SYSTEMIC EFFECTS OF MINOR EXPRESSION IN ADIPOCYTES

by

ROSICKA GRACE WALTON

W. TIMOTHY GARVEY, COMMITTEE CHAIR

YUCHANG FU TIMOTHY R. NAGY PHILIP A. WOOD

A THESIS

Submitted to the graduate faculty of the University of Alabama at Birmingham, In partial fulfillment of the requirements for the degree of

Master of Science

BIRMINGHAM, ALABAMA

2008

SYSTEMIC EFFECTS OF MINOR EXPRESSION IN ADIPOCYTES

ROSICKA GRACE WALTON

NUTRITION SCIENCES

ABSTRACT

The NR4A family of orphan nuclear receptors function as early response

genes with numerous stimuli. The NR4A family consists of three known

members, Nur77, Nurr1, and Mitogen-inducible nuclear orphan receptor

(MINOR). Our lab has previously demonstrated that stable over-expression of

MINOR in 3T3-L1 adipocytes enhances insulin-stimulated glucose uptake. In

order to assess the in vivo affect of MINOR on adipocytes, we generated

transgenic mice with MINOR over-expression driven by the adipocyte- and

macrophage-specific AP2 promoter (AP2-MINOR mice). We initially

hypothesized that AP2-MINOR mice would display increased glucose tolerance

and insulin sensitivity. However, AP2-MINOR transgenic male and female mice

have increased fasting glucose and respond poorly to glucose challenge, with

males developing insulin resistance late in life. AP2-MINOR males are larger

than wild-type due to increases in both lean and fat mass. When males are

maintained on high fat chow, transgenic animals are larger, but do not differ from

wild-type when challenged with glucose or insulin. These results led us to

hypothesize that MINOR over-expression induces adipocyte hypersensitivity to β-

adrenergic agonism, leading to increased serum non-esterified fatty acids and

impairment of the pancreatic β-cell response to glucose. Indeed, insulin fails to

inhibit transgenic NEFA production below the level of wild-type in spite of

ii

significantly reduced serum catecholamines. Oddly, fasting serum total

cholesterol and LDL were increased and NEFA was decreased in AP2-MINOR

animals versus wild-type. Thus, it appears that MINOR over-expression effects

both glucose and lipid homeostasis. As a whole, these observations are

consistent with a MINOR-mediated down-regulation of Liver X receptor (LXR)

and its target, sterol regulatory element binding protein-1c (SREBP-1c).

iii

ACKNOWLEDGEMENTS

Firstly, I would like to express my gratitude to my committee members, W.

Timothy Garvey, M.D., Yuchang Fu, Ph.D., Timothy R. Nagy, Ph.D., and Philip A.

Wood, D.V.M. Dr. Garvey has been incredibly generous with his time and he has

provided steadfast support and mentorship. Dr. Fu oversaw preliminary work on

MINOR, enabling this project to exist. Doctors Garvey and Fu formulated our

original hypotheses, and their scientific rigor and vision are appreciated. I would

also like to offer special thanks Dr. Nagy, who has mentored me for 8 years, and

encouraged me to pursue graduate studies. As a member of my committee, Dr.

Nagy’s advice and expertise regarding body composition and statistics have

been invaluable. Finally, Dr. Wood has also mentored me for many years and

served as an irreplaceable committee member, providing unparalleled expertise

in murine lipidology.

I would also like to thank the members of the Garvey lab. In particular,

Liehong Luo, Nanlan Luo, and Xiaolin Zhu performed many of the background

experiments on MINOR. However, each member of the lab has contributed to the

project by helping with animal sacrifices, molecular biology, and numerous other

essential tasks. These lab members include Pedro del Corral, Helliner Hill,

Cristina Lara-Castro, Jiarong Liu, Kerry Lok, Doug Moellering, Sasha Smith, Ling

Tian, and Xuxia Wu. I would like to specifically thank Dr. B. Hong Chung for his

technical and physiological expertise in the area of apolipoproteins.

iv

I am also thankful to the following scientists who work in other labs: from

the Nagy lab, Maria Johnson, Meghana Gavand, and Amanda Watts for

performing DEXA scans; from the lab of Dr. Barbara Gower, Maryellen Williams

who performed insulin radio-immuno assays; Ray Johnson from the CMN/KC

Neurochemistry Core Lab, Vanderbilt University, performed catecholamine

assays; Kate Kosmac from William Britt’s lab helped with molecular biology and

thesis development; the members of Dr. Robert Kesterson’s lab and the

members of Dr. Wood’s lab were generous with advice and equipment. Many

others have had some hand in this project and I regret that I cannot name them

all.

Lastly, my family has been unfailing in their support of my academic

endeavors and I am forever grateful.

After all, it takes a village to raise a mouse.

v

TABLE OF CONTENTS

Page ABSTRACT ...........................................................................................................ii ACKNOWLEDGEMENTS.....................................................................................iv LIST OF TABLES ................................................................................................. v LIST OF FIGURES ...............................................................................................vi INTRODUCTION.................................................................................................. 1 METHODS............................................................................................................ 8 rtPCR............................................................................................................. 8 Western blotting............................................................................................. 8 Animals.......................................................................................................... 8 Serum Assays ............................................................................................. 10 RESULTS........................................................................................................... 12 Verification of transgene insertion and function ........................................... 12 Female AP2-MINOR transgenic mice.......................................................... 13 Male AP2-MINOR transgenic mice—standard chow ................................... 14 Male AP2-MINOR transgenic mice—high fat chow ..................................... 26 DISCUSSION ..................................................................................................... 29 REFERENCES................................................................................................... 34 APPENDIX: IACUC APPROVAL ........................................................................ 39

vi

LIST OF TABLES Table Page 1 Body composition in 8-week old female AP2-MINOR and wild-type mice maintained on standard chow............................................................................. 14 2 Body composition in 8-week old AP2-MINOR and wild-type mice................... 17 3 Body composition in 16-week old AP2-MINOR and wild-type mice................. 18 4 Body composition in 30-week old AP2-MINOR and wild-type mice................. 18 5 Fasting serum lipids in transgenic and wild-type mice maintained on standard chow ................................................................................................................... 23 6 Body composition in 16-week old, high-fat fed AP2-MINOR and wild-type Mice.................................................................................................................... 27 7 Summary of hypothesized protein expression or function changes in AP2-MINOR mice ....................................................................................................... 31

vii

LIST OF FIGURES Figure Page 1 Verification of transgene insertion and function............................................... 12 2 Fasting glucose is increased in female AP2-MINOR mice versus wild-type littermates........................................................................................................... 13 3 Body weight and food intake in AP2-MINOR and wild-type mice .................... 15 4 Glucose tolerance in 17-week old mice ........................................................... 19 5 At age 18 weeks, no differences were observed in insulin tolerance............... 20 6 Glucose and insulin parameters in 31 week-old mice...................................... 21 7 Insulin tolerance in 32-week old mice.............................................................. 22 8 Serum lipids in AP2-MINOR and wild-type mice.............................................. 24 9 Insulin’s ability to suppress lipolysis in AP2-MINOR and wild-type mice ......... 25 10 Body weight and food intake in mice fed high fat chow ................................. 26 11 Glucose and insulin tolerance tests in animals maintained on high fat chow 28 12 Fasting serum triglycerides............................................................................ 29

viii

1

INTRODUCTION

Worldwide, the number of people affected by diabetes has greatly

increased in the past 3 decades. The international Diabetes federation estimates

that diabetes care consumes 5-10% of total healthcare costs in many nations.

