New CR vs Alzheimer's Rodent Study

[Guest guest]

Hi All,

It was previously written:

>

>Hot on the heels of this report:

>

>http://lists.calorierestriction.org/cgi-bin/wa?

A2=ind0412 & L=crsociety & P=R34529

>

>((1) below), a new FASEB Journal study (2) confirms the result and

gives

>us new insight into the mechanism. The original paper noted that

>

> " We report that a CR dietary regimen prevents Abeta peptides

generation

>and neuritic plaque deposition in the brain of a mouse model of AD

>neuropathology through mechanisms associated with promotion of

>anti-amyloidogenic alpha-secretase activity. "

>

>The deal here: the body routinely makes a substance called " amyloid

>precursor protein " (APP), whose normal physiological function is

>unknown. Beta-amyloid -- the gunk that forms the famous amyloid

plaques

>-- is formed when APP is snipped free of the membrane by two

enzymes:

>beta and gamma secretase. Alpha-secretase also snips APP , but the

>resulting peptide is not pathogenic.

>

>This means that CR is not (or not only) protecting neurons from the

>damage of beta-amyloid leading to cell lysis and plaque formation,

but

>is actively reducing beta-amyloid formation. " Study findings

support

>existing epidemiological evidence indicating that caloric intake may

>influence risk for AD and raises the possibility that CR may be used

in

>preventative measures aimed at delaying the onset of AD amyloid

>neuropathology. " (2)

>1. Nilay V. Patel, Marcia N. Gordon, E. Connor, A. Good,

> W. Engelman, Jerimiah Mason, G. , Todd E.

>Caloric restriction attenuates Abeta-deposition in Alzheimer

transgenic

>models

>Neurobiology of Aging

>Article in Press, Corrected Proof

>http://dx.doi.org/10.1016/j.neurobiolaging.2004.09.014

>

>2. Caloric restriction attenuates β-amyloid neuropathology in a

mouse

>model of Alzheimer's disease

>Jun Wang, Lap Ho, Weiping Qin, Anne B. Rocher, Ilana Seror,

>Humala, Kruti Maniar, Georgia Dolios, Rong Wang, R. Hof, and

>Giulio Pasinetti

>FASEB Journal Express Article doi:10.1096/fj.04-3182fje

>http://www.fasebj.org/cgi/content/abstract/04-3182fjev1

A related paper is below (1). It is a study done in real live people.

It and these two background papers (2, 3) for leptin and

amyloid protein are pdf-available, and (2, 3) are:

2: Fewlass DC, Noboa K, Pi-Sunyer FX, ston JM, Yan SD,

Tezapsidis N.

Obesity-related leptin regulates Alzheimer's Abeta.

FASEB J. 2004 Dec;18(15):1870-8.

PMID: 15576490 [PubMed - in process]

3: Trayhurn P, Wood IS.

Adipokines: inflammation and the pleiotropic role of white adipose

tissue.

Br J Nutr. 2004 Sep;92(3):347-55. Review.

PMID: 15469638 [PubMed - indexed for MEDLINE]

For two definitions, see:

amyloid protein saa: A serum protein believed to be a circulating

precursor to amyloid protein aa. It is present in low concentrations

in normal sera, but found in much higher concentrations in sera of

older persons and in patients with amyloidosis or with diseases known

to predispose to amyloidosis. Very high levels of this protein have

been reported during acute inflammatory episodes. Antisera to amyloid

protein aa cross-react with protein saa.

leptin <protein> Product (16 kD) of the ob (obesity) locus. Found

in plasma of mouse and man: reduces food uptake and increases energy

expenditure.

Here now is the new pdf-available CR/leptin/amyloid protein

paper. It is hoped that the format of the Fig. 4 data presentation

is presentable.

1. Diabetologia. 2005 Feb 24; [Epub ahead of print]

Serum amyloid A: production by human white adipocyte and regulation

by obesity and nutrition.

