Reduction in glucose levels in STZ diabetic rats by 4-(2,2-dimethyl-1-oxopropyl)benzoic acid: a prodrug approach for targeting the liver.

The overproduction of glucose by the liver in NIDDM patients markedly contributes to their fasting hyperglycemia and is a direct consequence of the increased oxidation of excess free fatty acids (FFA) being released from the adipocyte. 2-(1,1-Dimethylethyl)-2-(4-methylphenyl)[1,3]dioxolane (SAH51-641, 1) has previously been demonstrated to reduce glucose levels in animal models of diabetes by reducing fatty acid oxidation and hence depriving the system of the energy and cofactors necessary for gluconeogenesis. However, attempts at lowering glucose levels in vivo with 1 have been associated with toxicity in other organs such as the testes. An approach was developed utilizing the natural processing of triglyceride-like intermediates as a basis for selectively targeting the absorption, processing, and delivery of a prodrug to the liver. Compounds were identified by this method which lowered glucose levels in vivo without releasing toxic amounts of the active metabolites of 1 into circulation.

Substituted tetrahydropyrrolo[2,1-b]oxazol-5(6H)-ones and tetrahydropyrrolo[2,1-b]thiazol-5(6H)-ones as hypoglycemic agents.

A series of substituted tetrahydropyrrolo[2,1-b]oxazol-5(6H)-ones and tetrahydropyrrolo[2,1-b]thiazol-5(6H)-ones was synthesized from amino alcohols or amino thiols and keto acids. A pharmacological model based on the results obtained with these compounds led to the synthesis and evaluation of a series of isoxazoles and other monocyclic compounds. These were evaluated for their ability to enhance glucose utilization in cultured L6 myocytes. The in vivo hypoglycemic efficacy and potency of these compounds were evaluated in a model of type 2 diabetes mellitus (non-insulin-dependent diabetes mellitus), the ob/ob mouse. 25a(2S) (SDZ PGU 693) was selected for further pharmacological studies.

Hypoglycemic effects of a novel fatty acid oxidation inhibitor in rats and monkeys.

Increased fatty acid oxidation contributes to hyperglycemia in patients with non-insulin-dependent diabetes mellitus. To improve glucose homeostasis in these patients, we have designed a novel, reversible inhibitor of carnitine palmitoyl-transferase I (CPT I) that potently inhibits fatty acid oxidation. SDZ-CPI-975 significantly lowered glucose levels in normal 18-h-fasted nonhuman primates and rats. In rats, glucose lowering required fatty acid oxidation inhibition of > or = 70%, as measured by beta-hydroxybutyrate levels, the end product of beta-oxidation. In cynomolgus monkeys, comparable glucose lowering was achieved with more modest lowering of beta-hydroxybutyrate levels. SDZ-CPI-975 did not increase glucose utilization by heart muscle, suggesting that CPT I inhibition with SDZ-CPI-975 would not induce cardiac hypertrophy. This was in contrast to the irreversible CPT I inhibitor etomoxir. These results demonstrate that SDZ-CPI-975 effectively inhibited fatty acid oxidation and lowered blood glucose levels in two species. Thus reversible inhibitors of CPT I represent a class of novel hypoglycemic agents that inhibit fatty acid oxidation without inducing cardiac hypertrophy.

Muscle subcellular localization and recruitment by insulin of glucose transporters and Na+-K+-ATPase subunits in transgenic mice overexpressing the GLUT4 glucose transporter.

Insulin-stimulated glucose uptake in skeletal muscle is mediated through the GLUT4 glucose transporter. Transgenic (TG) mice overexpressing human GLUT4 in skeletal muscle show an increased ability to handle a glucose load. Here, the participation of the overexpressed GLUT4 in the response to insulin was examined. In TG mouse muscle, the GLUT4 protein content was 10-fold higher in crude membrane (CM), sevenfold higher in internal membrane (IM), and 15-fold higher in a plasma membrane (PM)-rich fraction, relative to non-TG littermates. This suggested partial saturation of the normal sorting mechanisms. The distribution and abundance of the GLUT1 glucose transporter was not affected. Insulin injection (4.3 U/kg body wt) increased GLUT4 in the PM-rich fraction; the increase was threefold higher in TG than in non-TG mice. Insulin decreased the GLUT4 content of the IM in both animal groups and of a second, heavier intracellular membrane fraction only in TG mice. The net content of Na+-K+-pump subunits was 40-65% lower in CM from TG compared with non-TG littermates. In spite of this, insulin caused a three- to sixfold higher translocation of the alpha2 and beta1 subunits of the Na+-K+-pump in TG compared with non-TG animals. The results suggest that overexpression of GLUT4 confers to the muscle increased ability to translocate subunits of the Na+-K+-pump either as a direct consequence of the recruitment of glucose transporters or as an adaptation to the more demanding metabolic state.

