Biomedical subjects

D K Kreutter

Publications and source records attributed to D K Kreutter.

13 recordsLinked to original sources

PubMed1998

Possibility of distinct insulin-signaling pathways beyond phosphatidylinositol 3-kinase-mediating glucose transport and lipogenesis.

The aim of this study was to compare the effects of insulin and the insulinomimetic agent, englitazone, on functional end points and putative mediators of insulin action in 3T3-L1 adipocytes. Cells were incubated with englitazone for 48 h or with insulin for 10 or 30 min, or both, and 2-deoxy-D-[3H]glucose (2DG) uptake and lipogenesis (from [14C]glucose) were measured. Tyrosine phosphorylation of the insulin receptor (IR), insulin receptor substrates 1 and 2 (IRS-1 and IRS-2), and pp60, and phosphatidylinositol (PI) 3-kinase activity (using PI as substrate) and mitogen-activated protein kinase (MAPK) activity were assayed in cell lysates. Englitazone increased 2DG uptake in a concentration-dependent (10-100 micromol/l) manner by up to sixfold, and preincubation with englitazone significantly enhanced insulin-stimulated 2DG uptake. However, englitazone had a biphasic effect on lipogenesis (163 +/- 13% basal at 10 micromol/l vs. 96 +/- 14% at 100 micromol/l), but when acetate was used as substrate, only concentration-dependent inhibition of lipogenesis occurred. In addition, englitazone decreased insulin-stimulated lipogenesis in a concentration-dependent manner. Englitazone did not increase IR, IRS-1/IRS-2, pp60, or MAPK phosphorylation, nor did it enhance insulin's stimulation of these parameters. Although englitazone alone did not activate PI 3-kinase, it did enhance the stimulation of the enzyme produced by a submaximally effective insulin concentration. Significant (63%) inhibition of insulin-stimulated lipogenesis occurred at a concentration of englitazone (30 micromol/l) that did not affect MAPK activation, which suggests that the drug's inhibitory effect on lipogenesis is not mediated by this pathway. Englitazone did not affect the expression of the peroxisome proliferator response element-containing fatty acyl CoA synthase gene, although it cannot be ruled out that expression of other lipogenic enzymes are altered by englitazone via peroxisome proliferator activated receptor-gamma activation or by an alternate pathway. Thus englitazone stimulates 2DG uptake without affecting PI 3-kinase, but it can enhance both insulin-stimulated 2DG uptake and PI 3-kinase activity. However, englitazone inhibits insulin-stimulated lipogenesis without inhibiting PI 3-kinase activity. Assuming activation of PI 3-kinase mediates insulin-stimulated 2-DG and lipogenesis, then the signaling pathways for each process diverge beyond PI 3-kinase.

3T3 Cells↗

PubMed1998

Islet amyloid-associated diabetes in obese A(vy)/a mice expressing human islet amyloid polypeptide.

We have previously shown that hemizygous transgenic mice expressing human islet amyloid polypeptide (hIAPP) in pancreatic beta-cells have no diabetic phenotype, whereas in the homozygous state, they developed severe, early-onset hyperglycemia associated with impaired insulin secretion and beta-cell death. We investigated the possibility that when the hemizygous mice are crossed onto an obese, insulin-resistant strain such as agouti viable yellow (A(vy)/a), they would exhibit a phenotype more akin to human type 2 diabetes. The hIAPP-expressing A(vy) males (TG-Y) displayed fasting hyperglycemia at 90 days of age and by 1 year progressed to severe hyperglycemia relative to their nontransgenic counterparts. Plasma insulin concentrations and pancreatic insulin content dropped 10- to 20-fold, suggesting severe impairment of beta-cell function. Histopathological findings revealed beta-cell degeneration and loss consistent with the drop in the plasma insulin concentration. In addition, large deposits of IAPP amyloid were present in TG-Y islets. We conclude that in transgenic mice expressing hIAPP, insulin resistance can induce overt, slow-onset diabetes associated with islet amyloid and decreased beta-cell mass.

Aging↗

PubMed1996

Spontaneous diabetes mellitus in transgenic mice expressing human islet amyloid polypeptide.