Poitou C, Viguerie N, Cancello R, De Matteis R, Cinti S, Stich V,

Coussieu C, Gauthier E, Courtine M, Zucker JD, Barsh GS, Saris W,

Bruneval P, Basdevant A, Langin D, Clement K.

Abstract

Aims/hypothesis The acute-phase proteins, serum amyloid As (SAA),

are precursors of amyloid A, involved in the pathogenesis of AA

amyloidosis. This work started with the characterisation of systemic

AA amyloidosis concurrent with SAA overexpression in the subcutaneous

white adipose tissue (sWAT) of an obese patient with a leptin

receptor deficiency. In the present study a series of

histopathological, cellular and gene __expression studies was

performed to assess the importance of SAA in common obesity and its

possible production by mature adipocytes.

Materials and methods Gene __expression profiling was performed in

the sWAT of two extremely obese patients with a leptin receptor

deficiency. Levels of the mRNAs of the different SAA isoforms were

quantified in sWAT cellular fractions from lean subjects and from

obese subjects before and after a very-low-calorie diet. These values

were subsequently compared with serum levels of SAA in these individu

als. In addition, histopathological analyses of sWAT were performed

in lean and obese subjects.

Results In sWAT, the __expression of SAA is more than 20-fold higher

in mature adipocytes than in the cells of the stroma vascular

fraction (p<0.01). Levels of SAA mRNA __expression and circulating

levels of the protein are sixfold (p<0.001) and 3.5-fold (p<0.01)

higher in obese subjects than in lean subjects, respectively. In lean

subjects, 5% of adipocytes are immunoreactive for SAA, whereas the

corresponding value is greater than 20% in obese subjects. Caloric

restriction results in decreases of 45–75% in levels of the

transcripts for the SAA isoforms and in circulating levels of the

protein.

Conclusions/interpretation The results of the present study indicate

that SAA is expressed by sWAT, and its production at this site is

regulated by nutritional status. If amyloidosis is seen in the

context of obesity, it is possible that production of SAA by

adipocytes cou ld be a contributory factor.

PMID: 15729583 [PubMed - as supplied by publisher]

Keywords Adipocyte - Amyloidosis - Calorie restriction - Gene

__expression profiling - Leptin receptor - Obesity - Serum amyloid A -

Stroma vascular fraction - Very-low-calorie diet

Abbreviations FDR False discovery rate - LEPR Leptin receptor -

SAA Serum amyloid A - SAM Significance analysis of microarray -

SVF Stroma vascular fraction - sWAT Subcutaneous white adipose

tissue - VLCD Very-low-calorie-diet

-------------------------------------------------------------

Introduction

Obesity is associated with increased circulating levels of

inflammatory markers such as acute-phase proteins and cytokines [1–

5]. Increasing evidence suggests that cytokines released from adipose

tissue contribute to the development of complications related to

obesity, including metabolic [6, 7] and cardiovascular diseases [8,

9]. Indeed, adipose cells have been shown to secrete a wide number of

proinflammatory cytokines (i.e. TNF-, TGF-, interferon and

interleukins) and chemokines (macrophage inflammatory protein-1,

monocyte chemotactic protein-1) [10–13] . It was recently shown that

macrophage infiltration is increased in the adipose tissue of obese

rodents and represents an important source of inflammation in this

tissue [14, 15]. Like atherosclerosis [16], obesity may be viewed as

a metabolic as well as an inflammatory disease.

At present there is no clinical evidence of a direct relationship

between obesity and inflammation-related diseases in humans. It is

not known whether the production of inflammatory mediators by adipose

tissue in extreme forms of adiposity may increase susceptibility to

specific pathogenic processes [17]. Gene __expression profiling of

human genetic forms of obesity characterised by extreme adiposity may

help to identify the consequences of early-onset fat mass

accumulation on adipose tissue gene __expression and the development

of inflammation-related complications.

We have previously reported the clinical and biological features

associated with extreme obesity due to a leptin receptor (LEPR)

deficiency [18]. Since the identification of the mutation, the two

sisters who participated in this study have been under regular

clinical care, and adipose tissue studies were continued in order to

gain further insight into this very severe clinical situation that

was observed to progressively deteriorate.

