# Acute-Phase Serum Amyloid A as a Marker of  Insulin Resistance in Mice

Authors: Ludger Scheja; Barbara Heese; Heike Zitzer; Mervyn D. Michael; Angela M. Siesky; Heike Pospisil; Ulrike Beisiegel; Klaus Seedorf
Journal: Experimental Diabetes Research
 (2008)

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## Abstract

Acute-phase serum amyloid A (A-SAA) was shown recently to correlate with obesity and insulin resistance in humans. However, the mechanisms linking obesity-associated inflammation and elevated plasma A-SAA to insulin resistance are poorly understood. Using high-fat diet- (HFD-) fed mice, we found that plasma A-SAA was increased early upon HFD feeding and was tightly associated with systemic insulin resistance. Plasma A-SAA elevation was due to induction ofSaa1 and Saa2 expression in liver but not in adipose tissue. In adipose tissue Saa3 was the predominant isoform and the earliest inflammatory marker induced, suggesting it  is important for initiation of adipose tissue inflammation. To assess the potential impact of A-SAA on adipose tissue insulin resistance, we treated 3T3-L1 adipocytes with recombinant A-SAA. Intriguingly, physiological levels of A-SAA caused alterations in gene expression closely resembling those observed in HFD-fed mice. Proinflammatory genes (Ccl2, Saa3) were induced while genes critical for insulin sensitivity (Irs1, Adipoq, Glut4) were down-regulated. Our data identify HFD-fed mice as a suitable model to study A-SAA as a biomarker and a novel possible mediator of insulin resistance.


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## Body

## 1. Introduction

An important aspect of obesity-linked insulin
resistance is chronic, subclinical inflammation taking place in adipose tissue
and other major metabolic tissues. Local activation of proinflammatory pathways
results in the suppression of insulin signaling thereby contributing to
impaired glucose metabolism (see [1] for a recent review). The molecular and
cellular mechanisms linking obesity-associated inflammation to insulin
resistance are under intense investigation. Within these studies, plasma
inflammation markers such as acute-phase proteins [2] draw special attention as
they are important both for understanding the relative contribution of
inflammation to insulin resistance and their immediate biological activities on
insulin signaling as well as for the development of clinical biomarkers.One acute-phase protein reported to be
increased in plasma of obese and insulin resistant humans is serum amyloid A
(SAA) (reviewed in [3]). Acute-phase SAA (A-SAA) consists of two closely
related isoforms, SAA1 and SAA2, which can be induced over a wide expression
range in response to proinflammatory stimuli. Additional members of the SAA family
are SAA3 and SAA4 [3]. SAA3 is expressed in mice and other mammalian species
but apparently not in humans who have a defective Saa3 gene [4]. SAA3 is also induced under proinflammatory
conditions and may share physiological
functions with the A-SAA isoforms. SAA4 is a constitutively expressed member of
the family and responds only moderately to inflammatory stimuli.Chronic systemic elevation of A-SAA has been
linked to metabolic disease and is a well-established risk factor of
atherosclerosis [5–7]. It also appears to correlate with insulin resistance and
body mass index in humans, as demonstrated in recent studies. A-SAA levels in
patients fell when either fat mass was reduced or insulin sensitivity was
restored by treatment with a PPARγ
agonist [8–10]. Interestingly, although liver is the organ believed to be most
important for A-SAA secretion in the acute-phase response, in insulin
resistance-related human studies Saa1 and Saa2 expression was higher in
adipose tissue than in liver [9, 11, 12] and also the correlation of A-SAA gene
expression with plasma levels was higher in this tissue [11]. Thus, it appears
that the regulation of A-SAA in the context of obesity and insulin resistance
has some unique features as compared to the acute-phase response. A-SAA
induction in that context is triggered primarily by cytokines such as
interleukin (IL)-1 and tumor necrosis factor-a (TNF a), but also by
IL-6 and related cytokines  [2, 13].In addition to their biomarker properties, SAA
isoforms may also be causally involved in the development of metabolic
diseases. In the case of atherosclerosis, a couple of mechanisms have been
proposed. A-SAA is an apolipoprotein predominantly associated with high-density
lipoprotein (HDL) particles and, when increased, it can influence
antiatherogenic functions of HDL, especially such involving cholesterol uptake
and efflux [14–16]. Also, as shown in mice, A-SAA binds to
atherosclerotic lesions and, thereby, may cause retention of lipoproteins and
lipid deposition [17]. Biological functions of A-SAA potentially contributing
to both atherosclerosis and insulin resistance are proinflammatory properties
normally involved in pathogen defense and tissue repair such as chemotaxis,
cytokine induction, and the secretion of extracellular matrix-degrading
proteases [18–20]. Chronic activation of these inflammatory processes may lead
to infiltration of immune cells into the tissues. In obesity, massive infiltration
of adipose tissue by macrophages takes place which can be detected through increased
expression of myeloid cell markers such as F4/80, CD68, and CD11b [21, 22]. This
infiltration is believed to be triggered by increased secretion by adipose
tissue cells of chemoattractant molecules including the chemokine CCL2 (also
known as MCP1) [23]. Accumulation of activated macrophages leads to sustained
production of proinflammatory mediators, entailing adipocyte insulin resistance,
and deteriorated glucose metabolism.Studies relating to A-SAA induction in rodents
have been performed in mouse atherosclerosis models and have offered valuable
clues regarding A-SAA regulation by lipids [24–26]. However, they were not
designed to study defects or mechanisms relating to glucose homeostasis. In
order to address the role of SAA in glucose metabolism, we designed a study
using a common model of insulin resistance, the diet-induced obesity (DIO)
mouse. We induced distinct degrees of overweight and insulin resistance by
feeding a high-fat diet (HFD) for different periods of time and compared plasma
A-SAA levels and expression of SAA isoforms with parameters of insulin
sensitivity and inflammation in liver, adipose tissue and muscle. Furthermore,
using cultured adipocytes we investigated whether A-SAA may directly promote
the expression of proinflammatory genes and interfere with insulin signaling.


## 2. Materials and Methods

### 2.1. Materials

Recombinant A-SAA was obtained from Peprotech (Hamburg, Germany).
DMEM, BSA, and calf serum were from Invitrogen (Karlsruhe, Germany).
FCS was from Biochrom-Seromed (Berlin,
Germany). All
other reagents (mouse TNFα,
insulin, dexamethasone, IBMX, LPS, PMB) were purchased from Sigma-Aldrich (Munich, Germany).


### 2.2. Animal Diet Regimens and Necropsy

Age-matched male C57Bl/6 mice (Taconic, Germantown, NY)
on distinct diets were used as models of insulin resistance and as the
respective controls. Severe insulin resistance and obesity were induced by
weaning animals on an HFD (Bioserv3282, 59 cal% fat) and maintaining them on
the diet for 16 weeks. Mildly insulin-resistant mice were generated by
randomizing lean mice based on fasting plasma glucose and insulin, and
subsequently feeding one group the HFD for one week while the other mice were
kept as controls on a standard rodent chow (Purina5001).For randomization, animals were bled by tail clip method at 19 weeks of
age after overnight fast. At 20 weeks of age, all mice were subjected to
necropsy. The animals were fasted overnight (16 hours), anesthetized with
isoflurane, and cardiac stick was performed to collect blood as EDTA plasma
from the right ventricle of the heart. After bleeding, the animals were
perfused (10 U/L heparinized saline) through the right ventricle of the heart,
allowing the blood to exit the animal through a cut in the inferior vena cava.
Organs were then removed. Selected organs and blood were snap frozen in liquid
nitrogen and stored at −80°C.Animals were maintained on a 12-hour light, 12-hour dark cycle and
received house water ad libitum. All animals were kept in accordance with the
Institutional American Association for the Accreditation of Laboratory Animal
Care guidelines.


