Figure 1

0

20

40

60

80

100

120

IR lox IGF1R lox

QPC

R n

orm

aliz

ed to

TB

P D

NA

(AU

)

Muscle DNA

IR lox

M-IR-/-

IGF1R lox

M-IGF1R-/-

IR lox IGF1R lox

MIGIRKO

IRlox/lox

M-IR-/-

IGF1Rlox/lox

M-IGF1R-/-

IRlox/loxIGF1Rlox/lox

MIGIRKO

** *

IR+ or IRlox IGF1R+ or IGF1Rlox

** **

0

20

40

60

80

100

120

IR lox IGF1R lox

Liver DNA

IR+ or IRlox IGF1R+ or IGF1Rlox

A. B.

C.

Figure S1. Recombination of IR and IGF1R locus is specific to muscle tissue in M-IR-/-, M-IGF1R-/-, and MIGIRKO mice. QPCR for IR+ or IRlox and IGF1R+ or IGF1Rlox on genomic DNA isolated from quadriceps muscle (A) and liver (B) in 8 week old mice (n=3-4). QPCR for IR and IGF1R mRNA from tibialis anterior muscle from randomly fed mice (C). (n=4-8) (*-p<0.05, **-p<0.01 vs. respective lox control, t-test)

0

0.2

0.4

0.6

0.8

1

1.2

1.4

IR IGF1R

mR

NA

(Fol

d of

Lox

Con

trol

)

IR lox

M-IR-/-

IGF1R lox

M-IGF1R-/-

IRloxIGF1Rlox

MIGIRKO ** **

** **

Muscle mRNA

IRlox/lox

M-IR-/-

IGF1Rlox/lox

M-IGF1R-/-

IRlox/loxIGF1Rlox/lox

MIGIRKO


Insulin

p-IR/IGF1R

p-AktS473

IR-β

IGF1R-β

Akt

Control MIGIRKO - - + + - - + +

eWAT

α-tubulin

Control MIGIRKO - - + + - - + +

sWAT Control MIGIRKO - - + + - - + +

BAT

0

100

200

300

400

500

600

sWAT eWAT BAT

Tiss

ue w

eigh

t (m

g)

Control M-IR-/- M-IGF1R-/- MIGIRKO

** **

D.

F.

* E.

G.

Figure S1. Fat tissue weights (D) and other tissue weights (E) were measured in control (n=22), M-IR-/- (n=8), M-IGF1R-/- (n=5), and MIGIRKO (n=9) mice . IR and IGF1R levels and insulin signaling was determined by western blot in fat tissues (F) as well as heart and liver (G) from MIGIRKO and control mice (n=2 per group). (*-p<0.05, **-p<0.01 vs. control, ANOVA) sWAT, subcutaneous white adipose tissue; eWAT, epididymal white adipose tissue; BAT, brown adipose tissue.

0

200

400

600

800

1000

1200

Heart Liver Kidney Brain

Tiss

ue W

eigh

t (m

g)

*

*

*

Heart Liver Control MIGIRKO

- - + + - - + + Insulin Control MIGIRKO

- - + + - - + +

p-AktS473

Akt

p-IR/IGF1RIR-β

IGF1R-β

α-tubulin

No Bands

2 mm

Control M-IR-/-

M-IGF1R-/- MIGIRKO

0

1

2

3

4

5

6

CSA

of T

A m

uscl

e (m

m2)

Control

M-IR-/-

M-IGF1R-/-

MIGIRKO

**

*

0%

10%

20%

30%

40%

50%

60%

70%

% Oxidative % Glycolytic

% o

f tot

al F

iber

s

**

**


Figure S2. Reduced myofiber size and a shift toward oxidative fibers in MIGIRKO. Representative cross sections of TA muscle stained for SDH to demonstrate oxidative (purple) and glycolytic (gray/white) muscle fibers from control, M-IR-/-, M-IGF1R-/-, and MIGIRKO mice (A) (boxes are represented in Figure 1H). Quantification of cross sectional area (CSA) of TA sections in mm2 (B) was performed using Image J as demonstrated in panel A. Total fiber number from whole TA cross sections (C). Quantification of the percent of oxidative and glycolytic fibers per TA section (D) (n=3-6 per group). (*-p<0.05, **-p<0.01 vs. control, ANOVA)

A.

