Metformin severely impairs in vivo muscle oxidative capacity in a rat model of type 2 diabetes

Objective: To investigate the effects of metformin on in vivo and in vitro skeletal muscle mitochondrial function in Zucker diabetic fatty (ZDF) rats using 31P magnetic resonance spectroscopy (MRS) and high-resolution respirometry (HRR), respectively. Methods: 12-week old healthy (fa/+) and diabetic (fa/fa) ZDF rats were treated with metformin (0, 30, 100 or 300 mg/kg body weight/day) for 15 days by oral gavage. At day 14, in vivo31P MRS was performed on the tibialis anterior (TA) muscle to measure PCr recovery. At day 15, animals were killed and TA muscles were excised for in vitro HRR measurements. Results: Metformin treatment decreased PCr recovery rates in a dose-dependent manner in both healthy fa/+ and diabetic fa/fa rats. Whereas, the clinical dose of 30 mg/kg/day had no significant effect, PCr recovery rates were ~22% and ~47% decreased at 100 and 300 mg/kg/day. HRR measurements showed a similar, but less pronounced effect of metformin on in vitro mitochondrial function

In vivo postprandial lipid partitioning in liver and muscle of diabetic rats is disturbed

Objective: To study in vivo lipid partitioning in insulin-resistant liver and muscle of diabetic rats using magnetic resonance spectroscopy (MRS). Methods: Four groups of n=6 male Zucker diabetic fatty rats were used for this study: obese, pre-diabetic fa/fa rats and lean, non-diabetic fa/+ littermates at the age of 6 weeks, and obese, diabetic fa/fa rats and lean, non-diabetic fa/+ littermates at the age of 12 weeks. 1H-[13C] MRS measurements were performed in liver and tibialis anterior muscle at baseline and 4, 24 and 48 h after oral administration of 1.5 g [U-13C] Algal lipid mixture per kg body weight. Results: At baseline, total lipid content was higher in fa/fa rats compared with fa/+ rats in both liver and muscle, and at both ages. Both in pre-diabetic and in diabetic fa/fa rats, hepatic lipid uptake was increased compared with non-diabetic fa/+ rats. Likewise, in muscle of diabetic fa/fa rats, lipid uptake was higher than in muscle of fa/+ rats. In contrast, lipid uptake in muscle of younger, pre-diabetic fa/fa rats was lower than in controls. Conclusion: In the pre-diabetic state, muscle appeared to be protected from massive lipid uptake, whereas lipid uptake in the liver was largely increased. In contrast, after developing full-blown diabetes, lipid uptake was highly elevated in both liver and muscle. This research was funded by a VIDI grant from the Netherlands Organisation for Scientific Research (NWO)

In vivo magnetic resonance spectroscopy of lipid handling in steatotic rat liver

Objective: Examine lipid handling in liver of rats fed with different high-fat diets using 1H-[13C] magnetic resonance spectroscopy (MRS) together with oral administration of 13C labeled lipids. Methods: 6 male Wistar rats (11 weeks old; 348 ± 8g) were divided into three diet groups: low-fat (10% fat, CON), high-fat lard (45% fat, HFL), and high-fat palm oil (45%, HFP). After 10 weeks of diet, MRS experiments were performed at baseline, and 4 and 24 h after oral administration of 1.5 g [U-13C] Algal lipid mixture per kg body weight. Results: At 4 h after administration of the 13C labeled lipids, 13C enrichment of intracellular liver lipids was similarly increased in all three groups compared to baseline (CON: 0.031 ± 0.017 %; HFL: 0.045 ± 0.022 %; HFP: 0.033 ± 0.013 %), demonstrating that lipid uptake was not affected by the diet regimen. At 24 h, on the other hand, 13C enrichment of liver lipids decreased in CON, whereas in both high-fat diet groups the 13C enrichment did not change compared to 4 h, indicating a lower turnover of the stored liver lipids. Conclusion: High-fat diet feeding did not alter liver lipid uptake in rats, but resulted in a decreased turnover of the lipids stored in the liver. This research was funded by the Netherlands Consortium for Systems Biology (NCSB) which is part of the Netherlands Genomics Initiative/Netherlands Organisation for Scientific Research

