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The Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201
Address all correspondence and requests for reprints to: Roger D. Cone, Ph.D., Vollum Institute, Oregon Health Sciences University, Portland, Oregon 97201. E-mail: cone{at}ohsu.edu
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Introduction |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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Hyperinsulinemia is one of the common features among all the animal models of obesity and human obesity, and also one of the earliest metabolic alterations observed in melanocortin obesity models. A pancreatic ß-cell hyperplasia has been found before any observed obesity in the AVY strain as one of the earliest changes, for example (17). Late-onset development of hyperglycemia has also been reported in male Ay and MC4-R knockout (MC4-RKO) mice, which seems to be quite similar to the pathophysiological process of type 2 diabetes (12). These data suggest that the central melanocortin system may be independently involved in the regulation of glucose homeostasis in addition to its regulation of energy intake and energy expenditure (1, 2). However, existing data do not address whether the hyperinsulinemia in the melanocortin obesity syndromes is primary to MC4-R blockade, a developmental consequence of MC4-R blockade, or secondary to the hyperphagia and obesity that develops in this model. To test the potential role of central melanocortin signaling in glucose homeostasis, we have examined the effects of central melanocortin administration on basal plasma/serum insulin levels and glucose tolerance, and studied the development of hyperinsulinemia and in the MC4-R KO mouse.
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Materials and Methods |
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4% fat by mass) and tap water ad libitum.
MC4-RKO/C57BL/6J x SW129 F1 mice (12) were backcrossed three times into
the C57BL/6J background, and maintained as described above. All studies
were conducted according to the NIH Guide for the Care and Use of
Laboratory Animals and approved by the Animal Care and Use Committee of
the Oregon Health Sciences University. The animals were anesthetized with halothane and placed in a stereotaxic apparatus (CARTESIAN Research, Inc., Sandy, OR). A sterile guide cannula with obdurator stylet was stereotaxically implanted into one side of the PVH (0.67–0.77 mm relative to bregma, 0.38–0.48 mm lateral to midline, and 4.69–4.75 mm below the surface of the skull; for icv injection in the mouse, the cannula was implanted with the coordinates of 0.5 mm posterior to the bregma, 1–1.6 mm lateral to the midline and 2 mm blow the bregma. The cannulla was then fixed in place using dental cement. The animals were housed separately after surgery at least one week for recovery before experiments. The positions of the cannulae were verified at the end of experiments by histological analysis; in some animals the position of the cannulae were tested by dye administration before sacrifice. Positioning of the cannulae in the more dorsal aspect of the PVH was found to ensure the integrity of the third ventricle and prevent dye from entering the cerebrospinal fluid.
RIA assay for serum or plasma insulin
ACSF, the synthetic 
-MSH analog MTII (0.1–3 nmol) or leptin
(1 µg) were infused in a total volume of 2 µl over 30 sec in
lateral ventricle-cannulated mice. In these experiments, the icv
dose-response to MTII or the icv response to leptin, or ip response to
phentolamine were completed using the same icv-cannulated mice. These
animals were allowed a washout period of at least 1 week between
treatments and were rerandomized between experiments. These experiments
were carried out at the beginning of the dark cycle (1800 h) with the
food withdrawn and water available ad libitum. 0.5–1 h
following the icv injection of the drugs, the blood samples were
collected by cutting the tail or by decapitating the animals. RIA of
serum insulin was performed as described (Linco Research, Inc., St. Charles, MO). In treated animals, the icv injections
were immediately administered following ip administration of
phentolamine or saline, then the blood samples were collected for
insulin RIA at the 0.5–1 h after the icv injection. For experiments
analyzing MTII effects on insulin in lean animals, the food was
withdrawn for 3–4 h before the experiment. Blood was drawn from the
retro-obital sinus using a heparinized microcapillary tube 45–60 min
after icv injection. Plasma insulin levels of MC4-RKO and wild-type
littermates were measured using the rat sensitive insulin kit
(Linco Research, Inc., St. Charles, MO) or Mercodia Rat
Insulin ELISA, (ALPCO, Windham, NH) from the retro-obital sinus or tail
blood samples.
Blood glucose and glucose tolerance tests
Lean female control mice implanted with a cannula in the lateral
ventricle or PVH were fasted from 1300–1700 h. The blood glucose level
was measured with a blood glucose meter and test strips (Glucometer
Elite, Bayer Corp., Elkhart, IN) from the tail blood of
the animals. Glucose (1 g/kg BW) was administered ip at 30 min after
icv or PVH injection of MTII or ACSF, and then the blood glucose level
was measured at the time points indicated in the text (15, 30, 45, 60,
90, 120 min) following the ip glucose challenge.
Nonesterified fatty acid assay
A colorimetric in vitro enzymatic assay was used to
quantitate nonesterified FFA, according to the manufacturer’s
specifications (Wako Pure Chemical Industries Ltd.,
Richmond, VA). Serum for this assay was obtained shortly after lights
on in mice fasted for the previous 16 h.