These costs are largely attributed to treatment of diabetes complications, which

may often be prevented through proper glycemic control. Many current anti-

hyperglycemic and insulin-sensitizing drugs have untoward side effects including

weight gain, resistance to weight loss, difficult dosing regimens, and

development of drug tolerance (Smyth & Heron, 2005). Insulin resistance is

strongly correlated to increased adipose tissue, particularly visceral adipose

tissue. Circulating and local levels of the relevant products of adipose tissue—

NEFA and adipocytokines—are often abnormal in patients with insulin resistance

and frank Type 2 diabetes (Farmer & Prusty, 2004). Thus, understanding the

regulation of adipocyte glucose and lipid metabolism provides one avenue for the

development of improved anti-diabetic drugs.

Mitogen-inducible nuclear orphan receptor (MINOR, also NOR-1, TEC,

CHN, NR4A3) is one of three known members of the NR4A subgroup of the

nuclear hormone receptor superfamily. Other NR4A members include Nur77

(TR3, NGFI-B, N10, DHR38, NAK-1, TIS1, NR4A1) and Nurr1 (HZF-3, RNR-3,

TINUR, NOT, NR4A2). NR4A family members are expressed in numerous

metabolically active tissues including brain, heart, skeletal muscle, kidney, liver,

certain leukocytes, and adipose. They are early response genes whose

expression is induced in a cell-type specific manner by numerous stimuli,

2

including: inflammation (LPS, oxidized lipids, cytokines), growth factors, HPA-

axis hormones, tyrosine-derived neurotransmitters, forskolin and cAMP analogs,

oxidized LDL, and many others. NR4A members modulate expression of

steroidogenic genes in both the HPA-axis and target tissues, with known mutual

transcriptional repression between NR4A members and the glucocorticoid

receptor (extensively reviewed by Maxwell & Muscat, 2006). They are also potent

stimulators of hepatic gluconeogenic enzyme transcription (Pei et al. 2006).

Finally, NR4A members are thought to have a high degree of transcriptional and

functional redundancy.

NR4A members appear to have atypical ligand binding domains. Two

activation functions are described for most nuclear receptors: the N-terminal

domain (AF1) and the ligand binding domain (LBD, AF2). The AF1 is highly

conserved across NR4A members and is likely to mediate the majority of their

protein-protein interactions. Indeed, MINOR AF1 can independently recruit

coactivators. NR4A members differ in their LBDs even though all members lack a

hydrophobic cleft typically found in the LBD of steroid hormone receptors.

However, a hydrophobic patch (distinct from the missing hydrophobic cleft) on

the LBD is believed to interact with cofactors in order to alter transcription of

target genes (Flaig et al. 2005). Nur77 and Nurr1 form functional heterodimers

with each other (Maira et al. 1999) as well as the retinoid X receptor (Perlmann &

Jansson, 1995), unlike MINOR, which does not appear to heterodimerize with

other nuclear receptors (Zetterstrom et al. 1996). NR4A members bind NGFI-B

3

response elements (NBRE) and Nur-responsive elements (an everted repeat of

the NBRE) as monomers or homodimers (Philips et al. 1997).

MINOR was first described in the mid-1990’s (Ohkura et al. 1994; Hedvat

& Irving, 1995). Two human MINOR transcripts have been described, with the

longer encoding a 626 amino acid residue (Ohkura et al. 1996). Both rats and

humans produce two isoforms of MINOR protein, the smaller of which has little

transcriptional activity (Ohkura et al. 1998). MINOR transcription can be induced

by the calcium/calmodulin-dependent protein kinase cascade (Inuzuka et al.

2002) and high levels of MINOR protein may repress MINOR transcription

(Hedvat & Irving, 1995). In adrenal fasciculata cells, MINOR transcription is

induced by ACTH and angiotensin II and is dependant on PKA and PKC

cascades (Fernadez et al. 2000). Platelet-derived growth factor (Nomiyama et

al. 2006) and oxidized LDL (Crespo et al. 2005) have also been shown to

increase MINOR transcription. Reported transactivators of MINOR include

prostaglandin A2 (Kagaya et al. 2005) and the homeotic protein Six3 (Laflamme

et al. 2003). Finally, inhibition of TNF-α in psoriatic skin decreases Nurr1

transcript and protein levels (O’kane et al. 2007). Since NR4A family members

are functionally related, it is reasonable to hypothesize that TNF-α increases

MINOR transcription.

No metabolic abnormalities have been reported in MINOR knockout mice.

Rather, one line of MINOR -/- exhibits abnormal hippocampal development,

increased predisposition to excitotoxic glutamate receptor kainic acid-induced

seizure (Ponnio & Conneely, 2004), inner ear defects, and aberrant circling

4

behavior (Ponnio et al. 2002). A second MINOR knockout was embryonic lethal

in homozygous mice due to incomplete gastrulation while heterozygous mice

appeared to be normal (DeYoung et al. 2003). Transgenic global over-expression

of MINOR produces dramatically reduced body weight (~50%), atrophy of the

spleen and thymus, and a live birth rate of less than 50% (Kagaya et al. 2005).

Adipocyte lipolysis is a major source of fuel during fasting. Lipolysis is

regulated by catecholamines, insulin, and natriuretic hormones in a species-

specific manner. Hormone sensitive lipase (HSL) is believed to mediate acute

changes in lipolytic activity, while adipocyte triglyceride lipase is believed to

mediate basal levels of lipolysis. Catecholamines generally stimulate lipolysis,

while insulin potently inhibits lipolysis. Human adipocytes express four

adrenoreceptors α2A, β1, β2, and β3, of which the first inhibits and the latter three

stimulate lipolysis. β1 and β2 are more active than β3 in humans. However, in

rodents, β3 is the primary mediator of adrenergic-induced lipolysis. β receptors

are Gs-coupled, leading to activation of PKA and finally to phosphorylation of

HSL, which hydrolyzes triglycerides into free fatty acids (NEFA) and glycerol

(Arner, 2005).