B. C. D.

0

500

1000

1500

2000

2500

3000

3500

Tota

l Fib

ers

* p=0.

06


Figure S3. Akt signaling is chronically increased in MIGIRKO mice and does not respond to insulin or IGF-1 treatment. Signaling in quadriceps from control and MIGIRKO mice treated with 1 mg/kg IGF-1 (A) or M-IR-/-, M-IGF1R-/-, and MIGIRKO treated with 5 U insulin via IVC (B) . QPCR for IRS and Akt isoforms in quadriceps (C). Densitometry of p-AktS473 from Fig 2D (D) (n=4). Total Akt levels and densitometric ratio of p-AktS473/Akt from Fig 2D (E). Densitometry of Akt targets GSK3β (F) and FoxO isoforms (G) in control and MIGIRKO mice from Fig 2D (n=3-4 per group). Total Epidermal Growth Factor Receptor (EGFR) in quadriceps (H). (†-p<0.05, ††-p<0.01 vs. basal of same genotype, #-p<0.05, ##-p<0.01 vs. control with same treatment, ANOVA)

p-AktS473

Akt

p-IR/IGF1R

GAPDH

p-ERK

ERK

Control MIGIRKO

- - + + - - + + 1mg/kg IGF-1

A.

0 10 20 30 40 50 60 70 80 90

Basal Insulin Basal Insulin

Control MIGIRKO

Den

sito

met

ry (A

U)

p-AktS473

††

## ##

0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8


Control MIGIRKO

p-FoxO1T24-3aT32/FoxO1

p=0.09

C. E. D. Control MIGIRKO - - + + - - + + Insulin

Total Akt

0

5

10

15


Control MIGIRKO Den

sito

met

ry (A

U)

p-AktS473/Akt ††

##

0 0.5

1 1.5

2 2.5

3 3.5


Control MIGIRKO

Den

sito

met

ry (A

U)

p-GSK3βS9/GSK3β††

## p=0.05 vs. Con Ins

F.

B. - + - + - + - +

p-AktS473

Akt1

p-IR/IGF1R

p-ERKERK

Insulin

p-IRS1Y608

IRS1

p-GSK3βGSK3β

Akt2

Control MIGIRKO Total EGFR

0.0

0.5

1.0

1.5

2.0

2.5

3.0

IRS1 IRS2 Akt1 Akt2

mR

NA

(Fol

d C

hang

e)

WT M-IR-/- M-IGFR-/- MIGIRKO

##

##

Control M-IR-/-

M-IGFR-/-

MIGIRKO

G. H.

Glut4-EGFP and Staining for HA-tagged TBC1D1 co-expression

GLUT4-GFP TBC1D1 (HA-tag) Overlay Figure S4. Insulin signaling, AMPK signaling, and Glut4 localization are altered in MIGIRKO muscle. Western blots for insulin signaling in EDL and soleus extracts from control, M-IR-/-, M-IGF1R-/-, and MIGIRKO mice treated with 5 U insulin via IVC (A). Western blots and densitometry for AMPK and ACC phosphorylation in mice treated with 5 U insulin via IVC (B) (n=4). Reference images of Glut4-EGFP patterns defined as puncta positive (C) and diffuse with membrane localization (D) in subsarcolemmal and intramyofibrillar areas. Bar = 10 µm. HA-tagged TBC1D1 by immunofluorescence together with Glut4-EGFP in fixed isolated fibers 5 days after co-infection by gene gun (E) (see Methods) Bar = 5µm. (*-p<0.05, **-p<0.01 vs. control with same treatment, ANOVA)

Figure S4. Relates to Figures 3 and 4

E.

