Importance of the route of intravenous glucose delivery to hepatic glucose balance in the conscious dog.

To assess the importance of the route of glucose delivery in determining net hepatic glucose balance (NHGB) eight conscious overnight-fasted dogs were given glucose via the portal or a peripheral vein. NHGB was measured using the arteriovenous difference technique during a control and two 90-min glucose infusion periods. The sequence of infusions was randomized. Insulin and glucagon were held at constant basal levels using somatostatin and intraportal insulin and glucagon infusions during the control, portal, and peripheral glucose infusion periods (7 +/- 1, 7 +/- 1, 7 +/- 1 microU/ml; 100 +/- 3, 101 +/- 6, 101 +/- 3 pg/ml, respectively). In the three periods the hepatic blood flow, glucose infusion rate, arterial glucose level, hepatic glucose load, arterial-portal glucose difference and NHGB were 37 +/- 1, 34 +/- 1, 32 +/- 3 ml/kg per min; 0 +/- 0, 4.51 +/- 0.57, 4.23 +/- 0.34 mg/kg per min; 101 +/- 5, 200 +/- 15, 217 +/- 13 mg/dl; 28.5 +/- 3.5, 57.2 +/- 6.7, 54.0 +/- 6.4 mg/kg per min; +2 +/- 1, -22 +/- 3, +4 +/- 1 mg/dl; and 2.22 +/- 0.28, -1.41 +/- 0.31, and 0.08 +/- 0.23 mg/kg per min, respectively. Thus when glucose was delivered via a peripheral vein the liver did not take up glucose but when a similar glucose load was delivered intraportally the liver took up 32% (P less than 0.01) of it. In conclusion portal glucose delivery provides a signal important for the normal hepatic-peripheral distribution of a glucose load.

Glycemic improvement in diabetic db/db mice by overexpression of the human insulin-regulatable glucose transporter (GLUT4).

The effects of increased GLUT4 (insulin-regulatable muscle/fat glucose transporter) expression on glucose homeostasis in a genetic model of non-insulin-dependent diabetes mellitus were determined by expressing a human GLUT4 transgene (hGLUT4) in diabetic C57BL/KsJ-db/db mice. A genomic hGLUT4 construct was microinjected directly into pronuclear murine embryos of db/+ matings to maintain the inbred background. Four lines of hGLUT4 transgenic mice were bred to homozygosity at the db locus and all showed a marked reduction of both fasted and fed plasma glucose levels (to approximately 50 and 360 mg/dl, respectively) compared with age-matched nontransgenic db/db mice (approximately 215 and 550 mg/dl, respectively), as well as an enhanced disposal of an oral glucose challenge. In situ immunocytochemical localization of GLUT4 protein in muscle from hGLUT4 db/db mice showed elevated plasma membrane-associated GLUT4 protein in the basal state, which markedly increased after an insulin/glucose injection. In contrast, nontransgenic db/db mice had low levels of plasma membrane-associated GLUT4 protein in the basal state with a relatively small increase after an insulin/glucose challenge. Since the intracellular GLUT4 levels in db/db mice were similar to nontransgenic db/+ mice, the glucose transport defect in db/db mice is at the level of glucose transporter translocation. Together, these data demonstrate that GLUT4 upregulation overcomes the glucose transporter translocation defect and alleviates insulin resistance in genetically diabetic mice, thus resulting in markedly improved glycemic control.

Lack of effect of somatostatin on the stimulation of hepatic glycogenolysis by epinephrine in isolated canine hepatocytes.

The effects of somatostatin on epinephrine's ability to stimulate glucose output have been examined in hepatocytes isolated from dogs fasted overnight. Half-maximal stimulation of phosphorylase a activity and glucose output occurred at an epinephrine concentration of approx. 5 X 10(-9) M. Somatostatin at 10, 100 or 1000 ng/ml had no effect on the ability of a maximal (1 X 10(-7) M) and a submaximal (1 X 10(-8) M) dose of epinephrine to activate phosphorylase at 2 min, or to stimulate glucose output over 20 min. Since the doses of somatostatin used in the present study are up to 50-fold higher than the blood concentrations commonly found when somatostatin is used in vivo to inhibit pancreatic hormone secretion, it seems unlikely that use of somatostatin in this way would affect stimulation of hepatic glycogenolysis by epinephrine in vivo.