### 2.3. Plasma Parameters

Plasma insulin was determined using Rat/Mouse Insulin Assay kit
(Mesoscale Discovery, Gaithersburg, Md) and Mouse Endocrine Lincoplex kit (Millipore, St. Charles, Mo)
for randomization and final study, respectively. Glucose was measured using a Hitachi 912 clinical chemistry analyzer (Roche, Indianapolis, Ind).
Adiponectin and A-SAA were determined using ELISAs from R&D Systems (Minneapolis, Minn)
and Biosource (Invitrogen), respectively.


### 2.4. Total RNA Extraction and Real-Time Quantitative PCR

Total RNA from frozen tissue samples was extracted using the
TissueLyser  and RNeasy system (Qiagen, Hilden, Germany)
according to the manufacturer’s instructions (RNeasy Lipid Tissue Kit and
RNeasy Fibrous Tissue Kit for adipose and muscle tissue, resp.). DNA
contaminants were removed via column DNase treatment (RNase-Free DNase set;
Qiagen). 1μg of RNA was reversely transcribed in a 50 μL reaction mixture using the High Capacity cDNA
Archive Kit (Applied Biosystems, Darmstadt, Germany)
according to the manufacturer’s instructions. Real time RT-PCR was performed as
described previously [27]. For detections of Saa2 and Irs2, Assay-by-Design Assay that mixes with the following sequences was used:Irs2-fwd GCGGCCTCATCTTCTTCACT,mIrs2-rev AACTGAAGTCCAGGTTCATATAGTCAGA,mIrs2-Pr CGACAGCCGGCAGCGCTCTC,mSaa2-fwd GAGTCTGCCATGGAGGGTTTT,mSaa2-rev TGTAGGCTCGCCACATGTC,mSaa2-Pr TCCAGCCCCTTGGAAAG.For all other genes Assay-on-Demand primer/probe sets supplied by
Applied Biosystems were used (Assay IDs are available upon request). Relative
expression was calculated by normalization to selected house keeper mRNA
(cyclophilin E for muscle, TBP for all others) byΔΔCt method [28]. Data are reported as copy
number relative to 104 copies of house keeper.


### 2.5. Differentiation and Treatment of  3T3-L1 Adipocytes

3T3-L1 cells were maintained in DMEM containing 10% calf serum. To
differentiate, cells were grown to confluence, switched to differentiation
medium (DMEM, 4.5 g/L glucose, supplemented with 10% FCS, 5μg/mL
insulin, 0.25 μM dexamethasone, 0.5 mM IBMX), and cultured for 2 days. Subsequently, cells were
kept in differentiation medium without dexamethasone and IBMX for 3 days and
further differentiated in medium also lacking insulin for at least 1 week before
experimentation. Before treatment, cells were washed once with PBS and starved
overnight in DMEM, 1 g/L glucose, containing 0.1% BSA. Reagents were then added
directly to wells.


### 2.6. Statistical Analysis

Unless indicated otherwise two-tailed Student’st-test was used
to assess statistical significance between groups. Pearson's correlation test
was used to evaluate the degree and the significance of association between
plasma A-SAA and obesity or insulin resistance parameters.


## 2.1. Materials

Recombinant A-SAA was obtained from Peprotech (Hamburg, Germany).
DMEM, BSA, and calf serum were from Invitrogen (Karlsruhe, Germany).
FCS was from Biochrom-Seromed (Berlin,
Germany). All
other reagents (mouse TNFα,
insulin, dexamethasone, IBMX, LPS, PMB) were purchased from Sigma-Aldrich (Munich, Germany).


## 2.2. Animal Diet Regimens and Necropsy

Age-matched male C57Bl/6 mice (Taconic, Germantown, NY)
on distinct diets were used as models of insulin resistance and as the
respective controls. Severe insulin resistance and obesity were induced by
weaning animals on an HFD (Bioserv3282, 59 cal% fat) and maintaining them on
the diet for 16 weeks. Mildly insulin-resistant mice were generated by
randomizing lean mice based on fasting plasma glucose and insulin, and
subsequently feeding one group the HFD for one week while the other mice were
kept as controls on a standard rodent chow (Purina5001).For randomization, animals were bled by tail clip method at 19 weeks of
age after overnight fast. At 20 weeks of age, all mice were subjected to
necropsy. The animals were fasted overnight (16 hours), anesthetized with
isoflurane, and cardiac stick was performed to collect blood as EDTA plasma
from the right ventricle of the heart. After bleeding, the animals were
perfused (10 U/L heparinized saline) through the right ventricle of the heart,
allowing the blood to exit the animal through a cut in the inferior vena cava.
Organs were then removed. Selected organs and blood were snap frozen in liquid
nitrogen and stored at −80°C.Animals were maintained on a 12-hour light, 12-hour dark cycle and
received house water ad libitum. All animals were kept in accordance with the
Institutional American Association for the Accreditation of Laboratory Animal
Care guidelines.


## 2.3. Plasma Parameters

Plasma insulin was determined using Rat/Mouse Insulin Assay kit
(Mesoscale Discovery, Gaithersburg, Md) and Mouse Endocrine Lincoplex kit (Millipore, St. Charles, Mo)
for randomization and final study, respectively. Glucose was measured using a Hitachi 912 clinical chemistry analyzer (Roche, Indianapolis, Ind).
Adiponectin and A-SAA were determined using ELISAs from R&D Systems (Minneapolis, Minn)
and Biosource (Invitrogen), respectively.


## 2.4. Total RNA Extraction and Real-Time Quantitative PCR

Total RNA from frozen tissue samples was extracted using the
TissueLyser  and RNeasy system (Qiagen, Hilden, Germany)
according to the manufacturer’s instructions (RNeasy Lipid Tissue Kit and
RNeasy Fibrous Tissue Kit for adipose and muscle tissue, resp.). DNA
contaminants were removed via column DNase treatment (RNase-Free DNase set;
Qiagen). 1μg of RNA was reversely transcribed in a 50 μL reaction mixture using the High Capacity cDNA
Archive Kit (Applied Biosystems, Darmstadt, Germany)
according to the manufacturer’s instructions. Real time RT-PCR was performed as
described previously [27]. For detections of Saa2 and Irs2, Assay-by-Design Assay that mixes with the following sequences was used:Irs2-fwd GCGGCCTCATCTTCTTCACT,mIrs2-rev AACTGAAGTCCAGGTTCATATAGTCAGA,mIrs2-Pr CGACAGCCGGCAGCGCTCTC,mSaa2-fwd GAGTCTGCCATGGAGGGTTTT,mSaa2-rev TGTAGGCTCGCCACATGTC,mSaa2-Pr TCCAGCCCCTTGGAAAG.For all other genes Assay-on-Demand primer/probe sets supplied by
Applied Biosystems were used (Assay IDs are available upon request). Relative
expression was calculated by normalization to selected house keeper mRNA
(cyclophilin E for muscle, TBP for all others) byΔΔCt method [28]. Data are reported as copy
number relative to 104 copies of house keeper.


## 2.5. Differentiation and Treatment of  3T3-L1 Adipocytes

3T3-L1 cells were maintained in DMEM containing 10% calf serum. To
differentiate, cells were grown to confluence, switched to differentiation
medium (DMEM, 4.5 g/L glucose, supplemented with 10% FCS, 5μg/mL
insulin, 0.25 μM dexamethasone, 0.5 mM IBMX), and cultured for 2 days. Subsequently, cells were
kept in differentiation medium without dexamethasone and IBMX for 3 days and
further differentiated in medium also lacking insulin for at least 1 week before
experimentation. Before treatment, cells were washed once with PBS and starved
overnight in DMEM, 1 g/L glucose, containing 0.1% BSA. Reagents were then added
directly to wells.


