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Does a growth hormone (GH) receptor blockade
delay organism ageing processes?
(Czy zablokowanie receptora dla hormonu wzrostu (GH) opóźnia
procesy starzenia się organizmu?)
S Słuczanowska-Głąbowska 1,A,B,C,D , M Laszczyńska 2,C,E,F, K Piotrowska 1,B,C,D,
M Grabowska2,B, W Głąbowski 3,C, M Z. Ratajczak 1,E,F
Abstract – Ageing is a physiological process in which changes
affect many tissues and organs, including gonads and
endocrine system. It has been believed that one of the causes
of the ageing of human organism is the decrease in growth
hormone (GH) blood level. What is more, GH has been
supplemented to healthy people in order to delay that process.
That is related to direct and indirect functioning of GH, which
affects cell differentiation and proliferation, and by means of
that also the development and maturation of organism. It also
affects the development and functioning of male and female
genital systems. In female gonad, GH affects, in both direct
and indirect manner (by insulin-like growth factor – IGF-1),
the follicle recruitment, maturation (the maturation of oocyte,
granule and theca cell proliferation). It also makes the ovary
more sensitive to gonadotropins and inhibits apoptosis and
atresia of follicles.
Materials and methods. The ovary examinations were
conducted on mice with GH receptor shortage (Laron mice,
GHRKO). They showed delayed sexual maturation and
decreased ovulation number. In our histological study, the
follicles of older Laron mice (2-year-olds) were monitored at
every development stage. In the control group of mice of the
same age, follicles were not monitored and the ovary structure
was blurred, which is characteristic for ovaries after
menopause. To follow these observations, an experiment to
breed the Laron mice was successfully conducted.
The results indicate that low concentration of GH and IGF-1
leads to prolonged reproductive activity of female gonad and
is responsible for younger biological age of mice ovaries.
wpływ na proliferację i różnicowanie komórek, a więc rozwój
i dojrzewanie organizmu. Wpływa on także na rozwój i
funkcjonowanie układu płciowego zarówno męskiego jak i
żeńskiego. W gonadzie żeńskiej GH wpływa, zarówno
bezpośrednio jak i pośrednio poprzez insulinopodobny czynnik
wzrostu (IGF-1), na rekrutację pęcherzyków jajnikowych, ich
dojrzewanie (dojrzewanie oocytu, proliferacja komórek
ziarnistych i tekalnych), jak również uwrażliwia jajnik na
działanie gonadotropin oraz hamuje apoptozę i atrezję
pęcherzyków jajnikowych.
Materiał i metoda. Badania jajników przeprowadzone na
myszach z niedoborem receptora dla GH (myszy Laron,
GHRKO) wykazały opóźnione dojrzewanie płciowe i
zmniejszoną ilość owulacji. U starych myszy, typu Laron(2letnie myszy), w naszych badaniach histologicznych,
obserwowano pęcherzyki jajnikowe we wszystkich stadiach
rozwoju, natomiast u myszy kontrolnych, w tym samym wieku,
nie obserwowano już pęcherzyków, a struktura jajnika była
zatarta,
i
była
charakterystyczna
dla
jajnika
pomemopauzalnego. W wyniku tych obserwacji dokonano
eksperymentu z dopuszczeniem myszy typu Laron i otrzymano
żywe potomstwo.
Wyniki naszych badań wskazują na to, że niskie stężenie
hormonu wzrostu i IGF-1 powoduje przedłużenie czynności
rozrodczej gonady żeńskiej i odpowiada za młodszy
biologiczny wiek jajnika myszy.
Key words - growth hormone (GH) receptor blockade,
organism ageing processes
Author Affiliations:
Streszczenie – Starzenie się jest fizjologicznym procesem, w
którym zmiany dotyczą wielu tkanek i narządów, w tym gonad
i układu endokrynnego. Do tej pory uważano, że jedną z
przyczyn starzenia się organizmu jest spadek stężenia we krwi
hormonu wzrostu (GH), a nawet stosowano suplementację GH
u zdrowych osób w celu opóźnienia tego procesu. Związane
jest to z działaniem bezpośrednim i pośrednim GH, który ma
Słowa kluczowe - zablokowanie receptora dla hormonu wzrostu (GH), procesy starzenia się organizmu
1.
