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Department of Veterinary Physiology, Veterinary Medical Science, University of Tokyo (A.I., K.-T.C., Y.F., M.N., M.T.), 1–1-1 Yayoi, Bunkyo-ku, Tokyo 113; and the Department of Anatomy, Osaka Prefecture University College of Agriculture (Y.M., F.S.), 1-1 Gakuen-cho, Sakai, Osaka 591, Japan
Address all correspondence and requests for reprints to: Dr. Michio Takahashi, Department of Veterinary Physiology, Veterinary Medical Science, University of Tokyo, 1–1-1 Yayoi, Bunkyo-ku, Tokyo 113, Japan. E-mail: amtaka{at}hongo.ecc.u-tokyo.ac.jp
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Abstract |
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Investigations of transgenic mice secreting foreign GH in extrapituitary sites have contributed to the understanding of the effects of chronic exposure of high levels of GH (9, 10, 11, 12, 13). We have produced two lines of transgenic rats from two different founders, integrating different copy numbers of the transgene (14); one line displayed relatively higher levels of serum human (h) GH, extended body length, and increased weight gain (high line), whereas the other had relatively lower levels of serum hGH and normal body length, but was severely obese (low line). Reproductive phenotypes in these lines of transgenic female rats were completely different from those in normal females (15). Due to a decrease in the number of somatotrophs, endogenous GH secretion in both lines of transgenic rats was inhibited. Unlike serum hGH levels in high line rats, serum hGH levels in low line rats were low (14). These low line animals developed symptoms characteristic of obesity and insulin resistance around 12 weeks of age.
Therefore, in the present study, the development of these symptoms was
studied by measuring plasma glucose, insulin, triglyceride, and FFA
levels 5 weeks before and 5 weeks after the onset of insulin
resistance. The adipose tissue of these transgenic rats was
characterized by assessing gene expression of lipoprotein lipase and
glycerol-3-phosphodehydrogenase (G3PDH) at 17 weeks of age. In
addition, plasma tumor necrosis factor-
(TNF) levels were also
measured. Insulin resistance was further confirmed by glucose tolerance
tests and treatment with an antidiabetic agent, thiazolidinedione.
Finally, at 17 weeks of age, these transgenic rats were treated with
recombinant hGH for 1 week to evaluate the importance of pulsatile GH
secretion for diabetogenic events in metabolism.
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Materials and Methods |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Four or five each of the male transgenic rats and their nontransgenic male littermates were killed at 4, 7, 12, and 17 weeks of age; body weight, nose to tail length, and weight of the epididymal fat pad were recorded. Another group of male transgenic rats (four to eight rats each) and their littermates were killed at 7, 12, and 17 weeks of age, and plasma glucose, insulin, triglyceride, and FFA were measured with commercial kits (Wako, Tokyo). Plasma insulin was determined using a commercial RIA kit (Amersham Life Science, Tokyo, Japan).
Analysis of the volume and number of epididymal fat cells
Adipose tissue was obtained from the epididymal fat pad of the
transgenic and control rats or their nontransgenic littermates at 17
weeks of age, fixed in 10% neutral buffered formalin, dehydrated in a
graded series of alcohol solutions, and embedded in paraffin. The
tissues were cut sagittally into 6-µm thick sections. Measurements of
adipose tissue volume, cell size, and number of cells were based on the
methods reported by Sasaki et al. (16).
Troglitazone treatment and blood sampling
The male transgenic rats and control rats at 17 weeks of age
were treated for 1 week with troglitazone (an analog of
thiazolidinedione, Sankyo Co., Tokyo, Japan) at a concentration that
results in approximately 150 mg/kg·day. To accustom them to the
handling procedures, animals were carefully patted daily. On the final
day of treatment, the tip of the tail was cut using a razor blade while
animals were conscious, and oozing blood was drawn into four or five
hematocrit tubes (50 µl). The tubes were centrifuged, and the plasma
was collected and stored at -20 C until assayed for glucose, insulin,
triglyceride, and FFA.