## 2.6. Statistical Analysis

Unless indicated otherwise two-tailed Student’st-test was used
to assess statistical significance between groups. Pearson's correlation test
was used to evaluate the degree and the significance of association between
plasma A-SAA and obesity or insulin resistance parameters.


## 3. Results

### 3.1. High-Fat Diet Feeding Induces Insulin Resistance and Increases Plasma A-SAA Levels

One week HFD (1 w HFD) feeding
of mice resulted in enhanced body weight (27.0±0.7 g versus 23.0±0.5 g, mean
±SEM, P<.001) and induced significant fasting hyperinsulinemia and
hyperglycemia compared to controls maintained on chow diet (see Figure 1),
indicating mild insulin resistance already after a short period on the
obesigenic diet. As expected, 16 weeks of HFD (16 w HFD) feeding led to more
pronounced overweight (44.9±0.7 g; P<.001 versus controls) and
insulin resistance compared to controls (see Figure 1). Plasma leptin showed a
similar pattern (19 ± 5 pmol/L, control, 130 ± 25 pmol/L, 1 w HFD, 1205 ± 194 pmol/L, 16 w HFD; P<.001).(a) Fasting plasma insulin, (b) glucose,
(c) adiponectin, and (d) A-SAA levels in mice fed either control diet (Chow),
control diet for 15 weeks, and HFD for one week (1 w HFD), and HFD for 16
weeks (16 w HFD), respectively. Bar
graphs are presented as mean ±SEM (n= 5–8), statistics: *P<.05 versus Chow, **P<.01 versus Chow, (a)P<.05 versus 1 w
HFD, (b)P<.01 versus
1 w HFD; Mann-Whitney U-Test was used for insulin (7/8 measurements in Chow
group were below detection level, 56 pmol/L); two-tailed Student’s t-test
for all other measurements.
(a)(b)(c)(d)Plasma A-SAA was elevated in the 1 w HFD group (see
Figure1; 5.06±1.05 μg/mL versus 2.11±0.25 μg/mL in control animals, P<0.05)
and increased substantially further in the 16 w HFD group (31.4±7.0 μg/mL, P<.01 versus controls),
demonstrating that the plasma levels of A-SAA are associated with obesity and
insulin resistance in a quantitative fashion. This quantitative association was
confirmed by combining all experimental groups and performing a correlation
analysis for plasma A-SAA (Pearson correlation coefficients: 0.76, insulin;
0.75, glucose; 0.84, HOMA-IR; 0.78, leptin; P<.001). In contrast,
plasma adiponectin levels were not affected by 1 w HFD, however, they were
significantly reduced after 16 w HFD (see Figure 1).


### 3.2. Tissue-Specific Induction of SAA Isoforms

In order to understand the contribution of key
metabolic tissues to the observed A-SAA induction in plasma, we performed
real-time quantitative PCR experiments in liver, adipose tissue, and skeletal
muscle. As shown in Figure2 and Table 1, both A-SAA isoforms, Saa1 and Saa2, were highly expressed in liver and were induced by short- and
long-term HFD feeding (P<.15 for 1 w HFD). Saa3 was also well expressed in liver, however, it was not
significantly induced in this tissue by the HFD. By contrast, Saa3 was strongly expressed in adipose
tissue and markedly induced already after one week of HFD with no further
increase observed after 16 weeks (see Figure 2). Interestingly, expression of Saa1 and Saa2 was very low in adipose tissue (see Table 1) and neither of
them was induced by the HFD indicating that adipose tissue is not involved in
the elevation of plasma A-SAA by the HFD. In skeletal muscle basal expression
of all SAA isoforms was very low. While the mRNA expression of Saa1
and Saa2 was not affected by HFD, Saa3 was markedly induced
after 16 weeks of HFD (see Table 1).Table 1
Expression of  SAA isoforms and SAA inducers in liver, adipose tissue and skeletal muscle of
HFD-fed mice. Data are reported as copy numbers relative to104 copies of house keeper. Numbers of animals (n) are in
parentheses. Data are mean ±SEM;  *P<.05 versus Chow, **P<.01 versus Chow, (a)P<.05 versus 1 w HFD, (b)P<.01 versus 1 w HFD; two-tailed Student’s t-test.
Experimental GroupsChow1 w HFD16 w HFDCopy numberCopy numberCopy numberLiverSaa120,885 ± 3,746 (5)80,450 ± 27,367 (6)168,070 ± 24,441 (5)**(a)Saa296,460 ± 16,927 (5)370,729 ± 150,376 (6)998,406 ± 164,697 (5)**(a)Saa318,824 ± 4,753 (6)16,098 ± 3,374 (6)31,641 ± 7,118 (6)Il1b1,216 ± 138 (6)813 ± 87 (6)*1,885 ± 255 (6)*(b)Il630 ± 5 (6)22 ± 4 (6)21 ± 3 (6)Tnf296 ± 65 (6)339 ± 51 (6)1,159 ± 142 (6)**(b)Adipose tissueSaa1262 ± 86 (6)70 ± 21 (6)78 ± 17 (6)Saa21,544 ± 1,035 (5)615 ± 170 (4)355 ± 75 (5)Saa35,566 ± 1,086 (5)60,924 ± 25,363 (4)*48,600 ± 14,279 (5)*Il1b261 ± 88 (5)97 ± 21 (4)384 ± 142 (5)Il6253 ± 105 (6)139 ± 24 (6)618 ± 352 (6)Tnf648 ± 189 (6)1,015 ± 119 (6)6,009 ± 488 (6)**(b)Skeletal muscleSaa124 ± 4  (6)23 ± 6 (6)36 ± 4 (6)Saa292 ± 18 (6)75 ± 12 (6)96 ± 16 (6)Saa344 ± 21 (6)65 ± 23 (6)1,196 ± 210 (6)**(b)Il1b34 ± 6 (6)41 ± 8 (6)45 ± 5 (6)Il688 ± 10 (6)96 ± 13 (6)56 ± 5 (6)*(a)Tnf60 ± 20 (6)115 ± 30 (6)122 ± 7 (6)*(a), (b) Diet-induced expression of
SAA isoforms and (c), (d) SAA inducers in (a), (c) liver and (b), (d) adipose tissue,
quantified by real-time
quantitative PCR. Data are mean ±SEM normalized to control animals (fold over
Chow). Number of animals (n), see Table 1.
Statistics: *P<.05 versus Chow, **P<.01 versus Chow, (a)P<.05 versus 1 w
HFD, (b)P<.01 versus
1 w HFD; two-tailed Student’s t-test.
(a)(b)(c)(d)The cytokines IL-1β, IL-6, and TNFα are established as important inducers of Saa1 and Saa2 in liver during the
acute-phase response. In order to assess the role of these cytokines in Saa1 and Saa2 induction upon HFD feeding, we quantified also their
expression. In liver, the mRNAs of both IL-1β
and TNFα (Il1b, Tnf) were increased after 16 w HFD feeding. In contrast,
neither cytokine was induced after 1 w HFD feeding, suggesting that they
are not responsible for the early elevation of plasma A-SAA (see Figure 2).In adipose tissue,Tnf was strongly induced after 16 weeks of HFD, however, only a
trend toward an increase was observed in the 1 w HFD group. The expression
level of Il1b in adipose tissue was
not significantly changed by HFD feeding (see Figure 2).The mRNA of IL-6 (Il6) was not induced in any of the tissues examined in a
statistically significant manner (see Figure 2 and
Table 1).


