beta -Cell adaptation in 60% pancreatectomy rats that preserves normoinsulinemia and normoglycemia

Ye Qi Liu, Peter W. Nevin, and Jack L. Leahy

Division of Endocrinology, Diabetes and Metabolism, University of Vermont, Burlington, Vermont 05405


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Islet beta -cells are the regulatory element of the glucose homeostasis system. When functioning normally, they precisely counterbalance changes in insulin sensitivity or beta -cell mass to preserve normoglycemia. This understanding seems counter to the dogma that beta -cells are regulated by glycemia. We studied 60% pancreatectomy rats (Px) 4 wk postsurgery to elucidate the beta -cell adaptive mechanisms. Nonfasting glycemia and insulinemia were identical in Px and sham-operated controls. There was partial regeneration of the excised beta -cells in the Px rats, but it was limited in scope, with the pancreas beta -cell mass reaching 55% of the shams (40% increase from the time of surgery). More consequential was a heightened glucose responsiveness of Px islets so that glucose utilization and insulin secretion per milligram of islet protein were both 80% augmented at normal levels of glycemia. Investigation of the biochemical basis showed a doubled glucokinase maximal velocity in Px islets, with no change in the glucokinase protein concentration after adjustment for the different beta -cell mass in Px and sham islets. Hexokinase activity measured in islet extracts was also minimally increased, but the glucose 6-phosphate concentration and basal glucose usage of Px islets were not different from those in islets from sham-operated rats. The dominant beta -cell adaptive response in the 60% Px rats was an increased catalytic activity of glucokinase. The remaining beta -cells thus sense, and respond to, perceived hyperglycemia despite glycemia actually being normal. beta -Cell mass and insulin secretion are both augmented so that whole pancreas insulin output, and consequently glycemia, are maintained at normal levels.

glucokinase; glucose metabolism; glycolysis; glucose 6-phosphate; islets of Langerhans; insulin secretion


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

THE ISLET beta -CELL through its secretion of insulin regulates the storage and metabolism of cellular fuels. Not surprisingly, the regulatory system is complex, with multiple factors affecting insulin secretion, proinsulin synthesis, and the mass of pancreatic beta -cells. The best studied factor is glucose; all of these functional aspects are glucose responsive, and it is well accepted that glycemia is the major regulator of beta -cell function. However, the primary effect of beta -cell activity is to maintain a normal metabolic milieu. When beta -cells function normally, insulin secretion precisely meets tissue insulin needs so that normoglycemia is maintained (16). Moreover, whole body insulin sensitivity varies throughout life (puberty, pregnancy, and aging are insulin-resistant states), yet most humans do not develop diabetes. As such, an unanswered question in regard to the glucose homeostasis system is how beta -cell adaptation occurs in the absence of ongoing changes in glycemia.

Insight has come from studies of rodents with genetic-based insulin resistance or pregnancy; their beta -cells are supersensitive to glucose so that insulin secretion is augmented at normoglycemia (4, 6-8, 11, 30). Kahn et al. (15) reported the same finding in healthy humans made insulin resistant by a 14-day nicotinic acid infusion. Thus a disassociation of the normal coupling between glucose concentration and insulin secretion characterizes the beta -cell adaptation to insulin resistance. The mechanism of this effect is unknown. We studied nondiabetic insulin-resistant spontaneously hypertensive rats (SHR) and found that catalytic activity of the beta -cell glucose sensor enzyme, glucokinase, was enhanced (6). However, alternate mechanisms have been reported in other models (3, 34, 40). In particular, a recent suggestion is that elevated beta -cell fatty acid (FA) metabolism mediates the heightened glucose sensing for insulin secretion, because hypertriglyceridemia commonly is found with insulin resistance (32), and culturing islets with FA shifts to the left the glucose concentration-insulin secretion curve (12, 28, 42). It remains unclear how to reconcile these different findings.

Little is known about how glucose homeostasis is preserved when the beta -cell mass is lowered. Biobreeding rats before onset of autoimmune diabetes have a left-shifted glucose concentration-insulin secretion curve (36). We made the same observation after a 60% pancreatectomy in rats (20). Thus a variable beta -cell sensitivity to glucose also operates when the beta -cell mass is lowered. Whether the biochemical details are the same as for insulin-resistant states is not known.

