In vitro lipolytic effect of bovine pituitary diabetogenic protein.

Bovine diabetogenic protein has been further purified by gel filtration yielding a fraction (Mr 25 000--28 000) having increased diabetogenic and in vitro lipolytic activity. Using rat epididymal fat pads, this fraction was shown to be lipolytic at concentrations as low as 1--10 mug/ml. The in vitro lipolytic effect was unaffected by the nutritional state of the animals, was not potentiated by dexamethasone, could be demonstrated in the presence and absence of glucose and was not mediated by alpha- and beta-adrenergic receptors. A lag phase of greater than 1 h was observed before diabetogenic protein induced lipolysis occurred, suggesting that protein synthesis might be involved. Cycloheximide (10 mug/ml), added initially, prevented the diabetogenic protein-induced lipolysis. This direct effect of the purified protein on adipose tissue helps explain the elevation of free fatty acids seen when bovine diabetogenic hormone is administered in vivo and suggests that this anterior pituitary protein may be a new lipid-mobilizing hormone.

A pharmacologic profile of McN-3495 [N-(1-methyl-2-pyrrolidinylidene)-N'-phenyl-1-pyrrolidinecarboximidamide], a new, orally effective hypoglycemic agent.

McN-3495, a new compound unrelated strucuturally to the sulfonylureas or phenformin, has been found to produce a hypoglycemic effect in nondiabetic rats, dogs, mice, and monkeys. The minimum effective dose of McN-3495 that lowers fasting blood glucose and improves glucose tolerance was found to be about 2.5 to 5 mg-per kilogram, per os, except in fasted monkeys, in which a tenfold greater potency was observed. When McN-3495 was given repeatedly for three to five days, no tolerance to the hypoglycemic activity occurred and no changes in other biochemical parameters were observed. In addition to being three to four times more potent than tolbutamide, McN-3495 also differs from the sulfonylureas in lowering blood glucose concentrations of streptozotocin-diabetic rats and db/db mice, and, moreover, oral administration to normal fasted dogs did not produce the characteristic rise in insulin concentrations observed with tolbutamide. Furthermore, unlike the biguanides, McN-3495 can lower dog and rat fasting blood glucose concentrations and can improve glucose tolerance whether the glucose is administered orally or parenterally. However, McN-3495, as phenformin, fails to work in totally depancreatized dogs.

Influence of the oral hypoglycemic agent linogliride (McN-3935) on insulin secretion from isolated rat islets of Langerhans.

The influence of a new orally effective hypoglycemic compound, linogliride (McN-3935), on insulin release from isolated perifused rat islets was investigated. At a concentration of 100 microM, linogliride was without effect on insulin secretion in the absence of glucose. While 5.5 mM glucose alone produced a weak secretagogue effect, the secretory response was dramatically (5- to 6-fold) increased by the addition of 100 microM linogliride. This concentration of linogliride did not affect the conversion of [5-3H]glucose to 3H2O, a measure of the rate of glycolysis by the islet. Insulin secretion in response to the combination of 100 microM linogliride and 5.5 mM glucose was abolished by the omission of extracellular calcium. Mannoheptulose (10 mM), an inhibitor of glucose phosphorylation and glucose-stimulated insulin secretion, markedly attenuated the insulinotropic effect of linogliride in parallel with reduced glucose usage. Paradoxically, in the presence of another metabolic inhibitor, 2-deoxyglucose (10 mM), the insulinotropic effect of linogliride (100 microM) in the presence of 5.5 mM glucose was not diminished despite the reduced glucose usage. Linogliride significantly increased the secretory response to other secretagogues, including 2.5 mM D-glyceraldehyde, 15 mM N-acetylglucosamine, 10 mM leucine, and 100 nM of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate. These data suggest that linogliride amplifies a cellular signal generated during beta-cell activation by various stimulants.

Resolution of (+/-)-2-tetradecyloxiranecarboxylic acid. Absolute configuration and chiral synthesis of the hypoglycemic R enantiomer and biological activity of enantiomers.