The islet in non-insulin-dependent diabetes mellitus (NIDDM) is characterized by loss of beta cells and large local deposits of amyloid derived from the 37-amino acid protein, islet amyloid polypeptide (IAPP). We have hypothesized that IAPP amyloid forms intracellularly causing beta-cell destruction under conditions of high rates of expression. To test this we developed a homozygous transgenic mouse model with high rates of expression of human IAPP. Male transgenic mice spontaneously developed diabetes mellitus by 8 weeks of age, which was associated with selective beta-cell death and impaired insulin secretion. Small intra- and extracellular amorphous IAPP aggregates were present in islets of transgenic mice during the development of diabetes mellitus. However, IAPP derived amyloid deposits were found in only a minority of islets at approximately 20 weeks of age, notably after development of diabetes mellitus in male transgenic mice. Approximately 20% of female transgenic mice spontaneously developed diabetes mellitus at 30+ weeks of age, when beta-cell degeneration and both amorphous and amyloid deposits of IAPP were present. We conclude that overexpression of human IAPP causes beta-cell death, impaired insulin secretion, and diabetes mellitus. Large deposits of IAPP derived amyloid do not appear to be important in this cytotoxicity, but early, small amorphous intra- and extracellular aggregates of human IAPP were consistently present at the time of beta-cell death and therefore may be the most cytotoxic form of IAPP.

Aging↗

PubMed1996

Age-related changes in amylin secretion.

Amylin (islet amyloid polypeptide) is a recently identified pancreatic peptide. It has been shown to affect glucose metabolism both in vitro and in vivo. A cross sectional analysis of the effects of age on amylin secretion following a 75 g glucose tolerance test in a young (20-40 years), middle aged (41-60 years) and older (61-90 years) group was performed. Thirty lean (BMI less than 25) non-diabetic individuals were studied. Amylin secretion exhibited a U-shaped curve with greater secretion in young and old subjects than in middle aged persons. Baseline levels were 7.2 +/- 1, 4.7 +/- 1, and 5.3 +/- 0.75 pM respectively, at 60 min 9.5 +/- 0.9 (y), 5.5 +/- 1 (m), 8.6 +/- 1 (o) pM; and at 120 min 10.3 +/- 2 (y), 4.4 +/- 0.5 (m), 10.9 +/- 1.5 (o) pM. Amylin production (area under the curve) was 1102 +/- 131, 619.5 +/- 79 and 1043 +/- 120 pM per min respectively (P < 0.05). Amylin secretion was similar in both sexes. Variability in the insulin to amylin ratio for each of the age groups at different time points following a glucose load was found, suggesting that insulin and amylin are not co-secreted in a fixed ratio. A significant association was found between both maximum amylin and rise in amylin (delta) and a glucose greater than 120 mg/dl at 2 h. (P < 0.001, P < 0.001). This correlation of glucose and amylin may be interpreted as suggestive of a counterregulatory role for amylin. However, aging is also associated with changes in glucose metabolism and amylin may merely be acting as a marker of impaired glucose metabolism.

Adult↗

PubMed1996

The antihyperglycemic agent englitazone prevents the defect in glucose transport in rats fed a high-fat diet.

The effects of englitazone in male Wistar rats fed a high-fat diet (59% of calories as fat) were compared with control rats fed a high-carbohydrate diet (69% of calories as carbohydrate) (5-15 animals per group). Insulin-stimulated (17 nmol/l) 2-deoxy-D-glucose (2-DG) uptake was inhibited 31% in adipocytes isolated from rats on the high-fat diet for 3 weeks, but englitazone (50 mg/kg for the last 7 days) normalized the response. There was a selective decrease in GLUT4 (54 +/- 5% of high-carbohydrate) in epididymal fat from rats on the high-fat diet for 3 weeks, but englitazone treatment did not reverse the defect in GLUT4 (43 +/- 8% of high-carbohydrate) or increase GLUT1 (81 +/- 12% of high-carbohydrate). Englitazone normalized oral glucose (1 g/kg body wt) intolerance and excessive (210% of high-carbohydrate) liver glycogen deposition (from [14C]glucose) caused by the high-fat diet. The high-fat diet tended to decrease insulin receptor substrate-1 (IRS-1) and phosphatidylinositol-3'-kinase (PI-3-kinase) expression in epididymal fat (26% decrease; P < 0.1). Englitazone did not reverse this decrease in IRS-1 and PI-3-kinase levels in fat from high-fat-fed rats (there was a further 25-30% decrease, P < 0.05), nor did it increase PI-3-kinase activity in 3T3-L1 adipocytes under conditions (48 h incubation) where it stimulated 2-DG uptake sixfold or enhanced insulin-stimulated 2-DG uptake. In summary, englitazone prevented the insulin resistance associated with a high-fat diet, but the mechanism of action does not involve changes in fat or muscle glucose transporter content and may not involve activation of the insulin signaling pathway via PI-3-kinase.

