M.-C. Beauvieux

Ethanol perfusion increases the yield of oxidative phosphorylation in isolated liver of fed rats.

The question arises as to the effect of ethanol on the actual yield of oxidative phosphorylation in the whole liver because of contradictory results reported in isolated hepatic mitochondria. The adenosine triphosphate (ATP) content of liver isolated from fed rats and perfused in the presence (10 mM) and absence of ethanol was continuously evaluated using 31P Nuclear Magnetic Resonance (NMR). An accurate estimation of mitochondrial ATP synthesis in the whole organ was obtained by subtracting the glycolytic ATP supply from the total ATP production. Simultaneously, the respiratory activity was assessed using O(2) Clark electrodes. The data indicate that ethanol enhanced the net consumption of ATP, leading to a new steady state of the ATP content. ATP synthesis was also found higher under ethanol [1.86+/-0.02 micromol/min g wet weight (min g ww)] than in control [1.44+/-0.18 micromol/min g ww]. However, mitochondrial respiration remained unchanged [2.20+/-0.13 micromol/min g ww] and, consequently, the in situ mitochondrial ATP/O ratio increased from 0.33+/-0.035 (control) to 0.42+/-0.015 (ethanol). The increase of the oxidative phosphorylation yield in the whole liver may be linked to the decrease in cytochrome oxidase activity induced by ethanol [FEBS Lett. 468 (2000) 239]. The significant raise (27%) of the ATP/O ratio was not sufficient to maintain the ATP level following ethanol-increased ATP consumption.

Insulin induces a positive relationship between the rates of ATP and glycogen changes in isolated rat liver in presence of glucose; a 31P and 13C NMR study.

BackgroundThere is an emerging theory suggesting that insulin, which is known to be the predominant postprandial anabolic hormone, is also a major regulator of mitochondrial oxidative phosphorylation in human skeletal muscle. However, little is known about its effects in the liver. Since there is a theoretical relationship between glycogen metabolism and energy status, a simultaneous and continuous investigation of hepatic ATP and glycogen content was performed in intact and isolated perfused liver by 31P and 13C nuclear magnetic resonance (NMR) The hepatic rates of ATP and glycogen changes were evaluated with different concentrations of insulin and glucose during continuous and short-term supply.ResultsLiver from rats fed ad libitum were perfused with Krebs-Henseleit Buffer (KHB)(controls) or KHB containing 6 mM glucose, 30 mM glucose, insulin alone, insulin + 6 mM glucose, insulin + 30 mM glucose. In the control, glycogenolysis occurred at a rate of -0.53 ± 0.021 %·min-1 and ATP content decreased at a rate of -0.28 ± 0.029 %·min-1. In the absence of insulin, there was a close proportional relationship between the glycogen flux and the glucose concentration, whereas ATP rates never varied. With insulin + glucose, both glycogen and ATP rates were strongly related to the glucose concentration; the magnitude of net glycogen flux was linearly correlated to the magnitude of net ATP flux: fluxglycogen = 72.543(fluxATP) + 172.08, R2 = 0.98.ConclusionOnly the co-infusion of 30 mM glucose and insulin led to (i) a net glycogen synthesis, (ii) the maintenance of the hepatic ATP content, and a strong positive correlation between their net fluxes. This has never previously been reported. The specific effect of insulin on ATP change is likely related to a rapid stimulation of the hepatic mitochondrial oxidative phosphorylation. We propose that variations in the correlation between rates of ATP and glycogen changes could be a probe for insulin resistance due to the action of substrates, drugs or pathologic situations. Consequently, any work evaluating insulin resistance on isolated organs or in vivo should determine both ATP and glycogen fluxes.

Rapid Adaptation of Rat Brain and Liver Metabolism to a Ketogenic Diet: An Integrated Study Using 1H- and 13C-NMR Spectroscopy:

