Riverine carbon dioxide evasion along a high-relief watercourse derived from seasonal dynamics of the water-atmosphere gas exchange.

The high-relief catchment of the Tavignanu River (Corsica Island, France) with an elevation range from sea level to 2622 m above sea level was investigated for its riverine carbon budget and stable carbon isotopes. Major riverine dissolved inorganic carbon (DIC or TCO2) sources depended on seasons and sub-catchment lithology. In winter δ13CDIC values of -2 to -7‰ (VPDB) indicated influences of atmospheric CO2. δ13CDIC values decreased gradually to values between -9 and -12‰ in July, which indicates elevated soil CO2 contribution. An observed downstream increase in the total amount of carbon species correlated with inputs from carbonate bearing tributaries and evaporation effects in summer. Main channel partial pressure of CO2 (pCO2) was seasonally highly variable in the upper silicate catchment and the lower coastal plain, where summer values exceed up to six times atmospheric levels. During winter, the central section of the Tavignanu River was found to be undersaturated with respect to atmospheric CO2 levels. The median values for main channel pCO2 were below atmospheric levels in winter and spring and above in summer and autumn. The annual carbon flux across the air-water boundary (FCO2) along the Tavignanu River was calculated with (0.77 ±â€¯0.24) Gg C yr-1, which is about seven times higher than the riverine TCO2 transport to the sea of about 0.11 Gg C yr-1. While large sections of the river experienced year-round atmospheric CO2 uptake or equilibrium, the river as a whole was a small but continuous net source of carbon to the atmosphere. This underlines the important, but so far not well-constrained, contributions of smaller streams and rivers to the terrestrial carbon flux and the need of incorporating them into future global carbon cycle models.

Chlorogenic acid and synthetic chlorogenic acid derivatives: novel inhibitors of hepatic glucose-6-phosphate translocase.

The enzyme system glucose-6-phosphatase (EC 3.1.3.9) plays a major role in the homeostatic regulation of blood glucose. It is responsible for the formation of endogenous glucose originating from gluconeogenesis and glycogenolysis. Recently, chlorogenic acid was identified as a specific inhibitor of the glucose-6-phosphate translocase component (Gl-6-P translocase) of this enzyme system in microsomes of rat liver. Glucose 6-phosphate hydrolysis was determined in the presence of chlorogenic acid or of new synthesized derivatives in intact rat liver microsomes in order to assess the inhibitory potency of the compounds on the translocase component. Variation in the 3-position of chlorogenic acid had only poor effects on inhibitory potency. Introduction of lipophilic side chain in the 1-position led to 100-fold more potent inhibitors. Functional assays on isolated perfused rat liver with compound 29i, a representative of the more potent derivatives, showed a dose-dependent inhibition of gluconeogenesis and glycogenolyosis, suggesting glucose-6-phosphatase as the locus of interference of the compound for inhibition of hepatic glucose production also in the isolated organ model. Gl-6-P translocase inhibitors may be useful for the reduction of inappropriately high rates of hepatic glucose output often found in non-insulin-dependent diabetes.

Alterations of carbohydrate and lipid intermediary metabolism during inhibition of glucose-6-phosphatase in rats.

S 4048 (1-[2-(4-Chloro-phenyl)-cyclopropylmethoxy]-3, 4-dihydroxy-5-(3-imidazo[4, 5-b]pyridin-1-yl-3-phenyl-acryloyloxy)-cyclohexanecarboxylic acid), a derivative of chlorogenic acid, specifically inhibits the glucose-6-phosphate translocating component T1 of the glucose-6-phosphatase system. Its pharmacological effect was studied on carbohydrate and lipid parameters in rats. In starved and fed rats, S 4048 caused a dose-dependent reduction of blood glucose levels with a corresponding increase in hepatic and renal glycogen and glucose-6-phosphate. The major quantitative route of carbon flux in the liver during S 4048-induced inhibition of the glucose-6-phosphatase activity seemed to be glycogenesis. Plasma free fatty acids were increased secondarily due to the S 4048-induced hypoglycemia. Hepatic triglycerides were increased possibly due to increased re-esterification of the readily available free fatty acids. Glucose-6-phosphate translocase inhibitors may be useful for experimentally studying aspects of type 1 glycogen storage disease in laboratory animals as well as for the therapeutic modulation of inappropriately high rates of hepatic glucose production in type 2 diabetes.

Hepatic uptake of synthetic chlorogenic acid derivatives by the organic anion transport proteins.