An in vivo magnetic resonance spectroscopy study of the effects of caloric and non-caloric sweeteners on liver lipid metabolism in rats

We aimed to elucidate the effects of caloric and non-caloric sweeteners on liver lipid metabolism in rats using in vivo magnetic resonance spectroscopy (MRS) and to determine their roles in the development of liver steatosis. Wistar rats received normal chow and either normal drinking water, or solutions containing 13% (w/v) glucose, 13% fructose, or 0.4% aspartame. After 7 weeks, in vivo hepatic dietary lipid uptake and de novo lipogenesis were assessed with proton-observed, carbon-13-edited MRS combined with13C-labeled lipids and13C-labeled glucose, respectively. The molecular basis of alterations in hepatic liver metabolism was analyzed in detail ex vivo using immunoblotting and targeted quantitative proteomics. Both glucose and fructose feeding increased adiposity, but only fructose induced hepatic lipid accumulation. In vivo MRS showed that this was not caused by increased hepatic uptake of dietary lipids, but could be attributed to an increase in de novo lipogenesis. Stimulation of lipogenesis by fructose was confirmed by a strong upregulation of lipogenic enzymes, which was more potent than with glucose. The non-caloric sweetener aspartame did not significantly affect liver lipid content or metabolism. In conclusion, liquid fructose more severely affected liver lipid metabolism in rats than glucose, while aspartame had no effect.</p

Adaptations in mitochondrial function parallel, but fail to rescue, the transition to severe hyperglycemia and hyperinsulinemia: a study in Zucker diabetic fatty rats.

Cross-sectional human studies have associated mitochondrial dysfunction to type 2 diabetes. We chose Zucker diabetic fatty (ZDF) rats as a model of progressive insulin resistance to examine whether intrinsic mitochondrial defects are required for development of type 2 diabetes. Muscle mitochondrial function was examined in 6-, 12-, and 19-week-old ZDF (fa/fa) and fa/+ control rats (n = 8-10 per group) using respirometry with pyruvate, glutamate, and palmitoyl-CoA as substrates. Six-week-old normoglycemic-hyperinsulinemic fa/fa rats had reduced mitochondrial fat oxidative capacity. Adenosine diphosphate (ADP)-driven state 3 and carbonyl cyanide p-trifluoromethoxyphenylhydrazone (FCCP)-stimulated state uncoupled (state u) respiration on palmitoyl-CoA were lower compared to controls (62.3 ± 9.5 vs. 119.1 ± 13.8 and 87.8 ± 13.3 vs. 141.9 ± 14.3 nmol O2/mg/min.). Pyruvate oxidation in 6-week-old fa/fa rats was similar to controls. Remarkably, reduced fat oxidative capacity in 6-week-old fa/fa rats was compensated for by an adaptive increase in intrinsic mitochondrial function at week 12, which could not be maintained toward week 19 (140.9 ± 11.2 and 57.7 ± 9.8 nmol O2/mg/min, weeks 12 and 19, respectively), whereas hyperglycemia had developed (13.5 ± 0.6 and 16.1 ± 0.3 mmol/l, weeks 12 and 19, respectively). This mitochondrial adaptation failed to rescue the progressive development of insulin resistance in fa/fa rats. The transition of prediabetes state toward advanced hyperglycemia and hyperinsulinemia was accompanied by a blunted increase in uncoupling protein-3 (UCP3). Thus, in ZDF rats insulin resistance develops progressively in the absence of mitochondrial dysfunction. In fact, improved mitochondrial capacity in hyperinsulinemic hyperglycemic rats does not rescue the progression toward advanced stages of insulin resistance

Acute cellular and vascular responses to photodynamic therapy using EGFR-targeted nanobody-photosensitizer conjugates studied with intravital optical imaging and magnetic resonance imaging

