(Received for publication, August 30, 1994; and in revised form, October 14, 1994)
From the
To elucidate the mechanism of the basal hyperinsulinemia of
obesity, we perfused pancreata from obese Zucker and lean Wistar rats
with substimulatory concentrations of glucose. Insulin secretion at 4.2
and 5.6 mM glucose was 10 times that of controls, whereas
-cell volume fraction was increased only 4-fold and DNA per islet
3.5-fold. We therefore compared glucose usage at 1.4, 2.8, and 5.6
mM. Usage was 8-11.4 times greater in Zucker islets at
1.4 and 2.8 mM and 4 times greater at 5.6 mM; glucose
oxidation at 2.8 and 5.6 mM glucose was >12 times lean
controls. To determine if the high free fatty acid (FFA) levels of
obesity induce these abnormalities, normal Wistar islets were cultured
with 0, 1, or 2 mM long chain FFA for 7 days. Compared to
islets cultured without FFA insulin secretion by FFA-cultured islets (2
mM) perifused with 1.4, 3, or 5.6 mM glucose was
increased more than 2-fold, bromodeoxyuridine incorporation was
increased 3-fold, and glucose usage at 2.8 and 5.6 mM glucose
was increased approximately 2-fold (1 mM FFA) and 3-fold (2
mM FFA). We conclude that hypersecretion of insulin by islets
of obese Zucker fatty rats is associated with, and probably caused by,
enhanced low K
glucose metabolism and
-cell hyperplasia, abnormalities that can be induced in normal
islets by increased FFA.
Hyperinsulinemia has long been recognized as a feature of
obesity-related insulin resistance in man (1, 2) and
in rodents(3) , but the mechanism of this association is
obscure. In perfused pancreata from one colony of obese
hyperinsulinemic Zucker (fa/fa) rats we have reported a
30-fold increase in insulin secretion at 5.6 mM glucose
compared with lean littermates (fa/+) (4) , a
difference that could not be accounted for by an increase in
-cells(5) . Rather the
-cells of fa/fa rats
appeared to be more sensitive than those of lean rats to glucose
concentrations in the normal fasting blood glucose range(6) .
The fact that insulin secretion by isolated pancreata of obese Zucker
diabetic fatty rats perfused with 5.6 mM glucose exceeded that
of pancreata of lean littermates (fa/+) or Wistar rats
perfused with 20 mM glucose (4) suggested that basal
hypersecretion of insulin at normoglycemic concentrations in obese rats
may be due, at least in part, to an increase in the high affinity
pathway of glucose metabolism in
-cells. In this study we show in
obese Zucker rats a leftward shift of the glucose-insulin
concentration-response curve of
-cells in the fasting and
subfasting range of glucose concentration that can be attributed to an
increased rate of glucose metabolism at low concentrations of the sugar
and to hyperplasia of
-cells. We provide evidence that the
hyperinsulinemia at low glucose concentrations, the hyperplasia, and
the increase in low K
glucose metabolism
can all be induced in normal islets by high concentrations of long
chain fatty acids (FFA) (
)that occur in obesity.
Male Wistar rats and obese Zucker female rats of 8-16 weeks of age, purchased from Charles River Laboratories, were used in the study. This colony differs from the University of Indiana Zucker diabetic fatty colony previously studied by our group(4, 5) .
The per min usage was calculated and expressed as picomoles of glucose/h/islet.
Glucose phosphorylation activity was measured in
islet cytosolic and mitochondrial suspensions utilizing the
radioisotopic method of Kuwajima et al.(18) . The
assay mixture contained 100 µCi of
[UC]glucose (New England Nuclear, Boston, MA)
resuspended in 1 ml of 2X assay buffer (200 mM Tris pH
= 7.4, 10 mM ATP, 20 mM MgCl
, 200
mM KCl, 0.5 mM dithiothreitol, and 100 mM glucose). The final reaction mixture consisted of 25 µl of 2
assay buffer, 20 µl of islet cytosol or mitochondria
(approximately 50-150 µg of protein), and 5 µl of either
100 mM glucose 6-phosphate or H
O. Samples were
incubated at 37 °C for 90 min. The reaction was stopped by the
addition of 100 µl of 97% ethanol, 3% methanol. A 30-µl aliquot
was then removed and spotted onto NA 45 DEAE-cellulose discs
(Schleicher and Schuell) and dried. The discs were washed three times
in a large volume of distilled water followed by a final overnight wash
with gentle agitation. The next day the discs were dried on Whatman No.
3MMchr paper. The residual radioactivity on the discs was detected by
liquid scintillation counting after addition of 10 ml of Ecolume
mixture (ICN). The activity measured in the presence of 10 mM glucose 6-phosphate was considered to be glucokinase. Hexokinase
activity was considered to represent the glucose
6-phosphate-inhibitable activity, obtained by subtracting
noninhibitable phosphorylation from total phosphorylation. Activities
were expressed as picomoles of glucose/min/µg of protein.
Glucopenia elicits a local release of catecholamines from adrenergic
nerve endings in the pancreatic islets (8) and catecholamines
inhibit insulin secretion(21) . We therefore considered the
possibility that the foregoing differences in the response of
-cells from lean and obese rats might have been caused by
differences in the release of norepinephrine from sympathetic nerve
endings in the islets. Since the inhibitory effect of glucopenia on
insulin secretion in perfused pancreata from normal rats can be largely
abolished by adrenergic blockade(8) , the experiments were
repeated with 10 µM phentolamine and propranolol added to
the perfusate. The combined
- and
-adrenergic blockade
increased insulin secretion in both groups, but the significant
differences between the insulin responses of lean and obese rats
remained (Table 1B).