Adipocytes↗

PubMed1995

Distribution of beta 3-adrenoceptor mRNA in human tissues.

The beta 3-adrenoceptor is a G protein-coupled receptor which mediates metabolic functions of the endogenous catecholamines epinephrine and norepinephrine. Questions exist regarding distribution of the beta 3-adrenoceptor in human tissue. In order to examine the distribution of beta 3-adrenoceptor mRNA in human tissues, we used sensitive and specific RNase protection assays without previous PCR amplification in an extensive list of human tissues. We confirm the presence of beta 3-adrenoceptor mRNA in human white fat from several locations, gall bladder, and small intestine, as well as extend the distribution of beta 3-adrenoceptor mRNA to previously uncharacterized human tissues such as stomach and prostate. The presence of beta 3-adrenoceptor mRNA in human white adipose tissue has important implications regarding possible use of beta 3-adrenoceptor selective agonists as anti-obesity agents, and the demonstration of beta 3-adrenoceptor mRNA in a number of gastrointestinal tissues and prostate raises the question of the role of the beta 3-adrenoceptor in motility and secretory processes.

Animals↗

PubMed1995

Human islet amyloid polypeptide expression in COS-1 cells. A model of intracellular amyloidogenesis.

Non-insulin-dependent diabetes mellitus is characterized by concurrent loss of beta-cells and deposition of islet amyloid derived from islet amyloid polypeptide (IAPP). We have previously demonstrated that IAPP-derived amyloid forms intracellularly in humans with chronic excess insulin expression (eg, insulinoma and insulin receptor antibody-induced insulin resistance). To determine whether overexpression of IAPP results in intracellular amyloid in mammalian cells, we transfected COS cells with vectors expressing amyloidogenic human IAPP or non-amyloidogenic rat IAPP. Transfected COS-1 cells secreted comparable amounts of human IAPP and rat IAPP (2.1 to 2.8 nmol/L/48 hours). After 96 hours, 90% of cells expressing human IAPP contained amyloid fibrils and were degenerating or dead, whereas cells transfected with rat IAPP lacked amyloid and were viable. Thus, overexpression of human IAPP can result in intracellular amyloid formation that is associated with cell death, suggesting that intracellular amyloid may play a role in beta-cell loss in non-insulin-dependent diabetes mellitus.

Amyloid↗

PubMed1993

Amylin and CGRP induce insulin resistance via a receptor distinct from cAMP-coupled CGRP receptor.

Amylin and calcitonin gene-related peptide (CGRP) inhibited insulin-stimulated 2-deoxyglucose uptake in L6 myocytes and isolated soleus muscle. Both peptides were maximally active at 10 pM in L6 cells and inhibited insulin action by 40-50%. In soleus muscle amylin and CGRP inhibited insulin-stimulated uptake by 65-85%. Amylin competed with 125I-CGRP for binding to L6 cells but with 100-fold lower potency than CGRP. Occupancy of the CGRP receptor in L6 cells is coupled to adenylyl cyclase. Amylin increased the cellular content of adenosine 3',5'-cyclic monophosphate (cAMP), but consistent with binding, amylin was 100-fold less potent than CGRP. In soleus muscle, 100 nM amylin, which maximally inhibited 2-deoxyglucose uptake, had no effect cAMP content, whereas CGRP at the same concentration increased cAMP by 50%. The effect of CGRP on cAMP levels was completely suppressed by the competitive antagonist, CGRP-(8-37). In contrast, the suppression of insulin-stimulated glycogen synthesis or 2-deoxyglucose uptake by amylin was unaffected by 1 microM CGRP-(8-37). Our results demonstrate that the inhibition of insulin-stimulated glucose transport by amylin is independent of cAMP and may be mediated by a unique receptor that is distinct from the adenylyl cyclase-coupled CGRP receptor.