C. D. “Puncta Positive” Prior to insulin

“Diffuse and Membrane Localized” 30 min of insulin stimulation

Subsarcolemma Intramyofibrillar Subsarcolemma Intramyofibrillar

Control MIGIRKO

- - + + - - + +

M-IR-/- M-IGF1R-/-

- - + + - - + + EDL Soleus EDL Soleus EDL Soleus EDL Soleus

Insulin

p-AktS473

Akt1

IR-βIGF1R-β

Ladd

er

Akt2

p-AktT308

A.

GAPDH

PAS 160

TBC1D1

p-AS160T642

AS160

0

1

2

3

Control MIGIRKO

Den

sito

met

ry (A

U)

p-AMPKT172/AMPK

Basal Insulin

0

0.5

1

1.5

Control MIGIRKO

p-ACCS79/ ACC

Basal Insulin

B. Control MIGIRKO - - + + - - + + Insulin

p-AMPKT172

p-ACCS79

AMPK

ACC

**

p=0.

2 p=0.

06

0

50

100

150

200

0 3 6 9

mg/

dl

Weeks on diet

Random fed glucoses

Ctrl CD MIGIRKO CD Ctrl HFD MIGIRKO HFD

0 5

10 15 20 25 30 35

IGF1R lox/lox

M-IGF1R-/-

Bod

y W

eigh

t (g)

CD HFD


0

100

200

300

400

500

600

0 30 60 90 120

Glu

cose

(mg/

dl)

Time (min)

IR lox/lox CD M-IR-/- CD

0

100

200

300

400

500

600

0 30 60 90 120

Glu

cose

(mg/

dl)

Time (min)

IGF1R lox/lox CD M-IGF1R-/- CD

0 100 200 300 400 500 600

0 30 60 90 120

Glu

cose

(mg/

dl)

Time (min)

IR lox/lox HFD M-IR-/- HFD

0

100

200

300

400

500

600

0 30 60 90 120

Glu

cose

(mg/

dl)

Time (min)

IGF1R lox/lox HFD M-IGF1R-/- HFD

0 5

10 15 20 25 30 35

IR lox/lox M-IR-/-

Bod

y W

eigh

t (g)

CD HFD

** **

**

A.

B. C.

Figure S5. Loss of IR or IGF1R does not worsen glucose intolerance with high fat diet (HFD). Weekly blood glucoses values were measured in MIGIRKO and control mice on chow diet (CD) or HFD (A). Body weight (B) and glucose tolerance (D) were measured in IRlox/lox and M-IR-/- mice fed either CD or HFD for 10 weeks. Body weight (C) and glucose tolerance (E) were measured in IGF1Rlox/lox and M-IGF1R-/- mice fed either CD or HFD for 10 weeks (C). (*-p<0.05, **-p<0.01 vs. control, student’s t-test)

*

** **

D. E.

0

50

100

150

200

250

300

350

400

450

0 10 20 30 40 50

Glu

cose

(mg/

dl)

Time (min)

MIGIRKO MKR-MIGIRKO

0

50

100

150

200

250

300

350

400

450

0 10 20 30 40 50

Glu

cose

(mg/

dl)

Time (min)

Control MKR


Figure S6. Metabolic Markers and IVGTT of MKR-MIGIRKO mice. Fasted serum triglyceride levels (A) and fasted/4-hour refed serum insulin (B) were measured in control, MKR, MIGIRKO, and MKR-MIGIRKO mice. Intravenous glucose tolerance test (IV GTT) with 1mg glucose/g BW was performed in control, MKR, MIGIRKO, and MKR-MIGIRKO mice (C). Glucose uptake into BAT, sWAT, and perigonadal WAT (pgWAT) was determined after IV GTT (D). (*-p<0.05, vs. control, ANOVA; ★-p<0.05 vs. control, #-p<0.05, vs. MIGIRKO ,student’s t-test)

0

10

20

30

40

50

60

Fast

Trig

lyce

rides

(mg/

dL)

Control

MKR

MIGIRKO

MKR-MIGIRKO

p=0.05

0

5

10

15

20

25

30

35

40

BAT sWAT pgWAT

IV G

TT G

luco

se U

ptak

e

(ng/

min

/mg)

Control

MKR

MIGIRKO

MKR-MIGIRKO

p=0.