[U-C]Glucose oxidation was >12 times
higher in islets of obese rats than in those of controls at both 2.8
and 5.6 mM glucose concentrations (Table 2), i.e. more than three times greater after correction for differences in
islet size.
Figure 1:
Effect of FFA on BrdUrd incorporation
by islet cells. BrdUrd incorporation was measured in islets isolated
from 6-week-old Wistar rats and cultured for 7 days with 2 mM FFA (FA+) or without FFA (FA-).
Adjacent sections were stained with either anti-insulin serum (upper panels) or anti-BrdUrd (lower panels) so as to
determine if a BrdUrd incorporation is inside or outside of the
-cell area.
This study was designed to characterize the -cells in
obesity and to identify the mechanism by which an increase in adipose
tissue results in an increase in basal insulin secretion. We observed a
striking shift to the left in the glucose dose-response curve for
insulin secretion in obese Zucker rats. The shift was not abolished by
- and
-adrenergic blockade, thus excluding the possibility
that intergroup differences in norepinephrine released from
peri-insular sympathetic nerve endings were responsible. In fact, at
the glucopenic concentrations at which norepinephrine release should be
greatest, adrenergic blockade magnified the difference between groups.
Insulin secretion in obese rats was as great at 1.4 mM glucose
(57 microunits/ml/min) as that in lean rats at 5.6 mM glucose
(55.4 microunits/ml/min).
Although the 4-fold increase in -cell
volume fraction in obese Zucker rats suggests that
-cell
hyperplasia may account for the 4-fold hypersecretion of insulin at 5.6
mM glucose in the normal fasting range, the >10-fold
increase in insulin secretion at glucopenic glucose concentrations
below 3 mM pointed to intrinsic differences in low K
glucose metabolism. At 1.4, 2.8, and 5.6 mM concentrations glucose usage in obese islets ranged from
4 to
11 times that of control islets from lean rats (Table 2). Glucose
oxidation in islets of obese rats was approximately 12 times greater
than in lean controls at both 2.8 and 5.6 mM glucose.
Becker et al.(25) have demonstrated that an
8-10-fold overexpression of recombinant hexokinase I in normal
islets via recombinant adenovirus doubles both glucose usage and
insulin secretion at 3 mM glucose. Overexpression of yeast
hexokinase in islets of transgenic animals has similar
effects(33) . Because of the similarity between the -cell
phenotypes caused by hexokinase overexpression and that of obesity, we
quantitated the hexokinase activity in the islets of obese rats. There
was a 3-fold increase in total enzyme activity in islets of Zucker rats
compared with lean controls. Mitochondrial hexokinase activity, which
is less inhibitable by glucose-6-PO
than the cytosolic
fraction(15) , was almost twice as high in islets of obese rats
as in lean controls.
The mechanism by which an increase in adipose
tissue increases insulin secretion in obese individuals with normal
glucose tolerance is unknown. There are reasons to suspect that the
elevated plasma FFA levels of obesity (26) may play a role;
certainly the glucose-fatty acid cycle of Randle (34) is widely
accepted as the cause of insulin resistance(35) , as are the
antilipolytic effect of insulin (36) and the insulinotropic
action of
FFA(27, 28, 29, 30, 31, 32) .
Nevertheless, the idea of an FFA-mediated adipocyte--cell feedback
relationship has not been entertained as the cause of obesity-related
changes in
-cells. The present study demonstrates that three of
the most dramatic abnormalities in the
-cells of obese rats can be
induced by culturing normal islets in FFA for 7 days. First, insulin
secretion at 1.4, 3.0, and 5.6 mM glucose is doubled after 7
days in medium containing 2 mM FFA (Table 4), confirming
by perifusion the work of Zhou and Grill (32) using a static
incubation technic. Second, the 3-fold increase in BrdUrd
incorporations in
-cells provides evidence that increased levels
of FFA are capable of enhancing
-cell replication and thus causing
or contributing to the
4-fold increase in the
-cell volume
fraction of obese rats. Finally, the 3-fold concentration-dependent
enhancement by FFA of glucose usage at a 2.8 and 5.6 mM glucose noted in islets cultured in 1 and 2 mM FFA for 1
week (Table 5) provides strong evidence that the FFA elevations
observed in obesity(26, 37) are capable of inducing
an increase in low K
glucose usage. Since insulin
secretion is coupled to glucose metabolism (22, 23, 24) , the FFA-induced increase in
low K
glucose metabolism could well be the cause
of the hyperinsulinemia at substimulatory glucose concentrations.
Finally, the inability of hyperinsulinemia of obesity to reduce FFA
levels to normal implies a resistance of adipocytes to the
antilipolytic action of insulin, as was reported in man(37) .
The potential implications of this relationship between adipocytes
and -cells are of clinical interest. If the twin abnormalities of
obesity, insulin resistance and hyperinsulinemia, are both secondary to
increased plasma levels of FFA, they will both vary in parallel with
changes in FFA levels. So long as insulin secretion is coupled to
insulin resistance, euglycemia will be maintained, and this may account
for the fact that most obese individuals remain normoglycemic. But if
FFA levels continue to rise and exceed a critical level,
-cells
may be unable to continue the insulin response required to match a
progressively increasing FFA-mediated insulin resistance; at this point
non-insulin-dependent diabetes mellitus (NIDDM) will ensue. We have
recently reported that when plasma FFA levels approach 2 mM in
Zucker prediabetic fatty rats, the
-cell response to hyperglycemia
disappears and NIDDM begins(38) . Also, obese Zucker rats are
much more vulnerable than lean rats to other diabetogenic factors such
as dexamethasone(39) , suggesting that the phenotype described
here represents a prediabetic state.