©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Pancreatic -Cells in Obesity
EVIDENCE FOR INDUCTION OF FUNCTIONAL, MORPHOLOGIC, AND METABOLIC ABNORMALITIES BY INCREASED LONG CHAIN FATTY ACIDS (*)

(Received for publication, August 30, 1994; and in revised form, October 14, 1994)

Joseph L. Milburn Jr. (1) (2) Hiroshi Hirose (1) (2) Young H. Lee (1) (2) Yoshitaka Nagasawa (1) (2) Atsushi Ogawa (1) (2) Makoto Ohneda (1) (2) Hector BeltrandelRio (1) (3) Christopher B. Newgard (1) (2) (3) John H. Johnson (1) (2) (4) Roger H. Unger (1) (2) (4)(§)

From the  (1)Center for Diabetes Research, Gifford Laboratories, the (2)Department of Internal Medicine and (3)Department of Biochemistry, University of Texas Southwestern Medical Center at Dallas, Dallas, Texas 75235, and the (4)Department of Veterans Affairs Medical Center, Dallas, Texas 75216

ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

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 beta-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 beta-cell hyperplasia, abnormalities that can be induced in normal islets by increased FFA.


INTRODUCTION

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 beta-cells(5) . Rather the beta-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 beta-cells. In this study we show in obese Zucker rats a leftward shift of the glucose-insulin concentration-response curve of beta-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 beta-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) (^1)that occur in obesity.


MATERIALS AND METHODS

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) .

Pancreatic Perfusion

Rat pancreata were perfused by the method of Grodsky and Fanska(7) , as modified by this laboratory(8) . In some experiments propranolol and phentolamine (10 µM) were added to block the effects of norepinephrine released from peri-insular sympathetic nerve endings during glucopenia(8) .

Morphometry

Bouins-fixed paraffin-embedded serial sections of perfused pancreata (5-µm thickness) were stained for insulin by indirect immunofluorescence (5) to determine the beta-cell volume fraction. The ratio of the area of insulin-positive cells to the total pancreatic area was determined using the method of Weibel(9) .

Measurements of Glucose Usage

Islets were isolated by a modification (10) of the method of Naber et al.(11) . Glucose usage was measured in freshly isolated islets using a modification of the method of Zawalich and Matchinsky(12, 13) . Fifty-microliter aliquots of islets were pipetted into individual wells of a 24-well cell culture plate. Glucose was added to Hanks' balanced salt solution (prepared according to the recipe of Whittaker Bioproducts, Walkersville, MD) to give final glucose concentrations of 1.4, 2.8, or 5.6 mM in a volume of 250 µl. After gentle shaking for 5 min at 37 °C, 5-[^3H]glucose (DuPont NEN) was added to give a final specific activity of 2.5-3.5 dpm/pmol. Samples were then incubated at 37 °C with continued shaking for 15 min. The reaction was stopped by the addition of 50 µl of 2 N perchloric acid or 10% trichloroacetic acid (Fisher). The background (blank) counts were determined by incubating a well without islets for each glucose concentration tested. After cooling to room temperature, 200 µl of the reaction mixture was transferred to a 1.5-ml microcentrifuge tube, placed in a scintillation vial containing 0.5 ml of unlabeled water, and incubated at 50 °C for 18 h. To determine the equilibration coefficient (EQC), 5 µl of tritiated water was added to 195 µl of unlabeled water in a 1.5-ml microcentrifuge tube and the foregoing procedure repeated in parallel with the experimental samples. Ten milliliters of Ecolume (ICN, Costa Mesa, CA) scintillation fluid was added to the vials, and the samples were counted in duplicate in a Beckman scintillation counter. Duplicates were averaged and glucose usage was derived from the following formula.

The per min usage was calculated and expressed as picomoles of glucose/h/islet.

Measurement of Glucose Oxidation

Glucose oxidation rates were determined by measuring the generation of ^14CO(2) from [U-^14C]glucose by the method of Bernstein and Wood(14) . Approximately 50 islets from Zucker obese female or Wistar rats were pipetted into vials containing either 2.8 or 5.6 mM glucose in Krebs-Ringer bicarbonate buffer supplemented with 10 mM Hepes (pH = 7.4), in a final volume of 1 ml. They were incubated at 37 °C for 5 min, followed by the addition of [U-^14C]glucose to a specific activity of 0.8 cpm/pmol. The samples were then incubated for 30 min, and the reaction was stopped by addition of 200 µl of 10% trichloroacetic acid. Each vial had a small well suspended from the rubber cap which contained 300 µl of benzethonium hydroxide (Sigma). After stopping the reaction with acid, the vials were left at room temperature for 3 h. Thereafter, the wells were removed and incorporation of ^14CO(2) was determined by liquid scintillation counting. Oxidation at each glucose concentration was measured in duplicate, and blank rates of ^14CO(2) evolution were measured by performing the same procedures on vials which contained no islets. The data was expressed as picomoles of glucose oxidized/h/islet.