Expression of human GLUT4 in mice results in increased insulin action.

Glucose metabolism was evaluated in transgenic mice expressing the human GLUT 4 glucose transporter. Fed GLUT 4 transgenic mice exhibited a 32% and 56% reduction in serum glucose and insulin and a 69% and 33% increase in non-esterified fatty acid and lactate levels, respectively. Transgenic mice exhibited a significant increase in whole-body glucose disposal during a euglycaemic-hyperinsulinaemic clamp. Insulin-stimulated glucose uptake in isolated soleus muscles and adipocytes was greater in transgenic compared to control mice due to increased basal glucose uptake. Transgenic mice displayed increased glycogen levels in liver and gastrocnemius muscle, and increased insulin-stimulated 14C-glycogen accumulation in isolated soleus muscle. We conclude that over-expression of the GLUT 4 glucose transporter in mice results in 1) an increase in whole-body glucose disposal and storage, and 2) an increase in both basal and insulin-stimulated glucose uptake and disposal in vitro. These changes resulted in the reduction of serum glucose and insulin levels. These results provide direct evidence that glucose transport (and GLUT 4 per se) plays a significant role in regulating whole-body glucose homeostasis. Additionally, these data support the idea that pharmacological strategies directed at increasing the expression of GLUT 4 protein may have beneficial (hypoglycaemic) effects in the diabetic state.

Limonene in expired lung air of patients with liver disease.

As part of an effort to examine the relationship between chemosensory disturbance and oral chemistry, we analyzed expired lung air samples from a series of 24 patients with liver disease and 24 healthy controls using gas chromatography-mass spectrometry. Compared to samples from controls, lung air from patients with liver disease contained unusually high levels of limonene, a monoterpene that is a major component of the essential oil of citrus fruits (0.1 vs 7.0 micrograms/20 liters for controls and patients). Only half the patients showed high levels of limonene. Patients with noncholestatic liver disease were significantly more likely to have elevated lung air limonene levels than those with cholestatic liver disease (0.2 vs 13.8 micrograms/20 liters). Responses to food frequency and dietary behavior questionnaires indicated a pattern of diet selection and food preferences that were consistent with a dietary origin for the limonene in these patients.

Relationship between liver biochemical tests and dietary intake in patients with liver disease.

Relationships between liver biochemical test values and reported frequency of consumption of various foods were examined using a principal-component analysis of data from 42 patients with chronic liver disease. The statistical procedure identified relationships among biochemical and dietary variables. One relationship included the variables albumin, bilirubin, and frequency of intake of fruits and vegetables, starch, and meats. A relationship was also found between serum alkaline phosphatase (ALP) levels and fat/oil intake. Data from patients with primary biliary cirrhosis (PBC) and noncholestatic liver disease were compared using a correlational analysis. In patients with PBC, serum ALP levels were positively correlated with frequency of intake of fat/oil (r = 0.59, p < 0.01) and meats (r = 0.46, p < 0.05), whereas serum bilirubin (Bili) and aspartate aminotransferase (AST) levels were significantly correlated with frequency of intake of dairy products (rs = 0.48 and 0.45, ps < 0.05 for Bili and AST, respectively), meats (rs = 0.59 and 0.65, ps < 0.01), and fat/oil (r = 0.54, p < 0.02 and r = 0.48, p < 0.05). In patients with noncholestatic liver disease, Bili levels were correlated with frequency of intake of fat/oil (r = 0.58, p < 0.01), and fruits and vegetables (r = 0.68, p < 0.01). These results suggest that the degree of elevation of some liver biochemical tests in patients with liver disease may be affected by dietary intake.

Insulin action on whole body glucose utilization and on muscle glucose transporter translocation in mice.