Insulin tolerance test
Insulin tolerance tests were performed by measuring blood
glucose levels following a single sc injection of regular human insulin
0.65–1 U/kg body wt (Humulin, Eli Lilly & Co.,
Indianapolis, IN) with food withdrawn for 3–4 h before the
experiment.
Statistical analyses
Data were expressed as mean ± SE. Statistical
analyses were performed using the Student’s t test
(Figs. 1
;
2; 3, A and
B; 4, A and D; and
5, C and D) or two-way ANOVA with
multiple measures (Figs. 3C; 4, B, C, E, and F; and 5, A and B).
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Results |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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), measured 30–60 min
after administration of the compound. 1 nmol MTII reduced serum insulin
levels by 72.8% (n = 10, P < 0.001) compared
with the ACSF control group. The effect of icv administration of leptin
(1 µg) is shown for comparison. In a separate experiment, the blood
glucose, at 60 min after 1 nmol of icv MTII administration, increased
to 351.8± 44.8 (mg/dl) from 201.4± 18.8 (mg/dl) (P <
0.05, n = 7), which paralleled the decreased blood insulin level,
indicating that the inhibitory effect of central MTII on insulin
release is not secondary to a lowering of the blood glucose (Fig. 1B).
Effect of the sympathetic system on MTII-induced lowering of serum
insulin in the ob/ob mouse
To test the hypothesis that the melanocortin agonist lowers serum
insulin by stimulating the sympathetic drive to the pancreas known to
inhibit insulin release, the effect of phentolamine, a nonspecific
-adrenoceptor antagonist, on the MTII-mediated reduction of serum
insulin was examined. Animals were first injected with phentolamine
(0.1–0.5 mg/kg in 0.2 ml saline ip) or saline (0.2 ml), then centrally
treated with either MTII (1nmol/2 µl, icv), leptin (1 µg/2 µl,
icv) or ACSF (2 µl, icv). As expected, administration of phentolamine
alone (0.1–0.5 mg/kg, ip) significantly elevated the basal insulin
level from 53.49 ± 4.5 to 83.94 ± 13.6 ng/ml, (Fig. 2, P < 0.01), due to its ability to block the inhibitory
effect of sympathetic tone on insulin release (18). Preadministration
of phentolamine completely blocked the ability of centrally
administered MTII to lower serum insulin in leptin-deficient animals
(Fig. 2, P < 0.001 n = 12). Phentolamine also
blocked the majority of leptin’s acute insulin-lowering effect on the
ob/ob mouse.
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), with
0.37 ± 0.04 ng/ml resulting from 3 nmol MTII treatment
vs. 0.50 ± 0.04 ng/ml in the ACSF treated group,
(P < 0.05). Significantly elevated fasting blood
glucose levels were seen at 60 min after treatment with 3 nmol MTII
(Fig. 3B, P < 0.05, n = 8). While icv injection
of 1 nmol of MTII was not found to significantly lower the base plasma
insulin level, however, the glucose tolerance in response to an ip
glucose challenge (1 g/kg) was significantly reduced in comparison with
ACSF-treated control animals (data not shown). Microinjection of MTII
(0.45 nmol/0.3 µl) into the paraventricular nuclei of hypothalamus
(PVH) also resulted in the similar effect on glucose tolerance after
glucose challenge (Fig. 3C, P < 0.01).
Increased basal plasma insulin and decreased peripheral insulin
tolerance in the young lean MC4-RKO mouse
To explore the potential direct pathophysiological consequences of
defective melanocortin signaling in the development of obesity and
diabetes, we examined the basal insulin level and insulin as well as
glucose tolerance in the young MC4-RKO mice in comparison with their
wild-type littermates. A higher fasting insulin level was seen in males
tested at 4 weeks in comparison to controls (Fig. 4A, P
< 0.05). At 6–7 weeks of age, there is no difference in the food
intake (data not shown) and body weights between the MC4-RKO mice and
wild-type controls (Fig. 4B, P > 0.05), however, an
impaired insulin tolerance was observed in both female and male MC4-R
KO mice compared with the wild types (Fig. 4, C and D,
P < 0.01).
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, P
< 0.01) with higher blood insulin level than the controls (data not
shown). The body weight of male MC4-R KO mice appeared a little heavier
than the controls at this time point but the difference was not
statistically significant (Fig. 4F). No significant change in glucose
tolerance was observed in male or female MC4-R KO mice compared with
the controls (Fig. 5, A and B). Because elevated nonesterified fatty
acids (NEFA, or FFA) induce insulin resistance (19), to determine if
the MC4-RKO mouse has a defect in trigyceride metabolism, serum NEFAs
were measured. No difference in serum FFA levels were observed between
MC4-R KO mice and their controls tested at 6–7 weeks (Fig. 5, C and D,
P > 0.05).