Intracellular digestion of saturated and unsaturated phospholipid liposomes by mucosal cells. Possible mechanism of transport of liposomally entrapped macromolecules across the isolated vascularly perfused rabbit ileum.

The mechanism of intestinal absorption of liposomally entrapped [14C]inulin and 125I-labelled poly(vinylpyrrolidone) was studied using the isolated rabbit intestinal loop with intact perfused vasculature, a system more closely resembling an in vivo system than the everted sac technique. [14C]Inulin or 125I-poly(vinylpyrrolidone) was entrapped in liposomes prepared from unsaturated egg phosphatidylcholine and soya phosphatidylcholine, and saturated distearoylphosphatidylcholine (18:0), dipalmitoylphosphatidylcholine (16:0) and dimyrostoylphosphatidylcholine (14:0). Free and liposomally entrapped macromolecules were introduced in the ileum and the transport of liposomes and entrapped macromolecules into the venous effluent was monitored by measuring the presence of the aqueous marker 125I-poly(vinylpyrrolidone) or [14C]inulin, and lipid marker [3H]cholesterol. The results show that intact liposomes are not transported across intestine into the venous effluent, but they are taken up by mucosal cells and digested intracellularly, releasing the entrapped markers 125I-poly(vinylpyrrolidone) and [14C]inulin. These markers are then transported into the venous effluent as free molecules. The absorption of liposomally entrapped [14C]inulin into the venous effluent is biphasic, first slow for 30 min (i.e., a lag period of 30 min), followed by a rapid linear increase. The duration of the lag period and the rate of absorption of the entrapped [14C]inulin are dependent on the degree of saturation and the transition temperature of the phospholipids used to prepare liposomes. The possible explanation of the lag period based on the evidence presented here is that it is the time required for the liposomes to be taken up by mucosal cells and digested intracellularly. Intracellular digestion of liposomes prepared from saturated phospholipids is more rapid than from those prepared from unsaturated phospholipids, and the greater the fatty acid chain length of the saturated phospholipids the more rapid the intracellular degradation of liposomes.

Prolonged hypoglycemic effect in diabetic dogs due to subcutaneous administration of insulin in liposomes.

The biologic action of insulin entrapped in liposomes (phospholipid vesicles) has been investigated following subcutaneous injection to dogs made diabetic with a combination of alloxan and streptozotocin. The fats of the liposomally entrapped material was determined by injecting rats subcutaneously with either 125I-insulin or the labeled polysaccharide 14C-inulin, incorporated in liposomes labeled with 3H-cholesterol. Injection of liposome insulin (0.75 U/kg) to five diabetic dogs resulted in a mean (+/- SEM) blood glucose fall from 16.4 +/- 0.8 to 2.9 +/- 0.4 mmol/L. The glucose level had still not returned to baseline after 24 h and, correspondingly, immunoreactive insulin (IRI) could still be detected in frozen and thawed plasma 24 h after injection. In contrast, the hypoglycemic effect of the same dose of free insulin with or without empty liposomes virtually ended within 8 h and IRI levels returned to baseline by 3 h after injection. In experiments on rats with liposomally entrapped 125I-insulin or 14C-inulin the proportion of the injected dose of tracer recoverable by excision of the injection site remained constant after about 1 h and 70% of the dose was still fixed in subcutaneous tissue for at least 5 h thereafter. When the plasma collected 3 h after subcutaneous injection of labeled liposomes containing 125I-insulin was passed through a column of Sepharose 6B, 50-75% of the 125I-activity was found in the fractions associated with intact liposomes. One possibility for the persistence of the hypoglycemic effect and of measurable IRI following injection of liposome insulin could be the presence of intact liposomes in the circulation for many hours after adsorption had ceased.

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.

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)

Regulation of ketogenesis by epinephrine and norepinephrine in the overnight-fasted, conscious dog.

