John H. Johnson

STZ Transport and Cytotoxicity: Specific Enhancement in GLUT2-Expressing Cells

The glucose analog streptozotocin (STZ) has long been used as a tool for creating experimental diabetes because of its relatively specific β-cell cytotoxic effect, but the mechanism by which systemic injection of STZ causes β-cell destruction is not well understood. In the current study, we have used insulinoma (RIN) and AtT-20ins cell lines engineered for overexpression of GLUT2 or GLUT1 to investigate the role of glucose transporter isoforms in mediating STZ cytotoxicity. The in vivo effects of STZ were evaluated by implantation of RIN cells expressing or lacking GLUT2 into athymic nude rats. The drug had a potent cytotoxic effect on RIN cells expressing GLUT2, but had no effect on cells lacking GLUT2 expression, as indicated by histological analysis and measurement of the blood glucose levels of treated animals. The preferential cytotoxic effect of STZ on GLUT2-expressing cell lines was confirmed by in vitro analysis of GLUT2-expressing and untransfected RIN cells, as well as GLUT2- and GLUTl-overexpressing AtT-20ins cells. Consistent with these data, only GLUT2-expressing RIN or AtT-20ins cells transported STZ efficiently. We conclude that expression of GLUT2 is required for efficient killing of neuroendocrine cells by STZ, and this effect is related to specific recognition of the drug as a transported substrate by GLUT2 but not GLUT1

Pancreatic beta-cells in obesity. Evidence for induction of functional, morphologic, and metabolic abnormalities by increased long chain fatty acids.

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

The tissue distribution of tumor necrosis factor biosynthesis during endotoxemia.

Tumor necrosis factor (TNF) is a protein hormone implicated in the development of septic shock and other pathologic states. However, complexities inherent in detecting TNF synthesis by individual tissues have left the precise origins of this protein undefined. In addition, the possibility that localized TNF production may contribute to the pathogenesis of organ-specific diseases such as type I diabetes has not been explored in vivo. We have developed a transgenic mouse line bearing a reporter gene construct in which the TNF coding sequence and introns are replaced by a chloramphenicol acetyltransferase (CAT) coding sequence. In normal transgenic animals, CAT activity is expressed only in the thymus. When endotoxin is administered to the animals, CAT activity is also evident in kidney, heart, islets of Langerhans, spleen, lung, fallopian tubes, and uterus, but not in other organs. The biosynthesis of CAT in vivo correlated with tissue capacity to secrete TNF in vitro. Thus, TNF was secreted by all the tissues that expressed CAT, including lung, spleen, thymus, uterus/fallopian tubes, pancreatic islets, renal glomeruli, and cultured cardiac cells after exposure to endotoxin.

Underexpression of beta cell high Km glucose transporters in noninsulin-dependent diabetes.

The role of defective glucose transport in the pathogenesis of noninsulin-dependent diabetes (NIDDM) was examined in Zucker diabetic fatty rats, a model of NIDDM. As in human NIDDM, insulin secretion was unresponsive to 20 mM glucose. Uptake of 3-O-methylglucose by islet cells was less than 19% of controls. The beta cell glucose transporter (GLUT-2) immunoreactivity and amount of GLUT-2 messenger RNA were profoundly reduced. Whenever fewer than 60% of beta cells were GLUT-2-positive, the response to glucose was absent and hyperglycemia exceeded 11 mM plasma glucose. We conclude that in NIDDM underexpression of GLUT-2 messenger RNA lowers high Km glucose transport in beta cells, and thereby impairs glucose-stimulated insulin secretion and prevents correction of hyperglycemia.

Roles of insulin resistance and beta-cell dysfunction in dexamethasone-induced diabetes.

The roles of insulin resistance and beta-cell dysfunction in glucocorticoid-induced diabetes were determined in Wistar and Zucker (fa/fa) rats. All Wistar rats treated with 5 mg/kg per d of dexamethasone for 24 d exhibited increased beta-cell mass and basal and arginine-stimulated insulin secretion, indicating insulin resistance, but only 16% became diabetic. The insulin response to 20 mM glucose was normal in the perfused pancreas of all normoglycemic dexamethasone-treated rats but absent in every diabetic rat. Immunostainable high Km beta-cell transporter, GLUT-2, was present in approximately 100% of beta-cells of normoglycemic rats, but in only 25% of beta cells of diabetic rats. GLUT-2 mRNA was not reduced. All Zucker (fa/fa) rats treated with 0.2-0.4 mg/kg per d of dexamethasone for 24 d became diabetic and glucose-stimulated insulin secretion was absent in all. High Km glucose transport in islets was 50% below nondiabetic controls. Only 25% of beta cells of diabetic rats were GLUT-2-positive compared with approximately 100% in controls. Total pancreatic GLUT-2 mRNA was increased twofold suggesting a posttranscriptional abnormality. We conclude that dexamethasone induces insulin resistance, whether or not it induces hyperglycemia. Whenever hyperglycemia is present, GLUT-2-positive beta cells are reduced, high Km glucose transport into beta cells is attenuated and the insulin response to glucose is absent.

Defective Fatty Acid-mediated -Cell Compensation in Zucker Diabetic Fatty Rats PATHOGENIC IMPLICATIONS FOR OBESITY-DEPENDENT DIABETES

Although obesity is associated with insulin resistance, most obese humans and rodents remain normoglycemic because of compensatory hyperinsulinemia. This has been attributed to β-cell hyperplasia and increased low K glucose metabolism of islets. Since free fatty acids (FFA) can induce these same β-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 β-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 β-cell function.

