Kinetics And Mechanisms Of The Aminolysis Of N-Hydroxysuccinimide Esters In Aqueous Buffers

Rate constants for the aminolysis of the N-hydroxysuccinimide (NHS) ester of ρ-methoxybenzoic acid, in aqueous buffer systems (20% dioxane), have been determined under pseudo-first-order conditions. For the amines studied (pKa = 7.60-11.1), the data fit the rate expression kobsd - KOH-⨯ [OH-] = k1[amine]free. This rate equation is in contrast to the two-term rate equation (kobsd = k1[amine] + k2[amine]2) obtained for this reaction in anhydrous dioxane (Cline, G. W.; Hanna, S. B. J. Am. Chem. Soc. 1987,109, 3087) and is suggestive of a disproportionate decrease in the catalyzed vs the uncatalyzed reaction path upon changing from a nonaqueous to an aqueous solvent system. The correlation of amine basicity with the nucleophilic rate constant, k1 yields a slope αnuc = 1.0. The magnitude of αnuc, in terms of a reaction mechanism where a tetrahedral intermediate is formed in a fast preequilibrium followed by rate-determining breakdown to products, reflects the sensitivity to changes in charge accumulation in the formation of the tetrahedral intermediate. The resultant increased rate constants, with increased basicity, are due to the effect of an increased concentration of the tetrahedral intermediate. A qualitative evaluation of the literature and current data concerning the leaving ability of N-hydroxy esters, in comparison to phenyl esters (equivalent acyl groups and nucleophiles), reveals that, with leaving groups of comparable basicity, the nucleophilic rate constants for N-hydroxy esters are about 2 orders of magnitude greater than that for phenyl esters. © 1988, American Chemical Society. All rights reserved

The Aminolysis Of N-Hydroxysuccinimide Esters. A Structure-Reactivity Study

Twelve amines, which vary substantially in basicity and in steric environment around N, have been allowed to compete—in anhydrous dioxane solution—in the aminolysis of the N-hydroxysuccinimide esters of unsubstituted, p-OCH3, p-N02, and 3,5-(N02)2 benzoic acids. The amines, which encompass a basicity range of 6.5 pk units, display a 10000-fold variation in reactivity in their reaction with the p-N02ester. for the sterically unhindered amines, a Bronsted-type plot of log kobsdvs. pKa has a slope of ~0.7. The data fit a model (Satterthwait, A. C.; Jencks, W. P. J. Am. Chem. Soc. 1974, 96, 7018–7044) in which reversible formation of a tetrahedral intermediate is followed by rate-determining breakdown to products. Appreciable sensitivity to steric factors, as evidenced from the depressed rates with a-methylbenzylamine and diethylamine, substantiates reversible formation of a crowded tetrahedral intermediate prior to the rate-determining step. The Hammett p values for the competitive acylation of aniline, a-methylbenzylamine, and benzylamine, by substituted N-succinimidyl benzoates, are 1.4,1.2, and 1.1, respectively. These values reflect the selectivity expected for these amines, and the substantial accumulation of charge density at the acyl C in the formation of the tetrahedral intermediate. Individual rate constants for the aminolysis of N-succinimidyl p-methoxybenzoate by n-butylamine and by piperidine, both show First-order and second-order terms in [amine]. The general-base catalysis term is suggestive of a path involving proton transfer in the rate-determining step. © 1987, American Chemical Society. All rights reserved

Regulation of Exogenous and Endogenous Glucose Metabolism by Insulin and Acetoacetate in the Isolated Working Rat Heart A Three Tracer Study of Glycolysis, Glycogen Metabolism, and Glucose Oxidation

Abstract Myocardial glucose use is regulated by competing substrates and hormonal influences. However, the interactions of these effectors on the metabolism of exogenous glucose and glucose derived from endogenous glycogen are not completely understood. In order to determine changes in exogenous glucose uptake, glucose oxidation, and glycogen enrichment, hearts were perfused with glucose (5 mM) either alone, or glucose plus insulin (40 U/ml), glucose plus acetoacetate (5 mM), or glucose plus insulin and acetoacetate, using a three tracer ( 3 H, 14 C, and 13 C) technique. Insulinstimulated glucose uptake and lactate production in the absence of acetoacetate, while acetoacetate inhibited the uptake of glucose and the oxidation of both exogenous glucose and endogenous carbohydrate. Depending on the metabolic conditions, the contribution of glycogen to carbohydrate metabolism varied from 20-60%. The addition of acetoacetate or insulin increased the incorporation of exogenous glucose into glycogen twofold, and the combination of the two had additive effects on the incorporation of glucose into glycogen. In contrast, the glycogen content was similar for the three groups. The increased incorporation of glucose in glycogen without a significant change in the glycogen content in hearts perfused with glucose, acetoacetate, and insulin suggests increased glycogen turnover. We conclude that insulin and acetoacetate regulate the incorporation of glucose into glycogen as well as the relative contributions of exogenous glucose and endogenous carbohydrate to myocardial energy metabolism by different mechanisms. ( J. Clin. Invest. 1997. 100:2892-2899.) Key words: citric acid cycle • NMR • isotopomer analysi

