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* Dipartimento di Medicina ed Oncologia Sperimentale, Sezione di Patologia Generale;
Dipartimento di Scienze Cliniche e Biologiche, Università di Torino;
** Istituto di Patologia Sperimentale, Università di Cagliari and
Centro CNR di Immunogenetica ed Oncologia Sperimentale, Torino.
 |
ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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KEY WORDS: • cachexia • insulin • lipoprotein lipase • lipid metabolism • cholesterol • metabolism • rats
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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).
Cancer cachexia frequently involves an impairment of both lipid and
cholesterol metabolism, with reduced adipose tissue mass and increased
plasma levels of free fatty acid
(FFA)4
and triglycerides (TG), (Beutler 1988
, Kern and Norton 1988
) and
reduced high density lipoprotein (HDL) cholesterol concentrations
(Dessì et al. 1991a
and b
, 1992
and 1994
).
The hyperlipidemia found in both human and experimental animal cancer
is generally reversible after tumor resection or clinical remission
(Spiegel et al. 1982
) and, in some cases, is associated with the
production of lipolytic factors by the tumor (McDevitt et al. 1995
).
Lipoprotein lipase (LPL), the enzyme responsible for the movement of
fatty acids from blood into adipocytes for triglyceride synthesis,
appears to play a role in the abnormalities of lipid metabolism
detected in tumor bearers (Kern et al. 1988
). In cancer hosts the
activity of LPL is decreased, and the decrement correlates with loss of
body fat (Carbò et al. 1994
, Lanza-Jacoby et al. 1984
).
A better understanding of the mechanisms responsible for the metabolic alterations associated with cancer cachexia might aid in the design of more effective therapies aimed at ameliorating the quality of life in cancer patients.
Previous data from our laboratories have shown that the above described
alterations in lipid and cholesterol metabolism also occur in rats
bearing the highly deviated ascites hepatoma, Yoshida AH130
(Carbò et al. 1994
, Dessì et al. 1995
, Tessitore et al. 1993
). Rats bearing this tumor have many features in common with human
cancer and have proved a very convenient model to study cancer cachexia
(Tessitore et al. 1993
).
This tumor is associated with a hypercatabolic state in the host,
evidenced by a rapid and progressive depletion of both skeletal muscle
and adipose tissue, that is similar to that in humans (Tessitore et al. 1987 and 1993
). The increased levels of glucagon, corticosterone, and
catecholamines in plasma and the decreased level of plasma insulin,
associated with the production of humoral mediators, such as tumor
necrosis factor-
(TNF), interleukin-1 (IL-1), and prostaglandin
E2 (PGE2), contribute to the severity of the
cachexia that accompanies tumor growth (Tessitore et al. 1993
).
Insulin, glucagon, catecholamines, and corticosterone are important in
regulating fuel metabolism. Insulin is the primary anabolic hormone
(associated with synthesis and storage of body fuels), whereas the
others have catabolic functions (the breakdown and oxidation of stored
fuels for the provision of energy in the absence of food intake). In
insulin-dependent diabetes, a wasting disease characterized by insulin
deficiency and an absolute or relative increase of counterregulatory
hormones, insulin administration can reverse the catabolic sequence and
the concomitant alterations in lipid metabolism. Human cancer cachexia
is often associated with a decreased insulin:glucagon ratio, and the
decrease in insulin was proposed as possibly being responsible for the
progressive catabolism that is characteristic of cancer cachexia
(Bartlett et al. 1994 and 1995
). Thus the question arises as to whether
insulin administration could also improve the metabolic state in
tumor-bearing individuals. We have previously demonstrated that insulin
administration to rats bearing the Yoshida AH130 hepatoma prevents the
onset of tissue protein hypercatabolism (Tessitore et al. 1994
). The
aim of the present work is to investigate whether insulin replacement
is also effective in modifying the alterations in lipid and cholesterol
metabolism observed in the AH130 hosts.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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The study was performed on male Wistar rats (Charles River, Como,