The resolution of the hypoglycemic agent (+/-)-2-tetradecyloxiranecarboxylic acid (3) as its d- and l-ephedrine salts is presented. The active enantiomer (R)-(+)-3 was also synthesized by the Sharpless chiral epoxidation procedure and its methyl ester (R)-(+)-4 was shown to be identical with the corresponding ester from the resolved acid. Single-crystal X-ray structure analysis of the diastereomeric salt of (+)-3 and (-)-ephedrine allowed assignment of (+)-3 as the R configuration. The effects on fatty acid oxidation and glucose tolerance of the racemic and enantiomeric forms of 3, 4, and the CoA ester of 3 are presented. A postulated mechanism of action for the active enantiomer as an enantioselective, active-site-directed, irreversible inhibitor of carnitine palmitoyl transferase is suggested.

Alkylglycidic acids: potential new hypoglycemic agents.

A series of alkylglycidic acid analogues and derivatives were synthesized and tested for their ability to inhibit long-chain fatty acid oxidation in vitro and to lower blood sugar in rats. The extent of inhibition of carnitine acyl transferase, the enzyme at the mitochondrial membrane necessary to transport long-chain fatty acids into the mitochondria for subsequent beta-oxidation, was determined for the series. Structure-activity relationships using in vitro inhibition of [1-14C]palmitic acid oxidation in rat hemidiaphragm muscle indicate that potent activity resides mainly in 2-alkyl (C12-C16) glycidates. Replacement of the oxirane ring with cyclopropyl, thiirane, or other rings diminishes activity, as does substitution of the glycidate ring at the 3-position. In vivo potency in the rat glucose tolerance test roughly parallels the hemidiaphragm results. The lead compound, methyl 2-tetradecylglycidate (8), is a potent hypoglycemic agent following oral administration to several animal species. The hypoglycemic analogues interfere with fatty acid oxidation by specific and irreversible inhibition of mitochondrial carnitine palmitoyl transferase-A.

Hypoglycaemic and hypoketonaemic effects of single and repeated oral doses of methyl palmoxirate (methyl 2-tetradecylglycidate) in streptozotocin/alloxan-induced diabetic dogs.

1. The hypoglycaemic and hypoketonaemic effects of orally administered methyl palmoxirate were studied in streptozotocin/alloxan-induced diabetic dogs. 2. Single oral 50 mg doses (approximately 7.5 mg kg-1) of methyl palmoxirate produced statistically significant reductions of plasma glucose (32 +/- 6% maximum reduction from baseline) and ketones (74 +/- 12% maximum reduction from baseline), with the peak effect on plasma ketones (3.5 h) preceding that for plasma glucose (6.0 h). 3. Lower doses (0.7-2.0 mg kg-1 daily) of methyl palmoxirate given repeatedly for seven days produced reductions of blood glucose and ketones equivalent to those produced with the higher single dose. Maximal reductions of plasma ketones were generally observed following the first dose of drug, whereas significant lowering of plasma glucose required several days of continuous dosing. 4. Repeated daily doses of methyl palmoxirate markedly reduced the overnight fasting ketone levels but not glucose levels of diabetic dogs. 5. In conclusion, administration of the fatty acid oxidation inhibitor methyl palmoxirate, in the absence of concomitant insulin therapy, was able to lower the plasma glucose and ketone levels of insulin-deficient streptozotocin/alloxan diabetic dogs. Only the plasma ketones were decreased to normal by this treatment.

Pharmacologic profile of methyl 2-tetradecylglycidate (McN-3716)--an orally effective hypoglycemic agent.

A specific inhibitor of fatty acid oxidation, methyl 2-tetradecylglycidate (McN-3716) has been found to produce a dose dependent hypoglycemic effect when administered orally to rats, mice, and dogs. In addition to being more potent than other inhibitors of fatty acid oxidation, McN-3716 was also found to be 15--20 times more potent than tolbutamide in lowering the blood glucose of fasting rats. Furthermore, evidence is presented that McN-3716 produces hypoglycemia by a mechanism which differs from that of other oral hypoglycemic agents, the biguanides and the sulfonylureas. As predicted by the Randle glucose-fatty acid cycle, McN-3716 lowered glucose concentrations only under conditions where fatty acids were being used as the major energy substrates (fasting, diabetes, and feeding of high fat diets) but not under conditions where energy was derived mainly from carbohydrate (fed state or following hypophysectomy). Administration of McN-3716 produced a remarkable lowering of the plasma glucose and the glycosuria of depancreatized dogs but did not result in complete normalization of glucose, especially the excursions of blood glucose following feeding. It did, however, produce virtually complete reversal of the ketoacidosis of alloxan diabetic rats and depancreatized dogs without worsening the plasma lipid profile. Thus, McN-3716 may have potential utility as an oral therapeutic agent for the treatment of ketosis-prone juvenile or maturity-onset diabetes.