The ketogenic diet (KD) is an effective alternative treatment for refractory epilepsy in children, but the mechanisms by which it reduces seizures are poorly understood. To investigate how the KD modifies brain metabolism, we infused control (CT) and 7-day KD rats with either [1-13C]glucose (Glc) or [2,4-13C2]β-hydroxybutyrate (β-HB). Specific enrichments of amino acids (AAs) measured by 1H- and 13C-NMR in total brain perchloric acid extracts were similar between CT and KD rats after [1-13C]Glc infusion whereas they were higher in KD rats after [2,4-13C2]β-HB infusion. This suggests better metabolic efficiency of ketone body utilization on the KD. The relative rapid metabolic adaptation to the KD included (1) 11%-higher brain γ-amino butyric acid (GABA)/glutamate (Glu) ratio versus CT, (2) liver accumulation of the ketogenic branched-chain AAs (BCAAs) leucine (Leu) and isoleucine (ILeu), which were never detected in CT, and (3) higher brain Leu and ILeu contents. Since Glu and GABA are excitatory and inhibitory neurotransmitters, respectively, higher brain GABA/Glu ratio could contribute to the mechanism by which the KD reduces seizures in epilepsy. Increased BCAA on the KD may also contribute to better seizure control.

Determination of myoglobin: comparative evaluation of the new automated VIDASR assay with two other immunoassays

OBJECTIVES Myoglobin provides the earliest indication of acute myocardial infarction. In this study, the new myoglobin assay for the VIDAS system (bioMérieux) was evaluated. DESIGN AND METHODS This assay, using an enzyme-linked fluorescent immunoassay (ELFIA) method, was compared with the Olympus immunoturbidimetric method and with another immunometric method (Immulite turbo) using an enzyme-linked chemiluminescent immunoassay (CLIA). RESULTS The CVs for within-and between-run reproducibility are very similar for the tested methods and acceptable linearity ranges were obtained. No significant interference of hemolysis, turbidity and icteria was observed. In the whole cohort, we obtained decreased values over the entire range of the assay with VIDAS and Immulite turbo methods compared to the Olympus assay; this is probably mainly linked to differences in standards used due to the absence of international standardization of the myoglobin determination. CONCLUSIONS The new VIDAS myoglobin automated assay provides biologists with a rapid, accurate and reliable determination of myoglobin in plasma samples collected during cardiac workup.

Early decrease in resting energy expenditure with bedtime insulin therapy

AIMS In type 2 diabetes (T2D), insulin-induced weight gain may stem from a reduction in resting energy expenditure (REE). We sought to determine the early effects of insulin introduction on REE in 20 poorly controlled T2D patients. METHODS After improving the glycaemia, REE was measured on Day 0 and Day 4 during two treatment regimens: bedtime insulin (n=10, group 1); and one off (3-day) intravenous insulin infusion (n=10, group 2). RESULTS Both groups were similar in age, gender, BMI, C-peptide, HbA(1c) and initial REE. By Day 4, fasting glycaemia had similarly improved in both groups: group 1: -5.3+/-2.7mmol/L vs group 2: -5.8+/-4.2 mmol/L. In group 2, the second REE was measured 12h after stopping the intravenous insulin infusion, whereas subcutaneous insulin was maintained in group 1. REE did not change in group 2 (-1.3+/-6.5%), whereas it decreased significantly in group 1 (-8.0+/-7.0%; P<0.05). CONCLUSION Bedtime insulin led to an early and specific reduction in REE.

Progression-related bias in the monitoring of kidney function in patients with diabetes and chronic kidney disease.

The Cockcroft and Gault (CG) and Modification of Diet in Renal Disease (MDRD) equations underestimate the glomerular filtration rate (GFR) decline in diabetes. Do this decline and the albumin excretion rate (AER) influence their validity? In 161 diabetic patients, isotopically determined GFR (i-GFR) (51Cr-EDTA) was compared with estimated GFR (e-GFR) by the CG, MDRD, and the new Mayo Clinic Quadratic (MCQ) equations. We searched for a relation between the error in e-GFR and the AER. An influence of the AER outcome on the e-GFR decline was evaluated in 63 subjects followed up over 3 years. The MDRD and the MCQ were more precise and accurate than the CG, but they were biased. The error increased with AER for the CG (r = 0.25, P = .001) and the MDRD (r = 0.20, P = .009), but not for the MCQ. For the 63 patients followed up, the e-GFR declines by the 3 estimations were related to the initial AER, whereas no relation with arterial blood pressure, hemoglobin A(1C), hemoglobin, and blood lipids emerged. The MCQ declines were more pronounced: -10.5% +/- 8.9% in the macroalbuminuric group (P < .05 vs both microalbuminuric [-2.6% +/- 10.1%] and normoalbuminuric [-0.1% +/- 6.6%] groups), and were related to the outcome of the AER (r = 0.33, P < .05). As chronic kidney disease progresses in diabetes, the declining GFR and rising AER influence the estimation of GFR by the CG and MDRD equations, underestimating the GFR decline and the benefit of reducing the AER. The less affected MCQ evidences a slower e-GFR decline with AER control.