The current study investigated rats after a 60% pancreatectomy (Px); these rats maintain normal plasma levels of insulin and glucose despite the considerable loss of beta -cells (14, 19, 29), making them an excellent model for the investigation of beta -cell adaptive mechanisms. Rats were studied 4 wk after the Px surgery to minimize any possibility that the observed changes were in transition and not reflective of the completed beta -cell adaptation process.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

60% Px model and islet isolation. One hundred-gram Sprague-Dawley rats underwent 60% Px by use of our previously described method (19). Briefly, the portion of the pancreas bordered by the spleen and stomach extending to the small flap of pancreas attached to the pylorus was removed by use of gentle abrasion with cotton applicators. The removed portion was 57 ± 3% of the pancreas weight (19). Control (sham) rats underwent laparotomy and mobilization of the pancreas with gentle rubbing between the fingers. Postoperatively, all rats received standard chow and tap water ad libitum until being studied 4 wk after the surgery. Islets were isolated using an adaptation of the Gotoh method (10): pancreas duct infiltration with collagenase (Serva, Heidelberg, Germany), Histopaque gradient separation (Sigma, St. Louis, MO), and hand picking. Islet DNA content was measured by the Labarca method (17), protein by a commercial kit (Bio-Rad, Hercules, CA) with bovine albumin as standard, and insulin content after acid ethanol extraction with an insulin RIA (1). Freshly isolated islets were used in all experiments.

Oral glucose tolerance test and meal challenge. Both tests were preceded by an overnight fast. Px and sham rats were administered 1 g/kg of glucose (0.5 g/ml) by gavage tube. Blood for plasma glucose determination was obtained by tail snipping at 0, 30, 60, and 120 min. The meal challenge was performed 3 days later. Px and sham rats were given free access to chow at 9:00 AM (time 0), and plasma glucose values were measured at 0, 30, 60, and 120 min.

Islet insulin secretion and glucose utilization. Islets underwent 30 min of preincubation in warmed and oxygenated Krebs-Ringer buffer (KRB) with 2.8 mM glucose and 0.5% BSA. Insulin secretion was assessed using triplicate batches of 10 islets in glass vials containing 1 ml of KRB with 0.5% BSA and 2.8, 5.5, 8.3, or 16.7 mM glucose for 60 min in a 37°C shaking water bath. Medium was separated by gentle centrifugation and stored at -20°C pending insulin measurement by RIA (1). Islet glucose usage was measured as previously described (6) under the same experimental conditions with a method based on quantifying conversion of D-[5-3H]glucose (NEN, Boston, MA) to [3H]H2O (2).

Islet glucokinase/hexokinase kinetics. Glucose phosphorylation was measured in islet extracts as previously described (6) with a method based on quantifying conversion of NAD+ to NADH by exogenous glucose-6-phosphate dehydrogenase (22). Islet homogenates were centrifuged at 12,000 g for 10 min, and the supernatants were incubated at 10 glucose concentrations (0.03-100 mM) to measure glucose phosphorylation. Maximal velocity (Vmax) and Michaelis-Menten constant (Km) values for glucokinase and hexokinase were calculated by linear regression from an Eadie-Scatchard plot (volume/substrate concn) and 10 cycles of the method of Spears et al. (35) to identify each enzyme's activity.

Glucokinase immunoblots. Glucokinase immunoblots were performed as described (6) using sheep antiserum raised against an Escherichia coli-derived B1 isoform of rat glucokinase (gift from Dr. Mark Magnuson, Vanderbilt University). Bound antibody was detected by chemiluminescence and quantified by densitometry. Islets used under denaturing conditions were homogenized in 80 mM Tris (pH 6.8), 0.5% NP-40, 0.5% Triton X, 5 mM EDTA, 0.2 mM N-ethylmaleimide, and 1 mM phenylmethylsulfonyl fluoride, followed by heating at 95°C for 5 min in Laemmli sample buffer that contained 2% SDS and by running on a 10% acrylamide gel with stacking and separating buffers that contained 0.4% SDS. Nondenaturing gels were performed using islets that were homogenized in 20 mM K2HPO4, 1 mM EDTA, 110 mM KCl, and 2 mM dithiothreitol, placed in Laemmli sample buffer without SDS, and run on a 15% acrylamide gel with buffers without SDS.

