Obese mice (C57BL/6J ob/ob) and their lean littermates were studied at various ages from immediately post weaning until 62 weeks of age, at which mortality increased markedly. Several age-related changes were noted. 1) Plasma glucose levels were elevated in obese mice 5-20 weeks and 62 weeks of age, but were similar to those in the lean mice at 20-60 weeks of age. Plasma insulin levels were elevated in obese mice, and there were no age-related differences. 2) Brain serotonin was elevated in obese mice at all ages and increased with age in both obese and lean animals. 3) Pituitary contents of ACTH and beta-endorphin were elevated in young obese mice and increased further as these mice approached their life expectancy. 4) The ratios of ACTH to beta-endorphin immunoreactivities were similar in obese and lean mice, except in obese mice over 50 weeks of age where this ratio was increased. We conclude that: 1) the obese mouse is characterized by hyperinsulinemia and hyperadrenocorticism throughout its life; 2) the insulin resistance of the obese mouse improves at 20 weeks of age, yet deteriorates as its life expectancy is approached; 3) the obese mouse has an elevated brain serotonin content similar to previously described elevations of the putative neurotransmitters dopamine and norepinephrine in these mice; and 4) as the obese mouse approaches its life expectancy, abnormalities may occur in the synthesis, processing, or secretion of ACTH and/or beta-endorphine.

1. The oral antidiabetic glycodiazine (2-benzene sulfonamido-5-~-methoxyethoxy pyrimidine), an inhibitor of hepatic lipolysis, was used to investigate the metabolic role of liver lipid in glucogenesis in the perfused livers of fasted (24-30 h) rats. I n a concentration of 10 mM, glycodiazine inhibited almost completely endogenous ketone body formation.2 . Perfusion with pyruvate as substrate, in the presence of glycodiazine, resulted in a higher rate of pyruvate oxidation and suppressed the rate of glucose synthesis in comparison to livers perfused with pyruvate alone. Thus, maximal rates of gluconeogenesis from pyruvate would seem to require an adequate supply and oxidation of fatty acids.3. Oleate, even in the presence of glycodiazine, stimulates glucose synthesis from pyruvate. Uptake of and ketone body formation from oleate were also not affected by glycodiazine. These results allow the conclusion that glycodiazine specifically inhibits liver lipolysis.4. Glucagon (57 nM), although having no significant effect on gluconeogenesis from pyruvate a t this concentration, overcame the inhibitory effects of glycodiazine on glucose formation and suppressed the elevated pyruvate oxidation which results from the inhibition of endogenous lipid mobilization.5. Glucagon, in the absence of added substrates, stimulated gluconeogenesis, urea production and ketogenesis. The increase in gluconeogenesis due to glucagon was not impaired by glycodiazine. This indicates that intermediates arising from fatty acid oxidation do not limit the rate of gluconeogenesis under these conditions.

After intravenous injections, we have found the immunological half-life of purified porcine proinsulin to be more prolonged than purified porcine single-component insulin in both swine (22 and 9 min, respectively) and in baboons (18 and 8 min, respectively). Studies in humans have also indicated a longer half-life of proinsulin than insulin (1). These findings have prompted the present investigation of the in vitro degradation of insulin, proinsulin, and the connecting peptide that links the A and B chains of insulin in the proinsulin molecule, using an isolated liver perfusion system.Methods. Intact livers, weighing 4.5-6.8 g, from male Wistar rats, fasted for 48 hr, were cyclically perfused by the method of Hems et al. ( 2 ) . The perfusion fluid consisted of washed human erythrocytes, 2 g/lOO ml bovine serum albumin (Cohn, Fraction V ) , and Krebs-Henseleit buffer, pH 7.4, with a hemoglobin concentration of approximately 3 g/lOO ml. After complete isolation from the circulation, the livers were perfused in situ by adjusting the hydrostatic pressure of the perfusate to give maximal perfusion rates without liver swelling. This was approximately 2-3 ml/g wet weight of liver. The total perfusion volume was 150 ml at the beginning of the experiment. After a 40-min equilibration period, purified porcine single-component insulin (Lilly ), proinsulin (Lilly ), or con-