### 3.3. Association of A-SAA Levels with Induction of Macrophage Markers and Chemoattractant Molecules in Insulin Target Tissues

The observed tissue- and isoform-selective patterns of SAA expression in response to the high-fat
diet prompted us to further characterize the underlying inflammatory processes.
To this end, we assessed the expression of inflammatory markers including the
chemoattractant molecules CCL2 and CXCL1 (also known as KC) as well as
macrophage infiltration markers (CD68, CD11b, F4/80). As shown in Figure3 and Table 2, one week feeding of HFD slightly induced the expression of the chemokine
genes (Ccl2, Cxcl1) in liver (P<.15) while neither chemokine was increased in adipose tissue and only Ccl2 was induced in muscle. The genes of
the myeloid cell markers CD68, CD11b, and F4/80 (Emr1) were moderately but significantly induced in adipose tissue
after one week of HFD, demonstrating incipient adipose infiltration by
macrophages, while in the other tissues significant induction of these markers
could not be observed, except for Cd68 in liver.Table 2
Expression of
inflammatory genes and genes related to insulin sensitivity in liver, adipose
tissue and skeletal muscle of HFD-fed mice. Data
are reported as copy number relative to104 copies of house keeper. Numbers of animals (n) are in
parentheses. Data are mean ±SEM;  *P<.05 versus Chow, **P<.01 versus Chow,
(a)P<.05 versus 1 w HFD, (b)P<.01 versus 1 w
HFD; two-tailed Student’s t-test.
Experimental GroupsChow1 w HFD16 w HFDCopy numberCopy numberCopy numberLiverCcl2590 ± 179 (6)1,813 ± 692 (6)4,769 ± 677 (6)**(a)Cxcl12,074 ± 529 (6)4,465 ± 1,016 (6)13,709 ± 1,489 (6)**(b)Cd688,969 ± 880 (6)13,192 ± 1,621 (6)*28,417 ± 3,828 (6)**(b)Cd11b2,129 ± 223 (6)1,300 ± 278 (6)*2,317 ± 156 (6)(b)Emr12,789 ± 588 (6)3,525 ± 646 (6)5,186 ± 977 (6)Adipoq61 ± 23 (6)67 ± 11 (6)33 ± 3 (6)(a)Glut480 ± 15 (6)84 ± 15 (6)212 ±  44 (6)*(a)Insr9,416 ± 1,909 (4)5,795 ± 223 (4)7,275 ± 380 (4)(a)Irs119,376 ± 1,196 (6)20,830 ± 905 (6)18,087 ± 355 (6)(a)Irs255,531 ± 4,549 (6)31,257 ± 4,180 (6)**13,435 ± 832 (6)**(b)Pgc1a7,065 ± 1,584 (6)3,357 ± 460 (6)*6,348 ± 559  (6)(b)Pgc1b6,257 ± 1,012 (6)9,217 ± 1,576 (6)12,543 ± 1,083 (6)**Adipose tissueCcl210,700 ± 6,564 (5)6,628 ± 628 (5)32,055 ± 8,522 (6)(a)Cxcl11,861 ± 752 (6)802 ± 129 (6)1,859 ± 284 (6)(b)Cd6851,947 ± 5,446 (6)92,213 ± 15,472 (6)*1,492,178 ± 435,981(6)**(b)Cd11b19,011 ± 3,357 (6)35,227 ± 4,969 (6)*227,310 ± 49,105 (6)**(b)Emr12,187 ± 381 (6)6,618 ± 1,032 (6)**55,752 ± 7,298 (6)**(b)Adipoq1,658,946 ± 598,784 (6)2,028,698 ± 485,799 (6)803,801 ± 248,995 (6)(a)Glut419,418 ±  6,662 (6)78,678 ± 11,679 (6)**25,965 ± 7,010 (6)(b)Insr18,149 ± 2,080 (4)17,441 ± 1,248 (4)13,880 ± 477 (4)(a)Irs160,068 ± 12,861 (6)36,282 ± 2,983 (6)11,888 ± 2,316 (6)**(b)Irs288,190 ± 14,430 (6)81,351 ± 6,149 (6)24,811 ± 3,661 (6)**(b)Pgc1a1,776 ± 441 (6)1,009 ± 87 (6)1,070 ± 388 (6)Pgc1b7,558 ± 1,261 (5)6,282 ± 737 (4)4,042 ± 299 (5)*(a)Skeletal muscleCcl2204 ± 46 (6)554 ± 131 (6)*1,224 ± 111 (6)**(b)Cxcl189 ± 9 (6)70 ± 13 (6)129 ± 15 (6)*(a)Cd683,927 ± 643 (6)4,237 ± 445 (6)10,178 ± 422 (6)**(b)Cd11b2,239 ± 361 (6)3,463 ± 521 (6)3,956 ± 773 (6)Emr1387 ± 25 (6)421 ± 57 (6)724 ± 41 (6)**(b)Adipoq94,837 ± 17,077 (6)116,610 ± 56,998 (6)114,194 ± 19,422 (6)Glut4512,548 ± 27,756 (6)379,390 ± 30,641 (6)**423,423 ± 22,047 (6)*Insr57,791 ± 2,283 (6)42,298 ± 1,673 (6)**50,984 ± 2,188 (6)(a)Irs158,141 ± 7,790 (6)45,326 ± 6,154 (6)47,738 ± 5,014 (6)Irs251,152 ± 9,399 (6)27,292 ± 4,061 (6)*21,470 ± 1,374 (6)*Pgc1a39,951 ± 24,961 (6)17,510 ± 8,171 (6)12,085 ± 1,107 (6)Pgc1b6,003 ± 1,476 (6)7,408 ± 1,327 (6)10,150 ± 271 (6)*(a) Diet-induced expression of
inflammatory genes in liver and (b) adipose tissue, quantified by real-time quantitative PCR.  Data are mean ±SEM normalized to control
animals (fold over Chow). Number of animals (n), see Table 2. Statistics: *P<.05 versus Chow, **P<.01 versus Chow, (a)P<.05 versus 1 week
HFD, (b)P<.01 versus
1 w HFD, two-tailed Student’s t-test.
(a)(b)After 16 weeks of HFD, pronounced expression of
inflammatory markers was observed in all tissues examined (see Figure3 and
Table 2). The macrophage markers Cd68, Cd11b, and Emr1 were highly induced in adipose, moderately increased in
muscle, while in liver only Cd68 was
induced significantly.Taken together, the temporal expression pattern
of liverCcl2, Cxcl1, and Cd68 as well
as adipose tissue Cd68, Cd11b, and Emr1 corresponded to liver Saa1 and Saa2 mRNA, plasma A-SAA protein,
and insulin resistance in our experiment. Interestingly, none of the inflammatory markers in adipose tissue showed
an expression pattern similar to Saa3.


### 3.4. Reduced Expression of Genes Implicated in Insulin Sensitivity in Liver, Adipose
Tissue, and Skeletal Muscle of  HFD-Fed Mice

Next, we determined the effect of HFD feeding
on the expression of signaling molecules known to regulate insulin sensitivity
(see Figure4 and Table 2). Consistent with unaltered plasma adiponectin levels
after one week and reduced plasma adiponectin levels after 16 w HFD
feeding, the mRNA levels of the adiponectin gene (Adipoq) in adipose tissue were not changed after one week and
significantly reduced after 16 w HFD feeding (see Figure 4). Adipoq levels in liver showed a similar
temporal pattern upon HFD feeding, however expression was very low compared to
adipose tissue (see Table 2). Expression of the insulin-responsive glucose
transporter Glut4 (also known as
Slc2a4) was induced 6-fold after 1 w HFD in adipose tissue and returned to
basal levels after 16 w HFD while in muscle it was moderately but
significantly suppressed already after 1 w HFD (see Table 2).(a) Diet-induced expression of
genes related to insulin sensitivity in liver and (b) adipose tissue,
quantified by real-time
quantitative PCR. Data are mean ±SEM normalized to control animals (fold over
Chow). Number of animals (n), see Table 2. Statistics: *P<.05 versus Chow, **P<.01 versus Chow, (a)P<0.05 versus 1 week
HFD, (b)P<.01 versus
1 w HFD, two-tailed Student’s t-test.
(a)(b)The expression of insulin receptor substrate-1 (Irs1) was largely unaffected in
liver and skeletal muscle while it was progressively down-regulated in adipose
tissue. By contrast, Irs2 expression
was reduced in all three tissues in a
tissue-specific manner (see Figure 4 and Table 2).To examine potential HFD-mediated tissue
specific shifts in metabolic capacity, we determined the expression of PGC-1α
and PGC-1β,
two transcriptional regulators of mitochondriogenesis and oxidative metabolism. As shown in Figure 4 and Table 2, Pgc1a expression was
significantly reduced after 1 w HFD in liver and recovered to control
levels after 16 w HFD. In adipose tissue and skeletal muscle, only a trend
towards downregulation could be observed. The expression of Pgc1b was upregulated in liver and
skeletal muscle after 16 w HFD with a trend towards upregulation evident
already after 1 w HFD in liver. In adipose tissue, Pgc1b was reduced after 1 w HFD diet and expression was
further impaired after 16 w HFD.In summary, significant tissue-specific downregulation of genes critical for maintaining insulin
sensitivity was observed in the HFD-fed mice.