Islet glucose 6-phosphate concentration. Px and sham islets (20 per tube) were incubated in prewarmed and oxygenated KRB with 0.5% BSA and 2.8, 8.3, or 16.7 mM glucose for 60 min in a 37°C shaking water bath, followed by rapid lysis in 10 µl of 40 mM NaOH. Then they were placed on ice for 10 min, and 3 µl of 0.15 M HCl were added with incubation at 75°C for 20 min to destroy cellular enzymes to ensure stability of glucose 6-phosphate (G-6-P) content. G-6-P was measured using an adaptation of the Lowry and Passonneau method (24) as previously described (23).

Analytical methods. Plasma glucose was measured with a Beckman Glucose Analyzer II (Beckman, Fullerton, CA). The insulin RIA used charcoal separation (1) and rat insulin standards (Lilly, Indianapolis, IN). Plasma triglycerides and free fatty acids were measured by commercial kits (Sigma and Wako Chemicals, Richmond, VA, respectively).

Data analysis and statistical methods. Data are expressed as means ± SE. The listed n values for the isolated islet data are the numbers of experiments that were performed using islets from separate isolation days. Islet data are expressed per protein or DNA content to adjust for the difference in cell mass of isolated islets from Px and sham rats. Western blots were performed using islets from single Px and sham rats, so the listed n value is the number of animals studied. The densitometry results from Western blots were expressed in relative terms by comparing the Px band on each gel to the sham band (assigned a value of 100%). Statistical significance was determined with the unpaired Student's t-test, except for the Western blot results, for which the one-tailed t-test was used.


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

General characteristics. Body weight and nonfasting glycemia, insulinemia, triglyceridemia, and plasma free fatty acids were equal in the 60% Px and sham-operated rats (Table 1). An oral glucose tolerance test and a meal challenge were performed to confirm the normoglycemia of the 60% Px rats; glycemic responses were identical in the two groups (Fig. 1).

                              
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Table 1.   General characteristics of rats 4 wk after sham operation or 60% Px



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Fig. 1.   Rats 4 wk after 60% (, n = 8) or sham (open circle , n = 4) pancreatectomy underwent an oral glucose tolerance test (OGTT, left) followed 3 days later by a meal challenge (right). Both tests were preceded by an overnight fast.

One possibility for these results was regeneration of the excised beta -cells. We had previously addressed that issue 7 wk after 60% Px: beta -cell mass was 55% of the sham rats, which is a 40% increase from the postsurgery period, and islet non-beta -cell mass was unchanged at 45% of control, which suggests that the islet regeneration was beta -cell specific (19). We now report identical findings in isolated islets from 60% Px rats 4 wk after the surgery. Islet DNA, protein, and insulin content were 40% increased compared with the sham-operated animals (Table 2). Consequently, the 60% Px induced some beta -cell regeneration, but it was incomplete.

                              
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Table 2.   Characterization of isolated islets from rats 4 wk after sham operation or 60% Px

Islet glucose sensing/responsiveness. We assessed insulin secretion and glucose utilization in isolated islets from the 60% Px and sham-operated rats (Fig. 2); results are expressed per milligram of protein to compensate for the different islet cell mass. Both parameters showed an upregulated response in the Px islets, with no change in the dose giving a half-maximal response (ED50), and a 70-80% increase at 8.4 mM glucose, which approximates the usual nonfasting plasma glucose level of the Px and sham rats (Table 1).


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Fig. 2.   Insulin secretion (A, n = 3 experiments) and glucose utilization (B, n = 4 experiments) in isolated islets from rats 4 wk after 60% () or sham (open circle ) pancreatectomy.