More recently, we showed that the __expression of key

transcription factors involved in adipose tissue differentiation was

normal in these two subjects [19]. In the present investigation we

performed adipose tissue gene __expression studies on a larger scale

with no a priori hypothesis using DNA microarray technology in this

human model of extreme obesity. This led to the unexpected finding of

serum amyloid A (SAA) overexpression in subcutaneous white adipose

tissue (sWAT) in both patients, concurrent with clinical and

biological features of systemic amyloidosis. Based on these findings

we performed a series of histopathological, cellular and gene _ex

pression studies to assess for the first time the importance of SAA

in common forms of obesity and to determine whether the increased SAA

__expression was specific to extreme obesity due to LEPR deficiency.

-------------------------------------------------------------

Materials and methods

Clinical investigation protocols

The study included several groups of subjects. All subjects were

premenopausal Caucasian women. None of the subjects had a familial or

personal history of diabetes, and none were taking medication. All of

the subjects had stable body weights, were at their maximal peak

weight, and were not on a restrictive diet at entry to the different

studies (Table 1). None were involved in an exercise programme. All

clinical investigations were performed according to the Declaration

of Helsinki and were approved by the ethical committees of the Hôtel-

Dieu Hospital (Paris, France), Toulouse University Hospitals

(Toulouse, France) , the Third Faculty of Medicine (

University, Prague, Czech Republic) and Maastricht University

(Maastricht, The Netherlands). Informed consent was obtained from all

patients.

Table 1 Clinical characteristics of obese subjects and control

subjects

-------------------------------

Gene profiling Obese and control subjects Response to a very-low-

calorie diet

-----------------------------------

Patient 1 (n=1) Patient 2 (n=1) Reference pool (n=14) Control

subjects (n=27) Obese subjects (n=34) Control subjects, basal (n=9)

Obese subjects before diet (n=21) Obese subjects after diet (n=21)

Sex (ratio of women/men) 1:0 1:0 14:0 21:6 29:5 9:0 21:0

Age (year) 22 17 38±2 [24–47] 35.5±.5 39.5±1.8 32±3.2 37±1.2

Weight (kg) 180 207 146.5±7 [116–189] 65.8±5 109±4.5a 62±2.0 95±0.3a

89±2.5b

BMI (kg/m2) 74 85 51.5±2 [43–65] 23.5±0.5 38.1±1.4a 22.5±2.0

34.0±0.7a 31.0±0.8b

----------------------------------------------------------

Data are presented as means±SEM [ranges]

ap<0.001 vs baseline values of non-obese subjects

bp<0.001 vs baseline values in obese subjects (non-parametric

Wilcoxon signed-rank test)

In the first set of experiments, the transcriptional profiles of

sWAT samples from the two LEPR-mutated women (Patient 1 and Patient

2) were compared with those of sWAT samples from a group of healthy

obese women (denoted as reference pool) who had circulating leptin

levels consistent with their corpulence (range 44–98 ng/ml) (Gene

profiling column in Table 1). This group of morbidly obese women were

followed by the Department of Nutrition of the Hôtel-Dieu Hospital.

The clinical characteristics of the LEPR-mutated subjects have been

described previously [18, 19]. For Patient 1, sWAT abdominal biopsies

were performed and metabolic parameters were determined in 1997 and

2000, when the patient was 19 and 22 years old, respectively. Samples

of sWAT were collected from Patient 2 during an abdominal lipectomy

performed in 2001, when the patient was 17 years old, justified by

the mechanical consequences of extreme adiposity.

In the second set of experiments we investigated the regulation of

SAA in weight-stable commonly obese patients and in non-obese

subjects (Obese and control subjects column in Table 1), as well as

during a nutritional challenge in obese women (Response to a very-low-

calorie diet column in Table 1). The group of 34 healthy obese

patients were prospectively recruited by the Department of Nutrition

at the Hôtel-Dieu Hospital. Patients in this group were different to

the obese women constituting the reference pool mentioned above. The

27 lean subjects (control subjects) were volunteers recruited by the

Obesity Research Unit at Toulouse Hospital and the Department of

Nutrition at the Hôtel-Dieu Hospital. Biopsy specimens of sWAT were

obtained from a subset of nine control subjects (Control subjects,

basal column in Table 1).