The ip glucose tolerance test
Male transgenic and control rats at 7 and 15–17 weeks of age
were fasted overnight for 12 h and received an ip injection of 0.5
g/kg BW D-glucose (20% solution). Blood samples were
obtained from the tail vein without anesthesia, and glucose
concentrations were determined before (0 min) and 15, 30, 60, 90, 120,
and 150 min after the glucose injection.
Preparation of complementary DNA (cDNA) probes for lipoprotein
lipase (LPL) and G3PDH
cDNA probes were synthesized by RT-PCR using the following
oligonucleotide primers: LPL forward primer
(5'-CGCGCTCTAGTCCTCTGACG-3'; rat LPL cDNA; nucleotides 64–83) and LPL
reverse primer (5'-TTCTTCCTCCAGCCAGTTGA-3'; nucleotides 590–571),
G3PDH forward primer (5'-TGAAGGTCGGTGTCAACGGATTTGGC-3'; rat G3PDH cDNA;
nucleotides 35–60) and G3PDH reverse primer
(5'-CATGTAGGCCATGAGGTCCACCAC-3'; nucleotides 1017–994).
Total RNA was extracted from the adipose tissues of mature intact male rats by the method described previously (17). In brief, 1 µg total RNA was incubated at 42 C for 60 min in a final volume of 20 µl with 50 U cloned Moloney murine leukemia virus reverse transcriptase (Gene AmpTM RNA PCR kit, Perkin-Elmer, Norwalk, CT). After reaction, 20 µl of the RT mix were added to 100 µl of the PCR solution (containing 5 U AmpliTaq DNA polymerase) and each of the forward and reverse primers (50 pmol each). The PCR conditions were as follows: denaturation, annealing, and elongation at 94, 60, and 72 C, respectively, for 1 min each for 35 cycles, followed by a 10-min final extension at 72 C. PCR products were analyzed by electrophoresis on a 1% agarose Tris-acetate EDTA (buffer) gel. cDNA fragments of LPL and G3PDH were ligated into T7Blue T-vector (Novagen, Madison, WI), transformed in NovaBlue strain Escherichia coli, cloned, and proliferated. The plasmid DNA was recovered from each transformant. The insert of each clone was sequenced, and nucleotide sequences were confirmed. For hybridization probes, EcoRI/PstI fragments of each clone were purified and radiolabeled using the random primed method.
Northern hybridization for LPL and G3PDH
An aliquot (10 µg) of the total RNAs from each transgenic or
control male rat at 17 weeks of age was denatured at 65 C for 15 min
and electrophoresed in a 1% 3-(N-morpholino)propanesulfonic
acid (MOPS)-formaldehyde agarose gel, and the bands of rRNAs were
detected by ethidium bromide staining (2 µg/ml). After washing with
distilled water, the agarose gel was photographed, and the RNA was
transferred to a nylon membrane (Biodyne B Membrane, Pall BioSupport,
Port Washington, NY). The membranes were prehybridized with salmon
sperm DNA and then hybridized with the specific probes described above.
The membrane washed once with 2 x SSC (standard saline
citrate)-0.1% SDS for 10 min at room temperature, twice with 0.5
x SSC-0.1% SDS for 5 min at 45 C, and then twice with 0.1 x
SSC-0.1% SDS for 5 min at 45 C. The transfer membrane was exposed to
x-ray film (Eastman Kodak, Rochester, NY) for 72 h at -70 C. The
pictures of ribosomal RNA (rRNA) and autoradiographs were scanned
(GT-6500, Epson, Tokyo, Japan), and the relative intensity of each band
was analyzed using NIH imaging software.
LPL activity and TNF concentration
Total LPL activity in the fat pad of transgenic or control male
rats at 17 weeks of age was measured as previously described (18).