The pattern of the increase suggested augmented glucokinase activity in the Px islets, which was confirmed by measuring glucose phosphorylation in islet extracts over a range of 0.03-100 mM glucose (Fig. 3; derived enzyme kinetics shown in Table 3). Glucose phosphorylation over the range of glucose concentrations that approximated normoglycemia was 60% increased in the Px islets, in close agreement with the results in Fig. 2. The reason was a twofold increase in glucokinase Vmax (P < 0.002) with no change in ED50. Hexokinase Vmax also was somewhat increased. However, the absence of increased basal glucose usage in the Px islets (Fig. 2) suggested that its activity within the intact cell was not substantially changed. Neither was there a difference in G-6-P concentration between the Px and sham islets at 2.8, 8.3, and 16.7 mM glucose (Fig. 4), which is a potent allosteric inhibitor of hexokinase (9). The reason for the raised basal insulin secretion in the Px islets was thus not clear from these results.


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Fig. 3.   Glucose phosphorylation (n = 5 experiments) measured after 90-min incubations at the shown glucose concentrations in islet extracts from rats 4 wk after 60% () or sham (open circle ) pancreatectomy. The reaction was measured at 30°C as the appearance of NADH from NAD+ by exogenous glucose-6-phosphate dehydrogenase. Left: incubations carried out at 0.03-0.5 mM glucose, primarily representing hexokinase activity; right: incubations carried out at 6-100 mM glucose, primarily representing glucokinase activity.


                              
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Table 3.   Hexokinase and glucokinase kinetics in islet extracts from rats 4 wk after sham operation or 60% Px



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Fig. 4.   Glucose 6-phosphate (G-6-P) concentration (n = 3 experiments) of isolated islets from rats 4 wk after 60% () or sham (open circle ) pancreatectomy. Freshly isolated islets were incubated in KRB and 2.8, 8.3, or 16.7 mM glucose for 60 min, followed by measurement of G-6-P content, as described in the text.

Glucokinase immunoblot. Glucokinase immunoblots were performed in Px and sham islets; Fig. 5A shows a standard denaturing gel with samples from 2 Px and 2 sham rats. A small increase in glucokinase was noted in the Px islets, which averaged 142 ± 12% of control (P < 0.02 based on 6 Px and 6 sham rats). However, the gels were performed using equivalent amounts of islet protein from Px and sham islets and thus failed to adjust for the 40% beta -cell enrichment of the Px islets (Table 1); the close agreement between these figures suggested that the increased band intensity of the Px islets on immunoblot simply reflected differences in how much beta -cell protein was loaded. Additional support for this conclusion is shown in Fig. 5B, which is a nondenaturing gel with comparable Px and sham islet extracts. Nondenaturing conditions leave relatively intact protein-protein interactions or allosteric influences that modulate a protein's function. Striking differences were seen compared with the denaturing gel; the Px band intensity was now less than that of the sham, confirming the presence of a glucokinase posttranslational change in Px islets. Thus the doubled glucokinase Vmax per kilogram DNA in the Px islets stemmed from an enhanced catalytic activity of this enzyme, as opposed to a changed beta -cell expression.


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Fig. 5.   Glucokinase Western blots of isolated islets from rats 4 wk after 60% (Px) or sham (C) pancreatectomy. A: 30-µg protein aliquots from 2 separate sham-operated and 60% Px rats run under denaturing conditions, as described in text. The top band eluted at 52 kDa and is glucokinase. B: 40-µg protein aliquots from a sham and a 60% Px rat run under nondenaturing conditions, as described in text.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

