Fátima O. Martins

Restoration of direct pathway glycogen synthesis flux in the STZ-diabetes rat model by insulin administration.

Type 1 diabetes subjects are characterized by impaired direct pathway synthesis of hepatic glycogen that is unresponsive to insulin therapy. Since it is not known whether this is an irreversible defect of insulin-dependent diabetes, direct and indirect pathway glycogen fluxes were quantified in streptozotocin (STZ)-induced diabetic rats and compared with STZ rats that received subcutaneous or intraperitoneal insulin (I-SC or I-IP). Three groups of STZ rats were studied at 18 days post-STZ treatment. One group was administered I-SC and another I-IP as two daily injections of short-acting insulin at the start of each light and dark period for days 9-18. A third group did not receive any insulin, and a fourth group of nondiabetic rats was used as control. Glycogen synthesis via direct and indirect pathways, de novo lipogenesis, and gluconeogenesis were determined over the nocturnal feeding period using deuterated water. Direct pathway was residual in STZ rats, and glucokinase activity was also reduced significantly from control levels. Insulin administration restored both net glycogen synthesis via the direct pathway and glucokinase activity to nondiabetic control levels and improved the lipogenic pathway despite an inefficient normalization of the gluconeogenic pathway. We conclude that the reduced direct pathway flux is not an irreversible defect of insulin-dependent diabetes.

2H enrichment distribution of hepatic glycogen from 2H2O reveals the contribution of dietary fructose to glycogen synthesis

Dietary fructose can benefit or hinder glycemic control, depending on the quantity consumed, and these contrasting effects are reflected by alterations in postprandial hepatic glycogen synthesis. Recently, we showed that ²H enrichment of glycogen positions 5 and 2 from deuterated water (²H₂O) informs direct and indirect pathway contributions to glycogenesis in naturally feeding rats. Inclusion of position 6(S) ²H enrichment data allows indirect pathway sources to be further resolved into triose phosphate and Krebs cycle precursors. This analysis was applied to six rats that had fed on standard chow (SC) and six rats that had fed on SC plus 35% sucrose in their drinking water (HS). After 2 wk, hepatic glycogenesis sources during overnight feeding were determined by ²H₂O administration and postmortem analysis of glycogen ²H enrichment at the conclusion of the dark period. Net overnight hepatic glycogenesis was similar between SC and HS rodents. Whereas direct pathway contributions were similar (403 ± 71 μmol/g dry wt HS vs. 578 ± 76 μmol/g dry wt SC), triose phosphate contributions were significantly higher for HS compared with SC (382 ± 61 vs. 87 ± 24 μmol/g dry wt, P < 0.01) and Krebs cycle inputs lower for HS compared with SC (110 ± 9 vs. 197 ± 32 μmol/g dry wt, P < 0.05). Analysis of plasma glucose ²H enrichments at the end of the feeding period also revealed a significantly higher fractional contribution of triose phosphate to plasma glucose levels in HS vs. SC. Hence, the ²H enrichment distributions of hepatic glycogen and glucose from ²H₂O inform the contribution of dietary fructose to hepatic glycogen and glucose synthesis.

Gender-dependent metabolic remodeling during heart preservation in cardioplegic celsior and histidine buffer solution.