Plasma TNF concentrations in transgenic or control male littermates
(n = 5) at 12 weeks of age were measured by ELISA with a
commercial kit (BioSource International, Camarillo, CA). The standard
curve for TNF concentrations was linear between 2.3–150 pg/ml.
hGH treatment
One group of 12-week-old transgenic rats was injected ip with
recombinant hGH (Pharmacia, Uppsala, Sweden) at a dose of 100
µg/rat·0.3 ml vehicle, four times daily at 4-h intervals between
0700–1900 h for 7 days. Another group of 12-week-old transgenic and
control rats was treated with vehicle only for 7 days. Changes in the
plasma hGH concentration after the first single injection of hGH or
vehicle to transgenic rats were determined by RIA. The mean daily body
weight gain during the 7-day treatment period was monitored. Tissue
samples were collected 3 h after the final injection, and weights
of the liver, epididymal fat pad, and kidney fat pad were recorded.
Statistical analysis
The data presented in Tables 1–4
, and
Figs. 2–4 were analyzed
by ANOVA followed by Student’s t test. The data presented
in Fig. 6 were analyzed by ANOVA followed by Duncan’s multiple range
test. In all statistical tests, the difference was considered
significant at P < 0.05.
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), although they
had the same head to tail length as their nontransgenic littermates
(Table 1
). Body weights of the transgenic
rats did not differ from those of the controls at 7 or 12 weeks of age,
but increased thereafter and were significantly heavier than those of
the controls by 17 weeks of age (Table 1). The higher rate of body
weight gain due to fat accumulation continued through 25 weeks of age,
as shown in Fig. 1.
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). Growth of the
epididymal fat pad between 4–17 weeks was much faster in the
transgenic rats (Fig. 2B). The average size of adipocytes at 17 weeks
of age (Table 2) was approximately 3
times that in the controls and was calculated to be 5.6-fold in volume
larger in the transgenic rats. The numbers of cells per adipose tissue
pad were similar in the two groups.
Plasma concentrations of lipids and TNF, and glucose-insulin
homeostasis
At 7 weeks of age, plasma glucose and insulin levels in the male
transgenic rats were not different from those in their nontransgenic
littermates. At 12 weeks of age, however, the transgenic animals had
significantly higher glucose and insulin levels. Plasma triglyceride
and FFA levels in the transgenic rats were also higher than those in
their nontransgenic littermates, but FFA levels at 7 weeks of age were
not significantly different. Plasma TNF levels in the transgenic
rats at 12 weeks of age were lower than those in controls (Table 3).
The results of an ip glucose tolerance test are presented in Fig. 3. After a 12-h fast, the transgenic rats
at 7 weeks of age were not hyperglycemic (Fig. 3, inset),
but at 15–17 weeks of age, transgenic rats were modestly, but
significantly, hyperglycemic compared with their littermates. In the
transgenic rats 90, 120, and 150 min after an ip glucose load, elevated
plasma glucose remained at a significantly higher level than that in
the nontransgenic controls.
LPL activity and its gene expression, and G3PDH gene expression in
adipose tissue
LPL activity per whole epididymal fat pad activity in the
transgenic rats (729 ± 31 U/tissue) was also not significantly
different from that in their nontransgenic littermates (812 ± 42
U/tissue). LPL gene expression in the adipose tissue of the male
transgenic rats or their nontransgenic littermates at 17 weeks of age
was quantified by Northern hybridization (Fig. 4A). LPL gene expression in the
transgenic rats was not significantly different from that in normal
rats when expressed after normalization with quantified rRNA (Fig. 4B).
G3PDH gene expression in the adipose tissue of the male transgenic or
their nontransgenic littermates at 17 weeks of age was quantified by
Northern hybridization (Fig. 4, C and D). G3PDH gene expression in the
male transgenic rats was about 5-fold higher than that in their
nontransgenic littermates when expressed after normalization with
quantified rRNA.
Effects of troglitazone on glucose and lipid metabolism
The male transgenic rats were treated with troglitazone (an
antidiabetic agent enhancing postinsulin receptor signal transduction)
for 1 week from 17 weeks of age. Plasma glucose (nonfasting state),
insulin, triglyceride, and FFA concentrations were determined at the
end of the treatment and are summarized in Table 4. Plasma glucose, insulin, and
triglyceride concentrations decreased significantly by 16%, 36%, and
71%, respectively, whereas only FFA levels increased significantly by
51%.