This study has identified the beta -cell adaptive responses in 60% Px rats that kept insulinemia, and thus glycemia and triglyceridemia, indistinguishable from sham-operated rats. One element was a limited regeneration of the excised beta -cells, which brought the pancreas beta -cell mass from 40% of normal at the time of surgery to slightly more than one-half of normal several weeks later (19). The more consequential effect was a changed relationship between the glucose concentration and islet glucose utilization so that normal levels of glycemia elicited a raised flux of islet glucose metabolism, and consequently greater than normal insulin secretion. The latter reflects beta -cell glycolytic flux being a key regulator of glucose-induced insulin secretion through well characterized effects on beta -cell ion channels and membrane potential (27, 31). The heightened glycolytic flux stemmed from an enhanced activity of glucokinase, which is the main regulator of beta -cell glucose usage and is termed the beta -cell glucose sensor (26). The mechanism was an increased catalytic activity of this enzyme, as opposed to a change of its beta -cell concentration, which was confirmed by the novel observation of divergent denaturing and nondenaturing glucokinase immunoblots in the Px islets. Crucial to the workings of this regulatory system was the observation that the degree of change in glucokinase activity was small; glucose phosphorylation and, consequently, glucose metabolism were 60-80% increased in Px islets at the physiologically relevant 8.4 mM glucose, compared with the multifold changes in gene expression that make up many cellular regulatory systems, such as the fivefold increase in proinsulin gene expression reported after short-term exposure of beta -cells to a high glucose concentration (21). The reason is that glucokinase is the rate-limiting beta -cell glycolytic enzyme and is without allosteric or end-product influences (26, 27). Its activity thus directly modulates beta -cell glucose utilization and insulin secretion (38). This effect was evident in the current study by the close agreement between the degree of changes in glucokinase activity, glucose utilization, and insulin secretion in the Px islets. As such, insulin release at 8.4 mM glucose also increased 80%, which is very significant physiologically. By this reasoning, it is of interest that the rise in beta -cell mass to 55% of normal (19), together with the 92% increased glucokinase activity per cell mass shown in Table 3, results in whole pancreas glucokinase activity in the Px rats being identical to that in the sham rats. Our results are consistent with the recent study of Martín et al. (25), which concluded that an upregulation of islet glucose metabolism was the basis for the beta -cell glucose hypersensitivity in normoglycemic 60% Px mice, although the study provided no insight as to the mechanism (25). Note, glucose utilization was assessed in this study only as conversion of [5-3H]glucose to [3H]H2O, and a measure of affected pathways and metabolites is needed to fully understand how the change in glucokinase activity affects islet-cell glucose metabolism, analogous to the studies of others (38, 39).

Our finding of increased glucokinase catalytic activity in the Px islets agrees with our studies of normoglycemic insulin-resistant SHR rats (6) and rats that were hyperinsulinemic and normoglycemic secondary to glucose infusions (5). Making the same observation in three diverse rat models of successful beta -cell adaptation suggests that variable glucokinase activity is a core beta -cell adaptive response of the glucose homeostasis system that is initiated by changing metabolic demands for insulin secretion of multiple types. The basis for this effect is not known, although evidence for a protein interaction affecting beta -cell glucokinase activity has recently appeared (37). However, this is not the only mechanism that alters beta -cell glucose sensing in a compensatory fashion, because others have been identified in specialized states. Best studied is pregnancy, a state of insulin resistance, in which upregulation of beta -cell glucokinase and hexokinase cellular levels occurs, presumably on the basis of the distinctive mediators prolactin and placental hormones (30, 34). A second mode regards the compensatory hyperinsulinemia in models of insulin resistance with marked hypertriglyceridemia, which has been linked to high levels of islet FA metabolites (12, 28, 42). We showed that the mechanism is increased activity of phosphofructokinase that lowers the G-6-P level through accelerated G-6-P metabolism (23). G-6-P is a potent inhibitor of hexokinase (9), and our results thus focused on deinhibition of hexokinase as the basis for the enhanced basal beta -cell glucose sensing and insulin secretion with excess FA. Viewed in toto, there is a repertoire of beta -cell adaptive responses depending on the stimulus, which have in common an ability to alter insulin secretion through changes in the beta -cell sensitivity to glucose and thus do not require a change in glycemia. The importance of this effect reflects the myriad of cellular dysfunctions attributed to chronic hyperglycemia (so-called glucose toxicity) (20, 33, 41).