A role for foregut tyrosine metabolism in glucose tolerance

Objective: We hypothesized that DA and L-DOPA derived from nutritional tyrosine and the resultant observed postprandial plasma excursions of L-DOPA and DA might affect glucose tolerance via their ability to be taken-up by beta cells and inhibit glucose-stimulated b-cell insulin secretion. Methods: To investigate a possible circuit between meal-stimulated 3,4-dihydroxy-L-phenylalanine (L-DOPA) and dopamine (DA) production in the GI tract and pancreatic b-cells, we: 1) mapped GI mucosal expression of tyrosine hydroxylase (TH) and aromatic amino acid decarboxylase (AADC); 2) measured L-DOPA and DA content of GI mucosal tissues following meal challenges with different L-tyrosine (TYR) content, 3) determined whether meal TYR content impacts plasma insulin and glucose excursions; and 4) characterized postprandial plasma excursions of L-DOPA and DA in response to meal tyrosine content in rodents and a population of bariatric surgery patients. Next, we characterized: 1) the metabolic transformation of TYR and L-DOPA into DA in vitro using purified islet tissue; 2) the metabolic transformation of orally administrated stable isotope labeled TYR into pancreatic DA, and 3) using a nuclear medicine technique, we studied endocrine beta cells in situ release and binding of DA in response to a glucose challenge. Results: We demonstrate in rodents that intestinal content and circulatory concentrations L-DOPA and DA, plasma glucose and insulin are responsive to the tyrosine (TYR) content of a test meal. Intestinal expression of two enzymes, Tyrosine hydroxylase (TH) and Aromatic Amino acid Decarboxylase (AADC), essential to the transformation of TYR to DA was mapped and the metabolism of metabolism of TYR to DA was traced in human islets and a rodent beta cell line in vitro and from gut to the pancreas in vivo. Lastly, we show that b cells secrete and bind DA in situ in response to glucose stimulation. Conclusions: We provide proof-of-principle evidence for the existence of a novel postprandial circuit of glucose homeostasis dependent on nutritional tyrosine. DA and L-DOPA derived from nutritional tyrosine may serve to defend against hypoglycemia via inhibition of glucosestimulated b-cell insulin secretion as proposed by the anti-incretin hypothesis

Alterations in Postprandial Hepatic Glycogen Metabolism in Type 2 Diabetes

Decreased skeletal muscle glucose disposal and increased endogenous glucose production (EGP) contribute to postprandial hyperglycemia in type 2 diabetes, but the contribution of hepatic glycogen metabolism remains uncertain. Hepatic glycogen metabolism and EGP were monitored in type 2 diabetic patients and nondiabetic volunteer control subjects (CON) after mixed meal ingestion and during hyperglycemic-hyperinsulinemic-somatostatin clamps applying 13C nuclear magnetic resonance spectroscopy (NMRS) and variable infusion dual-tracer technique. Hepatocellular lipid (HCL) content was quantified by 1H NMRS. Before dinner, hepatic glycogen was lower in type 2 diabetic patients (227 ± 6 vs. CON: 275 ± 10 mmol/l liver, P < 0.001). After meal ingestion, net synthetic rates were 0.76 ± 0.16 (type 2 diabetic patients) and 1.36 ± 0.15 mg · kg−1 · min−1 (CON, P < 0.02), resulting in peak concentrations of 283 ± 15 and 360 ± 11 mmol/l liver. Postprandial rates of EGP were ∼0.3 mg · kg−1 · min−1 (30–170 min; P < 0.05 vs. CON) higher in type 2 diabetic patients. Under clamp conditions, type 2 diabetic patients featured ∼54% lower (P < 0.03) net hepatic glycogen synthesis and ∼0.5 mg · kg−1 · min−1 higher (P < 0.02) EGP. Hepatic glucose storage negatively correlated with HCL content (R = −0.602, P < 0.05). Type 2 diabetic patients exhibit 1) reduction of postprandial hepatic glycogen synthesis, 2) temporarily impaired suppression of EGP, and 3) no normalization of these defects by controlled hyperglycemic hyperinsulinemia. Thus, impaired insulin sensitivity and/or chronic glucolipotoxicity in addition to the effects of an altered insulin-to-glucagon ratio or increased free fatty acids accounts for defective hepatic glycogen metabolism in type 2 diabetic patients

