Plasma insulin concentrations in fed rats were altered acutely by administration of glucose or anti-insulin serum. Rates of fatty acid synthesis in adipose tissue and liver were estimated from the incorporation of 3H from 3H2O. In the adipose tissue dehydrogenase and acetyl-CoA carboxylase were evident. In liver, although changes in rates of fatty acid synthesis were found, the initial activity of pyruvate dehydrogenase did not alter, but small parallel changes in acetyl-CoA carboxylase activity were observed.

The incretins, glucose-dependent insulinotropic polypeptide (GIP) and glucagon-like peptide 1 (GLP-1) are responsible for >50% of nutrient-stimulated insulin secretion. After being released into the circulation, GIP and GLP-1 are rapidly inactivated by the circulating enzyme dipeptidyl peptidase IV (DP IV). The use of DP IV inhibitors to enhance these insulinotropic hormonal axes has proven effective on an acute scale in both animals and humans; however, the long-term effects of these compounds have yet to be determined. Therefore, we carried out the following study: two groups of fa/fa Zucker rats (n ‫؍‬ 6 each) were treated twice daily for 3 months with the DP IV inhibitor P32/98 (20 mg ⅐ kg ؊1 ⅐ day ؊1 , p.o.). Monthly oral glucose tolerance tests (OGTTs), performed after drug washout, revealed a progressive and sustained improvement in glucose tolerance in the treated animals. After 12 weeks of treatment, peak OGTT blood glucose values in the treated animals averaged 8.5 mmol/l less than in the controls (12.0 ؎ 0.7 vs. 20.5 ؎ 1.3 mmol/l, respectively). Concomitant insulin determinations showed an increased earlyphase insulin response in the treated group (43% increase). Furthermore, in response to an 8.8 mmol/l glucose perfusion, pancreata from controls showed no increase in insulin secretion, whereas pancreata from treated animals exhibited a 3.2-fold rise in insulin secretion, indicating enhanced ␤-cell glucose responsiveness. Also, both basal and insulin-stimulated glucose uptake were increased in soleus muscle strips from the treated group (by 20 and 50%, respectively), providing direct evidence for an improvement in peripheral insulin sensitivity. In summary, long-term DP IV inhibitor treatment was shown to cause sustained improvements in glucose tolerance, insulinemia, ␤-cell glucose responsiveness, and peripheral insulin sensitivity, novel effects that provide further support for the use of DP IV inhibitors in the treatment of diabetes. Diabetes 51: 943-950, 2002

Acetyl-CoA carboxylase (ACC) catalyses the formation of malonyl-CoA, an essential substrate for fatty acid synthesis in lipogenic tissues and a key regulatory molecule in muscle, brain and other tissues. ACC contributes importantly to the overall control of energy metabolism and has provided an important model to explore mechanisms of enzyme control and hormone action. Mammalian ACCs are multifunctional dimeric proteins (530-560 kDa) with the potential to further polymerize and engage in multiprotein complexes. The enzymatic properties of ACC are complex, especially considering the two active sites, essential catalytic biotin, the three-substrate reaction and effects of allosteric ligands. The expression of the two major isoforms and splice variants of mammalian ACC is tissue-specific and responsive to hormones and nutritional status. Key regulatory elements and cognate transcription factors are still being defined. ACC specific activity is also rapidly modulated, being increased in response to insulin and decreased following exposure of cells to catabolic hormones or environmental stress. The acute control of ACC activity is the product of integrated changes in substrate supply, allosteric ligands, the phosphorylation of multiple serine residues and interactions with other proteins. This review traces the path and implications of studies initiated with Dick Denton in Bristol in the late 1970s, through to current proteomic and other approaches that have been consistently challenging and immensely rewarding.