Abstract: Understanding heart metabolism during preservation is crucial to develop new effective cardioplegic solutions. We aim to investigate metabolic alterations during heart preservation in the clinically used Celsior (Cs) and histidine buffer solution (HBS). We also focused in gender-specific metabolic adaptations during ischemia. We followed energy metabolism in hearts from males and females preserved during 6 hours in Cs and HBS. Hearts were subjected to cold ischemia (4°C) in Cs or HBS, and aliquots of the cardioplegic solution were collected throughout preservation for nuclear magnetic resonance analysis. HBS-preserved hearts from males consumed glucose mostly between 240 and 360 minutes, whereas HBS-preserved hearts from females consumed glucose throughout the 6 hours of ischemia. Lactate production rates followed approximately the glucose consumption rates in HBS-preserved hearts. The lactate to alanine ratio, an indicator of the redox state, was increased in HBS-preserved hearts when compared with Cs-preserved hearts. Hearts from males presented a higher redox state than those from females preserved in Cs after 300 minutes. Both Cs and HBS were capable of preventing acidification in hearts from females but not in hearts from males, which decreased the extracellular pH. HBS-preserved hearts from males and females produced 0.1 ± 0.01 and 0.15 ± 0.03 &mgr;mol·min−1·gdw−1 of lactate, respectively. Those rates were significantly higher than in Cs-preserved hearts. Thus, Cs was more effective in preventing lactate production. We conclude that glycolysis and lactate production are stimulated in HBS-preserved hearts. HBS shows better overall results particularly in hearts from females, which presented less extracellular acidification and were more effective in recycling the metabolic subproducts.Understanding heart metabolism during preservation is crucial to develop new effective cardioplegic solutions. We aim to investigate metabolic alterations during heart preservation in the clinically used Celsior (Cs) and histidine buffer solution (HBS). We also focused in gender-specific metabolic adaptations during ischemia. We followed energy metabolism in hearts from males and females preserved during 6 hours in Cs and HBS. Hearts were subjected to cold ischemia (4°C) in Cs or HBS, and aliquots of the cardioplegic solution were collected throughout preservation for nuclear magnetic resonance analysis. HBS-preserved hearts from males consumed glucose mostly between 240 and 360 minutes, whereas HBS-preserved hearts from females consumed glucose throughout the 6 hours of ischemia. Lactate production rates followed approximately the glucose consumption rates in HBS-preserved hearts. The lactate to alanine ratio, an indicator of the redox state, was increased in HBS-preserved hearts when compared with Cs-preserved hearts. Hearts from males presented a higher redox state than those from females preserved in Cs after 300 minutes. Both Cs and HBS were capable of preventing acidification in hearts from females but not in hearts from males, which decreased the extracellular pH. HBS-preserved hearts from males and females produced 0.1 ± 0.01 and 0.15 ± 0.03 μmol·min·gdw of lactate, respectively. Those rates were significantly higher than in Cs-preserved hearts. Thus, Cs was more effective in preventing lactate production. We conclude that glycolysis and lactate production are stimulated in HBS-preserved hearts. HBS shows better overall results particularly in hearts from females, which presented less extracellular acidification and were more effective in recycling the metabolic subproducts.

Disposition of [U-2H7]glucose into hepatic glycogen in rat and in seabass

The stimulation of hepatic glycogenesis is a ubiquitous response to a glucose challenge and quantifying its contribution to glucose uptake informs its role in restoring euglycemia. Glycogenesis can be quantified with labeled water provided that exchange of glucose-6-phosphate hydrogen 2 (G6P-H2) and body water via glucose-6-phosphate isomerase, and exchange of positions 4, 5 and 6 hydrogens (G6P-H456) via transaldolase, are known. These exchanges were quantified in 24-h fasted rats (Rattus norvegicus; n=6) and 21-day fasted seabass (Dicentrarchus labrax; n=8) by administration of a glucose load (2000mg·kg(-1)) enriched with [U-(2)H7]glucose and by quantifying hepatic glycogen (2)H-enrichments after 2h (rats) and 48h (seabass). Direct pathway contributions of the glucose load to glycogenesis were also estimated. G6P-H2 and body water exchange was 61±1% for rat and 47±3% for seabass. Transaldolase-mediated exchange of G6P-H456 was 5±1% for rat and 10±1% for seabass. Conversion of the glucose load to hepatic glycogen was significant in seabass (249±54mg·kg(-1)) but negligible in rats (12±1mg·kg(-1)). Preload plasma glucose levels were similar for seabass and rats (3.3±0.7 and 4.4±0.1mmol·L(-1), respectively) but post-load plasma glucose was significantly higher in seabass compared to rats (14.6±1.8 versus 5.8±0.3mmol·L(-1), p<0.01). In conclusion, G6P-H2 and body water exchange is incomplete for both species and has to be accounted for in estimating hepatic glycogen synthesis and direct pathway activities with labeled water tracers. Transaldolase-mediated exchange is insignificant. Hepatic direct pathway glycogenesis plays a prominent role in seabass glucose load disposal, but a negligible role in the rat.