Reversal of Hypertriglyceridemia, Fatty Liver Disease, and Insulin Resistance by a Liver-Targeted Mitochondrial Uncoupler

SummaryNonalcoholic fatty liver disease (NAFLD) affects one in three Americans and is a major predisposing condition for the metabolic syndrome and type 2 diabetes (T2D). We examined whether a functionally liver-targeted derivative of 2,4-dinitrophenol (DNP), DNP-methyl ether (DNPME), could safely decrease hypertriglyceridemia, NAFLD, and insulin resistance without systemic toxicities. Treatment with DNPME reversed hypertriglyceridemia, fatty liver, and whole-body insulin resistance in high-fat-fed rats and decreased hyperglycemia in a rat model of T2D with a wide therapeutic index. The reversal of liver and muscle insulin resistance was associated with reductions in tissue diacylglycerol content and reductions in protein kinase C epsilon (PKCε) and PKCθ activity in liver and muscle, respectively. These results demonstrate that the beneficial effects of DNP on hypertriglyceridemia, fatty liver, and insulin resistance can be dissociated from systemic toxicities and suggest the potential utility of liver-targeted mitochondrial uncoupling agents for the treatment of hypertriglyceridemia, NAFLD, metabolic syndrome, and T2D

Membrane-Bound sn-1,2-Diacylglycerols Explain the Dissociation of Hepatic Insulin Resistance from Hepatic Steatosis in MTTP Knockout Mice

Microsomal triglyceride transfer protein (MTTP) deficiency results in a syndrome of hypolipidemia and accelerated NAFLD. Animal models of decreased hepatic MTTP activity have revealed an unexplained dissociation between hepatic steatosis and hepatic insulin resistance. Here, we performed comprehensive metabolic phenotyping of liver-specific MTTP knockout (L-Mttp(-/-)) mice and age-weight matched wild-type control mice. Young (10-12-week-old) L-Mttp(-/-) mice exhibited hepatic steatosis and increased DAG content; however, the increase in hepatic DAG content was partitioned to the lipid droplet and was not increased in the plasma membrane. Young L-Mttp(-/-) mice also manifested normal hepatic insulin sensitivity, as assessed by hyperinsulinemic-euglycemic clamps, no PKC epsilon activation, and normal hepatic insulin signaling from the insulin receptor through AKT Ser/Thr kinase. In contrast, aged (10-month-old) L-Mttp(-/-) mice exhibited glucose intolerance and hepatic insulin resistance along with an increase in hepatic plasma membrane sn-1,2-DAG content and PKC epsilon activation. Treatment with a functionally liver-targeted mitochondrial uncoupler protected the aged L-Mttp(-/-) mice against the development of hepatic steatosis, increased plasma membrane sn-1,2-DAG content, PKC epsilon activation, and hepatic insulin resistance. Furthermore, increased hepatic insulin sensitivity in the aged controlled-release mitochondrial protonophore-treated L-Mttp(-/-) mice was not associated with any reductions in hepatic ceramide content. Taken together, these data demonstrate that differences in the intracellular compartmentation of sn-1,2-DAGs in the lipid droplet versus plasma membrane explains the dissociation of NAFLD/lipid-induced hepatic insulin resistance in young L-Mttp(-/-) mice as well as the development of lipid-induced hepatic insulin resistance in aged L-Mttp(-/-) miceThis work was supported by National Institutes of Health Grants R01 DK116774, R01 DK119968, R01 DK114793, R01 DK113984, K23 DK10287, P30 DK045735, DK121490, and HL137202 and the Veterans Health Administration Merit Review Awards I01 BX000901 and BX004113. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health or the U.S. Department of Veterans Affair