We tested the hypothesis that activation of AMP-activated protein kinase (AMPK) promotes myocardial glycogenolysis by decreasing glycogen synthase (GS) and/or increasing glycogen phosphorylase (GP) activities. Isolated working hearts from halothane-anesthetized male Sprague-Dawley rats perfused in the absence or presence of 0.8 or 1.2 mM 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranoside (AICAR), an adenosine analog and cell-permeable activator of AMPK, were studied. Glycogen degradation was increased by AICAR, while glycogen synthesis was not affected. AICAR increased myocardial 5-aminoimidazole-4-carboxamide 1-β-d-ribofuranotide (ZMP), the active intracellular form of AICAR, but did not alter the activity of GS and GP measured in tissue homogenates or the content of glucose-6-phosphate and adenine nucleotides in freeze-clamped tissue. Importantly, the calculated intracellular concentration of ZMP achieved in this study was similar to the K m value of ZMP for GP determined in homogenates of myocardial tissue. We conclude that the data are consistent with allosteric activation of GP by ZMP being responsible for the glycogenolysis caused by AICAR in the intact rat heart.

Stimulus-response coupling through protein kinase C (PKC) was shown to be defective in mononuclear phagocytes (MO) infected with Leishmania donovani. Phorbol 12-myristate 13-acetate (PMA)-induced oxidative burst activity and protein phosphorylation were markedly attenuated in infected M+. These results were not explained either by quantitative alterations in amounts of PKC or by altered phorbol ester binding but were related to defects in kinase activation. Analysis in vitro of the kinetic properties of PKC from infected MO revealed an -2-fold increase in the concentration of 1,2-dioleoyl-rac-glycerol required to achieve halfmaximal kinase activation. Evidence for abnormal PKC activation in vivo was reflected by attenuation of PMA-induced translocation of enzyme to the particulate fraction of infected cells. These results provide direct evidence that infection with Leishmania inhibits activation of, and therefore intracellular signaling dependent on, PKC. Inhibition of stimulus-response coupling through PKC provides a basis for understanding impairment of cellular activation by Leishmania and may contribute to chronic infection.Numerous intracellular pathogens including Leishmania (1-3), Yersinia (4), human immunodeficiency virus 1 (5, 6), and others promote mononuclear phagocyte (MO) dysfunction and this may contribute to chronic infection. For example, infection of MO with Leishmania donovani results in defective responses to -y-interferon for the expression of major histocompatibility complex class II genes (1) as well as to decreased responsiveness both to lipopolysaccharide for the induction of interleukin 1 production (2) and to phorbol esters for the induction of c-fqs gene expression (3) and the oxidative burst (7). These MO responses to extracellular stimuli may require activation of protein kinase C (PKC) (8, 9), and this suggests that defective stimulus-response coupling in MO infected with Leishmania may be brought about by altered activation of PKC. To examine this possibility, PKCdependent cell signaling was studied in MO infected with L. Mops (25 mM), EGTA (2 mM), EDTA (5 mM), P-glycerophosphate (50 mM), benzamidine (2.5 mM), leupeptin (2 kug/ml), phenylmethylsulfonyl fluoride (0.2 mM), pepstatin (2 ,qg/ml), soybean trypsin inhibitor (20 pug/ml), 2-mercaptoethanol (0.036%, vol/vol), and Nonidet P-40 (0.1%, vol/vol) were scraped and transferred into microcentrifuge tubes and extracted by sonication (30 W) for 15 s at 0C with a Branson sonifier. Extracts were centrifuged (60 s at 1000 x g), and the supernatants were added to 9 vol of acetone and kept at -20'C for 2 hr. Protein precipitates were collected by centrifugation (10 min at 12,000 x g), dried, and subjected to two-dimensional SDS/PAGE based on the method of O'Farrell (12). Protein samples were digested (20 min at 200C) in buffer (pH 9.5) containing urea (9 M), Nonidet P-40 (1%; vol/vol), ampholytes (20 pul/ml, pH 5-8), 2-mercaptoethanol (5%; vol/vol), and the same phosphatase and proteinase