The above discussion has focused on normoglycemic states, i.e., when the beta -cell adaptive mechanism is successful. Studies of hyperglycemic states have suggested that there is the occurrence of an additional element that affects beta -cell glucose sensing. We studied 90% Px rats that develop mild hyperglycemia, and we observed that increased hexokinase Vmax was the dominant change affecting islet glucose phosphorylation (13), in tandem with a substantial rise in basal insulin secretion (20). We also studied 48-h glucose-infused rats and showed that islet hexokinase Vmax and basal insulin secretion were increased in hyperglycemic rats but not in those that were normoglycemic/hyperinsulinemic (5). Cockburn et al. (7) studied diabetes-prone Zucker diabetic fatty rats before and after the onset of diabetes; the prediabetes time point was characterized by increased islet glucokinase activity (analogous to our results in 60% Px rats), as opposed to hexokinase activity and basal insulin secretion being markedly increased when hyperglycemia was established (7). These results suggest that hyperglycemia or an associated metabolic defect upregulates beta -cell hexokinase activity, and consequently basal insulin secretion, through an undefined mechanism. Moreover, we have argued that a hyperstimulated insulin secretion, leading to the beta -cell insulin content falling below a required level for normal secretory responses, is a mechanism of beta -cell dysfunction with chronic hyperglycemia (18). It may be that this increased hexokinase activity is an important causative factor of this sequence.

In summary, our results provide an explanation for the conundrum regarding the glucose homeostasis system whereby beta -cells are known to be glucose responsive at multiple functional levels, so that glycemia is considered their main regulatory influence, coupled with the understanding that glycemia varies little throughout life because of offsetting compensations of insulin secretion. The regulatory system observed herein is based on a changeable rate of beta -cell glucose metabolism so that beta -cells are "tricked" to perceive altered glycemia in the face of normoglycemia, allowing glucose-regulated beta -cell adaptive responses to occur when glycemia is unaltered.


    ACKNOWLEDGEMENTS

This work was supported by a grant from the American Diabetes Association (to J. L. Leahy).


    FOOTNOTES

Address for reprint requests and other correspondence: J. L. Leahy, Univ. of Vermont College of Medicine, Given C331, Burlington, VT 05405 (E-mail: jleahy{at}zoo.uvm.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Received 9 August 1999; accepted in final form 25 January 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Albano, JDM, Ekins RP, Maritz G, and Turner RC. A sensitive, precise radioimmunoassay of serum insulin relying on charcoal separation of bound and free hormone moieties. Acta Endocrinol 70: 487-509, 1972[ISI][Medline].

2.   Ashcroft, SJH, Weerasinghe LCC, Bassett JM, and Randle PJ. The pentose cycle and insulin release in mouse pancreatic islets. Biochem J 126: 525-532, 1972[ISI][Medline].

3.   Chan, CB. beta -Cell stimulus-secretion coupling defects in rodent models of obesity. Can J Physiol Pharmacol 73: 1414-1424, 1995[ISI][Medline].

4.   Chan, CB, MacPhail RM, and Mitton K. Evidence for defective glucose sensing by islets of fa/fa obese Zucker rats. Can J Physiol Pharmacol 71: 34-39, 1993[ISI][Medline].

5.   Chen, C, Bumbalo LM, Hosokawa H, and Leahy JL. Increased catalytic activity of glucokinase in isolated islets from hyperinsulinemic rats. Diabetes 43: 684-689, 1994[Abstract].

6.   Chen, C, Hosokawa H, Bumbalo LM, and Leahy JL. Mechanism of compensatory hyperinsulinemia in normoglycemic insulin resistant SHR rats: augmented enzymatic activity of glucokinase in beta -cells. J Clin Invest 94: 399-404, 1994[ISI][Medline].

7.   Cockburn, BN, Ostrega DM, Sturis J, Kubstrup C, Polonsky KS, and Bell GI. Changes in pancreatic islet glucokinase and hexokinase activities with increasing age, obesity, and the onset of diabetes. Diabetes 46: 1434-1439, 1997[Abstract].

8.   Curry, DL, and Stern JS. Dynamics of insulin hypersecretion by obese Zucker rats. Metabolism 34: 791-796, 1985[ISI][Medline].

9.   Giroix, M-H, Sener A, Pipeleers DG, and Malaisse WJ. Hexose metabolism in pancreatic islets. Inhibition of hexokinase. Biochem J 223: 447-453, 1984[ISI][Medline].

10.   Gotoh, M, Maki T, Satomi S, Porter J, Bonner-Weir S, O'Hara CJ, and Monaco AP. Reproducible high yield of rat islets by stationary in vitro digestion following pancreatic ductal or portal venous collagenase injection. Transplantation 43: 725-730, 1987[ISI][Medline].