### 3.5. Effects of Recombinant A-SAA on Gene Expression and Metabolism in 3T3-L1 Adipocytes

The observed concurrence of elevated plasma A-SAA with hyperinsulinemia in this mouse model prompted us to study a potential causal role
of A-SAA in the development of peripheral insulin resistance, using
differentiated 3T3-L1 cells as a model system. Treatment with physiological
concentrations of recombinant A-SAA for 24 hours led to a pronounced induction
ofCcl2 and Saa3 in a dose-dependent manner (see
Figure 5).Effects of recombinant A-SAA and
TNFα on gene expression in differentiated 3T3-L1 adipocytes. (a)
Dose-dependent induction of Saa3 and Ccl2 by recombinant A-SAA.
Differentiated 3T3-L1 adipocytes were washed with PBS and starved overnight in
DMEM containing 0.1% BSA. Subsequently, cells were treated for 24 hours with
various concentrations of recombinant A-SAA (0, 0.125 1.25, 12.5, 25 μg/mL).
(b) Regulation of signaling genes by A-SAA and TNFα: Differentiated 3T3-L1 adipocytes were treated as described under (a) and subsequently incubated with A-SAA (12.5 μg/mL, closed bars) and TNFα (10 ng/mL, hatched bars) for 24 hours. (c) Effect of polymyxin B (PMB) on induction of Saa3 and Ccl2. PMB at 10 μg/mL was added alone or in combination with
lipopolysaccharide (LPS, 2.5 ng/mL) and A-SAA (1.25 μg/mL), respectively. Experimental
conditions were as described under (a). Copy numbers are relative to one copy of TBP mRNA. Representative
data from at least three independent experiments are shown. Data are mean ±
standard deviation relative to untreated controls.
(a)(b)(c)These effects were unlikely to be caused by endotoxin contamination of
the A-SAA preparation, as they were unaffected by the
addition of the endotoxin neutralizer polymyxin B (see Figure5 and [29]). In contrast, genes involved in insulin signaling,
glucose transport, and mitochondriogenesis (Adipoq, Irs1, Glut4, Pgc1a, Pgc1b) were significantly downregulated
by A-SAA in a degree similar to TNFα
treatment (see Figure 5). Interestingly, the alterations in 3T3-L1 gene
expression resembled but were not identical to those observed in vivo in
adipose tissue after chronic HFD feeding (see Figure 4). Specifically, Irs2 was downregulated in vivo but not
in vitro, while Glut4 was downregulated
in vitro but not in vivo.Next, we checked whether the observed changes in gene expression had
effects on glucose transport, an important insulin-regulated metabolic function
of adipocytes. Treatment of 3T3-L1 adipocytes with A-SAA for 24 hours had no
significant effect on insulin-stimulated glucose transport either at submaximal
or maximum insulin concentrations (data not shown).


## 3.1. High-Fat Diet Feeding Induces Insulin Resistance and Increases Plasma A-SAA Levels

One week HFD (1 w HFD) feeding
of mice resulted in enhanced body weight (27.0±0.7 g versus 23.0±0.5 g, mean
±SEM, P<.001) and induced significant fasting hyperinsulinemia and
hyperglycemia compared to controls maintained on chow diet (see Figure 1),
indicating mild insulin resistance already after a short period on the
obesigenic diet. As expected, 16 weeks of HFD (16 w HFD) feeding led to more
pronounced overweight (44.9±0.7 g; P<.001 versus controls) and
insulin resistance compared to controls (see Figure 1). Plasma leptin showed a
similar pattern (19 ± 5 pmol/L, control, 130 ± 25 pmol/L, 1 w HFD, 1205 ± 194 pmol/L, 16 w HFD; P<.001).(a) Fasting plasma insulin, (b) glucose,
(c) adiponectin, and (d) A-SAA levels in mice fed either control diet (Chow),
control diet for 15 weeks, and HFD for one week (1 w HFD), and HFD for 16
weeks (16 w HFD), respectively. Bar
graphs are presented as mean ±SEM (n= 5–8), statistics: *P<.05 versus Chow, **P<.01 versus Chow, (a)P<.05 versus 1 w
HFD, (b)P<.01 versus
1 w HFD; Mann-Whitney U-Test was used for insulin (7/8 measurements in Chow
group were below detection level, 56 pmol/L); two-tailed Student’s t-test
for all other measurements.
(a)(b)(c)(d)Plasma A-SAA was elevated in the 1 w HFD group (see
Figure1; 5.06±1.05 μg/mL versus 2.11±0.25 μg/mL in control animals, P<0.05)
and increased substantially further in the 16 w HFD group (31.4±7.0 μg/mL, P<.01 versus controls),
demonstrating that the plasma levels of A-SAA are associated with obesity and
insulin resistance in a quantitative fashion. This quantitative association was
confirmed by combining all experimental groups and performing a correlation
analysis for plasma A-SAA (Pearson correlation coefficients: 0.76, insulin;
0.75, glucose; 0.84, HOMA-IR; 0.78, leptin; P<.001). In contrast,
plasma adiponectin levels were not affected by 1 w HFD, however, they were
significantly reduced after 16 w HFD (see Figure 1).