In order to assess the changes in the __expression of the gene

encoding SAA and circulating levels of the protein after caloric

restriction, a group of 21 obese vo lunteers received a very-low-

calorie diet (3.35 kJ/day) for 28 days at the Department of Sports

Medicine and Obesity Unit at University. Biopsy specimens of

sWAT were obtained at Day 1 and Day 28 in a subset of ten patients

(Response to a very-low-calorie diet column in Table 1).

The third set of experiments aimed to analyse the effects of a

leptin injection on inflammatory markers and SAA __expression. Six

healthy male volunteers (mean age 25±5 years, mean BMI 24±1.7 kg/m2)

recruited at the Department of Human Biology at Maastricht University

(Maastricht, The Netherlands) received an injection of 60 mg of

pegylated recombinant leptin (PEG-leptin) for 3 days. Blood and sWAT

samples were obtained before and after leptin treatment (when plasma

leptin concentrations were at their peak).

...

------------------------------------------------------

Results

Overexpression of SAA in LEPR-deficient women & nbs p; The sWAT

transcriptional profiles of the two patients with extreme obesity due

to LEPR mutation were compared with those of 14 morbidly obese women

(see Table 1 for clinical data). The SAM statistical procedure was

used to control for multiple testing [22]. Of 21,471 cDNAs with

signals recovered in all experiments and an estimated FDR of <1.2%,

SAM isolated 645 cDNAs, among which 349 were overexpressed (mean fold

change 1.8, range 1.5–3.5) and 296 were underexpressed (mean fold

change 1.6, range 1.3–4.5) in the two LEPR-mutated patients. Fifty-

four percent of the differentially expressed genes were expressed

sequence tags (ESTs) or hypothetical proteins. Genes encoding immune

and inflammatory response proteins were among the main categories

represented; 10% of the differentially expressed genes were members

of this category (see Table 1 of the electronic supplementary

material). Four transcripts of plasma acute-phase proteins (SAA1,

SAA2, SAA4 and haptoglobin) were overex pressed (fold change range

2.1–3.5), and SAAs were among the most highly overexpressed

transcripts. The changes in concentrations of SAA1, SAA2 and SAA4

mRNAs were assessed using quantitative RT-PCR. The results confirmed

the microarray data for SAA2 (fold change 1.3 and 1.7) and SAA4 (fold

change 1.5 and 1.9) in Patient 1 and Patient 2, respectively.

Combined detection of SAA1 and SAA2 mRNA showed that levels were

increased (fold change 1.3) in Patient 2.

Overexpression of SAA in sWAT and systemic amyloidosis Samples

of sWAT were obtained from Patient 1 (LEPR-mutated subject) in 1997

and in 2000. Over this period of time the clinical condition of the

patient deteriorated. In 1997, Patient 1 had proteinuria levels of <1

g/24 h; however, she rapidly developed a nephrotic syndrome in 2000,

and developed renal insufficiency leading to dialysis 1 year later.

An examination of the kidney biopsy under light (Fig. 1a), by

polarised microscopy (data not shown) , and by immunohistochemistry

(Fig. 1b) using specific anti-SAA antibodies led unambiguously to the

diagnosis of kidney AA amyloidosis. Amyloid A deposits were found in

the duodenum and antrum (Fig. 1c, d). Exhaustive clinical, biological

and radiological investigations did not detect any common cause of

secondary amyloidosis (infection, neoplasia, inflammatory disease

such as arthritis) at the time of diagnosis or during the 9 years of

follow-up. The patient did not experience recurrent attacks of fever,

pain or skin lesions, or any other symptoms indicative of hereditary

periodic fever [26]. We found no evidence of mutations associated

with hereditary periodic syndromes [27, 28].