Targeted photodynamic therapy (PDT) has the potential to selectively damage tumor tissue and to increase tumor vessel permeability. Here we characterize the tissue biodistribution of two EGFR-targeted nanobody-photosensitizer conjugates (NB-PS), the monovalent 7D12-PS and the biparatopic 7D12-9G8-PS. In addition, we report on the local and acute phototoxic effects triggered by illumination of these NB-PS which have previously shown to lead to extensive tumor damage. Methods: Intravital microscopy and the skin-fold chamber model, containing OSC-19-luc2-cGFP tumors, were used to investigate: a) the fluorescence kinetics and distribution, b) the vascular response and c) the induction of necrosis after illumination at 1 or 24 h post administration of 7D12-PS and 7D12-9G8-PS. In addition, dynamic contrast enhanced magnetic resonance imaging (DCE-MRI) of a solid tumor model was used to investigate the microvascular status 2 h after 7D12-PS mediated PDT. Results: Image analysis showed significant tumor colocalization for both NB-PS which was higher for 7D12-9G8-PS. Intravital imaging showed clear tumor cell membrane localization 1 and 2 h after administration of 7D12-9G8-PS, and fluorescence in or close to endothelial cells in normal tissue for both NB-PS. PDT lead to vasoconstriction and leakage of tumor and normal tissue vessels in the skin-fold chamber model. DCE-MRI confirmed the reduction of tumor perfusion after 7D12-PS mediated PDT. PDT induced extensive tumor necrosis and moderate normal tissue damage, which was similar for both NB-PS conjugates. This was significantly reduced when illumination was performed at 24 h com

NMR studies of Fusarium solani pisi cutinase: structure-mobility-function relationships

Contains fulltext : 145588.pdf (Publisher’s version ) (Open Access)159 p

Reorientational eigenmode dynamics : a combined MD/NMR relaxation analysis method for flexible parts in globular proteins

An approach is presented for the interpretation of heteronuclear NMR spin relaxation data in mobile protein parts in terms of reorientational eigenmode dynamics. The method is based on the covariance matrix of the spatial functions of the nuclear spin interactions that cause relaxation expressed as spherical harmonics of rank 2. The approach was applied to characterize the dynamics of a loop region of ubiquitin. The covariance matrix was determined from a conformational ensemble generated by a 5 ns molecular dynamics simulation. It was found that the time correlation functions of the dominant eigenmodes decay in good approximation with a single correlation time. From the reorientational eigenmodes, their eigenvalues, and correlation times, NMR relaxation data were calculated in accordance with Bloch-Wangsness-Redfield relaxation theory and directly compared with experimental 15N relaxation parameters. Using a fitting procedure, agreement between calculated and experimental data was improved significantly by adjusting eigenvalues and correlation times of the dominant modes. The presented procedure provides detailed information on correlated reorientational dynamics of flexible parts in globular proteins. The covariance matrix was linked to the covariance matrix of backbone dihedral angle fluctuations, allowing one to study the motional behavior of these degrees of freedom on nano- and subnanosecond time scales

Dynamic and structural analysis of isotropically distributed molecular ensembles

An efficient new method is presented for the characterization of motional correlations derived from a set of protein structures without requiring the separation of overall and internal motion. In this method, termed isotropically distributed ensemble (IDE) analysis, each structure is represented by an ensemble of isotropically distributed replicas corresponding to the situation found in an isotropic protein solution. This leads to a covariance matrix of the cartesian atomic positions with elements proportional to the ensemble average of scalar products of the position vectors with respect to the center of mass. Diagonalization of the covariance matrix yields eigenmodes and amplitudes that describe concerted motions of atoms, including overall rotational and intramolecular dynamics. It is demonstrated that this covariance matrix naturally distinguishes between "rigid" and "mobile" parts without necessitating a priori selection of a reference structure and an atom set for the orientational alignment process. The method was applied to the analysis of a 5-ns molecular dynamics trajectory of native ubiquitin and a 40-ns trajectory of a partially folded state of ubiquitin. The results were compared with essential dynamics analysis. By taking advantage of the spherical symmetry of the IDE covariance matrix, more than a 10-fold speed up is achieved for the computation of eigenmodes and mode amplitudes. IDE analysis is particularly suitable for studying the correlated dynamics of flexible and large molecules