Murine models of insulin resistance and diabetes are versatile and have been used to investigate genetic and metabolic disorders. However, the principal assays to assess insulin action, i.e., the euglycemic-hyperinsulinemic clamp and subcellular distribution of glucose transporters, have not been implemented in this species. Here we describe procedures which allow these methods to be adapted to mice. When normal C57bl/6j mice were infused with graded doses of insulin (1, 3, 10 or 30 mU/kg/min) during a euglycemic-hyerinsulinemic clamp, the glucose infusion rate necessary to maintain euglycemia increased in a dose-dependent manner (7.4 +/- 1.7, 13.1 +/- 3.6, 24.1 +/- 2.3 or 34.8 +/- 7.5 mg/kg/min), respectively. Hindlimb muscles were isolated and samples of 2-3 g were subjected to subcellular fractionation finalizing on 25%, 30% and 35% sucrose gradients. Fraction F25 (plasma membranes) was enriched in alpha 2 Na+/K(+)-ATPase and GLUT1 glucose transporters, whereas fraction F35 (intracellular membranes) was enriched in Ca(2+)-ATPase and GLUT4 glucose transporters. Following insulin treatment, GLUT4 increased in F25 and decreased in F35. Insulin treatment had no effect on GLUT1 in F25. However, unlike in rat skeletal muscle, GLUT1 was detectable in F35 and its content decreased in this fraction following insulin treatment. The results demonstrate that whole-body glucose utilization can be assessed in mice using euglycemic-hyperinsulinemic clamps and demonstrate how subcellular fractionation procedures can be applied to murine muscle. Murine muscle GLUT4 translocates from an intracellular storage site to the plasma membrane in response to insulin.

Pancreatic glucagon suppresses gustatory responsiveness to glucose.

Peripheral administration of the gut peptide pancreatic glucagon (GGN) alters hepatic metabolism and suppresses feeding. Other physical (gastric distension) and chemical factors (hyperglycemia, hyperinsulinemia) that reduce food intake also suppress taste-evoked activity. This may attenuate the reinforcement derived from feeding and so promote termination of the meal. To determine whether this mechanism was operative with GGN administration, we studied the effect of hepatic portal infusions of 40 micrograms/kg pancreatic GGN on taste responses in the nucleus tractus solitarius of the rat. Taste activity was elicited by oral application of NaCl, glucose, HCl, and quinine HCl. Responses were monitored before and after injections of GGN or a control vehicle. Blood glucose levels were measured in separate groups of GGN- and vehicle-injected rats. Blood glucose increased significantly after GGN infusion and returned to control levels within 35 min. Taste responsiveness to glucose was significantly reduced after the GGN injection and recovered to preinjection levels by 36 min. Activity evoked by NaCl, HCl, and quinine HCl was unaffected. The suppression of responsiveness to sugars may reduce the hedonic appeal of tastants and so serve as a mechanism by which GGN could contribute to postprandial satiety.

Dietary fat exacerbates liver disease in bile duct-ligated rats.

Previous studies have suggested that the mortality of bile duct-ligated (BDL) rats is related to the amount of dietary fat consumed. We investigated the influence of dietary fat concentration on liver disease in BDL rats. Groups of rats were fed for 4 wk either a low fat diet (LF, 0.92 kJ/g; 3% of total energy from fat), a high fat diet (HF, 1.07 kJ/g; 30% fat), a high fat diet with energy density equivalent to that of the LF diet (HFIB, 0.92 kJ/g; 30% fat) or a diet based on the composition of commercial nonpurified diets (COMP, 0.90 kJ/g; 10% fat). Energy intake, body weight gain, plasma biochemical indices and hepatic histology were compared in BDL and sham-operated control rats. Bile duct-ligated animals consuming the LF diet showed a faster recovery of energy intake and greater body weight gain following surgery than did BDL animals fed the other three diets. Plasma alkaline phosphatase activity was significantly greater in BDL animals fed either of the high fat diets than in those fed the LF or COMP diet starting 1 and 2 wk, respectively, after surgery. Hepatic fibrosis and bile duct proliferation at d 28 post-ligation were greater in rats fed a 30% fat (HF) diet than in rats fed the 3% fat (LF) diet. These results suggest that dietary fat concentration can influence the severity of liver dysfunction in extrahepatic biliary obstruction.