2.
Chair and Department of Physiology, Pomeranian
Medical University in Szczecin
Chair and Department of Histology and
Developmental Biology, Pomeranian Medical
University in Szczecin
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3.
4.
Chair and Department of Histology and Embryology,
Pomeranian Medical University in Szczecin
Stem Cell Institute at James Graham Brown Cancer
Center, University of Louisville, USA
Authors’ contributions to the article:
A. The idea and the planning of the study
B. Gathering and listing data
C. The data analysis and interpretation
D. Writing the article
E. Critical review of the article
F. Final approval of the article
71
have a better functioning nervous system [6,7]. On the
other hand, individuals with increased blood GH in
adulthood can be observed to develop a multitude of
pathological changes, especially those related to liver,
kidneys, muscles, joints and other organs. A typical
symptom is a disorder of carbohydrate levels in
organism (insulin resistance) [5, 8].
The authors assessed the morphology of selected
organs and tissues, including male and female gonads,
liver and adipose tissue in dwarf 2-year-old mice with
blocked growth hormone receptor (GHRKO, Laron
mice) [9,10], relating it to the process of ageing.
Correspondence to:
Sylwia Słuczanowska-Głąbowska , Chair and Department of
Physiology, Pomeranian Medical University in Szczecin, Al.
Powstańców Wielkopolskich 72 Str., PL- 70-111 Szczecin,
email:e-mail: [email protected]
Accepted for publication: November 15, 2014.
I. INTRODUCTION
geing is a physiological process in which changes
affect many tissues and organs, including gonads
and endocrine system. It has been believed that one
of the causes of the ageing of human organism is the
decrease in growth hormone (GH) blood level. What is
more, GH has been supplemented to healthy people in
order to delay that process. That is related to direct and
indirect functioning of GH, which affects cell
differentiation and proliferation, and by means of that
also the development and maturation of organism [1,2].
The direct influence is related to the direct interaction
between GH and GHR (growth hormone receptor). The
indirect influence is connected with the stimulation of
Insulin-like Growth Factor (IGF-1 and IGF-2) [3]. The
study on mice mutants with GH-IGF-1 axial defects
showed that the decreased activity on that axis leads to
prolonged life. The basic characteristics of these mice
are dwarfism, low blood IGF-1, increased sensitivity to
insulin, increased immunity to stress factors and
longevity [4,5].
The GH blood level changes along with age. It is
high at the early stages of life, when rapid and intense
somatic growth takes place. After the physical and
mental maturity is reached, the level drops [1]. Mature
people who were supplied with GH were observed to
lose adipose tissue and gain muscle tissue as well as
A
In a female gonad, GH affects, in both direct and
indirect manner (by insulin-like growth factor – IGF-1),
the follicle recruitment, maturation (the maturation of
oocyte, granule and theca cell proliferation). It also
makes the ovary more sensitive to gonadotropins and
inhibits apoptosis and atresia of follicles [11,12,13,14].
In a male gonad, GH facilitates the maturation of
Leydig and Sertoli cells as well as differentiation and
maturation of spermatogonia and spermatocytes – cells
that participate in spermatogenesis. What is more, GH
also affects the mobility of mature sperm cells [13, 15,
16]. Additionally, it makes male gonad more sensitive
to gonadotropins [16]. In both ovaries and testicles, GH
affects and regulates the steroidogenesis processes [15,
17, 18].
In liver, the effects of GH are related mainly to the
production of IGF-1 in hepatocytes and metabolic
processes related to carbohydrates, proteins and fats. GH
increases
the
lipolysis
processes,
intensifies
glycogenolysis and increases protein production. The
most GH receptors are located on hepatocytes [19].
In white adipose tissue, GH causes the increase in
the adipose cells and produces diabetogenic effects
(opposite to that of insulin), i.e. it reduces the transport
of glucose to cells, decreases lipogenesis processes,
intensifies lipolysis, and reduces the activity of
lipoprotein lipase (LPL)[20].
The aim of the study was to compare the
morphological structure of ovaries, testicles, liver cells
and adipose tissue cells in 2-year-old mice with GH
receptor blocked (Laron mice) to the analogical
morphological structures in wild type (WT) mice in the
control group.