The effects on ketogenesis and lipolysis of a norepinephrine (0.04 microgram/kg-min), epinephrine (0.04 microgram/kg-min), or saline infusion were examined in the overnight-fasted, conscious dog. Plasma insulin and glucagon levels were maintained constant by means of a somatostatin infusion (0.8 microgram/kg-min) and intraportal replacement infusions of insulin and glucagon. In saline-infused dogs, plasma epinephrine (62 +/- 8 pg/ml), norepinephrine (92 +/- 29 pg/ml), blood glycerol (87 +/- 10 microM), and plasma nonesterified fatty acid (NEFA) (0.82 +/- 0.17 mM) levels did not change. Total blood ketone body levels tended to rise (62 +/- 10 to 83 +/- 11 microM) by 3 h as did total ketone body production (1.5 +/- 0.4 to 2.2 +/- 0.4 mumol/kg-min) over the same time interval. Norepinephrine infusion to produce plasma levels of 447 +/- 86 pg/ml caused a sustained 50% rise in glycerol levels (66 +/- 17 to 99 +/- 15 mumol/L, P less than 0.05) and 53% rise in nonesterified fatty acids (0.53 +/- 0.07 to 0.81 +/- 0.15 mumol/L, P less than 0.05). Total ketone body levels rose by 43% (51 +/- 8 to 73 +/- 10 mumol/L) and ketone body production rose by a similar proportion (1.5 +/- 0.2 to 2.2 +/- 0.3 mumol/kg-min), changes that did not differ significantly from control animals. A similar increment in plasma epinephrine levels (75 +/- 15 to 475 +/- 60 pg/ml) caused glycerol levels to rise by 82% (105 +/- 23 to 191 +/- 26 mumol/L) in 30 min, but this rise was not sustained and the level fell to 146 +/- 14 mumol/L by 120 min.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

GLUT4 overexpression in db/db mice dose-dependently ameliorates diabetes but is not a lifelong cure.

We previously reported that overexpression of GLUT4 in lean, nondiabetic C57BL/KsJ-lepr(db/+) (db/+) mice resulted in improved glucose tolerance associated with increased basal and insulin-stimulated glucose transport in isolated skeletal muscle. We used the diabetic (db/db) litter mates of these mice to examine the effects of GLUT4 overexpression on in vivo glucose utilization and on in vitro glucose transport and GLUT4 translocation in diabetic mice. We examined in vivo glucose disposal by oral glucose challenge and hyperinsulinemic-hyperglycemic clamps. We also evaluated the in vitro relationship between glucose transport activity and cell surface GLUT4 levels as assessed by photolabeling with the membrane-impermeant reagent 2-N-(4-(1-azi-2,2,2-trifluoroethyl)benzoyl)-1,3-bis(D-mannose-4-yloxy)-2-propylamine in extensor digitorum longus (EDL) muscles. All parameters were examined as functions of animal age and the level of GLUT4 overexpression. In young mice (age 10-12 weeks), both lower (two- to threefold) and higher (four- to fivefold) levels of GLUT4 overexpression were associated with improved glucose tolerance compared to age-matched nontransgenic (NTG) mice. However, glucose tolerance deteriorated with age in db/db mice, although less rapidly in transgenic mice expressing the higher level of GLUT4. Glucose infusion rates during hyperinsulinemic-hyperglycemic clamps were increased with GLUT4 overexpression, compared with NTG mice in both lower and higher levels of GLUT4 overexpression, even in the older mice. Surprisingly, isolated EDL muscles from diabetic db/db mice did not exhibit alterations in either basal or insulin-stimulated glucose transport activity or cell surface GLUT4 compared to nondiabetic db/+ mice. Furthermore, both GLUT4 overexpression levels and animal age are associated with increased basal and insulin-stimulated glucose transport activities and cell surface GLUT4. However, the observed increased glucose transport activity in older db/db mice was not accompanied by an equivalent increase in cell surface GLUT4 compared to younger animals. Thus, although in vivo glucose tolerance is improved with GLUT4 overexpression in young animals, it deteriorates with age; in contrast, insulin responsiveness as assessed by the clamp technique remains improved with GLUT4 overexpression, as does in vitro insulin action. In summary, despite an impairment in whole-body glucose tolerance, skeletal muscle of the old transgenic GLUT4 db/db mice is still insulin responsive in vitro and in vivo.