Italy), weighing ~150 g at the beginning of the treatment. They were
housed individually, maintained on a regular dark-light cycle (light
from 08:00 to 20:00), and weighed every day. Unless otherwise stated,
rats had free access to water and to a semisynthetic diet (Piccioni,
Brescia, Italy) of the following composition: 225 g protein,
45 g lipid, 620 g carbohydrate, 34 g fiber, 76 g
ash. All animals procedures were approved by the University of Cagliari
Institutional Animal Care and Use Committee. The amount of food
consumed by individually housed rats was calculated every day by
weighing the food remaining at noon. The mean of the daily food intake
of tumor bearers was measured to provide pair-fed controls with the
same amount of food. Rats were divided into six groups: controls with
free access to food (n = 4), controls with free access
to food plus insulin (daily subcutaneous injections of 107
µmol · kg body wt-1 · d-1,
n = 4) controls pair-fed to tumor-bearing rats
(n = 4), pair fed controls treated with insulin
(n = 4), tumor hosts (n = 9), and tumor
hosts treated with insulin (n = 6). Pair-fed tumor
hosts treated with insulin were not included in the study because most
of the animals died after 3 d of insulin injections. The Yoshida
ascites hepatoma cells (~108 cells/rat) were inoculated
intraperitoneally (Tessitore et al. 1987
). Insulin administration
started the day of tumor implantation and continued for 6 d
(Tessitore et al. 1994
). Just before being killed, rats were weighed
and anesthetized with diethyl ether. Blood was collected and plasma was
obtained by centrifugation (3,500 x g for 10 min at
4°C) and immediately stored at -80°C. Tumor cells were harvested
from the peritoneal cavity, their volume and cellularity evaluated,
then the cells were separated from the ascitic fluid by centrifugation
at 1,000 x g for 10 min. Liver and perirenal white
adipose tissue (WAT) were excised, weighed, frozen in liquid nitrogen,
and stored at -80°C until analysis.
DNA synthesis.
To measure DNA synthesis, Yoshida AH130 cells obtained from the tumor
host, either treated or untreated with insulin, were incubated in the
presence of 3H-thymidine as previously reported
(Dessì et al. 1992
). Tumor cells (109/L) were
placed into glass tubes containing Krebs' bicarbonate buffer and 370
kBq of 3H-thymidine (New England Nuclear; Boston, MA, 925
GBq/mmol) in an atmosphere of 95% O2/5% CO2,
and incubated at 37°C for 2 h. The cells were then recovered on
glass filters by using an automatic harvester (Flow; Irvine, Scotland),
and radioactivity was measured by a scintillation counter (Beckman;
Palo Alto, CA) using Ultima Gold as the scintillation fluid (Packard;
Meriden, CT).
Cholesterol and triglyceride synthesis.
Rates of cholesterol and triglyceride synthesis were determined by
measuring the incorporation of 14C-acetate (New England
Nuclear; specific activity 925 GBq/mmol) in both liver and AH130 tumor
cells. Livers were cut into 1 mm thick slices, and tumor cells were
processed as described above. For the assay, 500 mg of tissue slices or
2 x 107 tumor cells were placed in glass tubes
containing Krebs' bicarbonate buffer and incubated with 740 kBq or 370
kBq, respectively, of 14C-acetate at 37°C for 2 h in
an atmosphere of 95% O2/5% CO2. After
incubation, both the slices and the cells were washed twice with
phosphate-buffered solution, and lipids were extracted with
chloroform-methanol 2:1 according to Folch et al. (1957)
. After the
evaporation of the solvent, lipids were dissolved in chloroform, and
neutral lipids were separated by thin-layer chromatography (DC-Aufolien
Kiesegel 60; Merck; Darmstadt, Germany), using the solvent system
n-heptan/isopropylether/formic acid (60/40/2, v/v/v). The bands
corresponding to free and esterified cholesterol and triglycerides were
then visualized using iodine vapor and scraped into counting vials to
detect the incorporation of 14C-acetate (Folch et al. 1957
).
Analytical procedures.
To determine free and esterified cholesterol as well as triglyceride
content, total lipids were extracted as described above. The two
cholesterol fractions were measured as directed by Bowman and Wolf (1962)
, using cholesterol and cholesterol palmitate (Sigma Chemical,
St. Louis, MO) as standards, whereas triglyceride content was evaluated
by the method of Van Handel and Zilversmit (1968)
, using triolein as
standard.