No abstract

Glucagon (28 nM) stimulated gluconeogenesis, urea production and ketogenesis in perfused livers from fasted rats. Since the livers were perfused without added substrate, liver protein is the source of carbon of the glucose synthesis. Addition of N6, O2′‐dibutyryl cyclic adenosine 3′:5′‐monophosphate in concentrations of 100 and 10 μM to the perfusion medium resulted in a response similar to that of glucagon. It is suggested that cyclic adenosine 3′:5′‐monophosphate is the mediator of glucagon action in liver. Preperfusion of the isolated livers with insulin diminished the glucagon effects. The glucagon‐stimulation of glucose and urea production was completely suppressed, while the increased ketogenesis was inhibited by 60% in the presence of insulin. Since all three glucagon responses were inhibited, this supports the concept that insulin lowers, in some way, the elevation of cyclic adenosine 3′:5′‐phosphate which occurs following glucagon administration. That insulin does not exert its effect subsequent to cyclic adenosine 3′:5′‐phosphate is suggested by the observation that insulin, under the same conditions which inhibited the response of liver to glucagon, had little or no effect on the increases in gluconeogenesis, ureogenesis, and ketogenesis following, dibutyryl cyclic adenosine‐3′:5′‐phosphate. Since an effect of insulin on cyclic adenosine 3′:5′‐phosphate phosphodiesterase seems unlikely, it is suggested that the inhibition of glucagon responses by insulin is the result of an interaction at the adenyl cyclase or one of the step(s) between binding of the hormone and the enzyme.

Gluconeogenesis from [l-14C]pyruvate in the perfused liver from fasted rats was stimulated by oleic acid as indicated by an increase in medium glucose, liver glycogen and specific activity of medium [14C]glucose. A balance of carbon from pyruvate revealed that in the presence of oleic acid 2 moles of pyruvate were utilized for each mole of glucose synthesized in contrast to a ratio 4: 1 without fatty acid. From this and 14C0, data it is concluded that oleic acid inhibited pyruvate oxidation markedly. Glucagon increased glucose formation from pyruvate but not from glycerol.Tremendous interest in the factors controlling the rate of gluconeogenesis has been shown in recent years. Long-chain fatty acids have been found to stimulate gluconeogenesis in the perfused rat liver using lactate [l] or alanine as substrate [2,3], and caprylate stimulates glucose formation in liver slices [4]. Increased fatty acid oxidation and ketogenesis have also been implicated in the i n vivo stimulation of gluconeogenesis by glucagon [5,6]. This interrelationship between increased fatty acid oxidation and gluconeogenesis has pointed to an acetyl-CoA activation of pyruvic carboxylase [7] as one of the possible control points in gluconeogenesis [S]. Recently, caprylate [9] and palmitate [lo] have been observed to decrease the oxidation of pyruvate by rat liver mitochondria, which is possibly due to an inhibition of pyruvic dehydrogenase by acetylCoA [10,11].Although glucagon has been reported to stimulate glucose production from lactate [1,12,13], pyruvate [13], fructose [13], and glycerol [14], studies of hepatic intermediates [6,12] have indicated that glucagon may accelerate gluconeogenesis a t the ratelimiting level which exists between pyruvate and phosphoenolpyruvate.The present work is a continuation and extension of the studies on the control of gluconeogenesis initiated by Struck and co-workers in our laborato-' Y [151.These workers found a stimulation of gluconeogenesis from lactate by either glucagon or oleic acid. The present studies are concerned with gluconeogenesis from pyruvate and the effect of oleic acid on this rate. The effect of glucagon on gluconeogenesis from glycerol and pyruvate has also been investigated. MATERIALS AND METHODSMale albino rats (Sprague-Dawley, Gassner, Munchen), weighing between 180 and 250 g and previously fed on laboratory chow (Altromin R., Altrogge, Lage Lippe) were fasted for 18-20 h before use.All chemicals were reagent grade unless otherwise noted. L-lactic acid and DL-carnitine chloride were from Schuchardt (Munchen) and oleic acid from Riedel-DeHaen (Seelze-Hannover). The oleic acid was vacuum distilled prior to use. Glucagon was a preparation and a gift of Eli Lilly, Indianapolis, U.S.A. (Lot Nr. 258-234, B-167-1). Sodium [1-14C]-pyruvate was obtained from Radiochemical Center, Amersham, England. All substrates were prepared as neutral solutions. Glucagon was dissolved in 1/250 N HCI.The apparatus and technique of perfusion were similar to that previously described [16,17]. The perfusin...