Since the mRNAs of all SAA isoforms were overexpressed in Patient

2, the younger patient, relative to the reference pool of morbidly

obese women, we performed a morphological analysis of sWAT in order

to determine whether any early features of amyloidosis and/or

inflammation were present. Immunohistochemical investigations

revealed the presence of SAA proteins within well-differentiated

adipocyte cytoplasm (Fig. 2). Morphological features of inflammation

were demonstrated on histological examination. Approximately 40% of

the small arterioles had been infiltrated by lymphocytes, and most of

them had thickened walls (data not shown). Electron microscopy

revealed features unique to this patient compared with the 16

morbidly obese control subjects. Vascular walls and all intercellular

spaces, including the narrow intercellular space between adipocytes,

had been infiltrated by collagen fibrils (Fig. 3a), and activated

fibroblasts were identified (Fig. 3b). A clear increase in collagen

arranged in thick strands surrounding areas of variable size composed

of unilocular small adipocytes was also observed (Fig. 3c–e). In the

absence of any alteration in renal function in Patient 2, no renal

biopsy has been performed for this subject as yet.

In order t o document the low-grade inflammatory status of the two

LEPR-mutated subjects, we measured plasma circulating levels of SAA

on several occasions and evaluated other inflammation-related

parameters. Plasma SAA concentrations were moderately increased in

Patient 2 (range 12–30 g/ml) and Patient 1 (range 10–60 g/ml)

compared with those in non-obese subjects (4.9±2.3 g/ml, range 0.8–

16.6 g/ml), and were within the upper range of those in subjects with

common obesity (Fig. 4a). Circulating levels of IL6 were also

increased in the LEPR-mutated patients (5.7 and 6 pg/ml) compared

with those in control subjects (2.1±1.5 pg/ml) and obese subjects

(2.8±0.2 pg/ml) (Obese and control subjects in Table 1). Levels of C-

reactive protein were also increased in Patient 2 (range 16–22 mg/l)

relative to those in non-obese subjects (1.3±0.2 mg/l) and obese

subjects (5±2.5 mg/l).

Fig. 4

Protein/mRNA--------Lean--------Ad lib obese--------CR obese

----------------------------------------------

SAA protein--------5*--------17

SAA protein------------------------16--------8***

SAA 1+2 mRNA--------1.7--------6.8**--------3.6**

SAA 2 mRNA--------0.4--------3.2**--------0.7**

SAA 4 mRNA--------0.2--------0.8**--------0.3NS

-----------------------------------------------------

Protein/mRNA

Lean

Ad lib obese

CR obese

SAA protein

5*

17

SAA protein

16

8***

SAA 1+2 mRNA

1.7

6.8**

3.6**

SAA 2 mRNA

0.4

3.2**

0.7**

SAA 4 mRNA

0.2

0.8**

0.3NS

------------------------------------------------------------------

Circulating levels of SAA and SAA mRNA __expression in sWAT of

control subjects (open bars), obese subjects from the Obese and

control subjects group in Table 1 (grey bar), obese subjects from the

Response to a very-low-calorie diet group in Table 1 before the diet

(black bars) and after the diet (striped bars). The data are

means±SEM. a Concentration of SAA in 34 obese subjects, in 27 non-

obese subjects, and in 21 obese patients before and after a very-low-

calorie diet. *p<0·01; ***p<0.0002. b __Expression of transcripts for

the SAA isoforms (SAA1+2, SAA2, SAA4) in sWAT of nine control

subjects and 10 obese women before and after a very-low-calorie diet.