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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A role for the melanocortin system in other aspects of sympathetic
outflow has already been clearly shown. Icv administration of the MC4-R
agonist MTII produced a dose-dependent sympathoexcitation affecting
sympathetic nerve activity of brown adipose tissue (BAT) and renal and
lumbar beds in rat, while the antagonist SHU9119 completely blocked the
sympathoexcitation effects of MTII, as well as renal sympathoexcitation
by leptin (20). Central administration of SHU9119 also completely
inhibited the leptin-induced increase in UCP-1 messenger RNA (mRNA)
expression in the BAT of rat, which is mediated through the activation
of ß3-adrenoceptor (21). MC4-R and MC3-R mRNAs
have been found in many structures of the nervous system which have
been implicated in the central control of glucose homeostasis (22, 23, 24, 25).
For example, in the hypothalamus, MC4-R mRNA was found expressed in the
lateral hypothalamic area (LHA), ventromedial hypothalamus (VMH), and
paraventricular nucleus (PVN) (26). Because the inhibitory effect of
MTII on insulin release could be completely blocked by phentolamine, a
nonspecific -adrenoceptor antagonist, the data suggest that central
melanocortin peptides may act on melanocortin receptors to increase the
sympathetic drive to pancreas (or alter the relative effectiveness of
sympathetic vs. parasympathetic outflow to favor sympathetic
activity) to exert a tonic inhibitory effect on insulin secretion. It
is feasible that an increased basal plasma insulin in the young lean
MC4-R KO mouse before the onset of hyperphagia or obesity may be due to
the removal of the tonic sympathetic inhibitory effects on the pancreas
resulting from blockade of central MC4-R signaling.
There exist multiple proposals regarding the mechanisms by which
obesity increases the risk of diabetes. Several lines of evidence
demonstrate that an increase in body adiposity induces insulin
resistance, leading ultimately to diabetes, via the release by the
enlarged adipose tissue of one or more messengers including FFA and
tumor necrosis factor- (TNF- ), which interfere with insulin
action (19, 27). However, it has also been argued that under some
circumstances hyperinsulinemia and insulin resistance are primary
causes for the development of obesity and diabetes (28). Our results
show that a reduced insulin tolerance can be observed in the 6- to
7-week-old MC4-R KO mice, in which there are no detectable differences
in food intake, body weight or serum FFA level between knock out and
wild-type animals. These data, along with the hyperinsulinemia seen as
early as 4 weeks, suggest, but do not prove, that the insulin
resistance in this model is not solely due to the development of
obesity. It is possible, for example, that insulin receptor
desensitization occurs in part as a result of the early chronic
increase in insulin secretion, compounding the insulin resistance due
to the increased adiposity that develops at 8 weeks, and perhaps
earlier (29).
Central administration of leptin inhibits insulin secretion and increases the insulin sensitivity of peripheral tissue directly (30, 31, 32). Furthermore, leptin receptor is expressed in the arcuate POMC neurons (33), and the anorexic effect and thermoregulatory effects of leptin can be blocked by central MC4-R antagonist (21, 34). Thus, it is likely that the central melanocortin signaling system might be also involved in a component of leptin’s ability to lower insulin levels and increase peripheral insulin sensitivity. Interestingly, phentolamine did not completely block the reduction of serum insulin by leptin, suggesting that a component of leptin’s insulin-reducing activity in the ob/ob mouse may also be melanocortin independent.
Our pharmacological studies clearly show the ability of centrally administered melanocortins to act independently on feeding behavior (1), metabolism (2), and basal serum insulin levels. The data on insulin levels and altered insulin tolerance in the young MC4-RKO suggests, but does not yet prove, that insulin resistance may develop independently of increased adiposity in this model. Certainly, much additional work will be needed to test this hypothesis because the C57BL/6J strain is hypersensitive to the effects of adipose mass on insulin resistance (35), and adipose mass may increase long before it can be measured as an increase in total body mass, as reported here. Our data does, however, support the hypothesis that a central melanocortin signaling defect, likely through altered sympathetic nerve activity innervating the pancreas, leads to the hypersecretion of insulin. The direct effects of defective MC4-R signaling on feeding and metabolism, which rapidly lead to obesity, could be imagined to be compounded by independently elevated blood insulin levels perhaps desensitizing the insulin receptor. The normal glucose tolerance accompanied with the insulin resistance and hyperinsulinemia in younger MC4-R KO mice also suggest that the pancreas of animals at a young age may be able to appropriately augment insulin secretion to offset the defect in insulin action and maintain euglycemia and normal glucose tolerance, which is coincident with the clinical observation that insulin resistance can often be detected years before onset of clinical disease (36, 37). With time, however, when the ß cell fails to maintain its high rate of insulin secretion and sensitivity to blood glucose, the balance between insulin resistance and insulin secretion would be broken, and the impaired glucose tolerance and eventually overt diabetes mellitus develop (28).
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Acknowledgments |
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Footnotes |
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Received March 14, 2000.
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