Elevated serum NEFA is a common correlate to obesity, insulin

resistance, and Type 2 diabetes. Increased basal lipolysis likely accounts for a

large portion of the elevated NEFA observed in obesity. Increased adipocyte size

and chronically high TNFα levels in the adipose tissue have been implicated in

increasing NEFA production (Arner, 2005). Larger adipocytes display increased:

FAS and LPL activity, GLUT4 and leptin expression, and overall lipolytic capacity

5

(Farnier et al. 2003). Furthermore, there is a strong positive correlation between

adipocyte size and circulating TNFα (Winkler et al. 2003). The precise

mechanisms of chronic-TNFα-induced lipolysis are currently unknown. However,

there is evidence that NEFA released by adipocytes elicits TNFα production by

local macrophages, creating a chronic inflammatory cycle (Suganami et al.

2005). Obesity-related perturbations in NEFA metabolism are both species- and

fat pad specific. However, it is clear that C57BL/6J mice maintained on high fat

diet exhibit elevated NEFA and plasma glucose (Ahren & Scheurink, 1998).

Pancreatic β-cells undergo progressive deterioration in type 2 diabetes.

Although certain free fatty acids are known to acutely increase β-cell response to

glucose, many experts believe that chronically elevated NEFA are harmful to β-

cells. Evidence suggests that NEFA can harm β-cells at physiological glucose

concentrations (Dubois et al. 2004). However, other experiments indicate that

hyperglycemia is necessary for any deleterious effects of NEFA (Roberston et al.

2004; Goh et al. 2007). In any event, it is clear that a chronically hyperlipidemic

and hyperglycemic environment reduces glucose-stimulated insulin secretion.

Proposed mechanisms of β-cell lipotoxicity include inhibition of insulin gene

transcription (Poitout et al. 2006) and increased ceramide synthesis and NO

production (Newsholme et al. 2007).

Particular interest should be paid to data from George Muscat’s laboratory

imputing MINOR as a target of β-adrenergic signaling. MINOR expression is

increased in C2C12 skeletal muscle cells and mouse skeletal muscle tissue after

incubation with isoprenaline or specific β2 or β3 agonists. Suppression of MINOR

6

in C2C12 cells by siRNA causes reductions in UCP2 and CAV3 transcription and

induction of UCP3 and myostatin transcription. In vivo observations confirm

induction of UCP2 in mouse skeletal muscle after treatment with β-adrenergic

agonist (Pearen et al. 2006). Muscat’s group made similar observations about

Nur77. Notably, suppression of Nur77 caused decreased lipid oxidation in C2C12

cells (Maxwell et al. 2005). Another group examined whether NR4A members

participate in brown adipose tissue thermogenesis. Nur77 was transiently

induced in brown adipose tissue following β-adrenergic stimulation or cold-

induced thermogenesis. They also reported a “superinduction” of MINOR in the

brown adipose tissue of cold-exposed Nur77 knockout mice (Kanzleiter et al.

2005).

MINOR is naturally expressed in adipocytes and our lab has demonstrated

that MINOR increases insulin-stimulated glucose uptake when overexpressed in

vitro (Fu et al. 2007). However, the physiological effects of MINOR

overexpression in adipocytes are unknown. Thus transgenic AP2-MINOR mice

that overexpress MINOR in adipocytes were produced. Surprisingly, preliminary

data indicated that these animals have an impaired rather than improved

response to glucose. This observation could result from elevated circulating free

fatty acids which are known to impair pancreatic β-cell response to glucose.

Since: a) elevations in free fatty acids are the result of adipocyte lipolysis, which

can be stimulated by β-adrenergic agonism and b) MINOR is a known target of β-

adrenergic signaling, I hypothesize that MINOR enhances lipolysis in adipocytes.

7

This study sought to determine the effect of adipose tissue-specific over-

expression of MINOR on body composition parameters and physiological

responses to glucose and insulin. Experiments began with assessment of

MINOR transgene functionality and protein expression. I proceeded to determine:

body weight, body composition, and food intake in MINOR transgenic mice on

both low fat and high fat diets; differences in glucose tolerance and insulin

tolerance in transgenic versus wild-type mice on both diets; differences in insulin-

mediated suppression of adipocyte lipolysis in transgenic versus wild-type mice;

and serum lipid variations in transgenic versus wild-type mice.

8

METHODS

rtPCR RNA was extracted from WAT using TRIzol reagent (Invitrogen)

according to manufacturer’s protocol. Samples were treated with DNAse I (amp

grade, Invitrogen), and then used as template for the production of cDNA

(SuperScript III, Invitrogen). PCR was then performed on cDNA and amplification

product resolved on 1.5% agarose gel. Primers used were the same as those

used for genotyping mice. Primer sequences were: 5’-GGA TCC AAA CTC ATT

ACT AAC CGG TA-3’ and 5’-ATA TCC AAG CCT TAG CCT GCC TGT-3’.

Western blotting WAT protein was extracted with Sigma CelLytic

Mammalian Tissue Lysis Reagent and separated by SDS-polyacrylamide gel

electrophoresis. Protein was transferred onto nitrocellulose membranes and

incubated overnight at 4°C with 5% non-fat milk in TBS. Membranes were then

incubated with MINOR antibody (Perseus Proteomics, Tokyo, Japan) for 1 hour

followed by incubation with horseradish peroxidase secondary antibody for 1

hour. Membranes were washed with TBS (with 0.1% Tween 20) and protein was

detected by chemiluminescence (Enhance, NEN Life Science) and quantified

with appropriate software.

Animals In order to produce transgenic animals and wild-type littermate

controls, breeding pairs consisted of one heterozygous AP2-MINOR transgenic

animal and one pure C57/BL (Taconic Farms). All animals were maintained

under standard conditions (22±2°C, 12-hour light cycle) and given ad libitum

9

access to water and either standard rodent chow (Harlan Teklad 7913 Irradiated

Modified 6% Mouse/Rat Sterilizible Diet) or high fat chow (Research Diets D-

12492, 60% kcal from fat, 20% kcal from protein, 20% kcal from carbohydrate).

Body weight and food intake were measured weekly beginning at age 8 weeks

and ending at age 30 weeks.

Glucose tolerance test (GTT): 17 week old and 31 week old animals were

fasted overnight with free access to water. At 9:00 a.m., animals were weighed

and given an intraperitoneal injection of D-(+)-glucose (100g glucose/L; 10μl/g

body weight). Blood glucose was measured at baseline (approximately 1 minute

prior to injection), and at 30, 60, 90, and 180 minutes post-injection (HemoCue

glucose 201 glucometer, HemoCue USA).