**p<0.001; NS=0.1

__Expression of SAA in mature adipose cells of obese subjects We

wished to determine whether the SAA detected in adipose tissue was

produced by adipocytes or by the cells of the SVF, which include

preadipocytes, endothelial cells and monocytes/macrophages. We first

investigated the presence and relative mRNA levels of all SAA

isoforms. The __expression of all SAA isoform mRNAs was higher in

mature adipocytes than in SVF cells. The ratio of adipocyte/SVF

concentrations was 49±24 (range 10–192) for SAA1+SAA2, 70±35 (range

10–275) for SAA2, and 22±7 (range 5–62) for SAA4 (p<0.01). The

results of the gene __expression analysis were confirmed by

immunohistochemical detection of SAA in sections of sWAT. Strong

positive staining was observed in the perinuclear area where the

cytoplasm is richer in Golgi complexes and endoplasmic reticulum

(Figs. 2, 5). In contrast, all the other cell types (fibroblasts,

endothelial cells, smooth muscle cells of the vess el walls) tested

negative, clearly indicating that the SAA protein detected in adipose

tissue was derived from fully mature adipocytes.

__Expression of SAA in common forms of obesity and variation with

nutritional status We further investigated whether adipose cell SAA

overproduction was a feature specific to morbid obesity caused by

LEPR mutation or was more generally related to the excess fat mass

seen in common obesity. __Expression of SAA in nine non-obese

individuals was compared with that in ten weight-stable obese

subjects before caloric restriction (Response to a very-low-calorie

diet subjects in Table 1). As shown in Fig. 4b, the average levels of

the transcripts for the SAA isoforms were statistically different

between the obese and non-obese subjects. In all subjects,

significant correlations were found between the mRNA levels of the

three different SAA isoforms (r=0.9, p<0.001 for SAA1 vs SAA2;

r=0.84, p<0.001 for SAA2 vs SAA4; and r =0.89, p<0.001 for SAA1 vs

SAA4). Immunohistochemical analysis of sWAT samples obtained from

obese and non-obese subjects showed that, in non-obese subjects, <5%

of the adipose cells were immunoreactive for SAA, whereas the

corresponding frequency in obese subjects was >20% (Table 2).

Interestingly, the level of SAA immunoreactivity observed in morbidly

obese subjects was comparable to that observed in Patient 2 (Table

2). We also detected the SAA protein in the media in which adipose

tissue explants from three obese subjects undergoing plastic surgery

were incubated. Over a 24-h period, 31.4 ng of SAA were released per

gram of adipose tissue from explants from a morbidly obese subject

(BMI 49 kg/m2). The corresponding figures for two obese subjects, one

with a BMI of 30.9 kg/m2, the other with a BMI of 31 kg/m2, were 3.6

and 7.6 ng/g of adipose tissue, respectively.

Table 2 Quantification (%) of SAA and adipocytes in obese and

control subjects by immunohistochemistry

--------------------------------------

BMI (kg/m2) <25 30–45 >45

Number of subjects 2 5 5

SAA-positive cells (%) <2 14 22.5

------------------------------------

LEPR-mutated Patient 2 N=1 SAA-positive cells (%) 22

-----------------------------------------------

All immunohistochemistry-tested patients were grouped according to

BMI and the percentage of positive adipocytes counted

Over the course of a 4-week very-low-calorie diet that induced a

substantial decrease in body weight (Response to a very-low-calorie

diet column in Table 1) the __expression of the transcripts for all

of the SAA isoforms decreased by 45–75% (Fig. 4b). The data on SAA

__expression in sWAT were consistent with the plasma levels of SAA

(Fig. 4a). Circulating levels of SAA were significantly higher in the

34 obese patients (16.4±1.7 g/ml, range 0.9–67 g/ml) than in the 27

non-obese subjects (4.9±2.3 g/ml, range 0.8–16. 6 g/ml) (Fig. 4a;

Obese and control subjects in Table 1). There was a significant

correlation between circulating SAA levels and BMI (r=0.73, p<10–4).

No relationship was observed between the age of the subject and the

level of SAA __expression. There were no differences between non-

obese healthy men and women in terms of the concentration of

circulating SAA levels (p=0.7).

Caloric restriction significantly reduced SAA plasma levels (Fig.

4a) in the 21 obese subjects who followed the caloric restriction

protocol (Response to a very-low-calorie diet subjects in Table 1).

In addition, we observed a significant correlation between the amount

of weight loss and the change in circulating levels of SAA in this

group (r=0.55, p=0.01).