Amyloid↗

PubMed1991

Amylin-induced in vivo insulin resistance in conscious rats: the liver is more sensitive to amylin than peripheral tissues.

UNLABELLED: Amylin is a polypeptide of 37 amino acids, predominantly synthesized in pancreatic Beta cells. The peptide was suggested to be dysregulated in Type 2 (non-insulin-dependent) diabetes mellitus and it antagonized certain actions of insulin in vitro in rat muscle. This led to speculation that amylin is involved in the pathogenesis of Type 2 diabetes. We have examined the in vivo effects of rat amylin, amidated at the carboxy-terminus, on insulin-mediated carbohydrate metabolism in conscious rats, using the hyperinsulinaemic (+/- 1 nmol/l) euglycaemic (6 mmol/l) clamp technique combined with [3-3H]-glucose infusion. Basal plasma amylin levels were less than or equal to 75 pmol/l. Applied amylin levels of 220 +/- 75 pmol/l (infusion rate of 12.5 pmol/min) antagonized only the insulin action on liver, resulting in a 100% increase of hepatic glucose output. Amylin levels of 4750 +/- 750 pmol/l (infusion rate of 125 pmol/min) induced a 250% increase of insulin-inhibited hepatic glucose output and, in addition, a 30% decrease of insulin-stimulated peripheral glucose up-take. Amylin did not affect: 1) the metabolic clearance rate of insulin, 2) the levels of plasma glucagon, epinephrine, norepinephrine, and corticosterone, 3) in vitro insulin binding and insulin-stimulated receptor autophosphorylation. This suggests that amylin antagonizes insulin action via binding to a yet unknown receptor. IN CONCLUSION: amylin causes in vivo insulin resistance and the liver seems the predominant organ regulated by this hormone. The in vivo effects of amylin mimic the pathophysiological abnormalities of insulin action in Type 2 diabetes.

Amyloid↗

PubMed1991

Antidiabetic agent englitazone enhances insulin action in nondiabetic rats without producing hypoglycemia.

The new antihyperglycemic agent englitazone (CP-68,722) was examined in nondiabetic rats. Administration of englitazone at 50 mg/kg/d for 8 days did not produce overt hypoglycemia but it lowered basal plasma insulin by 59% and 41% in rats fed ad libitum and fasted overnight on the last day, respectively. Drug treatment also lowered (P less than .05) plasma nonesterified fatty acids (1.09 +/- 0.05 to 0.36 +/- 0.05 mmol/L) and cholesterol (2.41 +/- 0.08 to 2.06 +/- 0.07 mmol/L) in fasted rats, and glycerol (0.25 +/- 0.02 to 0.14 +/- 0.02 mmol/L) in fed rats but had no effect on 3-hydroxybutyrate or lactate levels despite the hypoinsulinemia. Disposition of an oral glucose load (1 g/kg) in drug-treated fed rats was identical to that in control rats despite a 40% reduction in the area under the plasma insulin curve. Insulin-stimulated 2-deoxy-D-3H-glucose uptake was significantly (P less than .05) enhanced in adipocytes prepared from both fasted and fed drug-treated rats (0.56 +/- 0.07 to 0.84 +/- 0.03 and 0.79 +/- 0.02 to 1.00 +/- 0.02 nmol/5 min, respectively, at insulin concentration of 2,500 microU/mL). There was also a significant increase in the basal rate of 2-deoxyglucose uptake (0.07 +/- 0.01 to 0.24 +/- 0.07 nmol/5 min) in adipocytes from fasted rats only. Insulin-stimulated lipogenesis from 3H-2-glucose was enhanced in adipocytes from drug-treated fed rats (7.72 +/- 0.09 to 10.19 +/- 0.10 nmol glucose/45 min at insulin concentration of 2,500 microU/mL) but no effect was observed in adipocytes from fasted rats (2.57 +/- 0.30 to 2.33 +/- 0.16 nmol glucose/45 min).(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗

PubMed1991

Substituted dihydrobenzopyran and dihydrobenzofuran thiazolidine-2,4-diones as hypoglycemic agents.