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II. MATERIALS AND METHODS
The research was conducted on 2-year-old female
and male mice bred in Southern Illinois University
Medical School [21] with unlimited access to food and
water. Two types of these animals were designated for
the study: wild mice (n=22, including 11 males and 11
females) and Laron type mice (GHR-knockout mice)
(n=22, including 11 males and 11 females). The tissues
were obtained from Professor A. Bartke and courtesy of
Doctor J.J. Kopchicka, who obtained Laron mice as a
result of removing the gene for growth hormone
receptor/growth hormone-binding protein (GHR/BP)
[9]. During the section performed on these animals,
ovaries, testicles, livers and adipose tissue around the
kidneys were collected and submitted for morphological
analysis. The reference number of the permission from
the NIH Local Ethics Committee for Animal
Experimentation is 86-23 and from the Local Ethics
Committee for Animal Experimentation in Szczecin, it
is 7/2012 [21].
The tissues received were preserved in 10% buffered
formalin. After 24 hours, the tissues were dehydrated
and embedded in paraffin blocks. Then, 3-µm pieces
were cut using a HM325 microtome.
The pieces were hydrated and stained with
haematoxylin, eosin and PAS (Periodic Acid Shiff) in
accordance with standard procedures. The specimens
obtained were assessed using a BX41 Olympus light
microscope.
III.
72
(primordial, primary, secondary, antral and graafian
follicles). A decreased amount of interstitial tissue was
observed alongside isolated instances of degenerative
follicles and macrophages [21]. See Fig. 1 A, B.
The testicles of 2-year-old WT mice showed the
presence of seminiferous tubules in which all
developmental stages of spermatogenic epithelium could
be observed. The interstitial tissue was clearly visible
and it contained a multitude of Leydig cells. In an
insignificant amount of tubules, vacuoles formed in the
basis part of spermatogenic epithelium were observed.
In the case of testicles obtained from 2-year-old Laron
mice
(GHRKO),
seminiferous
tubules
had
spermatogenic epithelium at all stages of development.
The interstitial tissue was well-developed and contained
a significant amount of Leydig cells. No vacuoles in the
spermatogenic epithelium were observed. See Fig. 1 C,
D.
The morphological analysis of liver pieces from 2year-old WT mice indicated a correct lobulated structure
with clearly visible sinusoidal capillaries. Hepatocytes
were distributed in a plaque structure around the central
vein and filled with lipid droplets (2nd degree steatosis
on the Brunt scale) [22]. The droplets were classified as
microvesicles and macrovesicles. There were isolated
instances of leukocyte infiltration. Liver pieces from
Laron mice showed a correct lobulated structure with
sinusoidal capillaries clearly visible. Hepatocytes were
distributed in a plaque structure around the central vein
and not many of them showed the presence of lipid
droplets. Even if they were present, they were
predominantly microvesicles [23]. See Figure 1 E, F.
RESULTS
The morphological studies of the ovaries obtained
from 2-year-old wild type (WT) mice (mice in the
control group) showed the presence of correct surface
epithelium, blurred borderline between ovarian cortex
and medulla, and the absence of follicles at all the
developmental stages [21]. What is more, increased
amount of interstitial tissue was observed. Some ovaries
had large cysts created as a result of antral follicles
degeneration. Also inflammatory cells, including
macrophages, were present. On the other hand, the
observation of the ovaries with GH receptor
insufficiency obtained from Laron mice showed that not
only was the surface epithelium well-developed, but also
follicles were present a every developmental stage
In the adipose tissue around the kidneys in the
control group animals, predominantly white adipose
tissue cells (WAT) and could be found – there was only
an insignificant amount of brown adipose tissue (BAT).
What is more, the lymphatic system cells were present.
On the other hand, in Laron mice, the adipose tissue
around kidneys was rich in brown adipose tissue (BAT)
and had no inflammatory infiltrations [23]. See Figure
1. G, H.
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Ovaries
Figure 1 description
The histological image of the selected organs
obtained from 2-year-old wild type (WT)
mice and 2-year-old mice with GH receptor
knocked out (Laron mice): Ovaries (A and B
panels; magnification x 10), testicles (C and
D panels; magnification x10), liver (E and F
panels; magnification x 20), adipose tissue
from kidney area (G and H panels;
magnification x 20).