Most (80–90%) of the cyclic 3′:5′‐nucleotide phosphodiesterase activity in 0.25 M sucrose homogenates of rat liver was found in the 100,000 ×g× 30 min supernatant. The non‐particulate nature of the liver phosphodiesterase activity was also confirmed with beef and human liver. The phosphodiesterase activity of rat liver supernatant showed a pH maximum in the region of 7.0–7.5 in Tris‐Cl or cacodylate buffer, with a Km of 62 μM in Tris‐Cl buffer. A dependency on added Mg++ was indicated for maximum phosphodiesterase activity in rat liver supernatant. High‐speed centrifugation and Sephadex G‐200 chromatography studies indicated that phosphodiesterase activity is associated with a protein of high molecular weight (>200,000). Cyclic 3′:5′‐nucleotides with a purine base were preferentially hydrolyzed in all fractions of rat liver. Dibutyryl adenosine 3′:5′‐monophosphate exhibited complete resistance to rat liver phosphodiesterase hydrolysis in the two assay systems employed. The role of liver phosphodiesterase activity as a regulatory enzyme of adenosine 3′:5′‐monophosphate concentration in the action of insulin on liver was also investigated. Neither prior treatment of perfused livers with insulin nor presence of insulin in two different assay systems affected the phosphodiesterase activity in high‐speed supernatants.

Alterations in lipid metabolism were examined in adult male Sprague-Dawley rats seven days after a single intraperitoneal injection of perfluorodecanoic acid (PFDA; 20, 40 or 80 mg/kg). Because PFDA treatment caused a dose-related reduction in feed intake, the response of vehicle-treated rats pair-fed to those receiving PFDA was monitored to distinguish direct effects of the perfluorinated fatty acid from those secondary to hypophagia. Carcass content of lipid phosphorus and free cholesterol decreased in dose-dependent fashion in both PFDA-treated and pair-fed rats. Carcass triacylglycerols diminished in a similar manner, yet PFDA-treated rats at each dose had a higher concentration of neutral acylglycerols than their vehicle-treated, pair-fed counterparts. In vehicle-treated, pair-fed rats at the 80 mg/kg dose level, lipid phosphorus and free cholesterol as a proportion of carcass fat increased, whereas the share of the triacylglycerols declined. Because of the higher concentration of triacylglycerols in the carcass of rats treated with 80 mg/kg PFDA, enrichment of lipid phosphorus and free cholesterol in carcass fat was less than in their pair-fed partners. The amount of lipid phosphorus and free cholesterol per hepatocyte was similar in both PFDA-treated rats and their pair-fed partners. Liver triacylglycerols were markedly increased in PFDA-treated rats. A similar but less extensive augmentary effect of PFDA on hepatic esterified cholesterol was found. Concentration of triacylglycerols in plasma was not elevated in PFDA-treated rats, in spite of hepatic accumulation of esterified compounds. Also, the plasma level of free fatty acids and 3-hydroxybutyrate was similar in all treatment groups, including those receiving PFDA.(ABSTRACT TRUNCATED AT 250 WORDS)