Insulin tolerance test (ITT): 18 and 32 week old animals were fasted for 4

hours and then weighed. Animals were then given an intraperitoneal injection of

1.5U (male mice) or 1.0U (female mice) Humalog lispro insulin/kg body weight.

Blood glucose was measured at baseline (approximately 1 minute prior to

injection), and at 30, 60, 90, and 180 minutes post-injection (HemoCue glucose

201 glucometer, HemoCue USA). Animals were not given access to food or

water during GTT and ITT.

In order to assess insulin’s ability to suppress adipocyte lipolysis, male

animals (age 33-35 weeks, maintained on standard chow) were fasted for 4

hours, weighed, and injected with 1.5U Humalog lispro insulin/kg body weight.

Mice were sacrificed exactly 60 minutes after insulin injection, and whole trunk

10

blood was collected for analysis. Analytes included insulin, NEFA, epinephrine,

and norepinephrine (methods below).

Animals were euthanized by decapitation without anesthesia. Male

animals were sacrificed at 33-35 weeks of age. Tissues and whole trunk blood

were harvested and perigonadal fat pads were weighed. All tissues except trunk

blood were quickly placed into liquid nitrogen and stored at -80°C until needed.

Whole trunk blood was centrifuged for 30 minutes at 5000xG, 4°C and serum

was collected.

DEXA scans were performed in the lab of Dr. Timothy R. Nagy (University

of Alabama at Birmingham), according to previously published protocols (Nagy &

Clair, 2000). Animals were scanned at age 8 weeks (females); age 8, 16, and 30

weeks (males fed standard chow); or age 16 weeks (males fed high fat chow).

Mice were anesthetized with 2% isoflourane and body composition was analyzed

using dual-energy X-ray absorptiometry (GE Lunar Piximus (Madison, WI),

software version 1.45).

Serum assays Serum insulin was measured by double-antibody

radioimmunoassay (Sensitive rat insulin RIA, Linco, St. Charles, MO) in the lab of

Dr. Barbara Gower (University of Alabama at Birmingham).

Serum NEFA, total cholesterol, and TAG were assessed by in vitro

enzymatic colorimetry (HR Series NEFA-HR(2), Cholesterol-E, and L-Type TG H

kits from Wako Diagnostics).

11

Serum catecholamines were measured in the CMN/KC Neurochemistry

Core Lab, Vanderbilt University, Nashville, TN. First, serum catecholamines are

adsorbed onto solid Al2O3, and then desorbed from the Al203 using 200 μl 0.1 N

acetic acid. Biogenic amines are determined by a specific HPLC assay utilizing

an Antec Decade II (oxidation: 0.5) electrochemical detector. Twenty μl samples

were injected using a Water 717+ autosampler onto a Phenomenex Nucleosil

(5u, 100A) C18 HPLC column (150 x 4.60 mm).

Apolipoprotein assays were performed on four pooled serum samples

from 16 transgenic mice and four pooled samples from 16 wild-type mice. Pooled

samples were analyzed using the lipoprotein autoprofiler method (Chung et al.

1981). First, a 2.5 hour density gradient ultracentrifugation was used to separate

the major lipoprotein fractions in the serum. Next, effluent was continuously

removed from the bottom of the density gradient and analyzed using enzymatic

colorimetric cholesterol and phospholipid kits from Wako Diagnostics.

Measurement of effluent absorbance at the correct wavelength indicates HDL,

LDL, and VLDL concentrations.

12

RESULTS

Verification of transgene insertion and function Transgenic mice were

genotyped by PCR, with the 3’ primer annealing to a V5 coding region in order to

preclude amplification of wild-type DNA. Transcriptional activity of the MINOR

transgene was verified by reverse transcriptase PCR (fig. 1a). Increased MINOR

protein levels were observed in white adipose tissue (fig. 1b and 1c) (p<0.05,

paired t-test). Preliminary quantitative PCR results indicate numerous transgene

copies (data not shown).

a

b

c

0

2

4

6

8

10

AP2-MINOR Wild-type

MIN

OR

sig

nal (

Odu

*mm

2)

WT TG WT TG WT TG WT TG TG WT

WT TG WT TG WT TG WT TG TG WT

Figure 1. Verification of transgene insertion and function. a) reverse transcriptase PCR showing amplification of transgene mRNA, b) western blot using MINOR antibody against protein extracted from gonadal fat, and c) quantification of western blot shown in panel b (p<0.05, paired t-test). Error bars represent SEM. WT=wild-type, TG=AP2-MINOR transgenic

*

13

Female AP2-MINOR transgenic mice Transgenic female body weight did not

differ from wild-type (age 8-16 weeks, p=0.8, repeated measures ANOVA). Nor

were there any differences in body composition at age 8 weeks (table 1). Body

composition was not assessed at age 16 weeks because no differences in body

weight were observed. Fasting glucose at age 17 weeks was significantly higher

in transgenic mice (fig. 2) (134.1 versus 109.5 mg/dL, p<0.05, Wilcoxon rank

sum). When GTT was assessed by AUC analysis, AP2-MINOR females tended

to have lower scores than wild-type (p=0.052, WIlcoxon rank sum). However,

repeated measures ANOVA indicated no between-group difference in glucose

tolerance. Likewise, no differences in insulin tolerance were observed.

Blo

od g

luco

se (m

g/dL

)

100

110

120

130

140

150

160

170

AP2-MINOR Wild-type

Genotype

Figure 2. Fasting glucose is increased in female AP2-MINOR mice versus wild-type littermates (P<0.05, Wilcoxon). AP2-MINOR N=8, Wild-type N=8

14

Table 1. Body composition in 8-week old female AP2-MINOR and wild-type mice maintained on standard chow

N=11/group Body weight (g) (95%CI)

Lean (g) (95%CI) Fat (g) (95%CI) % Fat

Mean, median

AP2-MINOR 18.9 (18.2-19.5)

15.3 (14.7-15.8)

1.77 (1.65-1.89) 10.4, 10.2

Wild-type 19.0 (18.4-19.7)

15.5 (14.9-16.0)

1.81 (1.69-1.93) 10.5, 10.9

P 0.68 0.60 0.67 0.83 (Wilcoxon)

Male AP2-MINOR transgenic mice—standard chow AP2-MINOR males

maintained on standard chow were significantly larger than their wild-type

littermates (fig. 3a) from age 8 to 16 weeks (p<0.05), with this pattern persisting

from age 20 to 30 weeks (fig. 3b) (p<0.01). Food intake tended to be slightly

higher in transgenic males from 9 to 16 weeks of age (fig. 3c) (p=0.1) and from

20 to 29 weeks of age (fig. 3d) (p=0.09). However, average weekly food intake

per gram body weight tended to be lower in transgenic animals from 9 to 16

weeks of age (p=0.07, Wilcoxon rank sum) (fig. 3e) and from 20 to 29 weeks of

age (p<0.01, Wilcoxon rank sum) (fig. 3f).