Role of gluconeogenesis in sustaining glucose production during hypoglycemia caused by continuous insulin infusion in conscious dogs.

The roles of glycogenolysis and gluconeogenesis in sustaining glucose production during insulin-induced hypoglycemia were assessed in overnight-fasted conscious dogs. Insulin was infused intraportally for 3 h at 5 mU.kg-1.min-1 in five animals, and glycogenolysis and gluconeogenesis were measured by using a combination of tracer [( 3-3H]glucose and [U-14C]alanine) and hepatic arteriovenous difference techniques. In response to the elevated insulin level (263 +/- 39 microU/ml), plasma glucose level fell (41 +/- 3 mg/dl), and levels of the counterregulatory hormones glucagon, epinephrine, norepinephrine, and cortisol increased (91 +/- 29 to 271 +/- 55 pg/ml, 83 +/- 26 to 2356 +/- 632 pg/ml, 128 +/- 31 to 596 +/- 81 pg/ml, and 1.5 +/- 0.4 to 11.1 +/- 1.0 micrograms/dl, respectively; for all, P less than .05). Glucose production fell initially and then doubled (3.1 +/- 0.3 to 6.1 +/- 0.5 mg.kg-1.min-1; P less than .05) by 60 min. Net hepatic gluconeogenic precursor uptake increased approximately eightfold by the end of the hypoglycemic period. By the same time, the efficiency with which the liver converted the gluconeogenic precursors to glucose rose twofold. Five control experiments in which euglycemia was maintained by glucose infusion during insulin administration (5.0 mU.kg-1.min-1) provided baseline data. Glycogenolysis accounted for 69-88% of glucose production during the 1st h of hypoglycemia, whereas gluconeogenesis accounted for 48-88% of glucose production during the 3rd h of hypoglycemia. These data suggest that gluconeogenesis is the key process for the normal counterregulatory response to prolonged and marked hypoglycemia.

Similar dose responsiveness of hepatic glycogenolysis and gluconeogenesis to glucagon in vivo.

This study was undertaken to determine whether the dose-dependent effect of glucagon on gluconeogenesis parallels its effect on hepatic glycogenolysis in conscious overnight-fasted dogs. Endogenous insulin and glucagon secretion were inhibited by somatostatin (0.8 micrograms X kg-1 X min-1), and intraportal replacement infusions of insulin (213 +/- 28 microU X kg-1 X min-1) and glucagon (0.65 ng X kg-1 X min-1) were given to maintain basal hormone concentrations for 2 h (12 +/- 2 microU/ml and 108 +/- 23 pg/ml, respectively). The glucagon infusion was then increased 2-, 4-, 8-, or 12-fold for 3 h, whereas the rate of insulin infusion was left unchanged. Glucose production (GP) was determined with 3-[3H]glucose, and gluconeogenesis (GNG) was assessed with tracer (U-[14C]alanine conversion to [14C]glucose) and arteriovenous difference (hepatic fractional extraction of alanine, FEA) techniques. Increases in plasma glucagon of 53 +/- 8, 199 +/- 48, 402 +/- 28, and 697 +/- 149 pg/ml resulted in initial (15-30 min) increases in GP of 1.1 +/- 0.4 (N = 4), 4.9 +/- 0.5 (N = 4), 6.5 +/- 0.6 (N = 6), and 7.7 +/- 1.4 (N = 4) mg X kg-1 X min-1, respectively; increases in GNG (approximately 3 h) of 48 +/- 19, 151 +/- 50, 161 +/- 25, and 157 +/- 7%, respectively; and increases in FEA (3 h) of 0.14 +/- 0.07, 0.37 +/- 0.05, 0.42 +/- 0.04, and 0.40 +/- 0.17, respectively. In conclusion, GNG and glycogenolysis were similarly sensitive to stimulation by glucagon in vivo, and the dose-response curves were markedly parallel.