Chlorogenic acid derivatives were recently identified as novel, potent, and specific inhibitors of the hepatic glucose 6-phosphate translocase. Inhibition of the glucose 6-phosphate translocase leads to a decrease in hepatic glucose production, rendering chlorogenic acid derivatives as potential novel therapeutics in patients with type 2 diabetes. The present study examines the hepatic uptake mechanism of the radiolabeled chlorogenic acid derivative S 1743 into freshly isolated rat hepatocytes. Initial uptake rates were Na(+)-independent and followed saturation kinetics with no superimposition of facilitated diffusion. Inhibition studies demonstrated that other chlorogenic acid derivatives inhibited uptake of the radiolabeled compound S 1743 into rat hepatocytes in the range of 1.1 to 11 microM, whereas the natural chlorogenic acid (up to 100 microM) had no effect at all. In addition, inhibition of S 1743 uptake into rat hepatocytes was found in the presence of sulfobromophthalein, sulfolithocholyltaurine, estrone-3-sulfate, cholyltaurine, verapamil, bumetanide, probenecide, phenol red, digoxin, and ouabain (in decreasing order) but not with N-methylnicotinamide, alpha-ketoglutarate, p-aminohippurate, geneticin sulfate, and 5-sulfosalicylate. The observed inhibition pattern suggested that members of the family of the organic anion transporting polypeptides (Oatps) could be involved in hepatic uptake of chlorogenic acid derivatives. Indeed, S 1743 uptake could be demonstrated in Oatp1- and Oatp2-expressing Xenopus laevis oocytes as well as in Oatp1-expressing Chinese hamster ovary cells. A comparison of the inhibition pattern obtained in hepatocytes compared with that obtained in Oatp1-expressing Chinese hamster ovary cells suggests that facilitated uptake by Oatp1 is a major contributor in total hepatic uptake of chlorogenic acid derivatives.

Pharmacodynamic profile of a novel inhibitor of the hepatic glucose-6-phosphatase system.

The glucose-6-phosphatase (G-6-Pase) system catalyzes the terminal enzymatic step of gluconeogenesis and glycogenolysis. Inhibition of the G-6-Pase system in the liver is expected to result in a reduction of hepatic glucose production irrespective of the relative contribution of gluconeogenesis or glycogenolysis to hepatic glucose output. In isolated perfused rat liver, S-3483, a derivative of chlorogenic acid, produced concentration-dependent inhibition of gluconeogenesis and glycogenolysis in a similar concentration range. In fed rats, glucagon-induced glycogenolysis resulted in hyperglycemia for nearly 2 h. Intravenous infusion of 50 mg . kg-1. h-1 S-3483 prevented the hyperglycemic peak and subsequently caused a further lowering of blood glucose. In 24-h starved rats, in which normoglycemia is maintained predominantly by gluconeogenesis, intravenous infusion of S-3483 resulted in a constant reduction of blood glucose levels. Intrahepatic concentrations of glucose-6-phosphate (G-6-P) and glycogen were significantly increased at the end of both in vivo studies. In contrast, lowering of blood glucose in starved rats by 3-mercaptopicolinic acid was accompanied by a reduction of G-6-P and glycogen. Our results demonstrate for the first time in vivo a pharmacologically induced suppression of hepatic G-6-P activity with subsequent changes in blood glucose levels.

Chlorogenic acid and hydroxynitrobenzaldehyde: new inhibitors of hepatic glucose 6-phosphatase.

We have studied the interactions of chlorogenic acid (CHL) and 2-hydroxy-5-nitrobenzaldehyde (HNB) with the components of the rat hepatic glucose 6-phosphatase (Glc-6-Pase) system. CHL and HNB are competitive inhibitors of glucose 6-phosphate (Glc-6-P) hydrolysis in intact microsomes with Ki values of 0.26 and 0.22 mm, respectively. CHL is without effect on the enzyme of fully disrupted microsomes or the system inorganic pyrophosphatase (PPiase) activity. HNB is a potent competitive inhibitor of the system PPiase activity (Ki = 0.56 mm) and a somewhat weaker noncompetitive inhibitor of enzyme activity (Ki = 2.1 mm). These findings indicate CHL binds to T1, the Glc-6-P transporter, and HNB inhibits through interaction with both T1 and T2 the phosphate (Pi)-PPi transporter. Binding of CHL and HNB is freely reversible. However, the inhibition of both PPiase and Glc-6-Pase by HNB becomes irreversible following incubation of HNB-exposed microsomes with 2.5 mm sodium borohydride, indicating that inhibition involves the formation of a Schiff base. The presence of CHL effectively protects T1, but not T2, against the irreversible inhibition by HNB. In contrast, PPi and Pi are effective in protecting T2, but not T1. This is the first report describing an effective inhibitor of the system PPiase activity (T2). CHL is the most specific T1 inhibitor described to date.

Direct evidence for the involvement of two glucose 6-phosphate-binding sites in the glucose-6-phosphatase activity of intact liver microsomes. Characterization of T1, the microsomal glucose 6-phosphate transport protein by a direct binding assay.