09 v

s. W

T

p=0.

08 v

s. W

T

p=0.

09vs

WT

★#

★

0

1

2

3

4

5

Fast 4hr Refed

Insu

lin (n

g/m

L)

A. B.

C.

D.


- + - - + - - + - - + - - - + - - + - - + - - +

Insulin IGF-1

p-ERK

p-AktS473

Akt

ERK

p-IR/IGF1R

IR-β

IGF1R-β

Quadriceps

0 0.2 0.4 0.6 0.8

1 1.2 1.4 1.6

Bas

al

Insu

lin

IGF-

1

Bas

al

Insu

lin

IGF-

1

Bas

al

Insu

lin

IGF-

1

Bas

al

Insu

lin

IGF-

1

WT MKR MIGIRKO MKR-MIGIRKO

Akt

0.0 1.0 2.0 3.0 4.0 5.0 6.0 7.0

Bas

al

Insu

lin

IGF-

1

Bas

al

Insu

lin

IGF-

1

Bas

al

Insu

lin

IGF-

1

Bas

al

Insu

lin

IGF-

1 WT MKR MIGIRKO MKR-

MIGIRKO

p-AktS473/Akt

p=0.

05

## ## ## ##

p=0.

08

p=0.

06 ##

#

††

† ## ## ## # p=

0.07

p=0.

06

Figure S7. Akt signaling remains increased under basal conditions and unresponsive to insulin or IGF-1 treatment in MKR-MIGIRKO mice. (A) IR and IGF1R signaling in quadriceps from control, MKR, MIGIRKO and MKR-MIGIRKO mice treated with 5U insulin or 1 mg/kg IGF-1 via IVC injection. Densitometry of p-AktS473 (B), total Akt (C), and p-Akt/Akt ratio (D) (n=3 per group). (†-p<0.05, ††-p<0.01 vs. basal of same genotype, #-p<0.05, ##-p<0.01 vs. control with same treatment, ANOVA)

0

0.2

0.4

0.6

0.8

1

1.2

Bas

al

Insu

lin

IGF-

1

Bas

al

Insu

lin

IGF-

1

Bas

al

Insu

lin

IGF-

1

Bas

al

Insu

lin

IGF-

1

WT MKR MIGIRKO MKR-MIGIRKO

p-AktS473

†† ##

##

p=0.

05

p=0.

18vs

. MKR

Ba

sal

A. B.

C.

Den

sito

met

ry

Den

sito

met

ry

D.

Den

sito

met

ry

Table S1. Primers for QPCR of mouse genes. Relates to Figures 3-‐5, S1, S3, and Table S4