Chemosensory function, food preferences and appetite in human liver disease.

We evaluated chemosensory function, food preferences, and appetite in 88 patients with liver disease including those with hepatitis, cirrhosis, primary biliary cirrhosis, and sclerosing cholangitis. Reported chemosensory disturbances were common in the patients with liver disease: over 40% reported recent taste changes, and 27% reported recent changes in smell, compared to only 6% of healthy, age- and gender-matched controls with no history of marked chemosensory malfunction. Compared to controls, a greater proportion of liver patients reported food cravings (47 vs. 17%) and food aversions (33 vs. 16%). Foods with a predominantly bitter taste were specifically less preferred in patients with liver disease compared to healthy controls. Patients were also more likely to report poor to fair appetite than controls (37 vs. 5%). Groups of patients with different types of liver pathology showed varying patterns of food preferences, suggesting that dietary recommendations to liver patients might be tailored to the altered preferences associated with a particular type of hepatic dysfunction.

Amylin or CGRP (8-37) fragments reverse amylin-induced inhibition of 14C-glycogen accumulation.

The peptides amylin and calcitonin-gene related peptide (CGRP) have been shown to have similar effects on glycogen metabolism in vivo and in vitro. However, it is not clear whether they act via separate receptors. Peptide fragments based on the amino acid sequence of amylin or CGRP were evaluated for their ability to inhibit the action of the peptides in vitro. Insulin-stimulated glycogen turnover, as measured by 14C-glycogen accumulation, was inhibited about 70% by amylin (10nM) and 85% by CGRP (10nM). In the absence of exogenous peptide, peptide fragments based on the 8-37 and 10-37 amino acid sequences of rat amylin (10 uM) had no affect on 14C-glycogen accumulation. In the presence of amylin (10nM), the 8-37 and 10-37 fragments blocked amylin-induced inhibition of 14C-glycogen accumulation 100% and 11.4%, respectively. The 8-37 and 10-37 amylin fragments blocked CGRP inhibition of 14C-glycogen accumulation by 23.2% or 28.6%, respectively. The CGRP 8-37 fragment was equally effective as the amylin 8-37 reversing the effects of amylin than at reversing the effects of CGRP. These results demonstrate that amylin (8-37) completely antagonizes the effects of amylin with limited ability to block CGRP. Removing the eighth and ninth amino acids reduced the effectiveness of the inhibitor by about 90%.

Amylin activates glycogen phosphorylase and inactivates glycogen synthase via a cAMP-independent mechanism.

Although the novel pancreatic peptide amylin has been shown to induce insulin resistance and decrease glucose uptake, the mechanism of amylin's actions is unknown. The following study evaluated the effect of amylin on glycogen metabolism in isolated soleus muscles in the presence and absence of insulin (200 microU/ml). Total glycogen, glycogen phosphorylase and glycogen synthases activities, and cAMP levels were measured. Total glycogen levels were significantly decreased by amylin (100 nM) in fed or fasted muscles under conditions of insulin stimulation. Amylin (100 nM) activated glycogen phosphorylase by as much as 100% and decreased glycogen synthase activity by over 60%, depending on the metabolic state of the muscles. These effects where comparable to those of the beta adrenergic agonist isoproterenol. A lower concentration of amylin (1 nM) did not significantly affect glycogen levels, glycogen phosphorylase, or glycogen synthase activity. Cyclic AMP levels were increased two-fold by isoproterenol but were unaffected by amylin. In conclusion, amylin induces glycogenolysis by decreasing glycogen synthesis and increasing breakdown. The effect of amylin on enzyme activity is consistent with a phosphorylation-dependent mechanism. It is likely that these events are mediated via a cAMP independent protein kinase.

Insulin modifies flavor aversions and preferences in real- and sham-feeding rats.