IT
A
B
Testes
The absence of follicles and interstitial tissue
in WT mice (A panel). Numerous follicles
(red arrows) at different stages of
differentiation in Laron mice (B panel).
Vacuoles in cells neighbouring the basis parts
of seminiferous tubules (black arrows) in WT
mice (C panel). Correct spermatogenic
epithelium without vacuolisation in the basis
part in Laron mice (D panel).
C
D
Liver
Numerous hepatocytes containing adipose
macrobubbles (blue arrows) in WT mice are
visible (E panel). Single macrobubbles visible
in hepatocytes of Laron type mice (F panel).
F
E
Adipose Tissue
Insignificant content of brown adipose tissue
(asterisk) in the tissue around kidneys in WT
mice (G panel). Significant amount of brown
adipose tissue (asterisk) in the tissue around
kidneys in Laron mice (H panel).
*
*
G
H
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IV.
DISCUSSION
The tests on animal mutant models of GH/IGF-1
axes clearly indicate the significance of the system in
organism ageing [1,4,5]. This is true regardless of the
mutant type; both mutants of spontaneous origin (such
as Ames or Snell mice) and genetically modified
animals (Laron mice with GHRKO) led to the same
conclusions. Basic characteristics of such mice include
dwarfism, low blood IGF-1, increased sensitivity to
insulin, increased immunity to stress factors and
longevity. On the other hand, transgenic mice with
overexpression of bovine growth hormone (bGH) were
observed to have shorter lifespan and increased body
mass [5]. This confirms that lower IGF-1 combined with
increased sensitivity to insulin (leading to lower
GH/IGF-1 axis activity) has a significant impact on
longevity [4, 5, 8, 13, 24].
The authors compared the morphological structure
of ovaries obtained from 2-year-old mice with GH
receptor insufficiency (Laron mice, GHRKO) to the
structure of ovaries from 2-year-old wild type mice
(control group). The structure of ovaries from Laron
mice was correct. Follicles at every developmental stage
(primordial, primary, secondary, antral and graafian
follicles), correct surface epithelium, insignificant
amount of interstitial tissue, correct blood vessels,
isolated instances of macrophages and degenerative
follicles were observed. Such a histological image of
mouse ovary is indeed characteristic, but for much
younger congeners. In their study, Slot et al. [12] noted
that 9-week-old Laron mice had more primordial and
primary follicles. Bachelot et al. [11], who studied
Laron mice aged from 6 to 28 weeks, also observed
correct morphological ovary structure that is
characteristic of young mice, with less developing
follicles and fewer ovulations even after gonadotropin
stimulation. Nevertheless, as they also observed, 18month-old congeners were capable of getting pregnant
and breeding. Other studies involving mice with GH
receptor knocked out proved Laron mice to be fertile, yet
showed that they may have irregular cycles, the number
of pre-ovulation follicles may be lower and ovulations
may be less numerous [9, 25].
In the ovaries of 2-year-old control group mice no
follicles were observed. The ovaries contained a lot of
interstitial tissue and multiple macrophages were
present. Some ovaries had large cysts formed from
degenerative follicles. Such ovary structure is
74
characteristic of old mice. In the case of women, it is
observed in the post-menopausal period [26,27]. As an
aftermath of our experiment and the observations made,
the 2-year-old Laron mice were bred, which yielded
healthy young (unpublished authorial data).
The testicles of 2-year-old WT mice showed the
presence of correct spermatogenic epithelium in most of
the tubules monitored. Only few spermatogenic tubules
had vacuoles in the basis part. The symptom is often
described as one of the first morphologically visible
signs of male gonad ageing. In subsequent phases, it
leads to the loss of genital cells and the development of
testicular atrophy [28,29]. The absence of this
phenomenon in GHRKO mice leads to the conclusion
that the morphological symptoms of male gonad ageing
are delayed in the case of the animals concerned.
In the liver pieces of the 2-year-old WT mice,
steatosis was observed – microvesicles and
macrovesicles and sporadic inflammatory infiltrations
were present. In the liver samples from Laron mice
(GHRKO), microvesicles and sporadic macrovesicles
could be observed. No inflammatory cells were noted.
