Differing mechanisms of hepatic glucose overproduction in triiodothyronine-treated rats vs. Zucker diabetic fatty rats by NMR analysis of plasma glucose.

The metabolic mechanism of hepatic glucose overproduction was investigated in 3,3'-5-triiodo-l-thyronine (T3)-treated rats and Zucker diabetic fatty (ZDF) rats (fa/fa) after a 24-h fast. 2H2O and [U-13C3]propionate were administered intraperitoneally, and [3,4-13C2]glucose was administered as a primed infusion for 90 min under ketamine-xylazine anesthesia. 13C NMR analysis of monoacetone glucose derived from plasma glucose indicated that hepatic glucose production was twofold higher in both T3-treated rats and ZDF rats compared with controls, yet the sources of glucose overproduction differed significantly in the two models by 2H NMR analysis.

Cyclen-based phenylboronate ligands and their Eu3+ complexes for sensing glucose by MRI.

Novel cyclen-based phenylboronate ligands and their corresponding Eu(3+) complexes have been examined as glucose sensors using chemical exchange saturation transfer (CEST) MR imaging for detection. Two isomeric bis-phenylboronate complexes, Eu(4) and Eu(10), and a mono-phenylboronate complex, Eu(12), had been prepared and characterized by UV and circular dichroism spectroscopy, mass spectrometry, and CEST imaging. Both the free ligands and their Eu(3+) complexes bind to simple sugars, but their selectivity and binding affinities vary with sugar structure.

Impaired tricarboxylic acid cycle activity in mouse livers lacking cytosolic phosphoenolpyruvate carboxykinase.

Liver-specific phosphoenolpyruvate carboxykinase (PEPCK) null mice, when fasted, maintain normal whole body glucose kinetics but develop dramatic hepatic steatosis. To identify the abnormalities of hepatic energy generation that lead to steatosis during fasting, we studied metabolic fluxes in livers lacking hepatic cytosolic PEPCK by NMR using 2H and 13C tracers. After a 4-h fast, glucose production from glycogenolysis and conversion of glycerol to glucose remains normal, whereas gluconeogenesis from tricarboxylic acid (TCA) cycle intermediates was nearly absent.

Understanding of basic mechanisms of beta-cell function and survival: prelude to new diabetes therapies.

Type 1 and type 2 diabetes are both diseases of insulin insufficiency, although they develop by distinct pathways. The recent surge in the incidence of type 2 diabetes and the chronic ailments confronted by patients with either form of the disease highlight the need for better understanding of beta-cell biology. In this review, we present recent work focused on this goal.

Synthesis, relaxometric and photophysical properties of a new pH-responsive MRI contrast agent: the effect of other ligating groups on dissociation of a p-nitrophenolic pendant arm.

Two gadolinium(III) chelates, GdNP-DO3A (1-methlyene-(p-NitroPhenol)-1,4,7,10-tetraazacycloDOdecane-4,7,10-triAcetate) and GdNP-DO3AM (1-methlyene(p-NitroPhenol)-1,4,7,10-tetraazacycloDOdecane-4,7,10-triacetAMide), containing a single nitrophenolic pendant arm plus either three acetate or three amide pendant arms were synthesized and characterized. The properties of the gadolinium, terbium, and dysprosium complexes of these ligands were examined as a function of pH. The extent and mechanism of the changes in water relaxivity with pH of each gadolinium complex was found to differ substantially for the two complexes.

Solution dynamics and stability of lanthanide(III) (S)-2-(p-nitrobenzyl)DOTA complexes.

Addition of a benzyl substituent to the macrocyclic ring of DOTA has a substantial impact on the conformational ring flipping motion of the macrocycle in the resulting LnDOTA complexes. The p-NO2-benzyl substituent in the Ln(p-NO2-Bn-DOTA)- complexes lies in an equatorial position and effectively "locks" the conformation of the ring into the deltadeltadeltadelta configuration. The presence of the p-NO2-benzyl group also increases the population of the square antiprismatic (SAP) coordination isomer for all Ln(p-NO2-Bn-DOTA)- complexes relative to that seen for the respective LnDOTA- complexes.

Biochemical mechanism of lipid-induced impairment of glucose-stimulated insulin secretion and reversal with a malate analogue.

Hyperlipidemia appears to play an integral role in loss of glucose-stimulated insulin secretion (GSIS) in type 2 diabetes. This impairment can be simulated in vitro by chronic culture of 832/13 insulinoma cells with high concentrations of free fatty acids, or by study of lipid-laden islets from Zucker diabetic fatty rats. Here we show that impaired GSIS is not a simple result of saturation of lipid storage pathways, as adenovirus-mediated overexpression of a cytosolically localized variant of malonyl-CoA decarboxylase in either cellular model results in dramatic lowering of cellular triglyceride stores but no improvement in GSIS.