Stimulation of glucose production through hormone secretion and other mechanisms during insulin-induced hypoglycemia.

To assess the role of counterregulatory hormones per se in the response to continuous insulin infusion, overnight-fasted dogs were given 5 mU.kg-1.min-1 insulin intraportally either alone (INS, n = 5), with glucose to maintain euglycemia (INS + GLU, n = 5), or with glucose and hormone replacement [i.e., glucagon, epinephrine, norepinephrine, and cortisol infusions (INS + GLU + HR, n = 6)]. The increases in counterregulatory hormones that occurred during insulin-induced hypoglycemia were simulated in the latter group. In this way, it was possible to separate the effects of hypoglycemia per se from those due to the associated counterregulatory hormone response. Glycogenolysis and gluconeogenesis were measured with a combination of tracer ([ 3-3H]glucose and [U-14C]alanine) and hepatic arteriovenous (AV) difference techniques during a 40-min control and a 180-min experimental period. Insulin levels increased similarly in all groups (to congruent to 250 microU/ml), whereas plasma glucose levels decreased in INS (115 +/- 3 to 41 +/- 3 mg/dl; P less than .05) and rose slightly in both INS + GLU (108 +/- 2 to 115 +/- 4 mg/dl; P less than .05) and INS + GLU + HR (111 +/- 3 to 120 +/- 3 mg/dl; P less than .05) due to glucose infusion. Glucagon, epinephrine, norepinephrine, and cortisol were replaced in INS + GLU + HR so that the increments in their levels were 102 +/- 6, 106 +/- 14, 117 +/- 9, and 124 +/- 37%, respectively, of their increments in INS. At no time was there a significant difference between the hormone levels in INS and INS + GLU + HR. The rise in the counterregulatory hormones per se accounted for only half (53 +/- 9% by the AV difference method and 54 +/- 10% by tracer method) of the glucose production associated with hypoglycemia resulting from insulin infusion. The rate and efficiency of alanine conversion to glucose in the hormone-replacement studies were only 29 +/- 10 and 50 +/- 27% of what occurred during hypoglycemia induced by insulin infusion. In conclusion, the counterregulatory hormones alone (i.e., without accompanying hypoglycemia) can account for only 50% of the glucose production that is present during insulin-induced hypoglycemia. The remaining 50%, therefore, must result from effects of hypoglycemia other than its ability to trigger hormone release.

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.

Actions of novel antidiabetic agent englitazone in hyperglycemic hyperinsulinemic ob/ob mice.

The effects of CP 68722 (racemic englitazone) were examined in ob/ob mice, in adipocytes and soleus muscles from ob/ob mice, and in 3T3-L1 adipocytes. Administration of englitazone at 5-50 mg.kg-1.day-1 lowered plasma glucose and insulin dose dependently without producing frank hypoglycemia in either the diabetic or nondiabetic lean animals. The glucose-lowering effect in ob/ob mice preceded the reduction in hyperinsulinemia. On cessation of drug, plasma insulin returned to untreated levels within 48 h, whereas plasma glucose rose slowly over 5 days. Englitazone (50 mg/kg) for 11 days lowered plasma glucose (22.2 +/- 1.4 to 14.0 +/- 1.9 mM), insulin (7.57 +/- 0.67 to 1.64 +/- 0.60 nM), nonesterified fatty acids (1813 +/- 86 to 914 +/- 88 microM), glycerol (9.20 +/- 0.98 to 4.94 +/- 0.03 mM), triglycerides (1.99 +/- 0.25 to 1.03 +/- 0.11 g/L), and cholesterol (6.27 +/- 0.96 to 3.87 +/- 0.57 mM), but no effects were observed 3 h after a single dose. Basal and insulin-stimulated lipogenesis were enhanced in adipocytes from ob/ob mice treated with 50 mg/kg englitazone for 11 days compared with lipogenesis in cells from vehicle-treated controls. Treatment of ob/ob mice with 50 mg/kg englitazone reversed the defects in insulin-stimulated glycolysis (from [3-3H]glucose) and glycogenesis and basal glucose oxidation (from [1-14C]glucose) in isolated soleus muscles. Englitazone (30 microM) stimulated 2-deoxy-D-glucose transport in 3T3-L1 adipocytes from 0.37 +/- 0.03 to 0.65 +/- 0.06 and 1.53 nmol.min-1.mg-1 protein at 24 and 48 h, respectively. Thus, englitazone has 1) insulinomimetic and insulin-enhancing actions in vitro and 2) glucose-, insulin-, triglyceride-, and cholesterol-lowering properties in an animal model of non-insulin-dependent diabetes mellitus (NIDDM) in which sulfonylureas have little or no effect. Thus, this new agent may have beneficial effects including a reduced risk of hypoglycemia in patients with NIDDM.