Mild steatosis with no inflammatory process visible is
described as process developing with age and is not
considered a disease symptom, but im may be qualified
as a symptom of liver tissue ageing [30,31]. On the
other hand, severe steatosis of liver tissue may occur in
type 1 diabetes and correlate with the emergence of
insulin immunity [32]. The isolated instances of
inflammatory cell agglomerations are also a sign of
ageing and do not indicate a disease in progress [30,31].
In GHRKO animals, no morphological changes in cells
related to age (such as testicular swelling indicating
increased cell ploidy) were observed.
The studies of older (2-year-old) mice with GH
insufficiency (Laron mice, GHRKO) showed increased
amount of brown adipose tissue (BAT) around the most
important organs (heart, kidneys) as compared to the
occurrence of the tissue in such areas in the old animals
from the control group. BAT has a very good blood
supply and transmits heat to the most vital organs
[33,34]. In the ageing process, BAT is gradually
replaced with white adipose tissue (WAT). An increased
BAT level has a positive correlation with higher life
expectancy and protects against developing insulin
immunity [35].
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V.
CONCLUSIONS
The results of the study show that low GH and IGF-1
levels decrease the biological age of ovaries, testicles as
well as liver and adipose tissue. It may be one of the
factors responsible for slowing down ageing processes
and prolonging organism’s life.
VII. REFERENCES
[1] Bartke A. Growth hormone and aging: A challenging
controversy. Clin Interv Aging 2008; 3: 659-665.
[2] Corpas E, Harman SM, Blackman S. Human growth
hormone and human aging. Endocr Rev 1993; 14:
20-39.
[3] Perrini S, Laviola L, Carreira MC, Cignarelli A,
Natalicchio A, Giorgino F. The GH/IGF1 axis and
signaling pathways in the muscle and bone:
mechanisms underlying age-related skeletal muscle
wasting and osteoporosis. J Endocrinol 2010;
205:201-210.
[4] Bartke A, Brown-Borg H, Mattison J, Kinney B,
Hauck S, Wright C. Prolonged longevity of
hypopituitary dwarf mice. Exp Gerontol 2001; 36:
21-28.
[5] Bartke A. Consequences of growth hormone (GH)
overexpression and GH resistance. Neuropeptides
2002; 36: 201-208.
[6] Giordano R, Bonelli L, Marinazzo E, Ghigo E, Arvat
E. Growth hormone treatment In human ageing:
benefits and risks. Hormones 2008; 7: 133 – 139.
[7] Harman SM, Blackman. Use of growth hormone for
prevention or treatment of effects of aging. J
Gerontol 2004; 59: 652 – 658.
[8] Bartke A. Growth hormone, insulin and aging: The
benefits of endocrine defects. Exp Gerontol 2011;
46: 108-111.
[9] Zhou Y, Xu BC, Maheshwari HG et al. A
mammalian model for Laron syndrome produced by
target disruption of the mouse growth hormone
receptor/binding protein gene (Laron mouse). Proc
Natl Acad Sci 1997; 94: 13215-13220.
[10] Laron Z. Laron syndrome (primary growth hormone
resistance or insensitivity): the personal experience
1958-2003. J Clin Endocrinol 2004; 89: 1031 –
1044.
[11] Bachelot A, Monger P, Imbert-Bolloré P i wsp.
Growth Hormone is required for ovarian follicular
growth. Endocrinology 2002; 143: 4104-4112.
[12] Slot KA, Kastelijn J, Bachelot A, Kelly PA, Binart
N, Teerds KJ. Reduced recruitment and survival of
primordial and growing follicles in GH receptordeficiant mice. Reproduction 2006; 131: 525-532.
75
[13] Chandrashekar V, Bartke A, Coschigano KT,
Kopchick JJ. Pituitary and testicular function in
Growth Hormone Receptor Gene Knockout Mice.
Endocrinology 1999; 140: 1082-1088.
[14] Silva JRV, Figuiredo JR, Van den Hurk R.
Involvment of growth hormone (GH) and insulinlike growth factor (IGF) system in ovarian
folliculogenesis. Theriogenology 2009; 71: 11931208.
[15] Sirotkin AV. Control of reproductive processes by
growth hormone: extra- and intracellular
mechanisms. Vet J 2005; 170: 307 – 317.