Common name

Gene name

5’ primer 3’ primer

DNA Primers IGF1Rlox or IGF1R+

Igf1r CTTCCCAGCTTGCTACTCTAGG CAGGCTTGCAATGAGACATGGG

IRlox or IR+ Insr CTG AAT AGC TGA GAC CAC AG GAT GTG CAC CCC ATG TCT TBP Tbp CTC TTT GCT TTC CAC AGG GCG GTG CCG TAA GGC ATC ATT GGA mRNA Primers AS160 TBC1D4 GACCTCACCTACTTTGCCTATTT GGATAACTGCCTGATGCTACTG Cidea Cidea GGCTGATAGGGCAGTGATTT GGCTACTTCGGTCATGGTTT CD11b Itgam GTTTGTTGAAGGCATTTCCC ATTCGGTGATCCCTTGGATT CHOP Ddit3 CTGCCTTTCACCTTGGAGAC CGTTTCCTGGGGATGAGATA DIO2 Dio2 CAGTGTGGTGCACGTCTCCAATC TGAACCAAAGTTGACCACCAG Elovl3 Elovl3 GGACTTAAGGCCCTTTTTGG TTCCGCGTTCTCATGTAGGT F4/80 Emr1 CTGGGATCCTACAGCTGCTC AGGAGCCTGGTACATTGGTG FNDC5 Fndc5 ATGAGGTGACCATGAAGGAGATGG CTGGTTTCTGATGCGCTCTTGGTT FGF21 Fgf21 CTGCTGGGGGTCTACCAAG CTGCGCCTACCACTGTTCC Glut1 Slc2a1 GGACCCTGCACCTCATTG GGCCACGATGCTCAGATAG Glut4 Slc2a4 CATTCCCTGGTTCATTGTGG GAAGACGTAAGGACCCATAGC IGFR Igf1r ATCGCGATTTCTGCGCCAACA TTCTTCTCTTCATCGCCGCAGACT IL-‐6 Il6 TAGTCCTTCCTACCCCAATTTCC TTGGTCCTTAGCCACTCCTTC IR Insr AAATGCAGGAACTCTCGGAAGCCT ACCTTCGAGGATTTGGCAGACCTT p58IPK Dnajc3 TCCTGGTGGACCTGCAGTACG CTGCGAGTAATTTCTTCCCC PGC1α Ppargc1a CCCTGCCATTGTTAAGACC TGCTGCTGTTCCTGTTTTC PRDM16 Prdm16 ACATCCGTGTAGCGTGTTCC GCACCAACAGTTCCTCTCCA TBP Tbp ACCCTTCACCAATGACTCCTATG TGACTGCAGCAAATCGCTTGG TBC1D1 TBC1D1 GCTACTTTGCTTGCCTCATTAAG GCTGATGATCTCAGGCACTT TNFα Tnf ACGGCATGGATCTCAAAGAC AGATAGCAAATCGGCTGACG UCP1 Ucp1 ACTGCCACACCTCCAGTCATT CTTTGCCTCACTCAGGATTGG Xbp1 Spliced Xbp1 AAGAACACGCTTGGGAATGG ACTCCCCTTGGCCTCCAC

Table S2. Dexa Measurements. (*p<0.05 vs. Control, **p<0.01 vs. Control). Relates to Figure 1


Lean Mass (g) 21.0 ± 0.4 19.2 ± 0.7* 21.6 ± 0.7 13.7 ± 0.3**

Fat Mass (g) 3.98 ± 0.28 3.82 ± 0.25 2.60 ± 0.14** 2.68 ± 0.16**

Femur Length

(cm)

1.370 ± 0.016 1.343 ± 0.019 1.350 ± 0.012 1.415 ± 0.019

Table S3. Lactate and Glycogen content in skeletal muscle from control (n=11-‐12), M-‐IR-‐/-‐ (n=6), M-‐

IGF1R-‐/-‐ (n=5), and MIGIRKO (n=6) mice. (**p<0.01 vs. Control). Relates to Figure 3


Lactate

(nmol/mg tissue)

27.8 ± 2.5 20.9 ± 2.3 22.6 ± 2.9 9.0 ± 0.5**

Glycogen

(nmol glucose/mg tissue)

17.8 ± 1.0 21.9 ± 1.7 19.0 ± 2.3 17.1 ± 1.4

Table S4. mRNA levels of Fndc5, inflammatory markers, and ER stress markers in MIGIRKO muscle. (n=4,

*-‐p<0.05 vs. control) Relates to Figure 5

MIGIRKO (fold vs. Control)

Browning Marker Fndc5 0.27 ± 0.04**

Inflammatory Markers

IL-6 1.03 ± 0.06

TNFα 1.23 ± 0.15

F4/80 2.25 ± 0.46*

CD11b 2.38 ± 0.48*

ER Stress Markers

CHOP 0.88 ± 0.15

p58IPK 0.67 ± 0.09*

Xbp1 spliced 1.46 ± 0.29

Supplemental Experimental Procedures

Animal Diets and Treatments

Animals were maintained on a standard chow diet (Lab Diet 9F, 5020) unless otherwise

specified. High fat diet studies used a diet with 60% calories from fat (Open Source Diet

#12492). Fed mice were allowed ad libitum access to food and sacrificed at 9:00 am. For fasting

studies, mice were transferred to a new cage without food for 16 hours and then sacrificed or

refed for 4 hours prior to blood collection or sacrifice.