## 3.2. Tissue-Specific Induction of SAA Isoforms

In order to understand the contribution of key
metabolic tissues to the observed A-SAA induction in plasma, we performed
real-time quantitative PCR experiments in liver, adipose tissue, and skeletal
muscle. As shown in Figure2 and Table 1, both A-SAA isoforms, Saa1 and Saa2, were highly expressed in liver and were induced by short- and
long-term HFD feeding (P<.15 for 1 w HFD). Saa3 was also well expressed in liver, however, it was not
significantly induced in this tissue by the HFD. By contrast, Saa3 was strongly expressed in adipose
tissue and markedly induced already after one week of HFD with no further
increase observed after 16 weeks (see Figure 2). Interestingly, expression of Saa1 and Saa2 was very low in adipose tissue (see Table 1) and neither of
them was induced by the HFD indicating that adipose tissue is not involved in
the elevation of plasma A-SAA by the HFD. In skeletal muscle basal expression
of all SAA isoforms was very low. While the mRNA expression of Saa1
and Saa2 was not affected by HFD, Saa3 was markedly induced
after 16 weeks of HFD (see Table 1).Table 1
Expression of  SAA isoforms and SAA inducers in liver, adipose tissue and skeletal muscle of
HFD-fed mice. Data are reported as copy numbers relative to104 copies of house keeper. Numbers of animals (n) are in
parentheses. Data are mean ±SEM;  *P<.05 versus Chow, **P<.01 versus Chow, (a)P<.05 versus 1 w HFD, (b)P<.01 versus 1 w HFD; two-tailed Student’s t-test.
Experimental GroupsChow1 w HFD16 w HFDCopy numberCopy numberCopy numberLiverSaa120,885 ± 3,746 (5)80,450 ± 27,367 (6)168,070 ± 24,441 (5)**(a)Saa296,460 ± 16,927 (5)370,729 ± 150,376 (6)998,406 ± 164,697 (5)**(a)Saa318,824 ± 4,753 (6)16,098 ± 3,374 (6)31,641 ± 7,118 (6)Il1b1,216 ± 138 (6)813 ± 87 (6)*1,885 ± 255 (6)*(b)Il630 ± 5 (6)22 ± 4 (6)21 ± 3 (6)Tnf296 ± 65 (6)339 ± 51 (6)1,159 ± 142 (6)**(b)Adipose tissueSaa1262 ± 86 (6)70 ± 21 (6)78 ± 17 (6)Saa21,544 ± 1,035 (5)615 ± 170 (4)355 ± 75 (5)Saa35,566 ± 1,086 (5)60,924 ± 25,363 (4)*48,600 ± 14,279 (5)*Il1b261 ± 88 (5)97 ± 21 (4)384 ± 142 (5)Il6253 ± 105 (6)139 ± 24 (6)618 ± 352 (6)Tnf648 ± 189 (6)1,015 ± 119 (6)6,009 ± 488 (6)**(b)Skeletal muscleSaa124 ± 4  (6)23 ± 6 (6)36 ± 4 (6)Saa292 ± 18 (6)75 ± 12 (6)96 ± 16 (6)Saa344 ± 21 (6)65 ± 23 (6)1,196 ± 210 (6)**(b)Il1b34 ± 6 (6)41 ± 8 (6)45 ± 5 (6)Il688 ± 10 (6)96 ± 13 (6)56 ± 5 (6)*(a)Tnf60 ± 20 (6)115 ± 30 (6)122 ± 7 (6)*(a), (b) Diet-induced expression of
SAA isoforms and (c), (d) SAA inducers in (a), (c) liver and (b), (d) adipose tissue,
quantified by real-time
quantitative PCR. Data are mean ±SEM normalized to control animals (fold over
Chow). Number of animals (n), see Table 1.
Statistics: *P<.05 versus Chow, **P<.01 versus Chow, (a)P<.05 versus 1 w
HFD, (b)P<.01 versus
1 w HFD; two-tailed Student’s t-test.
(a)(b)(c)(d)The cytokines IL-1β, IL-6, and TNFα are established as important inducers of Saa1 and Saa2 in liver during the
acute-phase response. In order to assess the role of these cytokines in Saa1 and Saa2 induction upon HFD feeding, we quantified also their
expression. In liver, the mRNAs of both IL-1β
and TNFα (Il1b, Tnf) were increased after 16 w HFD feeding. In contrast,
neither cytokine was induced after 1 w HFD feeding, suggesting that they
are not responsible for the early elevation of plasma A-SAA (see Figure 2).In adipose tissue,Tnf was strongly induced after 16 weeks of HFD, however, only a
trend toward an increase was observed in the 1 w HFD group. The expression
level of Il1b in adipose tissue was
not significantly changed by HFD feeding (see Figure 2).The mRNA of IL-6 (Il6) was not induced in any of the tissues examined in a
statistically significant manner (see Figure 2 and
Table 1).


## 3.3. Association of A-SAA Levels with Induction of Macrophage Markers and Chemoattractant Molecules in Insulin Target Tissues

The observed tissue- and isoform-selective patterns of SAA expression in response to the high-fat
diet prompted us to further characterize the underlying inflammatory processes.
To this end, we assessed the expression of inflammatory markers including the
chemoattractant molecules CCL2 and CXCL1 (also known as KC) as well as
macrophage infiltration markers (CD68, CD11b, F4/80). As shown in Figure3 and Table 2, one week feeding of HFD slightly induced the expression of the chemokine
genes (Ccl2, Cxcl1) in liver (P<.15) while neither chemokine was increased in adipose tissue and only Ccl2 was induced in muscle. The genes of
the myeloid cell markers CD68, CD11b, and F4/80 (Emr1) were moderately but significantly induced in adipose tissue
after one week of HFD, demonstrating incipient adipose infiltration by
macrophages, while in the other tissues significant induction of these markers
could not be observed, except for Cd68 in liver.Table 2
Expression of
inflammatory genes and genes related to insulin sensitivity in liver, adipose
tissue and skeletal muscle of HFD-fed mice. Data
are reported as copy number relative to104 copies of house keeper. Numbers of animals (n) are in
parentheses. Data are mean ±SEM;  *P<.05 versus Chow, **P<.01 versus Chow,
(a)P<.05 versus 1 w HFD, (b)P<.01 versus 1 w
HFD; two-tailed Student’s t-test.
Experimental GroupsChow1 w HFD16 w HFDCopy numberCopy numberCopy numberLiverCcl2590 ± 179 (6)1,813 ± 692 (6)4,769 ± 677 (6)**(a)Cxcl12,074 ± 529 (6)4,465 ± 1,016 (6)13,709 ± 1,489 (6)**(b)Cd688,969 ± 880 (6)13,192 ± 1,621 (6)*28,417 ± 3,828 (6)**(b)Cd11b2,129 ± 223 (6)1,300 ± 278 (6)*2,317 ± 156 (6)(b)Emr12,789 ± 588 (6)3,525 ± 646 (6)5,186 ± 977 (6)Adipoq61 ± 23 (6)67 ± 11 (6)33 ± 3 (6)(a)Glut480 ± 15 (6)84 ± 15 (6)212 ±  44 (6)*(a)Insr9,416 ± 1,909 (4)5,795 ± 223 (4)7,275 ± 380 (4)(a)Irs119,376 ± 1,196 (6)20,830 ± 905 (6)18,087 ± 355 (6)(a)Irs255,531 ± 4,549 (6)31,257 ± 4,180 (6)**13,435 ± 832 (6)**(b)Pgc1a7,065 ± 1,584 (6)3,357 ± 460 (6)*6,348 ± 559  (6)(b)Pgc1b6,257 ± 1,012 (6)9,217 ± 1,576 (6)12,543 ± 1,083 (6)**Adipose tissueCcl210,700 ± 6,564 (5)6,628 ± 628 (5)32,055 ± 8,522 (6)(a)Cxcl11,861 ± 752 (6)802 ± 129 (6)1,859 ± 284 (6)(b)Cd6851,947 ± 5,446 (6)92,213 ± 15,472 (6)*1,492,178 ± 435,981(6)**(b)Cd11b19,011 ± 3,357 (6)35,227 ± 4,969 (6)*227,310 ± 49,105 (6)**(b)Emr12,187 ± 381 (6)6,618 ± 1,032 (6)**55,752 ± 7,298 (6)**(b)Adipoq1,658,946 ± 598,784 (6)2,028,698 ± 485,799 (6)803,801 ± 248,995 (6)(a)Glut419,418 ±  6,662 (6)78,678 ± 11,679 (6)**25,965 ± 7,010 (6)(b)Insr18,149 ± 2,080 (4)17,441 ± 1,248 (4)13,880 ± 477 (4)(a)Irs160,068 ± 12,861 (6)36,282 ± 2,983 (6)11,888 ± 2,316 (6)**(b)Irs288,190 ± 14,430 (6)81,351 ± 6,149 (6)24,811 ± 3,661 (6)**(b)Pgc1a1,776 ± 441 (6)1,009 ± 87 (6)1,070 ± 388 (6)Pgc1b7,558 ± 1,261 (5)6,282 ± 737 (4)4,042 ± 299 (5)*(a)Skeletal muscleCcl2204 ± 46 (6)554 ± 131 (6)*1,224 ± 111 (6)**(b)Cxcl189 ± 9 (6)70 ± 13 (6)129 ± 15 (6)*(a)Cd683,927 ± 643 (6)4,237 ± 445 (6)10,178 ± 422 (6)**(b)Cd11b2,239 ± 361 (6)3,463 ± 521 (6)3,956 ± 773 (6)Emr1387 ± 25 (6)421 ± 57 (6)724 ± 41 (6)**(b)Adipoq94,837 ± 17,077 (6)116,610 ± 56,998 (6)114,194 ± 19,422 (6)Glut4512,548 ± 27,756 (6)379,390 ± 30,641 (6)**423,423 ± 22,047 (6)*Insr57,791 ± 2,283 (6)42,298 ± 1,673 (6)**50,984 ± 2,188 (6)(a)Irs158,141 ± 7,790 (6)45,326 ± 6,154 (6)47,738 ± 5,014 (6)Irs251,152 ± 9,399 (6)27,292 ± 4,061 (6)*21,470 ± 1,374 (6)*Pgc1a39,951 ± 24,961 (6)17,510 ± 8,171 (6)12,085 ± 1,107 (6)Pgc1b6,003 ± 1,476 (6)7,408 ± 1,327 (6)10,150 ± 271 (6)*(a) Diet-induced expression of
inflammatory genes in liver and (b) adipose tissue, quantified by real-time quantitative PCR.  Data are mean ±SEM normalized to control
animals (fold over Chow). Number of animals (n), see Table 2. Statistics: *P<.05 versus Chow, **P<.01 versus Chow, (a)P<.05 versus 1 week
HFD, (b)P<.01 versus
1 w HFD, two-tailed Student’s t-test.
(a)(b)After 16 weeks of HFD, pronounced expression of
inflammatory markers was observed in all tissues examined (see Figure3 and
Table 2). The macrophage markers Cd68, Cd11b, and Emr1 were highly induced in adipose, moderately increased in
muscle, while in liver only Cd68 was
induced significantly.Taken together, the temporal expression pattern
of liverCcl2, Cxcl1, and Cd68 as well
as adipose tissue Cd68, Cd11b, and Emr1 corresponded to liver Saa1 and Saa2 mRNA, plasma A-SAA protein,
and insulin resistance in our experiment. Interestingly, none of the inflammatory markers in adipose tissue showed
an expression pattern similar to Saa3.