15

a

20

25

30

35

40

45

50

8 9 10 11 12 13 14 15 16

Age (weeks)

Bod

y w

eigh

t (g)

AP2-MINOR

Wild-type

b

20

25

30

35

40

45

50

20 21 22 23 24 25 26 27 28 29 30

Age (weeks)

Bod

y w

eigh

t (g)

Figure 3, panels a and b. Body weight and food intake in AP2-MINOR and wild-type mice. a) transgenic mice are larger than wild type mice from ages 8 to 16 weeks (p<0.05), b) transgenic mice are larger than wild-type from 20 to 30 weeks of age (<0.01). All body weight and food intake analyses are repeated measures ANOVA. Error bars represent SD. AP2-MINOR N=21, Wild-type N=19

16

c

20.0

25.0

30.0

35.0

40.0

9 10 11 12 13 14 15 16

Age (weeks)

Food

inta

ke (g

)

AP2-MINOR

Wild-type

d

20.0

25.0

30.0

35.0

40.0

20 21 22 23 24 25 26 27 28 29

Age (weeks)

Food

inta

ke (g

)

e

Ave

rage

wee

kly

food

inta

ke (g

)/bod

y w

eigh

t (g)

0.9

1

1.1

1.2

1.3

1.4

AP2-MINOR Wild-type

Genotype f

Ave

rage

wee

kly

food

inta

ke (g

)/bod

y w

eigh

t (g)

0.75

0.8

0.85

0.9

0.95

1

AP2-MINOR Wild-type

Genotype

Figure 3, panels c, d, e and f. Transgenic mice tend to consume slightly more food from 9 to 16 weeks of age (p=0.1) (c), and (d) from 20 to 29 weeks of age (p=0.09). All body weight and food intake analyses are repeated measures ANOVA. Error bars represent SD. Average weekly food intake (grams)/body weight (grams) is decreased in AP2-MINOR mice from ages 9 to 16 weeks (p=0.07, Wilcoxon) (e), and from ages 20 to 29 weeks (f) (p<0.01, Wilcoxon). AP2-MINOR N=21, Wild-type N=19

17

Body composition data indicate that male AP2-MINOR mice fed standard

chow are larger than wild-type at 8 weeks of age, with trends toward greater total

mass (p=0.16) and lean mass (p=0.12). However, no differences in fat mass or

percent fat were observed at 8 weeks of age (table 2). At 16 weeks of age,

transgenic mice displayed more pronounced trends toward greater total mass

(p=0.08) and lean mass (p=0.054). At 16 weeks, transgenic mice also displayed

a trend toward greater fat mass (p=0.18), with no difference in percent fat (table

3). At 30 weeks of age, AP2-MINOR mice again displayed a trend toward greater

total mass, with lean mass becoming significantly greater than wild-type (p<0.05).

Transgenic animals continued to tend toward greater fat mass (p=0.1), with no

differences observed in percent fat (table 4). When animals were sacrificed

(between 33 and 35 weeks of age), AP2-MINOR mice tended to have larger

gonadal fat pads, but this difference was not statistically significant (AP2-MINOR

mean fat pad weight=0.92 g, wild-type mean fat pad weight=0.82 g, p=0.14).

Table 2. Body composition in 8-week old AP2-MINOR and wild-type mice Body weight (g)

(95% CI) Lean mass (g)

(95% CI) Fat mass (g)

(95% CI) % Fat (g)

Mean, median AP2-MINOR

(n=20) 24.9

(24.0-25.8) 19.6

(19.0-20.1) 3.1

(2.7-3.4) 13.4, 13.0

Wild-type (n=19)

24.0 (23.1-24.9)

18.9 (18.3-19.5)

2.9 (2.5-3.3)

13.1, 12.8

P 0.16 0.12 0.51 0.52

(Wilcoxon rank sum)

18

Table 3. Body composition in 16-week old AP2-MINOR and wild-type mice Body weight (g)

(95% CI) Lean mass (g)

(95% CI) Fat mass (g)

(95% CI) % Fat (g)

mean, median AP2-MINOR

(n=19) 34.2

(32.5-35.9) 23.1

(22.4-23.9) 8.2

(7.1-9.3) 25.7, 25.7

Wild-type (n=19)

32.0 (30.3-33.8)

22.1 (21.4-22.8)

7.2 (6.1-8.2) 23.9, 25.1

P 0.08 0.054 0.18 0.34

(Wilcoxon rank sum)

Table 4. Body composition in 30-week old AP2-MINOR and wild-type mice Body weight (g)

(95% CI) Lean mass (g)

(95% CI) Fat mass (g)

(95% CI) % Fat

Mean, median Transgenic

(n=16) 42.8

(40.4-45.2) 25.6

(23.7-25.4) 14.3

(12.7-15.9) 36.5, 37.6

Wild-type (n=17)

39.6 (37.3-41.9)

23.2 (22.5-24.0)

12.5 (11.0-14.0) 34.4, 36.3

P 0.06 0.02 0.10 0.26

(Wilcoxon rank sum)

AP2-MINOR transgenic mice maintained on standard chow had elevated

fasting glucose compared to wild-type littermates at age 17 weeks (p=0.01) (fig.

4a). Additionally, glucose tolerance was significantly impaired in transgenic

animals at age 17 weeks. Two outliers were removed from the following analyses

due to glucose tolerance test AUC blood glucose that was greater than two

standard deviations from the mean. Both outlier animals were transgenic, with

AUC glucoses equaling 200 and 1530. Statistical significance was reduced

following the removal of these outliers. After removing outliers, AUC blood

glucose was still significantly greater in transgenic animals compared to wild-type

(p<0.05) (fig. 4b). Accordingly, repeated measures ANOVA analysis indicated

significantly different glucose responses between groups (p=0.01) (fig. 4c).