Effects of repeated hGH injections on the male transgenic
rats
The male transgenic rats were treated repeatedly with recombinant
hGH to simulate pulsatile changes in peripheral GH concentrations. The
experimental schedule and a peripheral hGH profile after the first
single injection are shown in Fig. 5, A
and B. A single ip injection of hGH at a dose of 100 µg/rat resulted
in a peak serum hGH level of approximately 120 ng/ml at 60–90 min
after the injection; the concentration returned to basal levels within
3–4 h.
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). On the
other hand, the treatment slightly increased the relative weight of the
liver, but the increase was not significant. The treatment
significantly reduced 1-week body weight gain in the male transgenic
rats, rendering it comparable to that in nontransgenic littermates
(Fig. 6), although daily food intake did not significantly change (data
not presented). |
Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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10 ng/ml plasma) throughout the day
(14). Thus, peripheral GH concentrations in these transgenic rats
remained low during the experimental period. Nonetheless, somatic
growth in the male transgenic rats, as assessed by nose to tail length
and liver weight, occurred in an entirely normal way. This indicated
that the low GH levels in the peripheral circulation were sufficient to
support normal somatic growth. Various strains of GH transgenic mice
have been established and their phenotypes reported. With a few
exceptions, a marked increase in body size (nose to tail length) has
been reported (19, 20). The difference in body size among GH
transgenic animals seems to result from the difference in peripheral
levels of both endogenous and foreign GH. The GH gene, in contrast to other genes, linked to the whey acidic protein promoter in transgenic mice leads to expression in tissues other than the mammary gland, particularly in brain glial cells and to a limited extent in liver (22, 23). If this were also the case in the rat transgenic model, then ectopic expression of hGH in the brain would lead to high levels of pituitary exposure and result in inhibition of somatotroph development, as observed in this study. Thus, in addition to GH, the profiles of IGF and IGF-binding protein levels in cerebrospinal fluid need to be evaluated to obtain deeper insight into various phenotypes expressed in these obese transgenic rats.
It is well documented that obesity is frequently associated with hyperinsulinemia, hyperglycemia, and/or hyperlipidemia, the typical symptoms of insulin resistance (21, 24, 25). The transgenic rats in this study deposited excess body fat as they grew and manifested all of these symptoms together with impaired glucose tolerance. It is a matter of controversy whether obesity is the cause or the result of insulin resistance (26, 27). The order of the emergence of these symptoms offers an important clue (25). In this study, the significant fat deposition in the epididymal fat pad was already evident at 4 weeks of age, whereas the onset of hyperinsulinemia, hyperglycemia, and hyperlipidemia was delayed until 12 weeks of age. Thus, the present results suggest that obesity is the cause rather than the result of insulin resistance.
Troglitazone, an analog of thiazolidinedione, given orally has been shown to decrease blood insulin, glucose, triglyceride, and FFA concentrations in various insulin-resistant and obese animal models, such as the KKAy mouse, the obese (ob/ob) mouse, and the Zucker fatty rat (28). Recently, analogs of thiazolidinedione have been intensively studied and shown to bind to the peroxizome proliferator activator receptor, inducing adipocyte differentiation and enhancing transcription of many genes, including an ob (leptin) gene (29, 30, 31). Although ob/ob mice express a mutant leptin molecule, troglitazone is still effective in ameliorating the insulin-resistant symptoms; therefore, leptin is not the only gene product responsible for symptoms of insulin resistance. As troglitazone treatment improved most of the symptoms of insulin resistance in the transgenic rats used here, it appears that a lack of insulin responsiveness was the cause of the symptoms.
Troglitazone given genetically obese animals has been shown to decrease
blood FFA (28). In those animals, it appears that an elevation of FFA
is due to insulin insensitivity of the adipose tissue, and thus,
troglitazone is effective in restoring normal FFA levels. In the
present study, however, troglitazone was totally ineffective in
decreasing elevated FFA levels. Thus, it appears that the origin of
high plasma FFA levels in the transgenic rats was not related to
insulin resistance and hence differs basically from that in other
mutant animal models for obesity. In obese Zucker rats, fat
accumulation is thought to be mainly attributable to an elevated LPL
activity in the adipose tissue (32). Recent reports indicate that
TNF production from adipocytes is increased in those mutant animal
models, which at least in part causes insulin resistance (33, 34, 35).