## 3.4. Reduced Expression of Genes Implicated in Insulin Sensitivity in Liver, Adipose
Tissue, and Skeletal Muscle of  HFD-Fed Mice

Next, we determined the effect of HFD feeding
on the expression of signaling molecules known to regulate insulin sensitivity
(see Figure4 and Table 2). Consistent with unaltered plasma adiponectin levels
after one week and reduced plasma adiponectin levels after 16 w HFD
feeding, the mRNA levels of the adiponectin gene (Adipoq) in adipose tissue were not changed after one week and
significantly reduced after 16 w HFD feeding (see Figure 4). Adipoq levels in liver showed a similar
temporal pattern upon HFD feeding, however expression was very low compared to
adipose tissue (see Table 2). Expression of the insulin-responsive glucose
transporter Glut4 (also known as
Slc2a4) was induced 6-fold after 1 w HFD in adipose tissue and returned to
basal levels after 16 w HFD while in muscle it was moderately but
significantly suppressed already after 1 w HFD (see Table 2).(a) Diet-induced expression of
genes related to insulin sensitivity in liver and (b) adipose tissue,
quantified by real-time
quantitative PCR. Data are mean ±SEM normalized to control animals (fold over
Chow). Number of animals (n), see Table 2. Statistics: *P<.05 versus Chow, **P<.01 versus Chow, (a)P<0.05 versus 1 week
HFD, (b)P<.01 versus
1 w HFD, two-tailed Student’s t-test.
(a)(b)The expression of insulin receptor substrate-1 (Irs1) was largely unaffected in
liver and skeletal muscle while it was progressively down-regulated in adipose
tissue. By contrast, Irs2 expression
was reduced in all three tissues in a
tissue-specific manner (see Figure 4 and Table 2).To examine potential HFD-mediated tissue
specific shifts in metabolic capacity, we determined the expression of PGC-1α
and PGC-1β,
two transcriptional regulators of mitochondriogenesis and oxidative metabolism. As shown in Figure 4 and Table 2, Pgc1a expression was
significantly reduced after 1 w HFD in liver and recovered to control
levels after 16 w HFD. In adipose tissue and skeletal muscle, only a trend
towards downregulation could be observed. The expression of Pgc1b was upregulated in liver and
skeletal muscle after 16 w HFD with a trend towards upregulation evident
already after 1 w HFD in liver. In adipose tissue, Pgc1b was reduced after 1 w HFD diet and expression was
further impaired after 16 w HFD.In summary, significant tissue-specific downregulation of genes critical for maintaining insulin
sensitivity was observed in the HFD-fed mice.


## 3.5. Effects of Recombinant A-SAA on Gene Expression and Metabolism in 3T3-L1 Adipocytes

The observed concurrence of elevated plasma A-SAA with hyperinsulinemia in this mouse model prompted us to study a potential causal role
of A-SAA in the development of peripheral insulin resistance, using
differentiated 3T3-L1 cells as a model system. Treatment with physiological
concentrations of recombinant A-SAA for 24 hours led to a pronounced induction
ofCcl2 and Saa3 in a dose-dependent manner (see
Figure 5).Effects of recombinant A-SAA and
TNFα on gene expression in differentiated 3T3-L1 adipocytes. (a)
Dose-dependent induction of Saa3 and Ccl2 by recombinant A-SAA.
Differentiated 3T3-L1 adipocytes were washed with PBS and starved overnight in
DMEM containing 0.1% BSA. Subsequently, cells were treated for 24 hours with
various concentrations of recombinant A-SAA (0, 0.125 1.25, 12.5, 25 μg/mL).
(b) Regulation of signaling genes by A-SAA and TNFα: Differentiated 3T3-L1 adipocytes were treated as described under (a) and subsequently incubated with A-SAA (12.5 μg/mL, closed bars) and TNFα (10 ng/mL, hatched bars) for 24 hours. (c) Effect of polymyxin B (PMB) on induction of Saa3 and Ccl2. PMB at 10 μg/mL was added alone or in combination with
lipopolysaccharide (LPS, 2.5 ng/mL) and A-SAA (1.25 μg/mL), respectively. Experimental
conditions were as described under (a). Copy numbers are relative to one copy of TBP mRNA. Representative
data from at least three independent experiments are shown. Data are mean ±
standard deviation relative to untreated controls.
(a)(b)(c)These effects were unlikely to be caused by endotoxin contamination of
the A-SAA preparation, as they were unaffected by the
addition of the endotoxin neutralizer polymyxin B (see Figure5 and [29]). In contrast, genes involved in insulin signaling,
glucose transport, and mitochondriogenesis (Adipoq, Irs1, Glut4, Pgc1a, Pgc1b) were significantly downregulated
by A-SAA in a degree similar to TNFα
treatment (see Figure 5). Interestingly, the alterations in 3T3-L1 gene
expression resembled but were not identical to those observed in vivo in
adipose tissue after chronic HFD feeding (see Figure 4). Specifically, Irs2 was downregulated in vivo but not
in vitro, while Glut4 was downregulated
in vitro but not in vivo.Next, we checked whether the observed changes in gene expression had
effects on glucose transport, an important insulin-regulated metabolic function
of adipocytes. Treatment of 3T3-L1 adipocytes with A-SAA for 24 hours had no
significant effect on insulin-stimulated glucose transport either at submaximal
or maximum insulin concentrations (data not shown).