Effects of two treatment regimes with synthetic human parathyroid hormone fragment on bone formation and the tissue balance of trabecular bone in greyhounds.

The adult greyhound was found to be similar to adult man with respect to kinetic and histomorphometric indices of calcium metabolism. The relationship between trabecular bone tissue balance and the pattern of human PTH fragment 1-34 (hPTH 1-34) administration by daily injections or continuous sc infusions was investigated in this model and the results compared to those from a clinical trial of hPTH 1-34 in involutional osteoporosis (peptide administration by single daily injections). In the dogs, the daily injection regime elevated plasma levels of immunoreactive hPTH 1-34 for no more than 4 h/day. The greyhounds so treated showed significantly increased indices of bone formation (surface osteoid, plasma alkaline phosphatase activity, and skeletal accretion rate of calcium) and resorption (number of osteoclasts, resorption surfaces). Iliac trabecular bone volume increased significantly, as it did in the patients. The infusions did not significantly increase the trabecular bone volume or the 47Ca accretion rate, two parameters which increased in parallel in dogs and patients treated successfully by daily injections. The osteoclastic surfaces, however, were clearly increased by continuous infusions, while the increases in the osteoblastic surfaces were less statistically significant. Since hPTH 1-34 may inhibit osteogenesis in Friedenstein chambers, it is possible that the increased osteoblastic activity induced by the daily injection regime in trabecular bone is dependent on the noncontinuous nature of the PTH stimulus.

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.

Benzyloxazolidine-2,4-diones as potent hypoglycemic agents.

A series of benzyloxazolidine-2,4-diones, containing oxazole-based side chains, were found to lower blood glucose levels in the genetically obese ob/ob mouse. Incorporation of a benzofuran structural element in these compounds provides greatly enhanced in vivo potency. The syntheses and structure-activity relationships for this series are detailed.

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.

Evidence that protease inhibitors reduce the degradation of parathyroid hormone and calcitonin injected subcutaneously.

1 Agents known to delay absorption from a subcutaneous site were tested in chicks for their ability to prolong the hypercalcaemic response to parathyroid hormone (PTH). 2 Polyvinylpyrrolidone was found to enhance the response but gelatine greatly reduced the 2 h hypercalcaemia. 3 The reduction by gelatine was reversed when the protease inhibitor aprotinin was added to the injection vehicle, and hypercalcaemia then persisted for more than 8 h. 4 Of other protease inhibitors studied, epsilon-aminocaproic acid was also found to enhance the hypercalcaemic response to subcutaneous PTH and its fragments but, unlike aprotinin, it was ineffective in the presence of gelatine. 5 By radioimmunoassay and bioassay respectively, it was confirmed that aprotinin raised circulating levels of PTH and also of another peptide hormone, calcitonin, injected subcutaneously. 6 Addition of calcium to the solutions injected subcutaneously abolished the hypercalcaemic response to PTH while injection of calcium and PTH simultaneously but at separate sites left the response unaltered. 7 The two protease inhibitors, epsilon-aminocaproic acid and aprotinin, each restored the response to subcutaneous PTH despite the presence of calcium at the injection site. 8 It was concluded that protease inhibitors injected subcutaneously with PTH and calcitonin in the chick reduced the rate of degradation of these hormones and that the proteases responsible for hormone degradation at the subcutaneous injection site may be released or activated by calcium ions.