11.   Green, IC, and Taylor KW. Effects of pregnancy in the rat on the size and insulin secretory response of the islets of Langerhans. J Endocrinol 54: 317-325, 1972[ISI][Medline].

12.   Hosokawa, H, Hosokawa YA, and Leahy JL. Upregulated hexokinase activity in isolated islets from diabetic 90% pancreatectomized rats. Diabetes 44: 1328-1333, 1995[Abstract].

13.   Hosokawa, H, Corkey BE, and Leahy JL. Beta-cell hypersensitivity to glucose following 24-h exposure of rat islets to fatty acids. Diabetologia 40: 392-397, 1997[ISI][Medline].

14.   Jansson, L, and Sandler S. Pancreatic and islet blood flow in the regenerating pancreas after a partial pancreatectomy in adult rats. Surgery 106: 861-866, 1989[ISI][Medline].

15.   Kahn, SE, Beard JC, Schwartz MW, Ward WK, Ding HL, Bergman RN, Taborsky GJ, Jr, and Porte D, Jr. Increased beta-cell secretory capacity as mechanism for islet adaption to nicotinic acid-induced insulin resistance. Diabetes 38: 562-568, 1989[Abstract].

16.   Kahn, SE, Prigeon RL, McCulloch DK, Boyko EJ, Bergman RN, Schwartz MW, Neifing JL, Ward WK, Beard JC, Palmer JP, and Porte D, Jr. Quantification of the relationship between insulin sensitivity and beta -cell function in human subjects. Evidence for a hyperbolic function. Diabetes 42: 1663-1672, 1993[Abstract].

17.   Labarca, C, and Paigen KD. A simple, rapid, and sensitive DNA assay procedure. Anal Biochem 102: 344-352, 1980[ISI][Medline].

18.   Leahy, JL. beta -Cell dysfunction with chronic hyperglycemia: the "overworked beta -cell" hypothesis. Diabetes Rev 4: 298-319, 1996.

19.   Leahy, JL, Bonner-Weir S, and Weir GC. Minimal chronic hyperglycemia is a critical determinant of impaired insulin secretion after an incomplete pancreatectomy. J Clin Invest 81: 1407-1414, 1988[ISI][Medline].

20.   Leahy, JL, Bumbalo LM, and Chen C. Beta-cell hypersensitivity for glucose precedes loss of glucose-induced insulin secretion in 90% pancreatectomized rats. Diabetologia 36: 1238-1244, 1993[ISI][Medline].

21.   Leibiger, B, Moede T, Schwarz T, Brown GR, Kohler M, Leibiger IB, and Berggren PO. Short-term regulation of insulin gene transcription by glucose. Proc Natl Acad Sci USA 95: 9307-9312, 1988[Abstract/Free Full Text].

22.   Liang, Y, Najafi H, and Matschinsky FM. Glucose regulates glucokinase activity in cultured islets from rat pancreas. J Biol Chem 265: 16863-16866, 1990[Abstract/Free Full Text].

23.   Liu, YQ, Tornheim K, and Leahy JL. Fatty acid-induced beta cell hypersensitivity to glucose. Increased phosphofructokinase activity and lowered glucose-6-phosphate content. J Clin Invest 101: 1870-1875, 1998[Abstract/Free Full Text].

24.   Lowry, OH, and Passonneau JV. A Flexible System of Enzymatic Analysis. New York: Academic, 1972, p. 179-182.

25.   Martín, F, Andreu E, Rovira JM, Pertusa JAG, Raurell M, Ripoll C, Sanchez-Andrés JV, Montanya E, and Soria B. Mechanisms of glucose hypersensitivity in beta -cells from normoglycemic, partially pancreatectomized mice. Diabetes 48: 1954-1961, 1999[Abstract].

26.   Matschinsky, F, Liang Y, Kesavan P, Wang L, Froguel P, Velho G, Cohen D, Permutt MA, Tanizawa Y, Jetton TL, Niswender K, and Magnuson MA. Glucokinase as beta  cell glucose sensor and diabetes gene. J Clin Invest 92: 2092-2098, 1993[ISI][Medline].