However, gene expression and enzymatic activity of LPL in the adipose
tissue from low line rats in this study were not different from those
in the normal rats, and blood TNF levels in the transgenic rats at
12 weeks, the age at which hyperinsulinemia became evident, were lower
than those in normal rats. Thus, TNF did not play a role in the
symptoms of insulin resistance, suggesting that insulin resistance in
the transgenic rats must be different from that in other experimental
models.
It has been suggested that FFA, glycerol concentrations, and their turnovers are proportional to an increase in the adipose tissue mass without deteriorating glucose tolerance, and that high FFA induces insulin resistance in liver and muscle (26). Thus, it is also possible that an elevation of FFA levels in the transgenic rats may not result from insulin resistance of the adipose tissue, but it may be the cause of obesity. In addition, the level of G3PDH messenger RNA (mRNA) has previously been used as a marker of insulin sensitivity, as G3PDH mRNA levels in cultured neonatal rat adipocytes are regulated by insulin (36). In the adipose tissue from the transgenic rats at 17 weeks of age, G3PDH gene expression was strongly enhanced, but it may be premature to conclude that the adipose tissue in the transgenic rats was sensitive to insulin. It is not clear at present whether G3PDH gene expression can be used as an in vivo marker for insulin susceptibility of adipocytes in adult animals. It is possible that other factors may also affect G3PDH gene expression in adopocytes in vivo.
The pulsatile and sexually differentiated secretory patterns of GH observed in mature animals in various species are believed to be important for regulating growth and metabolic functions of liver and adipose tissue (6, 7, 8). A relationship between GH secretory pattern and obesity has been described in humans and rodents; GH treatment in GH-deficient patients alters body composition (37, 38, 39), dietary obesity is induced in the female GH-deficient dwarf rat (38), and monosodium glutamate-treated rats gain weight when peripheral GH concentrations are low (40, 41, 42). In the view of these observations, the results of this study suggest that obesity in hGH transgenic rats could have resulted from low GH secretion. This is further supported by the experiment in which the transgenic rats were treated with recombinant hGH intermittently for 1 week. This decreased adipose tissue weight as well as body weight gain during the treatment period without changing food intake. Thus, the treatment must have suppressed fat synthesis but enhanced utilization of accumulated fat.
It is well documented that GH-deficient human adults show obesity and
insulin resistance (27). The insulin resistance transgenic animal model
described here could provide a useful experimental basis for developing
diagnostic and therapeutic approaches to noninsulin-dependent diabetes.
A disease condition where insulin resistance has not propagated to the
adipose tissue can be ranked as an intermediate status, followed by
final progression into a wasting noninsulin-dependent diabetic status.
Based on the results of the present investigation, this intermediate
status can be diagnosed with high FFA levels insensitive to
troglitazone treatment and normal TNF levels. This situation could
possibly be improved by treatment with appropriate amounts of GH.
In summary, by using the hGH transgenic rats, it was demonstrated that low GH levels devoid of pulsatile secretion in the peripheral circulation are sufficient to support normal body growth, yet can induce a type of obesity that is responsive to simulation of pulsatile GH secretion. Further investigation is required to elucidate the molecular mechanism of this obesity induction and progression.
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Acknowledgments |
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Footnotes |
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Received December 2, 1997.
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References |
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2, lipid-activated transcription
factor. Cell 79:1147–1156[CrossRef][Medline]
12,14-prostaglandin
J2 is a ligand for the adipocyte determination factor
PPR2. Cell 83:803–812[CrossRef][Medline]
and promotes adipocyte
differentiation. Cell 83:813–819[CrossRef][Medline]
: direct role in
obesity-linked insulin resistance. Science 259:87–91-induced phosphorylation of insulin
receptor substrate-1 (IRS-1). J Biol Chem 270:23780–23784 and obesity-induced
insulin resistance. Science 271:665–668[Abstract]
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