Cytosolic and Mitochondrial Glucokinase and Hexokinase Activity

Mitochondrial and cytosolic fractions were prepared by the method of Rasschaert and Malaisse(15) . Approximately 4500 Wistar islets and 1000 Zucker islets were suspended in 200 µl of cold homogenization buffer consisting of 250 mM sucrose, 5 mM Hepes, and 0.5 mM EDTA at pH 7.4 and then homogenized with a hand-held motor-driven (Kontes, Vineland, NJ) Teflon pestle using 20 strokes at full speed followed by centrifugation at 750 times g, 4 °C for 5 min. The islet supernatant was removed, and the pellet was resuspended in 200 µl of homogenization buffer, homogenized as before, and centrifuged again at 750 times g, 4 °C for 5 min. The two islet supernatants were combined and centrifuged at 12,000 times g, 4 °C for 10 min. The supernatants from the higher speed centrifugation were saved as the cytosolic fraction, and the mitochondrial fraction (pellet) was resuspended in 200 µl of homogenization buffer. Protein concentrations were determined by the method of Bradford (16) using a Bio-Rad (Hercules, CA) kit. Citrate synthase, measured by the method of Srere(17) , served as a mitochondrial marker enzyme.

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 [U^14C]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(2), 200 mM KCl, 0.5 mM dithiothreitol, and 100 mM glucose). The final reaction mixture consisted of 25 µl of 2 times 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(2)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.

Islet Culture

Isolated islets were maintained in suspension culture in 60 mm glass Petri dishes at 37 °C in 5% CO(2) and 95% air for 7 days. The culture medium consisted of RPMI 1640 supplemented with 10% fetal calf serum, 200 units/ml of penicillin, 0.2 mg/ml streptomycin (Life Technologies, Inc.), and 2% bovine serum albumin (fraction V, Miles, Kankakee, IL) either with or without a 2 mM mixture of oleate:palmitate (2:1) (Sigma). The glucose concentration in the medium was 8 mM. The medium was changed every 2 days.

BrdUrd Incorporation

BrdUrd incorporation was quantitated in Bouins-fixed islets immobilized in gelatin and embedded in paraffin. Five-µm-thick sections were processed and serial sections stained by the previously described technic for insulin (5) and for BrdUrd. The latter was quantitated using an anti-bromodeoxyuridine(BrdUrd) antibody (19) (Boehringer Mannheim). Pooled islets from 20 Wistar rats were cultured in 8 different dishes for 7 days in medium containing either 0 or 2 mM FFA. BrdUrd was maintained at a 15 µM concentration throughout the 7 days.

DNA Measurement

DNA was measured by the method of Hopcroft et al.(20) .


RESULTS

Insulin Secretion at 1.4-5.6 mM Glucose Concentrations by the Perfused Pancreata of Obese Zucker (fa/fa) and Lean Rats

We have reported previously that base-line insulin secretion by pancreata of obese Zucker diabetic fatty (ZDF-drt) female rats perfused with 5.6 mM glucose averages approximately 30 times that of Wistar rats(4) . In the present study of isolated pancreata from a different colony of obese Zucker rats (Charles River), perfusion with 1.4 mM glucose resulted in insulin levels more than twice those of pancreata of lean control rats perfused with 5.6 mM glucose (Table 1A); progressive increases in glucose to 2.8, 4.2, and 5.6 mM elicited significant increases in insulin secretion (p < 0.05). By contrast, in age-matched lean Wistar rats insulin was virtually undetectable until glucose concentrations reached 4.2 mM and above (Table 1A).



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 beta-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 alpha- and beta-adrenergic blockade increased insulin secretion in both groups, but the significant differences between the insulin responses of lean and obese rats remained (Table 1B).

beta-Cell Volume Fractions and Islet DNA in Obese and Lean Rats

To determine if the hypersecretion of insulin in the obese rats was associated with an increased beta-cell mass, we compared the volume fraction of insulin-positive cells in pancreatic sections from the two groups. The mean volume fraction of beta-cells in six obese rats was 1.55 ± 0.2, compared with 0.39 ± 0.06 in six lean Wistar controls, a 4-fold difference. Islet DNA averaged 113.3 ± 25 ng/islet in the Zucker rats compared with 32 ± 7 ng/islet in controls, a 3.5-fold difference. These results were highly suggestive of beta-cell hyperplasia in obese rats.