Activation of stimulus-secretion coupling in pancreatic beta-cells by specific products of glucose metabolism. Evidence for privileged signaling by glycolysis.

The energy requirements of most cells supplied with glucose are fulfilled by glycolytic and oxidative metabolism, yielding ATP. In pancreatic β-cells, a rise in cytosolic ATP is also a critical signaling event, coupling closure of ATP-sensitive K channels (K) to insulin secretion via depolarization-driven increases in intracellular Ca ([Ca]). We report that glycolytic but not Krebs cycle metabolism of glucose is critically involved in this signaling process. While inhibitors of glycolysis suppressed glucose-stimulated insulin secretion, blockers of pyruvate transport or Krebs cycle enzymes were without effect. While pyruvate was metabolized in islets to the same extent as glucose, it produced no stimulation of insulin secretion and did not block K. A membrane-permeant analog, methyl pyruvate, however, produced a block of K, a sustained rise in [Ca], and an increase in insulin secretion 6-fold the magnitude of that induced by glucose. These results indicate that ATP derived from mitochondrial pyruvate metabolism does not substantially contribute to the regulation of K responses to a glucose challenge, supporting the notion of subcompartmentation of ATP within the β-cell. Supranormal stimulation of the Krebs cycle by methyl pyruvate can, however, overwhelm intracellular partitioning of ATP and thereby drive insulin secretion.

Inhibition of Glucose Transport into Rat Islet Cells by Immunoglobulins from Patients with New-Onset Insulin-Dependent Diabetes Mellitus

Because glucose-stimulated insulin secretion is selectively impaired during the development of insulin-dependent diabetes mellitus (IDDM), we tested the possibility that the glucose transporter of pancreatic islet beta cells is a target of the autoimmune process in patients with IDDM. We measured the uptake of 3-O-methyl-beta-D-glucose by dispersed islet cells from rats after a 15-minute incubation with purified IgG from 27 patients with newly diagnosed IDDM, 28 normal subjects, and 5 patients with non-insulin-dependent diabetes mellitus (NIDDM). The IgG fractions from 26 of the 27 patients with IDDM (96 percent), but from none of the 5 patients with NIDDM, reduced the initial rates of 3-O-methyl-beta-D-glucose uptake to at least 1 SD below the mean of the rates observed in the presence of IgG fractions from normal subjects (P less than 0.001). In contrast, the uptake of L-leucine by islet cells was not affected by any of the IgG fractions. The inhibitory activity of IgG from the patients with IDDM was abolished by preincubation with islet cells and membranes from hepatocytes, which contain the same glucose transporter as beta cells, but not with erythrocytes, which do not contain this transporter. We conclude that IgG from patients with IDDM of recent onset, but not from those with NIDDM, inhibits glucose uptake by rat islet cells. The results are consistent with the presence of an antibody against a protein involved in glucose transport by beta cells that would thereby impair glucose-stimulated insulin secretion.

GLUT2 Expression and Function in β-cells of GK rats with NIDDM: Dissociation Between Reductions in Glucose Transport and Glucose-Stimulated Insulin Secretion

GLUT2 underexpression has been reported in the +-cells of Zucker diabetic fatty rats and db/db mice, models of spontaneously occurring NIDDM with antecedent obesity. To determine whether the +-cells of a nonobese rodent model of NIDDM exhibit the same abnormalities in GLUT2, we studied Goto-Kakizaki rats. In these mildly diabetic animals glucose-stimulated insulin secretion was reduced at all ages examined from 8 to 48 wk. In normal control Wistar rats, immunostainable GLUT2 was present on all insulin-positive cells in the pancreatic islets. Only 85% of +-cells were GLUT2-positive in GK rats at 12 wk of age, and only 34% were positive at 48 wk of age. GLUT2 mRNA was 50% of normal in 12-wk-old GK rats. In the latter age-group, glucose-stimulated insulin secretion was only 28% of normal at a time when 85% of +-cells were GLUT2-positive and initial 3-O-methyl-D-glucose transport rate was 77% of the control value. We conclude that although GLUT2 is underexpressed, neither the magnitude of the underexpression of GLUT2 nor of the reduction in GLUT2 transport function in islets of GK rats is sufficient by itself to explain the profound reduction in glucose-stimulated insulin secretion.

GLUT-2 function in glucose-unresponsive beta cells of dexamethasone-induced diabetes in rats.

Spontaneous and dexamethasone-induced noninsulin-dependent diabetes mellitus (NIDDM) in rats is associated with loss of glucose-stimulated insulin secretion (GSIS) and a reduction in both GLUT-2-positive beta cells and high Km glucose transport. To determine if the chronology and correlation of these abnormalities is consistent with a causal relationship, Zucker (fa/fa) rats were studied longitudinally before and during 10 d of dexamethasone-induced (0.4 mg/kg per d i.p.) NIDDM. Within 24 h of dexamethasone treatment blood glucose rose and GSIS declined, becoming paradoxically negative (-87 +/- 12 microU/ml per min) on day 10. Blood glucose was negatively correlated with GSIS (r = -0.92; P < 0.001). 3-0-methyl-D-glucose (3MG) transport was unchanged at 12 h, 23% below normal on day 1, and declined further to a nadir 59% below normal. The GLUT-2-positive beta cell area did not decline until 48 h, reaching a nadir of 35% of normal at 10 d. The area of GLUT-2-positive beta cells was correlated with GSIS (r = 0.77; P < 0.005). We conclude that the chronology and correlation between GSIS loss and hyperglycemia is consistent with a cause-effect relationship, but that the subtotal impairment in glucose transport by itself cannot explain the total loss of GSIS if one assumes that normal beta cells are functionally homogenous.