In Vivo Glucose Uptake

Glucose uptake into tissue was measured as previously described (Stanford et al., 2013)

with slight modifications. Briefly, mice were fasted for 4 hours (9:00– 13:00) and then

anesthetized with avertin (180 mg/kg) by i.p. injection. After 30 minutes, blood was taken from

the tail to assess basal glucose concentrations and background radioactivity levels. Mice were

injected with either saline or 1 mU/g insulin in combination with 0.33 µCi [14C]2-

deoxyglucose/g mouse body weight administered via the retro-orbital sinus. Blood samples were

taken 5, 15, 30, and 45 minutes after injection for the determination of glucose and [14C] levels.

After the last blood draw, animals were sacrificed by cervical dislocation, and tissues were

harvested and immediately frozen in liquid nitrogen. Accumulation of [14C]2-deoxyglucose was

assessed in tissues using a perchloric acid and Ba(OH)2/ZnSO4 precipitation procedure.

Histology

Frozen cross sections of tibialis anterior (TA) muscle were stained for succinate dehydrogenase

(SDH) by immersing slides in staining buffer containing PBS with 0.5 M disodium succinate, 20

mM MgCl2, and 0.5 mg/ml of nitro blue tetrazolium for 15 minutes at 37oC. All slides were

stained at the same time for the same duration. Staining was terminated by immersing in PBS,

then slides were mounted. Images of TA muscles were quantified using ImageJ64 software and

manually counting fibers of entire cross-sections with <5% flaw/folding. Light to dark purple

staining on any portion of the fibers was considered “Oxidative” while white to grey fibers were

“Glycolytic”. Cross-section of entire TA muscles was performed by quantifying the length of

the scale bar in pixels, manually outlining sections (See Figure S2A for example) using

ImageJ64 software, then converting pixel area to mm2. Histology of adipose tissue was

performed as previously described (Lee et al., 2013).

Western Analysis

Powdered muscle tissue was homogenized in RIPA buffer (Millipore) with protease and

phosphatase 2 and 3 inhibitors (Sigma). Lysates were subjected to SDS-PAGE and blotted using

antibodies as detailed below.

Antibodies used for Western Blot Analysis (all used at 1:1000 dilution)

The following antibodies were used: phospho-insulin receptor/IGF receptor, IGF-1 receptor beta

XP, IRS-2, phospho-AktS473, Akt (pan), Akt1, Akt2, phospho-ERK, ERK, phospho-GSK3β,

GSK3β , phospho-FoxO3a, phospho-Akt substrate (PAS 160), TBC1D1, phospho-AMPKT172,

AMPK, β1-integrin, AS160, p-FoxO1T24/3aT32, FoxO1 (Cell Signaling), GAPDH, IRS-1, insulin

receptor beta (Santa Cruz), phospho-IRS-1Tyr608/612 (BD bioscience), Glut1, Ubc9 (Abcam),

Glut4 (Chemicon), or phospho-AS160T642 (Invitrogen) antibodies.

Quantitative RT-PCR

Total RNA was extracted from all tissues using Qiazol reagent (Qiagen) then reverse transcribed

into cDNA (Applied Biosystems) according to the manufacturer’s protocol. RT-PCR was

carried out using Sybr green (Bio-Rad) with primers as detailed in the Supplemental Table, and

normalized to TBP.