19

Insulin tolerance did not differ between groups at age 18 weeks when assessed

by either AUC or repeated measures ANOVA (fig. 5).

a

100110120130140150160170180190200

AP2-MINOR Wild-typeFast

ing

bloo

d gl

ucos

e (m

g/dL

)

b

400

600

800

1000

1200

1400

AP2-MINOR Wild-type

Glu

cose

tole

ranc

e te

stA

UC

*

*

c

100

150

200

250

300

350

400

450

0 30 60 90 180

Time (minutes)

Bloo

d gl

ucos

e (m

g/dL

)

AP2-MINOR

Wild-type

Figure 4. Glucose tolerance in 17-week old mice. a) transgenic mice display elevated fasting glucose at age 17 weeks (p=0.01), b) glucose tolerance test AUC is significantly higher in transgenic mice (p<0.05), c) AP2-MINOR mice display reduced glucose tolerance (p=0.01, repeated measures ANOVA). Error bars represent SD. AP2-MINOR N=15, Wild-type N=16

20

75100125150175200225250275

0 30 60 90 180

Time (minutes)

Blo

od g

luco

se (m

g/dL

)

AP2-MINOR

Wild-type

Figure 5. At age 18 weeks, no differences were observed in insulin tolerance (repeated measures ANOVA). Error bars represent SD. AP2-MINOR N=15, Wild-type N=16

At 31 weeks of age, transgenic animals continued to display increased

fasting glucose compared to wild-type littermates (p<0.05) (fig. 6a). At 33-35

weeks of age, fasting insulin was also slightly elevated in AP2-MINOR animals

compared to wild-type (mean values 0.93 versus 0.72 ng/ml, p=0.09) (fig. 6b).

Furthermore, transgenic animals were glucose intolerant compared to wild-type

(p<0.01), with a significant interaction between time and genotype (p<0.01) (fig.

6c). However, glucose tolerance test AUC was not significantly different at age

31 weeks (mean AUC value for transgenic animals=694.7, mean AUC value for

wild-type animals=604.2). Transgenic mice appeared to be slightly insulin

resistant at age 32 weeks; insulin tolerance test AUC absolute values were

significantly smaller in AP2-MINOR mice (mean AUC value for transgenic

animals=-237.5, mean AUC value for wild-type animals=-388.33, p<0.05) (fig.

7a). However, repeated measures ANOVA did not indicate a significant

difference in insulin tolerance (p=0.2) (fig. 7b).

21

a

100110120130140150160170180190200

AP2-MINOR Wild-type

Fast

ing

bloo

d gl

ucos

e (m

g/dL

)

b

0

0.2

0.4

0.6

0.8

1

1.2

1.4

AP2-MINOR Wild-type

Fast

ing

insu

lin (n

g/m

l)

*

c

100

150

200

250

300

350

400

450

0 30 60 90 180

Time (minutes)

Bloo

d gl

ucos

e (m

g/dL

)

AP2-MINOR

Wild-type

Figure 6. Glucose and insulin parameters in 31 week-old mice. a) fasting glucose in 31-week old mice. AP2-MINOR mice have elevated fasting glucose (p<0.05), b) mean fasting insulin is greater in AP2-MINOR males (p=0.09), and c) 32-week old transgenic mice have impaired glucose tolerance (p<0.01, repeated measures ANOVA). Error bars represent SD. AP2-MINOR N=17, Wild-type N=17

22

a-500

-400

-300

-200

-100

Blo

od g

luco

se A

UC

(mg/

dL)

AP2-MINOR Wild-type

*

b

75100125150175200225250275300

0 30 60 90 180

Time (minutes)

Blo

od g

luco

se (m

g/dL

)

AP2-MINOR

Wild-type

Figure 7. Insulin tolerance in 32-week old mice. a) AP2-MINOR mice display decreased absolute value AUC blood glucose following insulin challenge (p<0.05), b) blood glucose curves do not differ between groups following insulin delivery (p=0.2, repeated measures ANOVA). Error bars represent SD. AP2-MINOR N=17, Wild-type N=17

Fasting serum lipid values are summarized in table 5. We were surprised

to observe significantly decreased non-esterified fatty acids (NEFA) in transgenic

mice versus wild-type (p<0.05) (figure 8a). AP2-MINOR animals also tended to

have lower mean triglycerides (p=0.16). Paradoxically, total cholesterol was

significantly higher in transgenic mice (p=0.001) (fig. 8b), with increased LDL

accounting almost completely for the difference in total cholesterol (AP2-MINOR

23

mean LDL=35.05 mg/dL, wild-type mean LDL=22.0 mg/dL, p=0.0002 two-way

ANOVA, Brown-Forsythe test p=0.42) (fig. 8c). Mean VLDL was greater in AP2-

MINOR mice (p=0.16), and HDL was not different between groups.

Table 5. Fasting serum lipids in transgenic and wild-type mice maintained on standard chow

AP2-MINOR Wild-type P Triglycerides (N=15/group) 105.9 118.4 0.16

NEFA (N=15/group) 1.15 1.32 0.02

Total cholesterol (N=15/group) 117.9 96.1 0.001

HDL (N=4 pooled

samples/group) 73.7 69.6 0.59

LDL (N=4 pooled

samples/group) 35.05 22.0 0.0002

VLDL (N=4 pooled

samples/group) 6.475 5.4 0.16

24

a

00.20.40.60.8

11.21.41.61.8

AP2-MINOR Wild-type

Non

-est

erifi

ed fa

tty a

cid

(mm

ol/L

)

b

0

30

60

90

120

150

AP2-MINOR Wild-type

Tota

l ser

um c

hole

ster

ol

(mg/

dL)

* **

c

05

10152025303540

AP2-MINOR Wild-type

LDL

(mg/

dL)

***

Figure 8. Serum lipids in AP2-MINOR and wild-type mice. a) transgenic mice have significantly lower fasting serum NEFA (p<0.05), b) total cholesterol is significantly increased in transgenic animals (p=0.001), and c) increased total cholesterol is explained by highly increased low density lipoprotein (p=0.0002, 4 pooled samples/group). Error bars represent SD. AP2-MINOR N=15, Wild-type N=15

Insulin’s ability to suppress adipocyte lipolysis appears somewhat

impaired in AP2-MINOR transgenic mice. When injected with insulin and

sacrificed 1 hour later, transgenic animals had slightly higher serum NEFA than

wild-type (mean value 1.2 mmol/L versus 1.1 mmol/L, p=0.17) (fig. 9b). This

occurred in spite of significantly decreased serum catecholamines in transgenic

animals; mean serum norepinephrine was 35.7 pg/μl in transgenic mice versus

43.9 pg/μl in wild-type mice (p<0.05) (fig. 9c). Mean serum epinephrine was 5.1

25

pg/μl in transgenic mice versus 10.3 pg/μl in wild-type mice (p<0.001) (fig. 9d).