Some processes of energy saving and expenditure occurring during ethanol perfusion in the isolated liver of fed rats; a Nuclear Magnetic Resonance study.

BackgroundIn the isolated liver of fed rats, a 10 mM ethanol perfusion rapidly induced a rapid 25% decrease in the total ATP content, the new steady state resulting from both synthesis and consumption. The in situ rate of mitochondrial ATP synthesis without activation of the respiration was increased by 27%, implying an increased energy demand. An attempt to identify the ethanol-induced ATP-consuming pathways was performed using 31P and 13C Nuclear Magnetic Resonance.ResultsEthanol (i) transiently increased sn-glycerol-3-phosphate formation whereas glycogenolysis was continuously maintained; (ii) decreased the glycolytic ATP supply and (iii) diminished the intracellular pH in a dose-dependent manner in a slight extend. Although the cytosolic oxidation of ethanol largely generated H+ (and NADH), intracellular pHi was maintained by (i) the large and passive excretion of cellular acetic acid arising from ethanol oxidation (evidenced by exogenous acetate administration), without energetic cost or (ii) proton extrusion via the Na+-HCO3- symport (implying the indirect activation of the Na+-K+-ATPase pump and thus an energy use), demonstrated during the addition of their specific inhibitors SITS and ouabaïn, respectively.ConclusionVarious cellular mechanisms diminish the cytosolic concentration of H+ and NADH produced by ethanol oxidation, such as (i) the large but transient contribution of the dihydroxyacetone phosphate / sn- glycerol-3-phosphate shuttle between cytosol and mitochondria, mainly implicated in the redox state and (ii) the major participation of acetic acid in passive proton extrusion out of the cell. These processes are not ATP-consuming and the latter is a cellular way to save some energy. Their starting in conjunction with the increase in mitochondrial ATP synthesis in ethanol-perfused whole liver was however insufficient to alleviate either the inhibition of glycolytic ATP synthesis and/or the implication of Na+-HCO3- symport and Na+-K+-ATPase in the pHi homeostasis, energy-consuming carriers.

Glucose flux in controlled hyperglycaemia before and after oral glucose ingestion in men with mild type 2 diabetes.

AIMS This study aimed to determine how insufficiently suppressed endogenous glucose production vs. reduced peripheral glucose uptake contribute to postprandial hyperglycaemia in type 2 diabetes (T2D). METHODS Eight men with T2D (age: 52+/-7 years; BMI: 26.6+/-2.3 kg/m(2); fasting glycaemia: 7.1+/-1.5 mmol/L) were compared with eight non-diabetic controls (age: 51+/-5 years; BMI: 24.6+/-2.9 kg/m(2); fasting glycaemia: 4.9+/-0.4 mmol/L). Their glucose turnover rates and hepatic glucose cycles were measured by monitoring [2H7]glucose infusion, with m+7 and m+6 enrichment, 3 h before and 4 h after the ingestion of [6,6-2H2]-labelled glucose, while maintaining glycaemia at 10 mmol/L using the pancreatic clamp technique. RESULTS Of the 700 mg/kg oral glucose load, 71% appeared in the systemic circulation of the T2D patients vs. 63% in the controls (NS). Endogenous glucose production and hepatic glucose cycles did not differ from normal either before or after oral glucose ingestion, while peripheral glucose uptake was reduced by 40% in the T2D group both before (P<0.01) and after (P<0.05) ingestion of oral glucose. CONCLUSION When T2D patients were compared with non-diabetic subjects with similarly controlled levels of hyperglycaemia after oral glucose ingestion, they essentially differed only in peripheral glucose uptake, whereas endogenous glucose production was apparently unaltered.