A series of dihydrobenzofuran and dihydrobenzopyran thiazolidine-2,4-diones (compounds 3-26) was synthesized from the corresponding aryl aldehydes 1 in two steps. These compounds represent conformationally restricted analogues of the novel hypoglycemic ciglitazone. The series was evaluated by hypoglycemic effects in vitro by measuring stimulation of 2-deoxyglucose uptake in L6 myocytes and stimulation of expression of the glucose transporter protein in 3T3-L1 adipocytes. In vivo hypoglycemic effects were evaluated in the genetically obese ob/ob mouse, and structure-activity relationships are discussed. On the basis of this in vivo potency, we have selected the 2(R)-benzylbenzopyran derivative to be further studied in a clinical setting.

Adipose Tissue↗

PubMed1990

Insulinlike activity of new antidiabetic agent CP 68722 in 3T3-L1 adipocytes.

We examined the in vitro effects of CP 68722, a novel antidiabetic agent, in 3T3-L1 adipocytes. CP 68722 stimulated 2-deoxyglucose uptake in the absence of insulin. At least 30 min of incubation were required for stimulation of uptake. This effect increased over 5 h and was sustained up to 72 h. The stimulation of 2-deoxyglucose uptake by CP 68722 could be inhibited approximately 60% by inhibition of protein synthesis with cycloheximide. Half-maximal and maximal responses to CP 68722 at 72 h of incubation were observed at 10 and 100 microM of drug, respectively, with a threefold stimulation of uptake at 100 microM approximating the maximal response of these cells to acute insulin stimulation. CP 68722 was able to overcome insulin resistance induced by dexamethasone in 3T3-L1 cells. The effect of drug, like that of insulin, was primarily to increase the Vmax of 2-deoxyglucose uptake. The stimulation of uptake by CP 68722 or insulin could be prevented by incubating the cells at 10 degrees C, a temperature that impedes translocation of glucose transporters to the plasma membrane. Therefore, it appears that CP 68722, like insulin, stimulates glucose uptake by a mechanism that involves translocation of intracellular glucose transporters to the plasma membrane and de novo protein synthesis. We compared the effect of CP 68722 with the sulfonylureas, the primary drugs used in the treatment of non-insulin-dependent diabetes mellitus (NIDDM). CP 68722 was a more potent and effective stimulator of 2-deoxyglucose uptake in 3T3-L1 cells than either first- or second-generation sulfonylureas.(ABSTRACT TRUNCATED AT 250 WORDS)

Adipose Tissue↗

PubMed1983

Inhibition of oxidative phosphorylation by an oligomer of prostaglandin B1, PGBx.

PGBx is a synthetic, oligomeric derivative of prostaglandin B1 that has been shown to protect rat liver mitochondria from the deleterious effects of aging. In fresh mitochondria, PGBx inhibits reactions involving the F1F0-ATPase. It prevents the stimulation of respiration by ADP and inhibits ATP-driven Ca2+ transport. It has no effect, however, on Ca2+-stimulated respiration and associated proton movements, or on respiration-driven Ca2+ transport, indicating that PGBx is not an inhibitor of the electron transport chain. The ATPase activity of submitochondrial particles is inhibited by PGBx with mixed type kinetics in which both Km and V are affected. PGBx has no effect on the ATPase activity of soluble F1, but induces respiratory control in F1-deficient submitochondrial particles, indicating a mode of action similar to oligomycin and DCCD. The binding of DCCD to its proteolipid receptor is inhibited by PGBx, suggesting that this is the binding site for PGBx. It is concluded that PGBx inhibits reactions involving the F1F0-ATPase by binding at or near the DCCD-binding protein and blocking proton conduction through the F0 moiety of the complex.

Adenosine Triphosphatases↗