Post-injection insulin levels did not differ between groups (AP2-MINOR mean

insulin=3.9 ng/ml, wild-type mean insulin=3.8 ng/ml, p=0.9) (fig. 9a). There was

no correlation between serum insulin and NEFA for either group alone or for both

groups together. However, there was a slight negative correlation between serum

eprinephrine and NEFA, ostensibly due to deranged epinephrine values in

transgenic mice (R squared=0.19, p=0.07).

a

0

1

2

3

4

5

6

7

AP2-MINOR Wild-type

Post

-inje

ctio

n in

sulin

(n

g/m

l)

b

00.20.40.60.8

11.21.41.61.8

AP2-MINOR Wild-type

NEF

A (m

mol

/L)

c

0

10

20

30

40

50

60

AP2-MINOR Wild-typeSeru

m n

orep

inep

hrin

e (p

g/ul

)

d

0

2

4

6

8

10

12

14

16

AP2-MINOR Wild-typeSeru

m e

pine

phrin

e (p

g/ul

)

* ***

Figure 9. Insulin’s ability to suppress lipolysis in AP2-MINOR and wild-type mice. a) insulin levels attained following injection did not differ between groups (p=0.9), b) NEFA levels were slightly higher in transgenic animals (p=0.17). c) serum norepinephrine is significantly decreased in transgenic mice compared to wild-type (p<0.05), d) serum epinephrine is highly significantly decreased in AP2-MINOR mice compared to wild-type (p<0.001). Error bars represent SD. AP2-MINOR N=9, Wild-type N=9

26

Male AP2-MINOR transgenic mice—high fat diet Male AP2-MINOR mice

maintained on high fat chow were also larger than their wild-type littermates from

age 9 to 16 weeks (p<0.05) (fig 10a). From age 20 to 30 weeks, transgenic

animals had higher mean body weights that did not reach statistical significance

(p=0.15) (fig 10b). Food intake did not differ between groups (p=0.3) (fig. 10c).

a

2025303540455055

1 2 3 4 5 6 7 8

Age (weeks)

Bod

y w

eigh

t (g)

AP2-MINOR

Wild-type

b

20

25

30

35

40

45

50

55

20 21 22 23 24 25 26 27 28 29 30

Age (weeks)

Bod

y w

eigh

t (g)

Figure 10, panels and b. Body weight and food intake in mice fed high fat chow. a) AP2-MINOR body weight was significantly higher from age 9 to 16 weeks (p<0.05), b) body weight in AP2-MINOR mice was not significantly greater from age 20 to 30 weeks (p=0.15). Body weight and food intake analyses are repeated measures ANOVA. Error bars represent SD. AP2-MINOR N=13, Wild-type N=14

27

c

15171921232527

10 11 12 13 14 15

Week

Food

inta

ke (g

)

AP2-MINOR

Wild-type

Figure 10, panel c. Food intake did not differ between transgenic and wild-type mice (p=0.3) maintained on high fat chow (p=0.3, repeated measures ANOVA). Error bars represent SD. AP2-MINOR N=13, Wild-type N=14

Table 6 summarizes body composition measures in mice maintained on

high fat chow. At age 16 weeks, no significant differences were observed in body

weight (on the day of DEXA scan), lean mass, fat mass, or percent fat. Gonadal

fat pad weight at age 33-35 weeks of age also did not differ between groups

(p=0.15), although transgenic mice had lower mean fat pad weight than wild-type

littermates (0.52 g versus 0.57 g).

Table 6. Body composition in 16-week old, high-fat fed AP2-MINOR and wild-type mice

Body weight (g) (95% CI)

Lean mass (g) (95% CI)

Fat mass (g) (95% CI)

% Fat mean,

median AP2-MINOR

N=8 45.0

(43.0-46.9) 22.2

(21.0-23.4) 18.8

(17.8-19.8) 45.0, 45.0

Wild-type N=12

43.7 (42.1-45.3)

21.5 (20.5-22.5)

18.2 (17.3-19.0) 45.0, 46.0

P 0.29 0.38 0.32 1.0 (Wilcoxon)

28

Transgenic mice maintained on high fat chow exhibited no difference in

fasting glucose, fasting insulin, glucose tolerance, or insulin tolerance. Results of

glucose and insulin tolerance tests are shown in figure 11.

a

100150200250300350400450

0 30 60 90 180

Time (minutes)

Blo

od g

luco

se (m

g/dL

)

AP2-MINOR

Wild-type

b

75100125150175200225250275300

0 30 60 90 180

Time (minutes)

Blo

od g

luco

se (m

g/dL

)

Figure 11. Glucose (a) and insulin (b) tolerance tests in animals

maintained on high fat chow. Results are not significant when assessed by either AUC student’s t-test or repeated measures ANOVA. Error bars represent SD. AP2-MINOR N=10, Wild-type N=11

Additionally, fasting serum lipids did not differ significantly between AP2-

MINOR and wild-type animals raised on high fat chow. Thus, NEFA, total

cholesterol, HDL, LDL, and VLDL were virtually identical between groups.

However, fasting triglycerides displayed a significant difference in variance

29

(p<0.05, Brown-Forsythe), with transgenic animals having less variance about

the mean (fig. 12).

Tr

igly

cerid

es (m

g/dL

)

60

70

80

90

100

110

120

130

AP2-MINOR Wild-type

Genotype

Figure 12. Fasting serum triglycerides. Means do not differ significantly (p=0.16, Welch ANOVA), but variance is significantly different (p<0.05, Brown-Forsythe). AP2-MINOR N=11, Wild-type N=14

DISCUSSION AP2-MINOR transgenic mice exhibit abnormal glucose and lipid

homeostasis. At age 16 weeks, female mice maintained on standard chow have

elevated fasting glucose and impaired glucose tolerance. Male transgenic mice

maintained on standard chow were larger than wild-type, with no significant

differences in body composition at 8 or 16 weeks of age. For the duration of the

study, transgenic mice ate less per gram body weight in spite of significantly

increased lean mass at age 30 weeks. This finding is particularly interesting

when considering the fact that transgenic mice have slightly higher day-time body

temperatures (data not shown). So, it would be plausible to hypothesize that

AP2-MINOR mice are less active than wild-type. AP2-MINOR males fed standard

chow exhibited impaired glucose tolerance at ages 17 and 31 weeks, and

impaired insulin tolerance at age 32 weeks. Fasting serum triglyceride was

slightly decreased in transgenic animals while NEFA, total cholesterol, and LDL

were significantly elevated.