Moderate ethanol supply inhibits both glycogen synthesis and glycogenolysis in the perfused and isolated rat liver

In isolated and perfused rat liver a positive glucose-dependent linear correlation between the net fluxes (Fn) of ATP and glycogen (Glg) has been found only in presence of insulin (Ins; Fn(Glg)= 72.5Fn(ATP) + 172); this result indicates that Ins can control the Glg store via energy metabolism. Any change in this relationship in the presence of a substrate could then indicate a variation in insulin sensitivity. In the presence of a moderate ethanol (EtOH) supply (10 mM) the slope of the correlation is 4-fold higher (284.5Fn(ATP) + 2848), suggesting that for the same change in the consumption Fn of ATP Glg consumption is lower. Thus, the unidirectional hepatic fluxes (synthesis and lysis) of Glg were investigated in the presence of 10 mM-EtOH. Male Wistar rats (100 g) were fasted for 48 h in order to deplete the liver Glg store. Livers were then perfused with an isotonic buffer (5 ml/min per g; 37 C; O2–CO2, 95 : 5 (v/v)) containing 30 mM-glucose (enriched with 20% [1C]glucose) + Ins (120 mIU /l) + 2 mMfructose to induce Glg synthesis. In a second step [C]glucose was replaced by 30 mM-glucose to investigate glycogenolysis. EtOH (10 mM) was added either in the Glg synthesis or the glycogenolysis phase (n 3 for each dataset). The change in the Glg content was monitored by C NMR (Brucker DPX400, 9.4T; Brucker, Bremen, Germany); since ATP is consumed for Glg synthesis, its hepatic content was measured by P NMR. The 48 h fasting induced a dramatic decrease in liver Glg content (99%). In the Glg synthesis study perfusion with [C]glucose + fructose induced an increase in the liver [C]Glg content (synthesis rate 2.4 (SE 0.2)mmol/h per g)), followed after 25 5 min by a decreased rate (0.66 (SE 0.07)mmol/h per g; within 30 min). After the addition of EtOH at 25 min of the incorporation phase a plateau of [C]Glg content was observed, suggesting (a) inhibition of Glg synthesis or (b) an increase in glycogenolysis. In the glycogenolysis study replacement of [C]glucose with 30 mM-glucose resulted in a decrease in [C]Glg (0.69 (SE 0.08)mmol/h per g) indicating glycogenolysis. The subsequent addition of EtOH reduced glycogenolysis (0.09 (SE 0.01)mmol/h per g). A moderate EtOH supply in presence of Ins inhibits both hepatic Glg synthesis and glycogenolysis.

Prediction of mortality rate in type 2 diabetes: estimated glomerular filtration rate underestimates the true rate

To the Editor: In their paper published in Diabetologia [1], Bruno et al. assessed whether a reduction in estimated glomerular filtration rate (eGFR), calculated using the abbreviated Modification of Diet in Renal Disease (MDRD) study equation [2], predicted mortality in type 2 diabetes. Although an eGFR of <60 ml min 1.73 m was associated with a twofold increase in the mortality rate, further analyses using smaller eGFR categories (15–29, 30–44, 45–59 ml min 1.73 m) revealed that this was due to the increased risk in patients with eGFR values between 15 and 29 ml min 1.73 m, with hazard ratios even suggesting a benefit for the non-proteinuric, moderate renal failure strata. To investigate whether this unexpected finding was due to the inaccuracy of the abbreviated MDRD equation in estimating GFR, we compared eGFR values with GFR values determined by Cr-labelled EDTA clearance (isotopic GFR [iGFR]) in a group of volunteers, stratifying the results as per Bruno et al. [1]. In total, 128 patients with type 2 diabetes (age 67±9 years, BMI 28.8±4.8, HbA1c 8.5±1.6% [data presented as means ± SD], 53 women) gave informed consent to participate in this study, which was conducted in accordance with the Declaration of Helsinki. In the group as a whole, iGFR was 54.5±32.7 ml min 1.73 m. Although the eGFR (48.2±18.8 ml min 1.73 m) showed a strong correlation with i-GFR (r=0.80, p<0.001), it underestimated it (p<0.001). The comparisons of eGFR and iGFR for each eGFR stratum as defined by Bruno et al. are shown in Table 1. In the group as a whole, most of the subjects (55.5%) were wrongly classified by the MDRD-estimated GFR in the four GFR strata. This suggests that the majority of patients followed by Bruno et al. would have been classified in other strata if measured GFR rather than eGFR values had been used. In particular, many patients in the 45–60 and 60–89 ml min 1.73 m eGFR intervals, who had hazard ratios of <1.00 for all-cause and cardiovascular mortality in the paper [1], would have been in higher GFR strata. Although it is the best predictor of GFR in diabetic patients with renal insufficiency [3], the MDRD equation is well known to underestimate GFR values at the upper end of the normal range [4]. This explains the high proportion of patients with chronic kidney disease in the population Diabetologia (2007) 50:2410–2411 DOI 10.1007/s00125-007-0796-8