In separate, parallel experiments, rats were allowed to sham or real feed a flavored sweetened condensed-milk solution after a 17.5-h fast. Coinciding with administration of the milk were injections of insulin or vehicle (saline). Insulin had no effect on intake in the real feeding situation, and flavors paired with insulin (0.8, 2.5, or 5.0 U/rat) were avoided, relative to saline-paired flavors. In sham-feeding rats, insulin (0.8 U/rat) significantly reduced the amount of milk consumed, and flavors paired with insulin injection were preferred, relative to saline-paired ones. Insulin produced a relatively larger hypoglycemia during sham feeding than during real feeding. Thus it appears that insulin-paired flavors of milk may be preferred by animals when they are ingested in sham feeding but not in animals real feeding. Perhaps rapid gastric emptying, as would occur with a liquid diet, accompanied by increased insulin levels, leads to malaise and flavor aversion.

Effects of amylin on glucose metabolism and glycogenolysis in vivo and in vitro.

The effects of amylin on glucose metabolism and glycogenolysis were examined in vivo and in vitro. Eighteen-hour-fasted rats were infused with 5 nmol.kg-1.min-1 amylin and [3-3H]glucose for 120 min. Blood glucose levels increased an average of 45% during the infusion. Glucose turnover measurements indicated that the overall rate of glucose appearance (Ra) did not change, but the metabolic clearance rate of glucose was decreased by 42%. Samples of liver, gastrocnemius, and soleus muscles were freeze-clamped at the end of the infusion period and analyzed for glycogen and glucose 6-phosphate levels. Glycogen levels were decreased in all tissue samples, whereas glucose 6-phosphate was elevated in gastrocnemius and soleus muscles. Isolated soleus muscles were incubated in vitro with 200 microU/ml of insulin and 1, 10, or 100 nM amylin. Amylin treatment had no effect on 3-O-methyl-D-glucose transport; however, 2-deoxy-D-glucose uptake was inhibited by 33 or 48% at 10 or 100 nM amylin, respectively. Glycogen levels were also decreased after treatment with 10 and 100 nM amylin. Glucose 6-phosphate levels were not affected by amylin treatment in the presence of insulin but were increased nearly twofold in its absence. The data suggest that amylin stimulates glycogenolysis and inhibits glucose uptake both in vivo and in vitro and that the inhibition of glucose uptake is due to inhibition of glucose phosphorylation (i.e., hexokinase).

Macronutrient selection in an animal model of cholestatic liver disease.

Diet selection was investigated in an animal model of cholestatic liver disease produced by bile duct ligation. Animals self-selected diets from separate sources of macronutrients (protein, fat, carbohydrate). Diet selection was evaluated when the fat source was comprised of either a primarily medium-chain fat (coconut oil) or a primarily long-chain fat (Crisco vegetable shortening). Relative to intakes of control animals, bile duct ligated (BDL) animals consuming the long-chain fat decreased fat intake, decreased protein intake, and increased carbohydrate intake. Consumption of the fat source was decreased in BDL rats fed the medium-chain fat relative to intakes of control animals, however carbohydrate and protein intakes were not affected. Total caloric intake was comparable to control intakes by day 16 post-ligation in BDL rats fed the long-chain fat and by day 11 in BDL rats fed the medium-chain fat. Body weight gain was significantly greater in BDL rats fed the medium-chain fat than in those fed the long-chain fat. Mortality was 44% in BDL animals fed the long-chain fat, and 0% in those fed the medium-chain fat. The results suggest that BDL animals make dietary selections which may decrease the severity of liver disease. Differences between ligated animals consuming either medium- or long-chain fats suggest that some fat sources may be more beneficial during cholestasis.

Ingestion of low concentrations of sucrose or hydrochloric acid solutions accelerates mortality in rats with cholestatic liver disease.

Mortality rates were monitored in bile duct ligated (BDL) rats which were consuming flavor solutions in addition to water and chow. Following ligation of the common bile duct, animals were offered a single concentration of a flavor solution (200 mM NaCl; 400 mM sucrose; 100 mM urea; 5 mM HCl) in addition to water. During three weeks postligation, mortality rates were compared between groups of ligated animals consuming either one of the taste solutions or consuming only water. Within the first five days postligation, mortality was 70% in ligated animals drinking HCl and 50% in animals consuming sucrose, compared to 30% for urea, 10% for sodium chloride and 0% in animals drinking only water. Total mortality of ligated rats consuming HCl or sucrose solutions was significantly increased compared to animals consuming only water. The results suggest that consumption of low concentrations of sucrose or HCl solutions during the initial stages of liver disease in BDL rats may drastically accelerate their mortality rate.