DNA content was measured by the method of Boer et al. (1975)
and
protein was determined according to Lowry et al. (1951)
, using herring
sperm DNA and bovine serum albumin as standards, respectively.
Cholesterol, triglyceride, FFA, and phospholipid concentrations in plasma and ascitic fluid were estimated using enzymatic colorimetric tests obtained commercially (Boeringher, Mannheim, Germany). VLDL and LDL were isolated by precipitation with a mixture of phosphotungstic acid and magnesium ions. After standing for 10 min at room temperature, the mixtures were centrifuged at 10,000 x g for 10 min, the supernatant containing the HDL fraction was taken, and the levels of cholesterol, triglycerides, and phospholipids were determined. The precipitate containing the VLDL-LDL fraction was dissolved in 0.15 mol NaCl/L and cholesterol, triglycerides, and phospholipids were assayed as above.
Lipoprotein lipase activity.
Lipoprotein lipase activity in WAT was estimated by a modification of
the technique of Nilsson-Ehle and Ekman (1977)
. Tissues were dried to a
powder by subsequent passages in acetone and ether, then resolubilized
and used in an assay system containing 14C-triolein as
substrate; 14C-fatty acids released after a 30-min
incubation period were extracted and determined by the method of
Nilsson-Ehle and Ekman (1977)
. LPL activity was expressed as pmol of
fatty acid release/d (min/mg acetone-dried powder).
Other procedures.
Plasma glucose levels were determined using an enzymatic colorimetric test (Glu-Cinet; Sclavo; S.p.a., Siena Italy), and insulin levels were measured by radioimmunoassay using a commercially available kit, Insulin radioimmunassay (Corning, Medfield, MA).
Statistical analysis.
Significant differences induced by insulin in body weight, food intake, glucose, insulin concentrations, and plasma lipids in control rats, pair-fed rats, and AH130 hosts were determined by an overall ANOVA followed by the post-hoc Newman-Keuls test, when appropriate. The effect of insulin on cholesterol and triglyceride synthesis in AH130 cells of AH130 hosts were determined by using the Student's t-test. Differences were considered significant if P < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, P < 0.01). Insulin treatment was effective in
preventing body weight loss in tumor hosts (P < 0.01).
On the other hand, it did not modify body weight in control rats with
free access to food or in pair-fed rats (Table 1)
. The daily food
intake per rat amounted to 18–20 g for control rats with free access
to food, but gradually declined from 20 g on Day 0 to ~13 g at
Day 6 after tumor implantation. The insulin treatment restored normal
food intake in AH130 hosts, but no significant differences in control
rats given insulin with free access to food were observed (Table 1)
.
Plasma glucose concentrations did not differ in controls and AH130
hosts treated and not treated with insulin; pair-fed rats had slightly
lower glucose concentrations, whereas the pair-fed rats treated with
insulin developed severe hypoglycemia (Table 1)
. Plasma insulin
concentrations were significantly lower in tumor-bearing rats than in
controls (P < 0.01); insulin administration completely
restored normal insulin levels in AH130 hosts (Table 1)
.
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Insulin significantly enhanced both free cholesterol synthesis and
esterification in the AH130 cells (P < 0.01) without
affecting triglyceride synthesis (Table 2). Total cholesterol content in tumor cells from the untreated group was
higher than in cells from rats receiving insulin (0.19 ± 0.01 vs.
0.12 ± 0.01 µmol/106 cells). No differences were
observed in phospholipid and protein content, whereas significantly
lower triglyceride concentrations were detected only in insulin-treated
AH130 bearing rats compared to controls (0.19 ± 0.01 vs.
0.37 ± 0.01 mmol/106 cells).
|
, P < 0.01), whereas insulin administration to AH130
hosts significantly increased this value above the control levels
(P < 0.01). By contrast, hepatic triglyceride
synthesis was not affected by insulin treatment (Table 3)
.
|
, P < 0.05). TG and FFA were significantly lower in
AH130 hosts treated with insulin than in AH130 hosts, but
concentrations were still greater than in freely fed and pair-fed
controls. (P < 0.05).
|
.