Glucose production pathways by 2H and 13C NMR in patients with HIV-associated lipoatrophy.

Patients with HIV taking protease inhibitors were selected for the presence (five subjects) or absence (five subjects) of lipoatrophy. Following an overnight fast, subjects were given oral (2)H(2)O in divided doses (5 mL/kg body water), [U-(13)C(3)] propionate (10 mg/kg), and acetaminophen (1000 mg). Glucose (from plasma) or acetaminophen glucuronide (from urine) were converted to monoacetone glucose for (2)H NMR and (13)C NMR analysis.

Compartmentation of glycolysis and glycogenolysis in the perfused rat heart.

Developing methods that can detect compartmentation of metabolic pathways in intact tissues may be important for understanding energy demand and supply. In this study, we investigated compartmentation of glycolysis and glycogenolysis in the isolated perfused rat heart using (13)C NMR isotopomer analysis. Rat hearts previously depleted of myocardial glycogen were perfused with 5.

Glucose production, gluconeogenesis, and hepatic tricarboxylic acid cycle fluxes measured by nuclear magnetic resonance analysis of a single glucose derivative.

A triple-tracer method was developed to provide absolute fluxes contributing to endogenous glucose production and hepatic tricarboxylic acid (TCA) cycle fluxes in 24-h-fasted rats by (2)H and (13)C nuclear magnetic resonance (NMR) analysis of a single glucose derivative. A primed, intravenous [3,4-(13)C(2)]glucose infusion was used to measure endogenous glucose production; intraperitoneal (2)H(2)O (to enrich total body water) was used to quantify sources of glucose (TCA cycle, glycerol, and glycogen), and intraperitoneal [U-(13)C(3)] propionate was used to quantify hepatic anaplerosis, pyruvate cycling, and TCA cycle flux. Plasma glucose was converted to monoacetone glucose (MAG), and a single (2)H and (13)C NMR spectrum of MAG provided the following metabolic data (all in units of micromol/kg/min; n = 6): endogenous glucose production (40.

A (13)C isotopomer kinetic analysis of cardiac metabolism: influence of altered cytosolic redox and [Ca(2+)](o).

Rat hearts were perfused with mixtures of [3-(13)C]pyruvate and [3-(13)C]lactate (to alter cytosolic redox) at low (0.5 mM) or high (2.5 mM) Ca(2+) concentrations to alter contractility.

Solid-phase synthesis of DOTA-peptides.

A general synthetic route to two DOTA-linked N-Fmoc amino acids (DOTA-F and DOTA-K) is described that allows insertion of DOTA at any endo-position within a peptide sequence. Three model pentapeptides were prepared to test the general utility of these derivatives in solid-phase peptide synthesis. Both DOTA derivatives reacted smoothly by means of standard HBTU activation chemistry to the point of insertion of the DOTA amino acid, but extension of the peptide chain beyond the DOTA-amino acid insertion required the use of pre-activated C-pentafluorophenyl ester N-alpha-Fmoc amino acids.

A paramagnetic CEST agent for imaging glucose by MRI.

The europium(III) complex of a DOTA-tetraamide ligand (DOTA = 1,4,7,10-tetraazacyclododecane-N,N',N' ',N' ''-tetraacetic acids) containing two phenyl boronate pendent arms binds glucose reversibly with an association constant of 383 M-1 at pH 7. Glucose binding results in slowing of water exchange between a single Eu(III)-bound water molecule and bulk water, and this can be imaged by MRI using chemical exchange saturation transfer (CEST) imaging sequence. This metabolite-responsive paramagnetic CEST agent responds to changes in glucose over the physiologically important range (0-20 mM), and thus it offers the possibility of high-sensitivity MR imaging glucose in tissues using bulk water protons as antenna.

Silencing of phosphonate-gadolinium magnetic resonance imaging contrast by hydroxyapatite binding.

Rationale And Objectives: GdDOTP5- is a highly charged, bone-seeking paramagnetic complex that could potentially detect bone lesions by magnetic resonance imaging (MRI). To date, its pharmacokinetics, effects on organ relaxivity, and interaction with hydroxyapatite (HA) has not been described.

Methods: Liver, kidney, and bone MRI images were obtained on male white rabbits after the administration of GdDOTP5- or a gold standard MRI contrast agent, GdDTPA2-.