Highly purified human growth hormone fails to stimulate lipolysis in rabbit adipocytes in vitro or in rabbits in vivo.

To determine whether the rapid lipolytic effect observed with human growth hormone (hGH) preparations in rabbits and rabbit adipose tissue is an intrinsic property of the hormone, we examined the lipolytic effects in vivo and in vitro of clinical grade preparations, hGH purified by DEAE cellulose chromatography, and hGH prepared by recombinant DNA techniques. Using isolated rabbit perirenal adipocytes, ACTH and clinical-grade hGH preparations both stimulated glycerol release to the same maximal rate with half-maximal hGH effects observed between 8 and 50 micrograms/mL. Purification of the most potent lipolytic preparation of clinical grade hGH by DEAE cellulose chromatography yielded a preparation (2 IU/mg of growth activity that retained insulinlike effects on rat fat pad [U-14C] glucose metabolism) that, at concentrations up to 0.2 mg/mL, failed to stimulate lipolysis by adipocytes incubated for one or four hours in the presence or absence of dexamethasone or trypsin inhibitor or when preincubated for three hours prior to addition of hGH. Recombinant DNA-derived hGH did not stimulate glycerol release at 0.1 mg/mL. While antiserum to purified hGH blocked the increase in glucose oxidation in rat fat pads produced by clinical grade hGH, it did not inhibit its lipolytic effect using rabbit adipocytes. Purified hGH (0.1 mg/kg IV) was also unable to elicit a rise in serum free fatty acid (FFA) levels of conscious rabbits while, at the same dose, clinical grade hGH increased FFA levels to 900 microEq/L over basal. Rapid lipolytic stimulation in rabbit adipocytes by hGH preparations could not be attributed to the 20,000 molecular weight variant of hGH (hGH20K) or the peptide corresponding to positions 32 to 46 of hGH (deletion peptide).(ABSTRACT TRUNCATED AT 250 WORDS)

Diabetogenic activity of the anterior pituitary (a progress report).

The exact nature of the diabetogenic activity of the anterior pituitary gland remains a mystery. While growth hormone (GH) fractions clearly can produce a diabetogenic effect, there is increasing evidence that GH itself does not exert this effect. Several new theories that purport to explain the nature of diabetogenic activity are discussed. The current evidence supports the view that the activity is the result of a GH fragment or some other closely related protein such as the one ("diabetogenic protein") first described by LOUIS and CONN. A progress report of the research on this new "diabetogenic protein" is given.

Effects of phenformin and alpha-bromopalmitate on fatty acid and glucose oxidation in muscle.

Hemidiaphragms removed from fasted rats which were treated with the fatty acid oxidation inhibitor, alpha-bromopalmitate (125-250 mg/kg, i.p.) oxidized less glucose to CO2 than vehicle-treated rats. Hypoglycemia was noted and was preceded by reduction of muscle palmitate oxidation and by increased plasma free fatty acids (FFA). However, phenformin (100 mg/kg, i.p.) lowered plasma FFA and diaphragm oxidation of both palmitic acid and glucose, without altering glucose levels. These results fail to support the hypothesis that phenformin lowers blood glucose secondary to inhibiton of fatty acid oxidation.

Effect of the oral hypoglycemic agent pirogliride (McN-3495) on glycogen levels of normal and diabetic rats.