The HDL fraction in AH130 tumor hosts had higher triglycerides and
lower cholesterol and protein concentrations than control and pair-fed
rats (Table 5
, P < 0.01). Triglyceride and protein concentrations
were significantly higher in AH130 host treated with insulin than in
untreated tumor-bearing rats (P < 0.01). Insulin was
effective in normalizing the latter variable, while it further
significantly enhanced triglyceride concentrations (Table 5
, P< 0.01). In the VLDL + LDL fraction, cholesterol,
triglyceride, phospholipid, and protein concentrations were
significantly higher in untreated tumor hosts than in
non-tumor–bearing groups (P < 0.01). Insulin treatment
decreased cholesterol concentration (P < 0.01), without
affecting the high triglyceride, phospholipid, and protein levels
(Table 6). In the ascites fluid, all the lipid components (cholesterol,
triglyceride, and phospholipids) were significantly increased by
insulin treatment of AH130 tumor host (P < 0.05), whereas
no effect was observed on protein concentrations (Table 7). In the ascitic HDL fraction only cholesterol was significantly greater
in insulin-treated hosts (P < 0.05). Insulin treatment also
restored the normal LPL activity in WAT, which was significantly lower
in AH130 tumor–bearing rats than in controls (Fig. 1)
.
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|
DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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). This pattern is
associated with marked hormonal perturbations, in particular increased
plasma levels of counterregulatory hormones and decreased insulin
concentrations with normal glucose levels, accompanied by alterations
of cholesterol and lipid metabolism in both tumor cells and plasma
(Dessì et al. 1992
).
Most of these metabolic characteristics, including a decreased
insulin:glucagon ratio, are commonly observed in human cancer patients
and are considered important factors leading to their early death
(Bartlett et al. 1994 and 1995
, Rofe et al. 1994
).
In the present study, the restoration of normal insulin levels in insulin-treated tumor-bearing rats, without lowering glucose levels, partially prevented most of the metabolic alterations consequent to tumor growth, including those in lipid and cholesterol metabolism as well as the dramatic reduction of food intake and the loss of body weight. In addition, although insulin treatment did not reduce mortality in tumor-bearing rats, it prolonged survival.
It is noteworthy that insulin administration appears to be effective only during pathological conditions; in fact, it does not affect metabolism in rats fed normally, supporting the hypothesis that changes in metabolism observed in tumor hosts are probably related to the biochemical phenotype of the tumor cells.
We hypothesize that the metabolic alterations found in tumor-bearing
rats may resemble a stressful situation common to other pathological
conditions. In fact, the stress system activation leads to a series of
hormonal responses designed to preserve body homeostasis and increase
chances for survival. In particular, hormonal changes occur principally
to promote the direction of energy and nutrients mainly to stressed
body sites (Chrousos and Gold 1992
). In cancer such responses, by
providing substrates more readily oxidized, i.e. glucose, (Board et al. 1995
), might help the growth and/or the survival of tumor cells. We
have previously shown that high plasma levels of catabolic hormones
were already evident at Day 1 after tumor implantation preceding the
decrease of insulin concentrations observed only after 4 d
(Tessitore et al. 1993
). These data suggest that the low levels of
insulin and the activation of catabolic hormones probably ensure
sufficient fuel for the survival of tumor cells. The high rate of
glucose import and glycolysis by tumor cells may also help to explain
why the levels of glucose were normal despite the low insulinemia and
the high gluconeogesis (Board et al. 1995
).
It is unlikely that the reduction of food intake is the only cause of
the observed decrease in insulin levels; contrary to pair-fed rats,
tumor hosts did not have low plasma glucose (Tessitore et al. 1987
),
which is considered the factor responsible for the reduction of insulin
secretion during food restriction.
We suggest that insulin, by counteracting the catabolic hormone action, limits the use of endogenous substrates, preventing the loss of adipose and lean tissue, and it preserves an adequate food intake and promotes the utilization of exogenous substrates for energy expenditure. These effects contribute to normal body weight maintenance.