PARACEST agents: modulating MRI contrast via water proton exchange.

Scientific interest in optimizing the properties of gadolinium (III) complexes as MRI contrast agents has led to many new insights into lanthanide ion coordination chemistry in the last two decades. Among these was the surprising observation that water exchange in lanthanide (III) derivatives of DOTA can be modulated dramatically by judicious choice of ligand side chain and Ln(3+) ionic radii. This resulted in the discovery of paramagnetic CEST agents for altering MRI image contrast based upon the chemical exchange saturation transfer mechanism.

pH-sensitive modulation of the second hydration sphere in lanthanide(III) tetraamide-DOTA complexes: a novel approach to smart MR contrast media.

The lanthanide(III) complexes of three tetraamide DOTA bearing pyridyl, phenolic and hydroxypyridyl substituents have been studied by NMR, luminescence and cyclic voltammetry. The relaxivity profiles of the gadolinium complexes of the pyridyl and phenolic ligands were flat and essentially the same between pH 2 and 8. The hydroxypyridyl ligand, however, exhibited two regions of enhanced relaxivity.

Sources of plasma glucose by automated Bayesian analysis of 2H NMR spectra.

Sources of blood glucose can be determined after oral ingestion of (2)H(2)O followed by isolation of plasma glucose and measurement of the relative (2)H enrichments in select positions within the glucose molecule. Typically, (2)H enrichments are obtained by mass spectrometry but (2)H NMR offers an alternative. Here it is demonstrated that the entire analysis may be automated by Bayesian analysis of a (2)H free induction decay signal of monoacetone glucose to obtain a direct readout of the relative contributions of glycogenolysis, glycerol, and phosphoenol pyruvate to plasma glucose production.

Synthesis and luminescence studies of aryl substituted tetraamide complexes of europium(III): a new approach to pH responsive luminescent europium probes.

DOTA-tetraamide ligands having extended phenol or pyridine substituents have been synthesized. The (5)D(0) --> (7)F(J) deltaJ = 1 and 2 emission bands in the corresponding europium(III) complexes differ in their sensitivity to solution pH. This offers the potential for developing pH responsive probes for in vivo imaging that are independent of probe concentration.

Mechanisms by which liver-specific PEPCK knockout mice preserve euglycemia during starvation.

Liver-specific PEPCK knockout mice, which are viable despite markedly abnormal lipid metabolism, exhibit mild hyperglycemia in response to fasting. We used isotopic tracer methods, biochemical measurements, and nuclear magnetic resonance spectroscopy to show that in mice lacking hepatic PEPCK, 1) whole-body glucose turnover is only slightly decreased; 2) whole-body gluconeogenesis from phosphoenolpyruvate, but not from glycerol, is moderately decreased; 3) tricarboxylic acid cycle activity is globally increased, even though pyruvate cycling and anaplerosis are decreased; 4) the liver is unable to synthesize glucose from lactate/pyruvate and produces only a minimal amount of glucose; and 5) glycogen synthesis in both the liver and muscle is impaired. Thus, although mice without hepatic PEPCK have markedly impaired hepatic gluconeogenesis, they are able to maintain a near-normal blood glucose concentration while fasting by increasing extrahepatic gluconeogenesis coupled with diminishing whole-body glucose utilization.

Increased hepatic fructose 2,6-bisphosphate after an oral glucose load does not affect gluconeogenesis.

The generally accepted metabolic concept that fructose 2,6-bisphosphate (Fru-2,6-P2) inhibits gluconeogenesis by directly inhibiting fructose 1,6-bisphosphatase is based entirely on in vitro observations. To establish whether gluconeogenesis is indeed inhibited by Fru-2,6-P2 in intact animals, a novel NMR method was developed using [U-13C]glucose and 2H2O as tracers. The method was used to estimate the sources of plasma glucose from gastric absorption of oral [U-13C]glucose, from gluconeogenesis, and from glycogen in 24-h fasted rats.

Renal and systemic pH imaging by contrast-enhanced MRI.

Perturbations of renal and systemic pH accompany diseases of the kidney, such as renal tubular acidosis, and the ability to image tissue pH would be helpful to assess the extent and severity of such conditions. A dual-contrast-agent strategy using two gadolinium agents, the pH-insensitive GdDOTP(5-) and the pH-sensitive GdDOTA-4AmP(5-), has been developed to generate pH maps by MRI. The renal pharmacokinetics of the structurally dissimilar pH-insensitive contrast agents GdDTPA(2-) and GdDOTP(5-) were found to be similar.