Pirogliride, a new oral hypoglycemic agent unrelated structurally or mechanistically to either the sulfonylureas or biguanides, produced a dose-dependent increase in liver but not muscle glycogen levels of fasted non-diabetic rats and produced at 100 mg/kg, p.o. a modest increase in liver but not gastrocnemius muscle glycogen of fasted streptozotocin diabetic rats. This liver glycogenic effect of pirogliride was similar to that obtained using insulin but differed from the decrease of liver glycogen levels which occurred following acute phenformin treatment.

Relation of oxidation of long-chain fatty acids to gluconeogenesis in the perfused liver of the guinea pig: effect of 2-tetradecylglycidic acid (McN-3802).

1. The potent, specific inhibitor of long-chain fatty acid oxidation, 2-tetradecylglycidic acid (McN-3802), at 10 microM totally abolished ketogenesis from endogenous substrates in the isolated perfused guinea pig liver. This effect was accompanied by a marked inhibition of gluconeogenesis from lactate plus pyruvate and by a shift toward a more oxidized state of the mitochondrial (3-hydroxybutyrate/acetoacetate ratio) and cytoplasmic (lactate/pyruvate ratio) compartments. 2. The addition of octanoate (88-500 microM) almost completely reversed the inhibitory effect of 2-tetradecylglycidic acid on gluconeogenesis. Octanoate oxidation, measured by the rate of ketogenesis, was not inhibited. This protective effect of octanoate against inhibition of gluconeogenesis by 2-tetradecylglycidic acid was seen even though in some experiments the mitochondrial redox state remained two to three times the magnitude observed prior to octanoate addition. 3. Gluconeogenesis from 4 mM glycerol was not inhibited and gluconeogenesis from 4 mM propionate was only slightly inhibited by 2-tetradecylglycidic acid. 4. Thus, it would appear that fatty acid oxidation in guinea pig liver is essential for maintaining maximal rates of gluconeogenesis, especially from substrates dependent on pyruvate carboxylation for conversion to glucose. 5. A single dose of 2-tetradecylglycidic acid (orally, 10-25 mg/kg) given to guinea pigs previously fasted 72 h produced a highly significant decrease of total ketones, of the 3-hydroxybutyrate/acetoacetate ratio and of plasma glucose. A smaller hypoglycemic effect was seen when the drug was administered to animals fasted for only 24 h or 48 h. 6. It appears from evidence in vivo and in vitro that the guinea pig and rat respond similarly to inhibition of fatty acid oxidation. This may be important since it has been suggested that the role of fatty acid oxidation in glucose synthesis is markedly different in these two species.

Inhibition of mitochondrial carnitine palmitoyl transferase A in vivo with methyl 2-tetradecylglycidate (methyl palmoxirate) and its relationship to ketonemia and glycemia.

The oral hypoglycemic agent, methyl 2-tetradecylglycidate (Me-TDGA), which inhibits in vitro mitochondrial carnitine palmitoyl transferase A (CPT-A) was used to study the relationship of CPT inhibition to changes in ketonemia and glycemia in normal and diabetic rats. After oral administration of Me-TDGA, the CPT activity of isolated rat liver mitochondria was substantially reduced with only the presumed outer enzyme fraction CPT-A released by digitonin treatment showing reduced activity. Mitochondrial fatty acyl-CoA synthetase was not inhibited. Oral doses of 0.1-2.5 mg/kg Me-TDGA produced both a dose-dependent lowering of plasma ketones and an inhibition of liver CPT. With single doses in excess of 2.5 mg/kg, po, heart and skeletal muscle CPT were also consistently inhibited. The effect on the liver enzyme persisted for at least 48 hr following 1 mg/kg, po, while the effect on ketones disappeared by 36 hr. The degree of inhibition of liver CPT produced by Me-TDGA was not altered by diabetes or the dietary state. At low doses (0.05-0.25 mg/kg, po), the most sensitive parameter was inhibition of hepatic CPT. Both plasma ketones and CPT were lowered with doses 10-fold less (0.1 mg/kg) than were required for blood glucose lowering, thus making Me-TDGA the most potent hypoketonemic compound known. In conclusion, inhibition of liver beta-oxidation at the stage of CPT-A by Me-TDGA can explain the potent hypoketonemic effects of this compound in fasted normal and diabetic rats. Higher acute doses are needed for both inhibition of muscle CPT and lowering of blood glucose.