(Received for publication, October 5, 1995; and in revised form, December 20, 1995)
From the
Although obesity is associated with insulin resistance, most
obese humans and rodents remain normoglycemic because of compensatory
hyperinsulinemia. This has been attributed to -cell hyperplasia
and increased low K
glucose metabolism of
islets. Since free fatty acids (FFA) can induce these same
-cell
changes in normal islets of Wistar rats and since plasma FFA are
increased in obesity, FFA could be the signal from adipocytes that
elicits
-cell compensation sufficient to prevent diabetes. To
determine if FFA-induced compensation is impaired in islets of rats
with a diabetogenic mutation, the Zucker diabetic fatty (ZDF) rat, we
cultured islets from 6-week-old obese (fa/fa) rats that had
compensated for obesity and apparently normal islets from lean ZDF rats (fa/+) in 0, 1, or 2 mM FFA. Low K
glucose usage rose 2.5-fold in
FFA-cultured control islets from age-matched Wistar rats, but failed to
rise in either the precompensated islets of ZDF rats or in islets of
lean ZDF rats. Bromodeoxyuridine incorporation increased 3.2-fold in
Wistar islets but not in islets from obese or lean ZDF rats. Insulin
secretion doubled in normal islets cultured in 2 mM FFA (p < 0.01) but increased only slightly in islets from lean ZDF
rats (not significant) and declined in islets from obese ZDF rats (p < 0.05). We conclude that, unlike the islets of
age-matched Wistar rats, islets of 6-week-old heterozygous and
homozygous ZDF rats lack the capacity for FFA-induced enhancement of
-cell function.
Pancreatic islets from obese rodents are enlarged and exhibit a
marked increase in low K glucose
metabolism(1) , which may account for their high output of
insulin even at low concentrations of
glucose(1, 2, 3) . The hyperinsulinemia is
regarded as a compensatory response that prevents hyperglycemia despite
the insulin resistance that invariably accompanies obesity.
There is
evidence that free fatty acids (FFA) ()may be the signal
from adipocytes that mediates this compensatory insulin
secretion(1) . Plasma FFA are elevated in obesity (4, 5) and have long been known to stimulate insulin
secretion(6, 7, 8) . Moreover, the
compensatory triad observed in islets from obese rats can be induced in
normal islets by culturing them for 7 days in the presence of 1 or 2
mM FFA; low K
glucose metabolism
rises dramatically (1) , there is evidence of increased
-cell replication(1) , and insulin secretion
increases(1) , confirming the earlier description of
FFA-induced hyperinsulinemia(9) .
If the FFA-induced
compensation in obesity is, at least in part, responsible for
preventing diabetes, it follows that FFA-induced compensation may be
impaired at or before the onset of diabetes. In this study we assess
the ability of FFA to induce the compensatory triad of enhanced low K glucose metabolism, increased
-cell replication, and insulin hypersecretion in islets from obese
rats with a diabetogenic mutation, the Zucker diabetic fatty rat
(ZDF-drt). We observe that FFA induction of these compensatory changes
is impaired, not only in islets from obese homozygous rats, but also in
islets from lean heterozygous ZDF rats.
All rats received standard rat chow (Teklad F6 8664, Teklad, Madison, WI) ad libitum and had free access to water. All institutional guidelines for animal care and use were followed.
Figure 1:
Insulin secretion at 3 and 23 mM glucose in islets cultured for 7 days with 0
(-
), 1 (
-
), or 2
(
-
) mM long chain fatty acids. Islets
were isolated from normal male Wistar rats (A), lean male ZDF (fa/+) rats (B), obese female ZDF (fa/fa) rats that do not develop diabetes (C), and
obese male ZDF (fa/fa) rats that develop diabetes between the
ages of 8 and 10 weeks (D). The numbers in each panel
reflect the differences in insulin secreted (fmol/min/50 islets) by
islets cultured in the presence of 2 mM FFA or in the absence
of FFA during perifusion in 3 or 23 mM glucose.
To avoid the influence of precompensation, we examined the effects of FFA on the islets of lean heterozygous ZDF animals in which obesity, insulin resistance and compensatory hyperinsulinemia were absent and no prior compensatory changes in islets had occurred. As shown in Fig. 1B, the FFA-induced increase in basal insulin secretion after culture in 2 mM FFA was small and not statistically significant; glucose-stimulated insulin secretion was significantly reduced, suggesting an intrinsic resistance to the effects of FFA on islet function in rats with a single ZDF allele.
To exclude insulin depletion as the cause of the impaired FFA-mediated effects on insulin secretion, insulin content was measured in all islet groups (Table 1B). There were no significant differences in change of insulin content between the lean Wistar and ZDF groups. Moreover, since the secreted insulin in all groups (Table 1A) represented at most only 3.8% of the total insulin content, it is unlikely that differences in pancreatic insulin content account for the effect of FFA on insulin secretion.
To exclude the possibility that elevated concentrations of FFA in vitro might have killed a greater number of cells in the islets of obese and lean ZDF rats than in the lean Wistar controls, we tested cell viability using fluoroacetate incorporation. As indicated in Table 2, exposure to 2 mM FFA reduced viability by 7% in Wistar islets and 12% in ZDF islets, not nearly enough to account for the large differences in insulin secretion associated with exposure to FFA.