S 5627 is a synthetic analogue of chlorogenic acid. S 5627 is a potent linear competitive inhibitor of glucose 6-phosphate (Glc-6-P) hydrolysis by intact microsomes (Ki = 41 nM) but is without effect on the enzyme in detergent- or NH4OH-disrupted microsomes. 3H-S 5627 was synthesized and used as a ligand in binding studies directed at characterizing T1, the Glc-6-P transporter. Binding was evaluated using Ca2+-aggregated microsomes, which can be sedimented at low g forces. Aside from a modest reduction in K values for both substrate and S 5627, Ca2+ aggregation had no effect on glucose-6-phosphatase (Glc-6-Pase). Scatchard plots of binding data are readily fit to a simple "two-site" model, with Kd = 21 nM for the high affinity site and Kd = 2 microM for the low affinity site. Binding to the high affinity site was competitively blocked by Glc-6-P (Ki = 9 microM), whereas binding was unaffected by mannose-6-phosphate, Pi, and PPi and only modestly depressed by 2-deoxy-D-glucose 6-phosphate, a poor substrate for Glc-6-Pase in intact microsomes. Thus the high affinity 3H-S 5627 binding site fits the criteria for T1. Permeabilization of the membrane with 0.3% (3-[(chloramidopropyl)-dimethylammonio]-1-propanesulfonate) activated Glc-6-Pase and broadened its substrate specificity, but it did not significantly alter the binding of 3H-S 5627 to the high affinity sites or the ability of Glc-6-P to block binding. These data demonstrate unequivocally that two independent Glc-6-P binding sites are involved in the hydrolysis of Glc-6-P by intact microsomes. The present findings are the strongest and most direct evidence to date against the notion that the substrate specificity and the intrinsic activity of Glc-6-Pase in native membranes are determined by specific conformational constraints imposed on the enzyme protein. These data constitute compelling evidence for the role of T1 in Glc-6-Pase activity.

Chlorogenic acid analogue S 3483: a potent competitive inhibitor of the hepatic and renal glucose-6-phosphatase systems.

S 3483, a synthetic derivative of chlorogenic acid (CHL), was found to be a reversible, linear competitive inhibitor of the glucose-6-phosphatase (Glc-6-Pase) system in rat renal microsomes and rat and human liver microsomes. The Ki for S 3483 in rat liver microsomes (129 nM) is three orders of magnitude smaller than the Ki for CHL. S 3483 up to 100 microM had no effect on the Glc-6-Pase enzyme activity or on the system inorganic pyrophosphatase activity (i.e., on T2, the Pi/inorganic pyrophosphate transporter). Thus, like CHL, S 3483 appears to be a site-specific inhibitor of T1, the Glc-6-P transporter of renal and liver microsomes. The potency of S 3483 was unaffected when the ratio Vmax(T1):Vmax(enzyme) was altered over a 10-fold range by applying enzyme inhibition and selective inactivation of T1. The absence of T1-imposed rate restrictions on the potency of reversible T1 inhibitors contrasts markedly with the response of reversible Glc-6-Pase enzyme inhibitors, whose potency declines sharply as T1 becomes more rate controlling. The potency of S 3483, but not of CHL, decreased as the microsomal protein concentration in the assay medium was increased. This effect suggests that as the protein concentration was raised the concentration of T1 in the assay medium approached the order of magnitude of the Ki for S 3483. Thus, the microsomal content of T1 is likely to be on the order of 100 pmol/mg protein. S 3483 is the most potent inhibitor of the Glc-6-Pase system reported to date. It and other tight-binding inhibitors of T1 will provide useful new tools for investigating the molecular structure and physiology/pathology of the Glc-6-Pase system.

Identification of protein components of the microsomal glucose 6-phosphate transporter by photoaffinity labelling.

The glucose-6-phosphatase system catalyses the terminal step of hepatic glucose production from both gluconeogenesis and glycogenolysis and is thus a key regulatory factor of blood glucose homoeostasis. To identify the glucose 6-phosphate transporter T1, we have performed photoaffinity labelling of human and rat liver microsomes by using the specific photoreactive glucose-6-phosphate translocase inhibitors S 0957 and S 1743. Membrane proteins of molecular mass 70, 55, 33 and 31 kDa were labelled in human microsomes by [3H]S 0957, whereas in rat liver microsomes bands at 95, 70, 57, 54, 50, 41, 33 and 31 kDa were detectable. The photoprobe [3H]S 1743 led to the predominant labelling of a 57 kDa and a 50 kDa protein in the rat. Stripping of microsomes with 0.3% CHAPS retains the specific binding of T1 inhibitors; photoaffinity labelling of such CHAPS-treated microsomes resulted in the labelling of membrane proteins of molecular mass 55, 33 and 31 kDa in human liver and 50, 33 and 31 kDa in rat liver. Photoaffinity labelling of human liver tissue samples from a healthy individual and from liver samples of patients with a diagnosed glycogen-storage disease type 1b (GSD type 1b; von Gierke's disease) revealed the absence of the 55 kDa protein from one of the patients with GSD type 1. These findings support the identity of the glucose 6-phosphate transporter T1, with endoplasmic reticulum protein of molecular mass 50 kDa in rat liver and 55 kDa in human liver.