30

The adipocytes of AP2-MINOR transgenic mice appear to be either

exquisitely sensitive to serum catecholamines or resistant to the effects of insulin,

or both; insulin failed to decrease transgenic serum NEFA below the level of wild-

type animals in spite of significantly lower catecholamines in transgenics. It is

unclear whether decreased catecholamines following insulin injection indicate a

chronic depression of the sympathetic nervous system or a decreased response

to the acute stress associated with human handling. It also unclear whether this

change is mild or severe; reports on sympathetic function in adipocytes employ

synthetic β-adrenergic agonists and antagonists rather than epinephrine and

norepinephrine, preventing meaningful comparisons with our experiment. In any

event, alterations in either acute or chronic stress responses could have lasting

physiological consequences. Furthermore, perturbations in adrenergic tone could

account for elevated fasting glucose and poor glucose tolerance in transgenic

mice since β-adrenoreceptor agonism potentiates pancreatic production of insulin

and both α- and β-adrenoreceptors modulate glucagon secretion (Nonogaki

2000).

It would also be reasonable to hypothesize that the physiological

characteristics observed in AP2-MINOR mice are due to down-regulation of liver

X receptor (LXR) and its direct target, sterol response element binding protein-1c

(SREBP-1c), important transcriptional regulators of lipid, sterol, and glucose

metabolic enzymes in adipocytes. It is interesting to note that adenoviral-driven

over-expression of NR4A family member Nur77 inhibits SREBP-1c expression

and activity in mouse hepatocytes, inducing a 98% increase in serum LDL and a

67% increase in serum triglycerides (Pols et al. 2008). Pols et al. also observed

decreased expression of LXR target genes, ABCg5 and ABCg8, leading them to

hypothesize that down-regulation of SREBP-1c is effected through inhibition of

LXR. In vivo over-expression of SREBP-1c driven by the AP2 promoter induces a

severe lipodystrophic phenotype in mice, with enlarged fatty liver, atrophied white

adipose tissue depots, and “WAT” phenotype in intrascapular brown fat pads.

These changes are mediated by a host of protein expression abnormalities,

including: down-regulation of leptin, fatty acid synthase (WAT), PPAR-γ, C/EPB-

31

α, adipocyte FABP (AP2), Insulin receptor, IRS-1, IRS-2, GLUT-4, and UCP-1

(BAT), and concomitant up-regulation of LDL receptor, acetyl Co-A carboxylase,

HMG-CoA reductase, TNF- α, stearoyl Co-A desaturase (liver, BAT) and fatty

acid synthase (liver, BAT) (Shimomura et al. 1998). It is possible that

overexpression of MINOR in adipocytes is driving decreased expression or

activity of SREBP-1c, causing protein expression changes that are more subtle,

yet roughly inverse to those seen in AP2-SREBP-1c mice (summarized in table

7).

Table 7. Summary of hypothesized protein expression or function changes in AP2-MINOR mice Direction Protein Increased Leptin FAS (WAT) PPAR-γ C/EBP-α AP2 (FABP) UCP-1 (BAT) Insulin receptor IRS-1 IRS-2 GLUT-4 Decreased LDL receptor TNF-α ACC SCD HMGCoA reductase FAS (liver, BAT)

Thus, MINOR-mediated SREBP-1c down-regulation could cause

increased leptin expression leading to an overall decrease in sympathetic

nervous system tone and a blunted pancreatic response to glucose. Contrary to

observations in rats and humans, chronically elevated leptin in mice is known to

decrease sympathetic output through direct actions of leptin on the hypothalamus

(Malendowicz et al. 2007). Furthermore, pancreas-specific knockout of leptin

32

receptor in mice results in improved glucose tolerance, an effect which is

dampened by high-fat feeding (Morioka et al. 2007).

MINOR-induced SREBP-1c could also drive decreased TNF-α production

resulting in decreased sympathetic tone. Indeed, TNF-α administration to mice

(at doses too small to affect blood pressure, food intake, or prolactin release)

causes increased ACTH production within minutes (Dunn, 2000).

Furthermore, decreased SREBP-1c could account for increased serum

LDL and decreased NEFA in AP2-MINOR mice. SREBP-1c is a known

transcriptional activator of LDL receptor (Pols et al. 2008). Mice lacking LDL

receptor are well characterized, with increased serum LDL and IDL (Breslow,

1996). However, LDL levels observed in AP2-MINOR mice are not sufficiently

elevated to promote atherosclerotic plaque formation; in LDL-receptor knockout

and ApoE knockout mice, non-HDL cholesterol of approximately 300 mg/dL is

required for atherogenesis (Hsueh et al. 2007). Increased expression of FABP in

AP2-MINOR mice could account for decreased serum NEFA. Mice lacking FABP

display slightly increased NEFA and decreased triglycerides when reared on

standard chow. Additionally, transgenic AP2-endothelial-FABP mice display

increased serum glucose and decreased insulin sensitivity (Furuhashi &

Hotamisligil, 2008).

Since MINOR overexpression in cultured 3T3-L1 cells led to enhanced

insulin-stimulated glucose uptake (Fu et al. 2007), we were surprised to find that

AP2-MINOR mice responded poorly when challenged with glucose and insulin.

However, the apparent contradiction between our in vitro and in vivo findings

could be explained by SREBP-1c modulation of a wide array of proteins required

for lipid metabolism, insulin-signaling, and glucose metabolism in adipocytes. In

vitro, the effects of the lipid modulators, LDL-receptor and FABP, would be

assumed to be minimal, while the effects of insulin- and glucose-related

pathways would have more obvious effects. Thus decreased SREBP-1c function

in vitro could allow increased expression of insulin receptor, IRS-1, IRS-2, GLUT-

4, PPAR-γ, and C/EBP-α, producing increased insulin sensitivity.

33

The AP2 promoter is known to be functional in macrophages as well as

adipocytes. Expression of the AP2 gene product, adipocyte fatty acid binding

protein (FABP), is approximately 10,000X higher in adipocytes than THP-1

monocytes (Furuhashi & Hotamisligil, 2008). Yet, our lab has demonstrated that

oxidized lipid-mediated induction of foam cell formation is accompanied by

extreme up-regulation of FABP (Fu et al. 2006). Thus, AP2-driven transgene

expression in macrophages could be a significant contributor to the phenotype of

the AP2-MINOR mice.

Finally, it is clear that NR4A family members are not required for

adipogenesis; stable expression of a dominant negative Nur77 suppresses the

function of all NR4A family members without affecting lipid accumulation or

mature adipocyte gene production (Au et al. 2008). Thus it appears that MINOR

and other NR4A family members function as important modulators of other

nuclear receptors in differentiating and mature adipocytes. Over-expression of

MINOR in mouse adipocytes causes decreased glucose tolerance and mild

dislipidemia, and these effects are likely due to complex interactions between

MINOR and other nuclear receptors.

34

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APPENDIX: IACUC APPROVAL