SAA __expression after leptin administration We next studied the

influence of the injection of leptin on SAA __expression in sWAT. To

avoid the confounding effect of obesity-induced leptin resistance,

leptin was administered to six healthy subjects at a concentration

that would produce a supraphysiological blood level of leptin. After

3 days of treatment, the mean serum leptin concentration increased

from 3±1 mol/l to 2112±186 mol/l (p<0.016); however, the __expression

of SAA1+2, SAA2 and SAA4 did not change significantly (ratio of

after/before leptin 0.83±0.1, 0.93±0.3 and 0.93±0.15, respectively,

p>0.05). The measurements of circulating levels of SAA also showed no

significant change (p=0.13).

------------------------------------------------------------

Discussion

The presence of AA amyloidosis together with SAA overexpression in

the sWAT of a subject with extreme adiposity due to a LEPR mutation

prompted a series of investigations, the results of which indicate

that SAA is expressed by adipocytes and is regulated by nutritional

changes. This suggests that SAA could contribute to the systemic

complications of obesity in humans. The SAA proteins are a fam ily of

apolipoproteins that were, until now, thought to be mainly expressed

in hepatocytes. They may be involved in cholesterol transport and in

the early response to injury. The SAAs are precursors of amyloid A, a

constituent of the amyloid fibrils that are involved in AA

amyloidosis [29, 30]. This form of amyloidosis is a complication that

is associated with many inflammatory conditions (including rheumatoid

arthritis and neoplasia). Amyloid deposits have also been found in

some chronic diseases, such as Alzheimers disease, dialysis-related

amyloidosis and atherosclerosis [30]. The causes and effects of

amyloid deposition are uncertain in these pathologies, which are all

characterised by a low-grade inflammation.

Our data from gene __expression and immunohistochemical

experiments demonstrate that SAA mRNA and protein are expressed in

human adipocytes. Other cell types found in adipose tissue do not

seem to significantly contribute to SAA __expression. These findings

are in agreement with in vitro and in vivo studies performed in

rodents. Mouse adipose tissue expresses transcripts of the genes

encoding SAA4 and SAA3, which correspond to pseudogenes in humans

[31, 32]. The __expression of SAA3 in murine 3T3-L1 adipocytes has

been shown to be strongly induced by TNF- and lipopolysaccharides in

vitro [31].

In the present study we have demonstrated that the overproduction

of SAA in adipose tissue is not exclusive to LEPR-mutated subjects as

it is also observed in obese subjects with a functional leptin

receptor, particularly in morbidly obese subjects. In addition,

weight loss produced by calorie restriction in obese subjects is

associated with reductions in levels of SAA mRNA and protein,

confirming that SAA concentrations are highly regulated by the

nutritional status of the individual. Thus, the overexpression of SAA

in LEPR-mutated subjects is due to extreme early onset adiposity as

opposed to the absence of leptin signalling. Th is was confirmed by

the lack of SAA regulation observed in normal subjects following the

administration of a supraphysiological dose of leptin. In accordance

with these results, gene __expression profiling in the adipose tissue

of ob/ob mice showed that SAA overexpression was only partially

corrected by leptin [32].

The relative contributions made by adipose tissue and the liver to

the increase in circulating levels of SAA are not known. Using

immunohistochemistry we showed that, in mature adipocytes, SAA

protein is localised in cytoplasmic regions that are very rich in

rough endoplasmic reticulum/Golgi apparatus (the cytoplasmic

organelles involved in the synthesis of secreted proteins). Electron

microscopy did not reveal any amyloid deposits in the intercellular

spaces in sWAT samples from the LEPR-mutated patients. This suggests

that, even if SAA is synthesised in adipose tissue, it could be

delivered to the systemic circulation together with SAA from other

tissue s, particularly the liver. Finally, our preliminary

observations in three obese subjects revealed that SAA protein is

released into the media in which adipose tissue explants are

incubated. Together, these observations provide further evidence that

adipose tissue contributes to the increased circulating SAA levels

observed in morbidly obese subjects.

