In vitro, leptin secretion is regulated at the level of mRNA translation by the rapamycin-sensitive mammalian target of rapamycin (mTOR) and its agonist leucine (Leu). Studies were conducted on meal-trained rats to evaluate the potential physiological relevance of these in vitro findings and the role of Leu in affecting rises in plasma leptin observed after a meal. In the first study, we correlated changes in plasma insulin and Leu to mTOR-signaling pathway activation and plasma leptin at different times during meal feeding. Rapid rises in plasma insulin and Leu, along with mTOR signaling (phosphorylation of eIF4G, S6K1, rpS6, and 4E-BP1) in adipose tissue were observed during the 3-h meal and declined thereafter. Plasma leptin rose more slowly, peaking at 3 h, and was inhibited by rapamycin (0.75 mg/kg) pretreatment. In another experiment, oral Leu or norleucine was provided instead of a meal. Leu and norleucine stimulated a rise in plasma leptin; however, the magnitude was less than the response to a complete meal. In a third study, rats were provided a meal that lacked Leu, branched-chain amino acids, or all amino acids. Stimulation of leptin secretion was reduced approximately 40% in animals provided the Leu-deficient meal. Further reductions were not observed by removing the other amino acids. Thus Leu appears to regulate most of the effects of dietary amino acids on the postprandial rise in plasma leptin but is responsible only for part of the leptin response to meal feeding.

The purpose of this study was to identify a cell culture system in which the role of insulin in regulating albumin gene expression could be investigated. The system selected was rat hepatocytes maintained in primary culture in a chemically defined, serum-free medium. Under control conditions albumin secretion was nearly the same as the rate recorded in vivo and in perfused liver and was reasonably well maintained during 8 days of culture. Deletion of insulin from the culture medium for 3-6 days resulted in 40-60% reductions in albumin secretion. Furthermore, albumin secretion relative to the rate of total protein synthesis was reduced by approximately 50% as a result of insulin deficiency. Readdition of the hormone to insulin-deficient cultures restored secretion to the control rate. A maximal effect of insulin was observed within 3 days after readdition of the hormone, and a half-maximal response was obtained with a hormone concentration of approximately 3.0 nM. The relative abundance of albumin mRNA, as measured by solution hybridization using a complementary DNA probe, responded in a parallel fashion to the changes in albumin secretion. Thus rat hepatocytes maintained under appropriate culture conditions reflect the effects of diabetes and insulin treatment on albumin gene expression observed in vivo and provide an excellent model system in which to study the mechanism(s) of insulin action.

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Rates of total hepatic proteolysis were measured in normal and streptozotocin-diabetic mice during feeding and over 48 The protein content ofliver is highly labile and, in small animals such as the rat or mouse, 30-40% may be lost in 48 hr of starvation (1, 2). Because the loss is proteolytic and the degradative mechanism is acutely responsive to amino acids at physiological concentrations (3, 4), it could serve as an immediate source of amino acids for gluconeogenesis and other ongoing metabolic processes in the postabsorptive period (3). We know little, however, of the way protein content is regulated in vivo or the interplay between its determining functions, synthesis and degradation. The RNA content does diminish during starvation (5, 6); thus, absolute rates of protein synthesis per liver would be expected to decrease (5). Equivalent estimates of cellular protein breakdown have not yet been reported, but they too may decrease. However, because the cells steadily lose protein, degradative rates will remain higher than those of synthesis.These questions assume considerable importance in uncontrolled diabetes, in which the demand for glucogenic substrates is elevated and the steady-state content of liver protein quite possibly is reduced as a consequence of lower rates of protein synthesis (7-9) and increased rates ofbreakdown (7, 10). Here, the sudden withdrawal of food might accelerate existing proteolytic processes and even induce catabolic responses not usually manifest under normal circumstances. In this study, we have evaluated both the degradation of resident intracellular proteins and total rates of hepatic proteolysis in normal and streptozotocin-diabetic mice during 48 hr of starvation. The findings support the above predictions concerning intracellular protein degradation but also show that starvation can enhance hepatic degradation ofextrinsic proteins in both groups ofmice.In the diabetic mouse, this appears to become a major fraction of proteolysis in liver.EXPERIMENTAL PROCEDURES Animals. Male 7-week-old CD-1 mice (Charles River Breeding Laboratories), initially weighing 32-34 g, were used. They had free access to water and standard Charles River chow and were maintained in an environmentally controlled room (light on 7 a.m. to 7 p.m.). Diabetes was induced in fed animals by administration of streptozotocin (200 mg/kg of body weight), given intravenously by tail vein early in the day. The agent was dissolved to 20 mg/ml in ice-cold 0. 9% NaCl/0. 05 M Na citrate, pH 4.3, and used within 2 hr. Semiquantitative measurements of urine glucose (Tes-Tape, Eli Lilly) and ketones (Ketostix, Ames, Elkhart, IN) and of blood glucose (Dextrostix, Ames) were used to monitor the severity of the diabetes. Plasma glucose and insulin were measured by using a Technicon AutoAnalyzer and a radioimmunoassay kit (Becton Dickinson, Rutherford, NJ); plasma glucagon was determined as described by Cherrington et al (11).Liver Perfusion. Livers were cyclically perfused in situ (3, 12) at a flow of3 ml/min with ...

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