Original Article Fish Oil Regulates Adiponectin Secretion by a Peroxisome Proliferator-Activated Receptor-␥-Dependent Mechanism in Mice

Adiponectin has insulin-sensitizing, antiatherogenic, and anti-inflammatory properties, but little is known about factors that regulate its secretion. To examine the effect of fish oil on adiponectin secretion, mice were fed either a control diet or isocaloric diets containing 27% safflower oil or 27, 13.5, and 8% menhaden fish oil. Within 15 days, fish oil feeding raised plasma adiponectin concentrations twoto threefold in a dose-dependent manner, and the concentrations remained approximately twofold higher for 7 days when the fish oil diet was replaced by the safflower oil diet. Within 24 h, fish oil markedly induced transcription of the adiponectin gene in epididymal adipose tissue but not in subcutaneous fat. The increase of plasma adiponectin by fish oil was completely blocked by administration of the peroxisome proliferator-activated receptor (PPAR)␥ inhibitor bisphenol-A-diglycidyl ether. In contrast, there was no effect of fish oil feeding on adiponectin secretion in PPAR␣-null mice. These data suggest that fish oil is a naturally occurring potent regulator of adiponectin secretion in vivo and that it does so through a PPAR␥-dependent and PPAR␣-independent manner in epididymal fat. Diabetes 55: 924 -928, 200

Mitochondrial Glycerol-3-phosphate Acyltransferase-1 Is Essential in Liver for the Metabolism of Excess Acyl-CoAs

In vitro studies suggest that the mitochondrial glycerol-3-phosphate acyltransferase-1 (mtGPAT1) isoform catalyzes the initial and rate-controlling step in glycerolipid synthesis and aids in partitioning acyl-CoAs toward triacylglycerol synthesis and away from degradative pathways. To determine whether the absence of mtGPAT1 would increase oxidation of acyl-CoAs and restrict the development of hepatic steatosis, we fed wild type and mtGPAT1-/- mice a diet high in fat and sucrose (HH) for 4 months to induce the development of obesity and a fatty liver. Control mice were fed a diet low in fat and sucrose (LL). With the HH diet, absence of mtGPAT1 resulted in increased partitioning of acyl-CoAs toward oxidative pathways, demonstrated by 60% lower hepatic triacylglycerol content and 2-fold increases in plasma beta-hydroxybutyrate, acylcarnitines, and hepatic mRNA expression of mitochondrial HMG-CoA synthase. Despite the increase in fatty acid oxidation, liver acyl-CoA levels were 3-fold higher in the mtGPAT1-/- mice fed both diets. A lack of difference in CPT1 and FAS mRNA expression between genotypes suggested that the increased acyl-CoA content was not because of increased de novo synthesis, but instead, to an impaired ability to use long-chain acyl-CoAs derived from the diet, even when the dietary fat content was low. Hyperinsulinemia and reduced glucose tolerance on the HH diet was greater in the mtGPAT1-/- mice, which did not suppress the expression of the gluconeogenic genes glucose-6-phosphatase and phosphoenolpyruvate carboxykinase. This study demonstrates that mtGPAT1 is essential for normal acyl-CoA metabolism, and that the absence of hepatic mtGPAT1 results in the partitioning of fatty acids away from triacylglycerol synthesis and toward oxidation and ketogenesis

Prevention of Hepatic Steatosis and Hepatic Insulin Resistance by Knockdown of cAMP Response Element-Binding Protein

SummaryIn patients with poorly controlled type 2 diabetes mellitus (T2DM), hepatic insulin resistance and increased gluconeogenesis contribute to fasting and postprandial hyperglycemia. Since cAMP response element-binding protein (CREB) is a key regulator of gluconeogenic gene expression, we hypothesized that decreasing hepatic CREB expression would reduce fasting hyperglycemia in rodent models of T2DM. In order to test this hypothesis, we used a CREB-specific antisense oligonucleotide (ASO) to knock down CREB expression in liver. CREB ASO treatment dramatically reduced fasting plasma glucose concentrations in ZDF rats, ob/ob mice, and an STZ-treated, high-fat-fed rat model of T2DM. Surprisingly, CREB ASO treatment also decreased plasma cholesterol and triglyceride concentrations, as well as hepatic triglyceride content, due to decreases in hepatic lipogenesis. These results suggest that CREB is an attractive therapeutic target for correcting both hepatic insulin resistance and dyslipidemia associated with nonalcoholic fatty liver disease (NAFLD) and T2DM