In different experimental and human neoplasms, including the Yoshida
AH130 hepatoma, both high cholesterol synthesis in cancer cells and low
HDL cholesterol levels in plasma have mainly been ascribed to cell
proliferation (Dessì et al. 1991a and 1992
, Erikson et al.
1988
). Insulin administration to the AH130 hosts further enhances both
cholesterol synthesis and esterification in the tumor cells. In
addition, the treatment prevents the decrease of HDL cholesterol
levels, suggesting that the hormone increases the flux of cholesterol
from tumor cells to ascitic fluid and plasma.
Hyperlipidemia is the most prominent sign of altered lipid metabolism
in tumor bearers. The AH130 hosts have increased plasma levels of both
triglycerides and FFA associated with a profound lost of body fat and
altered adipose LPL activity (Kern and Norton 1988
).
Hypertriglyceridemia and depletion of FFA were observed in patients and
experimental animals during the growth of a wide variety of neoplasms.
Animal studies revealed that loss of fat is not caused by decreased
food intake alone because it precedes the onset of anorexia in mice and
is more severe in tumor-bearing rats than in pair-fed controls (Murase
and Inoue 1985). LPL regulates the clearance of triglycerides from the
blood to the tissues, and its activity in WAT is generally decreased in
cancer hosts, contributing at least in part to the hyperlipidemia
(Carbò et al. 1994
). In the AH130-bearing rats, insulin treatment
restores the mass of WAT and reduces the increase of both triglyceride
and FFA levels, with such effects being associated with an increased
LPL activity in WAT. Cytokines, such as TNF, which were shown to play a
role in this model system (Tessitore et al. 1993
) may inhibit LPL
activity (Carbò et al. 1994
). It is unlikely, however, that the
effect of insulin on this enzyme is exerted by modulating TNF action
because TNF plasma levels in the AH130 hosts are unaffected by the
hormone (Tessitore et al. 1994
).
These results suggest that an increased clearance of VLDL may be the main mechanism responsible for the observed decrease in plasma triglycerides. In fact, food intake and hepatic triglyceride synthesis, the two main sources of plasma triglycerides, were increased and unaffected, respectively, in insulin-treated tumor bearers. Moreover, the reduction of hypertriglyceridemia is evident in the VLDL + LDL fraction, further suggesting that increased triglyceride-rich lipoprotein clearance may be responsible for the lowering of plasma triglyceride levels caused by administration of insulin.
On the whole, these results suggest that, although insulin does not
affect tumor growth, it might play an important role in improving the
devastating condition that contributes to the death of cancer patients.
Because elevated levels of insulin-like growth factor (IGF) are
associated with an increased risk of some types of cancer (Chan et al. 1998
, Hankinson et al. 1998
), and insulin itself is a growth factor for
many breast cancer cells, caution is necessary in the use of insulin in
hormone-responsive cancers, such as those of the breast and prostate.
It is worthy to note that insulin can act only if an adequate food
intake is assured. In fact, the partial reversal of metabolic effects
caused by insulin administration is accompanied by an increased food
intake that is utilized for energy expenditure. These conclusions fit
well with the clinical observation that the association of insulin with
total parenteral nutrition improves the body composition of cancer
patients (Hunter et al. 1989
).
|
FOOTNOTES |
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1 This work was supported by the Ministero
dell'Università e della Ricerca Scientifica e Tecnologica, Roma,
CNR (Special Project ACRO), Roma, Associazione Italiana per la Ricerca
sul Cancro, Milano, and Regione Autonoma della Sardegna.
2 The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 USC section 1734 solely to indicate this fact.
4 Abbreviations used: FFA, free fatty acid; HDL,
high density lipoproteins; IGF, insulin-like growth factor; IL-1,
interleukin-1; LDL, low density lipoproteins; LPL, lipoprotein lipase;
PGE2, prostaglandin E2; TG, triglyceride; TNF,
tumor necrosis factor-; VLDL, very low density lipoproteins; WAT,
white adipose tissue.
Manuscript received May 26, 1998. Initial review completed July 15, 1998. Revision accepted November 24, 1998.
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