Muscle Glycogen Analysis

Gastrocnemius muscle was pulverized in a liquid nitrogen bath, then 20-30 mg of tissue was

weighed and hydrolyzed in 0.25 ml of 2 N HCl by heating at 95°C for overnight. The solution

was then neutralized with 0.25 ml of 2 N NaOH, and the resulting free glycosyl units were

assayed spectrophotometrically using a hexokinase-dependent assay kit from Eagle Diagnostics.

Tissue Fractionation for Plasma Membranes

Fractionation of skeletal muscle was performed using a combination of previously described

techniques (Frezza et al., 2007; McKeel and Jarett, 1970). Briefly, quadriceps and TA muscles

were dissected, combined and minced in ice cold PBS with 10 mM EDTA and 0.05% trypsin for

5 minutes. Tissue piece were washed 3 times with PBS plus 10 mM EDTA, then homogenized

in M1 buffer (0.25 M Sucrose, 1 mM EDTA, 10 mM Tris-HCl (pH 7.4), with protease and

phosphatase inhibitors (Sigma)) using a Potter-Elvehjem homogenizer. Homogenate was

centrifuged at 1000 x g to remove nuclei and unbroken cells, then at 16,000 x g to pellet

mitochondria and plasma membranes (P1). P1 was then resuspended in 0.5 mL of M1 buffer and

loaded on 5-25% linear ficoll gradient and centrifuged for 30 min at 24,000 rpm (SW. 41 rotor).

The upper band containing the plasma membrane was removed from the gradient, washed in 3

volumes of M1 buffer and re-centrifuged at 16,000 x g to obtain pure plasma membrane isolates

which were resuspended in 50 µl of RIPA buffer.

Plasmid and transfection procedures. The construction of GLUT4-EGFP (Lauritzen et al., 2008;

Lauritzen et al., 2002) and TBC1D1 (An et al., 2010) have been described previously. Mice were

transfected using the Helios gene gun system (Bio-Rad) as previously described (Lauritzen et al.,

2002; Lauritzen, 2010; Lauritzen and Schertzer, 2010). Briefly, mice were anaesthetized with 90

mg/kg pentobarbital, the skin was opened to expose the vastus lateralus to the bombardment of

DNA/gold particles using the gene gun. Each muscle was shot twice with 200 psi pressure with

1.4µg GLUT4-EGFP cDNA+1.4µg TBC1D1 cDNA/0.5 mg gold (0.6 µm gold particle size).

Intravital microscopy. Five days after transfection, random-fed mice were anesthetized and

mounted on the microscope stage with muscle exposed as previously described (Lauritzen et al.,

2002; Lauritzen et al., 2006; Lauritzen, 2010). Intravital images of superficial transfected

quadriceps muscle fibers were collected with a 63x, 1.2 NA Zeiss C-Apochromat objective on a

Zeiss-LSM-710 confocal microscope with 488 nm laser line for excitation of EGFP, 3-8 fibers

were imaged in each mouse. TIF images obtained with the Zeiss confocal software were

imported into MetaMorph Software (V. 6.1, Universal Imaging Corp) and image stacks were

created. Quantification of Glut4-EGFP vesicular depots were performed as previously described

(Lauritzen et al., 2010). Briefly, threshold and classifying settings in the MetaMorph software

were used to discriminate GLUT4-EGFP vesicle depots above 1 µm in size from the background

and measurement of average GLUT4-EGFP area were automatically generated.

Image analysis of GLUT4-EGFP and HA tagged TBC1D1 localization in fixed fibers.

Gene gun GLUT4-EGFP transfected quadriceps muscles were fixed, transfected fibers isolated

and immuno-stained as previously described (Lauritzen et al., 2008). Primary anti-HA tag

antibody (Abcam ab13834) against TBC1D1 was used in combination with a secondary goat-anti

rabbit Alexa Flour 594 (Life technologies). Confocal images were collected on the Zeiss

LSM710 using a 100x oil immersion objective (NA 1.4).

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