The biological function of SAA, particularly its role in glucose

and lipid metabolism, has yet to be fully established. When

associated with HDL particles, SAA protein may impair reverse

cholesterol transport, and it has been implicated in the decreased

HDL levels observed during inflammation [33]. Transgenic mice

overexpressing SAA have been shown to have similar lipoprotein

profiles to wild-type mice [34]. In humans, circulating SAA levels

were found to be correlated with atherogenic remnant-like lipoprotein

particles in type 2 diabetic patients with coronary heart disease

[35], and with HbA1c but not with HDL cho lesterol in diabetic

subjects [36]. The Tanis protein has recently been described as a

putative receptor for SAA, and has been demonstrated to be

deregulated in diabetic obese rats [37]. It has been shown that the

__expression of skeletal muscle and adipose tissue Tanis/SelS mRNA is

positively correlated with circulating levels of SAA in humans, and

that it is induced by insulin infusion in adipose tissue from type 2

diabetic subjects. These data suggest that SAA protein and its

putative receptor could be involved in insulin signalling processes

[38]. Our microarray data did not provide any evidence for Tanis/SelS

regulation in the two LEPR-mutated patients. However, preliminary

analysis in subjects with common obesity indicates trends towards

correlations between the __expression of SAA and fasting insulin

status and HDL cholesterol levels (data not shown). These results

need to be confirmed and extended to a larger cohort.

The link between the increase of SAA in the c ontext of morbid

obesity and the presence of AA amyloidosis in LEPR-mutated subjects

is questionable. Although an early study on ob/ob mice reported the

occurrence of amyloidosis in a model of massive obesity [39], our

observations in LEPR-mutated patients appear to contradict previous

findings linking AA amyloidosis to chronically high circulating

concentrations of SAA [40]. Repetitive measurements of circulating

SAA showed that this protein never reached extremely high levels in

Patient 1. This finding is contrast to the peak acute-phase response

and the markedly elevated levels of SAA (>1000 mg/l) associated with

chronic inflammatory disease. No symptoms indicative of hereditary

periodic fever [26, 27] were observed during 9 years of regular

exhaustive clinical and biological checkups. At the present time

classic inflammation sources of AA amyloidosis can be ruled out.

Whether extreme obesity could have contributed to the pathogenesis of

AA amyloidosis by creating a low-grade inflammation through the

exacerbated production of proinflammatory molecules by adipose and

liver tissues remains to be determined.

While increased concentrations of circulating SAA are considered

to be a prerequisite for AA amyloidosis, several other mechanisms

could be important. Enlargement of fat mass around organs could

contribute to a local effect of SAA, as could dysfunction of SAA

proteolytic processes and remodelling of the extracellular matrix

[30, 41, 42]. Histopathological analysis of sWAT samples from Patient

2 revealed signs of inflammation, including fibroblast activation and

infiltration by monocyte/macrophage cells (data not shown), as well

extracellular matrix components with an unusual morphology.

Increased SAA mRNA and protein __expression in adipocytes and the

liver are associated with increased circulating levels of SAA and the

increased __expression of associated proteins in subjects with common

obesity. This suggests that amyloid deposition may be a potential

complication of morbid obesity. Secondary amyloidosis leads to

proteinuria and microalbuminuria [43], which are urinary features

seen in morbid obesity [44, 45]. Histological studies are scarce,

particularly in morbid obesity where kidney biopsy is technically

difficult. Hence, the presence of amyloidosis has not been

systematically investigated.

In conclusion, our work reveals that adipocytes are able to

express SAA mRNA and protein. In the present paper we have described

a previously unsuspected link between extreme obesity and systemic AA

amyloidosis associated with kidney dysfunction. These data suggest

that the overproduction of the SAA family of proteins in human

adipose tissue may contribute to morbidity in obese individuals. A

systematic search for amyloidosis should be carried out on sWAT

biopsies when kidney dysfunction secondary to obesity is suspected.

Al Pater.