Figure 2: Comparison of BrdUrd incorporation in islets cultured for 3 days in 0 or 2 mM FFA. A, male Wistar rats; B, lean heterozygous male ZDF rats (fa/+); C, obese homozygous male ZDF rats (fa/fa). All rats were 6 weeks old at the time of islet isolation.
The results indicate that islets from homozygous and
heterozygous ZDF rats do not develop any of the ``compensatory
changes'' that are induced in islets from normal Wistar rats after
7 day of culture in 1 or 2 mM long chain fatty acids (FFA).
These changes include increased basal and glucose-stimulated insulin
secretion, increased BrdUrd incorporation, and increased low K glucose metabolism. Since compensatory
hyperinsulinemia is required to prevent hyperglycemia in the face of
worsening insulin resistance, it follows that the propensity for
obesity-dependent diabetes in ZDF rats could be the consequence of the
failure of FFA to induce the changes in
-cells that result in the
necessary degree of hyperinsulinemia.
In the case of islets from
6-week-old obese homozygous ZDF rats a measure of compensation had
already occurred in vivo prior to their isolation for the
culture experiments, making it impossible to differentiate between
precompensation that had reached a maximum and intrinsic resistance of
ZDF -cells to actions of FFA. The islets of 6-week-old obese rats
are 3.3 times larger than those of lean controls (12) and
their insulin secretion is 3.2 times greater(12) . Lean
heterozygous ZDF rats, by contrast, do not differ from Wistar islets
with respect to
-cell volume density, low K
glucose usage or insulin secretion. Nevertheless, despite the
absence of any antecedent compensatory changes in vivo, they
too were completely unresponsive to culture in FFA. BrdUrd
incorporation, which increased 3.2-fold in islets of Wistar males in
the presence of 2 mM FFA, did not increase at all in the
islets of lean ZDF rats. Similarly, low K
glucose
usage, which increased 2.5-fold in the Wistar animals, did not increase
in the lean ZDF group. Finally, insulin secretion at 3 mM glucose, which more than doubled in the Wistar islets, increased
only slightly (NS) in the lean ZDF group, and glucose-stimulated
insulin secretion was reduced by the 2 mM FFA. Thus, high FFA
levels comparable to those observed in plasma of prediabetic ZDF rats (14) did not elicit a normal compensatory insulin response. The
ZDF colony may thus have an intrinsic
-cell defect that interferes
with FFA-mediated compensation. In as much as islets from 6-week-old
obese homozygous ZDF rats appear to have reached a fully compensated
state in vivo prior to their isolation, either more than 7
days are required in vitro for FFA-mediated induction of
compensatory events in ZDF rats or, more likely in ZDF rats a time
window during which compensation can occur close before the age of 6
weeks.
The mechanism by which FFA induce the changes that in islets
of normal rats result in compensatory hyperinsulinemia has not as yet
been identified. In preadipocytes a fatty acid-activated receptor with
homology to the peroxisome proliferator-activated receptor (PPAR) was
recently cloned(20) . An isoform of PPAR is expressed in
islets. ()It may be involved in up-regulation of glycolytic
enzymes responsible for low K
glucose usage.
However, changes in alternative fuel use could be the cause of the
increase in low K
glycolysis(21) .
The mechanism of the normal FFA-induced increase in BrdUrd incorporation is also unknown. To our knowledge, a mitogenic effect of FFA in vitro has not been described previously in mammalian cells. However, intracellular levels of palmitoyl-CoA within the known physiologic range are able to potentiate protein kinase C activity in vitro(22, 23) and stimulate protein kinase C-catalyzed phosphorylation of epidermal-growth factor receptor(24) .
If the compensatory hyperinsulinemia in normal islets is the result of FFA-mediated induction of enzymes, what is the mechanism of the failed compensation in islets of ZDF rats? Given the fact that islets from obese prediabetic rats have an abnormally high lipid content in vivo(14) , lipid overload seems plausible. It has long been known that increased long-chain fatty acyl-CoA impedes glucose metabolism at multiple levels(25, 26, 27, 28, 29) , as recently reviewed by McGarry(30) . A novel additional mechanism, excessive acylation, also warrants consideration; just as unregulated overglycation resulting from hyperglycemia can modify the function of certain proteins, excessively high FFA levels causing overacylation might similarly alter protein functions(31) .
In summary, these results confirm our earlier report (1) that normal islets cultured in the presence of elevated FFA
levels develop the same changes in DNA synthesis, glucose usage and
insulin secretion that are present in vivo in nondiabetic
obese rats. We further demonstrated that islets from obese prediabetic
and nonprediabetic rats do not develop the foregoing compensatory
changes when cultured under these same conditions, perhaps because they
had already occurred earlier in vivo. Consequently, if insulin
resistance worsens, such islets would be incapable of further insulin
secretion and hyperglycemia would supervene. However, islets from lean
heterozygous ZDF rats without -cell precompensation also failed to
respond normally to FFA enriched culture. It is possible that the
-cell unresponsiveness to FFA present at 6 weeks of age and
thereafter accounts for the inability to compensate fully for the
insulin resistance and thereby prevent diabetes.