## 4. Discussion

We report here the effect of  HFD feeding on SAA in the
insulin resistance- and obesity-prone mouse strain C57Bl/6. Short-term HFD
feeding (1 w HFD) resulted in a moderate but significant increase in fasting
plasma insulin, glucose and leptin, indicating incipient, and mild insulin
resistance in this group. The chronically fed group (16 w HFD) was severely
insulin resistant, as shown by elevated fasting plasma insulin, glucose and leptin,
and reduced adiponectin levels. In order to relate the observed insulin
resistance to inflammatory processes, we measured plasma levels of  A-SAA and found that these were already
elevated after one week of HFD, further increasing after 16 weeks of HFD
feeding. A correlation of plasma A-SAA with insulin resistance and obesity in
humans has been reported  by several
groups, however, acute phase proteins as markers or mediators of insulin
resistance have not been addressed in animal models so far. We show for the
first time that plasma A-SAA is associated with obesity and insulin resistance
in the frequently used DIO mouse model.
Therefore, this model may be useful to unravel molecular mechanisms
relating to A-SAA and its potential significance to insulin resistance.In order to determine the contribution of metabolically relevant tissues to the
diet-induced rise in plasma A-SAA, we quantified the expression of  SAA genes. The expression of the genes
corresponding to A-SAA,Saa1, and Saa2, in liver correlated with plasma
A-SAA, while in adipose tissue and muscle expression of the A-SAA isoforms was
very low and no induction by HFD was observed. This is in contrast to recently
published human studies which reported that
the elevation of plasma A-SAA in metabolic disease is linked to
induction of Saa1 and Saa2 in adipose
tissue [9, 12] but apparently not in liver [11]. In adipose tissue of the
HFD-fed mice, the Saa3 gene was
highly induced suggesting that in mouse adipose tissue SAA3 plays a role
similar to adipose A-SAA in humans who do not have a functional Saa3 gene [4].  Further experiments with regard to Saa3 gene regulation and functions in murine
adipose tissue are needed to understand how closely murine adipose Saa3 relates to Saa1 and Saa2 in human
adipose tissue.The tissue specific
and temporal pattern of SAA expression prompted us to further delineate the
inflammatory responses in the tissues of the insulin resistant mice by
assessing the expression of inflammatory mediators as well as expression of
myeloid cell surface markers as indicators of macrophage activation and infiltration. These were then put into relation to plasma A-SAA
levels and to the observed SAA expression patterns. In agreement with published
data [1, 21, 22], fully insulin resistant mice
(16 w HFD) showed a substantial induction in adipose tissue of  the cytokine Tnf,  the chemokine Ccl2 as well as the macrophage markers Cd68, Cd11b, and Emr1. In liver
and in muscle, Tnf and Ccl2 expressions were also induced, however, less
pronounced than in adipose tissue while the macrophage markers were only
partially and moderately induced in these tissues. One exception was Cd68 which, like the chemokine Cxcl1, showed an expression pattern
similar to Saa1 and Saa2 in liver suggesting that they may
be regulated in a similar manner as the A-SAA isoforms. After one week of HFD feeding, moderate but significant induction of all three macrophage markers was
observed in adipose tissue, but not in the other tissues, indicating that
adipose macrophage infiltration [21, 22] is an early event related to
HFD-induced insulin resistance. Expression of the chemokine CCL2, which has
been implicated in obesity-linked macrophage infiltration [23], was, however,
not elevated in fat at this stage. These data suggest that other chemoattractants
may play an important role in the early phase of adipose macrophage
infiltration, while CCL2 may be more important at later stages of
obesity-induced adipose tissue inflammation. SAA3 which was profoundly induced
in the 1 w HFD group and which has very recently been shown to exert chemotactic
activity on monocytes [30] may actually be such a chemoattractant conferring
initiation of macrophage infiltration in adipose tissue. Expression of Saa3 in adipocytes is sensitive to
inflammatory stimuli as well as nutrient overflow [31], making it a good
candidate for the induction of macrophage infiltration in adipose tissue.
Studies involving loss of function of SAA3 or the not yet identified SAA3
receptor responsible for macrophage attraction are needed to definitely address
the role of SAA3 in insulin resistant adipose tissue.In light of the observations that adipose tissue exhibited the first detectable inflammatory
response under HFD feeding, and that plasma A-SAA correlates with the degree of
insulin resistance, we asked whether there may be a direct induction of insulin
resistance in adipocytes by A-SAA. For this purpose, we incubated 3T3-L1 adipocytes
with recombinant A-SAA and used the expression of inflammatory markers and
signaling genes as indicators for insulin resistance. Intriguingly,
physiological A-SAA concentrations led to an induction ofSaa3 and downregulation of selected signaling genes linked to
insulin sensitivity such as Adipoq, Glut4, Irs1,
Pgc1a, and Pgc1b. Reduction of these genes, with
the exception of Glut4 and Pgc1a, was also detected in adipose
tissue of the insulin resistant mice after 16 weeks on the HFD. However,
insulin-stimulated glucose transport was not altered after A-SAA treatment in
our experiments suggesting that A-SAA alone is not sufficient to induce
impaired insulin signaling in adipocytes. Interestingly, in contrast to adipose
tissue after chronic HFD feeding, the expression of Irs2 in the 3T3-L1 adipocytes was unaltered which is likely
explaining the preserved insulin sensitivity. Nevertheless, it is conceivable
that A-SAA secreted from the liver potentiates inflammatory and insulin
desensitization processes in adipose tissue. This mechanism would likely be
more important in an early phase of insulin resistance, comparable to our 1 week
HFD group, when tissue expression of inflammatory regulators such as TNFα,
which is well known to have the effects described above on adipocytes [32, 33]
and mediates insulin resistance in vivo [1], is not yet significantly induced.What could be the primary factor causing A-SAA induction in liver? Known inducers of the A-SAA
isoforms in this tissue are the cytokines IL-1β,
IL-6, and TNFα [3, 13]. Only the latter was induced to a meaningful degree in liver in our study,
with a strong rise after 16 weeks on the HFD, however, no change compared to
the controls after one week on the HFD. Since we found no significant change in
expression of these cytokines in the other tissues investigated, it is unlikely
that either of these cytokines plays a role in the early rise of Saa1 and Saa2 in liver, and hence the elevation of plasma A-SAA.An alternative
mechanism of SAA elevation in liver is lipid overload. Evidence for a role of
lipids in A-SAA induction was provided in dietary studies with mouse
atherosclerosis models showing an induction ofSaa1, Saa2, and Saa3 in liver by atherogenic diets
containing high fat and high cholesterol [24, 25, 34]. Similarly, in humans
plasma A-SAA could be induced by a high-cholesterol diet [35]. Since the diet
used in our experiment also contained cholesterol, albeit less than in a
typical atherogenic diet, it is possible that cholesterol played an important
role here as well. However, in our study total liver cholesterol and
triglycerides were not elevated in the 1 w HFD group (data not shown),
arguing against a simple lipid overload mechanism. Clearly, more studies are
warranted to unravel the nutritional mechanism of SAA induction in humans and
animal models in order to understand its respective contributions to
atherosclerosis and impaired glucose metabolism as well as its value as a
clinical biomarker.Taken together, we show that in the commonly used DIO mouse model plasma
A-SAA levels are associated with insulin resistance. Our data indicate that
A-SAA elevation is due toSaa1 and Saa2 induction in liver but not, as
reported for humans, in adipose tissue. Also, we identify Saa3 as a strong candidate for mediating the initiation of adipose
tissue inflammation in HFD-induced obesity. Furthermore, we found that
recombinant A-SAA at physiological concentrations regulates gene expression in
cultured adipocytes in a fashion similar to insulin resistant adipose tissue,
suggesting that A-SAA might be a contributor to the development of insulin
resistance and not merely a marker of inflammation. Future experiments will be
directed towards the triggering mechanisms for SAA induction in liver and
adipose tissue, and the importance of adipose tissue derived SAA3 in provoking
inflammatory responses and insulin resistance.



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*Source: 230837-2008-06-22.xml*