27.   Meglasson, MD, and Matschinsky FM. Pancreatic islet glucose metabolism and regulation of insulin secretion. Diabetes Metab Rev 2: 163-214, 1986[Medline].

28.   Milburn, JL, Jr, Hirose H, Lee YH, Nagasawa Y, Ogawa A, Ohneda M, BeltrandelRio H, Newgard CB, Johnson JH, and Unger RH. Pancreatic beta -cells in obesity. Evidence for induction of functional, morphologic, and metabolic abnormalities by increased long chain fatty acids. J Biol Chem 270: 1295-1299, 1995[Abstract/Free Full Text].

29.   Orland, MJ, Chyn R, and Permutt MA. Modulation of proinsulin messenger RNA after partial pancreatectomy in rats. Relationship to glucose homeostasis. J Clin Invest 75: 2047-2055, 1985[ISI][Medline].

30.   Parsons, JA, Brelje TC, and Sorenson RL. Adaption of islets of Langerhans to pregnancy: increased islet cell proliferation and insulin secretion correlates with the onset of placental lactogen secretion. Endocrinology 130: 1459-1466, 1992[Abstract].

31.   Piston, DW, Knobel SM, Postic C, Shelton KD, and Magnuson MA. Adenovirus-mediated knockout of a conditional glucokinase gene in isolated pancreatic islets reveals an essential role for proximal metabolic events in glucose-stimulated insulin secretion. J Biol Chem 274: 1000-1004, 1999[Abstract/Free Full Text].

32.   Prentki, M, and Corkey BE. Are the beta -cell signaling molecules malonyl-CoA and cytosolic long-chain acyl-CoA implicated in multiple tissue defects of obesity and NIDDM? Diabetes 45: 273-283, 1996[Abstract].

33.   Rossetti, L, Giaccari A, and DeFronzo RA. Glucose toxicity. Diabetes Care 13: 610-630, 1990[Abstract].

34.   Sorenson, RL, and Brelje TC. Adaptation of islets of Langerhans to pregnancy: beta-cell growth, enhanced insulin secretion and the role of lactogenic hormones. Horm Metab Res 29: 301-307, 1997[ISI][Medline].

35.   Spears, G, Sneyd GT, and Loten EG. A method for deriving kinetic constants for two enzymes acting on the same substrate. Biochem J 125: 1149-1151, 1971[ISI][Medline].

36.   Teruya, M, Takei S, Forrest LE, Grunewald A, Chan EK, and Charles MA. Pancreatic islet function in nondiabetic and diabetic BB rats. Diabetes 42: 1310-1317, 1993[Abstract].

37.   Tiedge, M, Steffeck H, Elsner M, and Lenzen S. Metabolic regulation, activity state, and intracellular binding of glucokinase in insulin-secreting cells. Diabetes 48: 514-523, 1999[Abstract].

38.   Wang, H, and Iynedjian PB. Modulation of glucose responsiveness of insulinoma beta-cells by graded overexpression of glucokinase. Proc Natl Acad Sci USA 94: 4372-4377, 1997[Abstract/Free Full Text].

39.   Wang, H, and Iynedjian PB. Acute glucose intolerance in insulinoma cells with unbalanced overexpression of glucokinase. J Biol Chem 272: 25731-25736, 1997[Abstract/Free Full Text].

40.   Weinhaus, AJ, Bhagroo NV, Brelje TC, and Sorenson RL. Role of cAMP in upregulation of insulin secretion during the adaptation of islets of Langerhans to pregnancy. Diabetes 47: 1426-1435, 1998[Abstract].

41.   Yki-Järvinen, H. Glucose toxicity. Endocr Rev 13: 415-431, 1992[ISI][Medline].

42.   Zhou, YP, and Grill VE. Long-term exposure of rat pancreatic islets to fatty acids inhibits glucose-induced insulin secretion and biosynthesis through a glucose fatty acid cycle. J Clin Invest 93: 870-876, 1994[ISI][Medline].


Am J Physiol Endocrinol Metab 279(1):E68-E73
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