[16] Childs GV. Growth Hormone Cells as cogonadotropes: partners in the regulation of
reproductive system. TEM 2000; 11: 168 – 175.
[17] Hull KR, Harvey S. Growth hormone: a reproductive
endocrine-paracrine regulator? Rev Reprod 2000; 5:
175-182.
[18] Sirotkin AV. Growth factors controlling ovarian
functions. J Cell Physiol 2011; 226: 2222 – 2225.
[19] Takahashi Y. Essential roles of growth hormone
(GH) and insulin – like growth factor-I (IGF-I) in
the liver. Endocrine J 2012; 59: 955 – 962.
[20] Nam SY, Lobie PE. The mechanism of effect of
growth hormone on preadipocyte and adipocyte
function. Obes Revs 2000; 1: 73-86.
[21] Słuczanowska-Głąbowska S, Laszczyńska M,
Piotrowska K . i wsp. Morphology of ovaries in
laron dwarf mice, with low circulating plasma levels
of insulin-like growth factor -1 (IGF-!), and bovine
GH-transgenic mice, with high circulating plasma
levels of IGF-1. J Ovarian Res 2012; 5: 18.
[22] Brunt EM, Tiniakos DG. Histopathology of
nonalcoholic fatty liver disease. World J
Gastroenterol 2010; 16: 5286-5296.
[23] Piotrowska K, Borkowska SJ, Wiszniewska B i wsp.
The effect of low and high plasma levels of insulinlike growth factor -1 (IGF-1) on the morphology of
major organs: studies of Laron dwarf and bovine
growth hormone transgenic (bGHTg) mice. Histol
Histopathol 2013; 28:1325 – 1336.
[24] Berryman DE, Christiansen JS, Johannsson G,
Thorner MO, Kopchick JJ. Role of the GH/IGF-1
axis in lifespan and health span: Lessons from
animal models. Growth Hormone & IGF Research
2008; 18: 455–471.
[25] Danilovich N, Wernsing D, Coschigano KT,
Kopchick JJ, Bartke A. Deficits in female
reproductive function in GH-R-KO mice; role of
IGF-1. Endocrinology 1999; 140: 2637 – 2640.
[26] Davis BJ, Dixon D, Herbert RA. Ovary, oviduct,
uterus, cervix and vagina. W: Pathology of the
mouse. Maronpot RR (red.). Cache River Press;
1999: 409-443.
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HEALTH,
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AND
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RESCUE
● ●No.4/2014
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HEALTH,
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AND
MEDICAL
RESCUE
2/2014 ● ●
[27] Laszczyńska M, Brodowska A, Starczewski A,
Masiuk M, Brodowski J. Human postmenopausal
ovary – hormonally inactive fibrous connective
tissue or more? Histol Histopathol 2008; 23: 219 –
226.
[28] Takano H, Abe K. Age - Related Histologic changes
in the Adult Mouse Testis. Arch histol jap 1987; 5:
533-544.
[29] Radovsky A, Mitsumori K, Chapin RC. The Male
Reproductive Track. W: Maronpot RR (red.)
Pathology of the Mouse. Cache River Press 1999;
381-405.
[30] Singh P, Coskun ZZ, Goode C, Dean A, ThompsonSnipes L, Darlington G. Lymphoid neogenesis and
immune infiltration in aged liver. Hepatology
2008;47: 1680-1690.
[31] Harada T, Enomoto A, Boorman GA, Maronpot RR.
Liver and gallbladder. W: Maronpot RR (red.).
Pathology of the Mouse. Cache River Press 1999;
119-171.
[32] Bulum T, Kolarić B, Duvnjak L, Duvnjak M.
Nonalcoholic Fatty Liver Disease Markers Are
Associated with Insulin Resistance in Type 1
Diabetes. Dig Dis Sci 2011; 56: 3655-3663.
[33] Cannon B, Nedergaard J. Brown adipose tissue:
function and physiological significance. Physiol Rev
2004; 84: 277-359.
[34] Cousin B, Cinti S, Morroni M. et al. Occurrence of
brown adipocytes in rat white adipose tissue:
molecular and morphological characterization. J Cell
Sci 1992; 103: 931-942.
[35] Mattson MP. Perspective: Does brown fat protect
against diseases of aging? Aging Res Rev 2010; 9:
69-76.
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