Glucose Usage and Oxidation by Isolated Islets from Obese Zucker and Lean Wistar Rats

In view of the well known relationship between glucose metabolism and insulin secretion(22, 23, 24) , the failure of glucopenia to suppress insulin secretion by pancreata of obese rats (Table 1) might reflect a higher rate of glucose metabolism at glucopenic levels of glucose. Therefore we measured glucose usage, at concentrations of 1.4, 2.8, and 5.6 mM glucose in islets of obese Zucker rats and in lean Wistar controls (Table 2). Obese Zucker female rats exhibited, respectively, 8-, 11.4-, and 3.9-fold increases in glucose usage/islet/h compared with age-matched Wistars. At 5.6 mM glucose, the normal fasting level, the increased glucose usage in islets of obese rats was exactly proportional to the increase in cells; however, at lower glucose concentrations glucose usage was >8 times that of controls, more than could be explained by the beta-cell hyperplasia (Table 2).



[U-^14C]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.

Hexokinase Activity in Islets of Obese and Lean Rats

Overexpression of hexokinase I in rat islets results in enhanced glucose metabolism and insulin secretion at basal glucose concentrations, i.e. a beta-cell phenotype similar to that of obesity(25) . To determine if the increase in glucose usage at low glucose concentrations in islets of obese rats reflected an increase in the activity of low K(m) enzymes of glucose metabolism such as hexokinase I, cytosolic and mitochondrial fractions were prepared from pooled islets of both groups of rats. Total hexokinase activity was approximately three times greater in islets of obese rats, and mitochondrial hexokinase activity was twice as high (Table 3).



Effects of 2 mM Long Chain Fatty Acids on Insulin Secretion of Normal Islets at Fasting and Subfasting Glucose Concentrations

Plasma FFA levels have long been known to be high in obesity(26) , and FFAs are known to stimulate insulin secretion acutely(27, 28, 29, 30, 31) . More recently Zhou and Grill (32) have reported that insulin secretion at 3 mM glucose is increased after removal of islets from FFA-containing culture medium. To confirm this effect of FFA on beta-cell function, we cultured islets of Wistar rats for 7 days in the presence or absence of a 2 mM concentration of a 2:1 mixture of oleate:palmitate and then perifused them at 1.4, 3, and 5.6 mM glucose (Table 4). At all 3 mM glucose concentrations, insulin secretion by the FFA-cultured islets was more than twice that of control islets cultured without FFA.



Effects of 2 mM Long Chain Fatty Acids on BrdUrd Incorporation by Normal Islets

To determine if the islet hyperplasia observed in obese rats could also be attributed to the increased plasma FFA levels of obesity, we compared BrdUrd incorporations in islets of normal Wistar rats measured after 7 days of culture in either 0 or 2 mM FFA. BrdUrd incorporations were 3.2-fold greater in the FFA-cultured islets than in control islets averaging 10.7 ± 1.2 versus 3.3 ± 0.4 positive cells per islet in the controls (n = 4; p < 0.001). The incorporations appeared to be predominantly within beta-cells (Fig. 1).


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 beta-cell area.



Effects of Long Chain Fatty Acids on Glucose Usage by Normal Islets

To determine if FFA can induce in normal rat islets an increase in low K(m) glucose metabolism similar to that observed in the islets of obese Zucker rats (Table 2), we measured glucose usage at concentrations of 2.8 and 5.6 mM glucose in Wistar rat islets cultured for 7 days in either 0, 1, or 2 mM FFA. Glucose usage at both 2.8 and 5.6 mM glucose was between 2- and 3-fold greater (p < 0.01) in islets cultured in 1 or in 2 mM FFA (Table 5).




DISCUSSION

This study was designed to characterize the beta-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 alpha- and beta-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 beta-cell volume fraction in obese Zucker rats suggests that beta-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(m) 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 beta-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(4) 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-beta-cell feedback relationship has not been entertained as the cause of obesity-related changes in beta-cells. The present study demonstrates that three of the most dramatic abnormalities in the beta-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 beta-cells provides evidence that increased levels of FFA are capable of enhancing beta-cell replication and thus causing or contributing to the 4-fold increase in the beta-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(m) glucose usage. Since insulin secretion is coupled to glucose metabolism (22, 23, 24) , the FFA-induced increase in low K(m) 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 beta-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, beta-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 beta-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.


FOOTNOTES

*
This work was supported by National Institutes of Health Grants DK02700 and 1-P01-DK42582 and by Veterans Administrations Research Support Grant 549-8000. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
To whom correspondence should be addressed: Center for Diabetes Research, 5323 Harry Hines Blvd., Dallas, TX 75235-8854. Tel.: 214-648-3488; Fax: 214-648-9191.

(^1)
The abbreviations used are: FFA, free fatty acid; BrdUrd, bromodeoxyuridine.


ACKNOWLEDGEMENTS

We thank Kay McCorkle, Linda Kappler, Joan McGrath, and Chris McAllister for outstanding technical contributions and Teresa Autrey for excellent secretarial assistance. We thank Dr. J. D. McGarry for helpful advice.


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