2H2O incorporation into hepatic acetyl-CoA and de novo lipogenesis as measured by Krebs cycle-mediated 2H-enrichment of glutamate and glutamine

Deuterated water is widely used for measuring de novo lipogenesis on the basis of quantifying lipid 2H‐enrichment relative to that of body water. However, incorporation of 2H‐enrichment from body water into newly synthesized lipid molecules is incomplete therefore the true lipid precursor enrichment differs from that of body water. We describe a novel measurement of de novo lipogenesis that is based on a true precursor‐product analysis of hepatic acetyl‐CoA and triglyceride methyl enrichments from deuterated water. After deuterated water administration to seven in situ and seven perfused livers, acetyl‐CoA methyl enrichment was inferred from 2H nuclear magnetic resonance analysis of hepatic glutamate/glutamine (Glx) enrichment and triglyceride methyl enrichment was directly determined by 2H nuclear magnetic resonance of triglycerides. Acetyl‐CoA 2H‐enrichment was 71% ± 1% that of body water for in situ livers and 53% ± 2% of perfusate water for perfused livers. From the ratio of triglyceride‐methyl/acetyl‐CoA enrichments, fractional de novo lipogenesis rates of 0.97% ± 0.09%/2 hr and 7.92% ± 1.47%/48 hr were obtained for perfused and in situ liver triglycerides, respectively. Our method reveals that acetyl‐CoA enrichment is significantly less than body water both for in situ and perfused livers. Furthermore, the difference between acetyl‐CoA and body water enrichments is sensitive to the experimental setting. Magn Reson Med, 2011.

Mechanisms by which the thiazolidinedione troglitazone protects against sucrose-induced hepatic fat accumulation and hyperinsulinaemia.

Thiazolidinediones (TZD) are known to ameliorate fatty liver in type 2 diabetes. To date, the underlying mechanisms of their hepatic actions remain unclear.

Direct analysis of [6,6-(2)H2]glucose and [U-(13)C6]glucose dry blood spot enrichments by LC-MS/MS.

A liquid chromatography tandem mass spectrometry (LC-MS/MS) using multiple reaction monitoring (MRM) in a triple-quadrupole scan mode was developed and comprehensively validated for the determination of [6,6-(2)H2]glucose and [U-(13)C6]glucose enrichments from dried blood spots (DBS) without prior derivatization. The method is demonstrated with dried blood spots obtained from rats administered with a primed-constant infusion of [U-(13)C6]glucose and an oral glucose load enriched with [6,6-(2)H2]glucose. The sensitivity is sufficient for analysis of the equivalent to <5μL of blood and the overall method was accurate and precise for the determination of DBS isotopic enrichments.

Intestinal Microbial and Metabolite Profiling of Mice Fed with Dietary Glucose and Fructose

1 CNC – Center for Neuroscience and Cell Biology, University of Coimbra, Portugal; 7 john.griffith.jones@gmail.com 8 2 Institute of Microbiology, Faculty of Medicine, University of Coimbra, Portugal 9 3 CEDOC, NOVA Medical School, Universidade NOVA de Lisboa, Rua Camara Pestana, no6, 6A, edifício II, 10 piso 3, 1150-082 Lisbon, Portugal 11 4 CICECO-Department of Chemistry, University of Aveiro, Campus de Santiago, 3810-193 Aveiro, Portugal; 12 e-mail: agil@ua.pt 13 5 UCIBIO@REQUIMTE/Toxicological Laboratory, Biological Science Department, Faculty of Pharmacy, 14 Univeristy of Porto, 4050-313 Porto, Portugal 15 6 Department of Cardiothoracic Surgery and Physiology, Faculty of Medicine, Porto 4200-319, Portugal 16 7 APDP – Portuguese Diabetes Association, Lisbon, Portugal 17 18 † these authors contributed equally to this paper. 19 * Correspondence: john.griffith.jones@gmail.com; Tel.: +351231249181; agil@ua.pt, tel:+351234370707 20 21 22 Abstract: Increased sugar intake is implicated in Type-2 diabetes and fatty liver disease. 23 Mechanisms by which glucose and fructose components promote these conditions are unclear. We 24 hypothesize that alterations in intestinal metabolite and microbiota profiles specific to each 25 monosaccharide are involved. Two groups of six adult C57BL/6 mice were fed for 10-weeks with a 26 diet where either glucose or fructose was the sole carbohydrate component (G and F, respectively). 27 A third group was fed with normal chow (N). Fecal metabolites were profiled every 2-weeks by 1H 28 NMR and microbial composition was analysed by real-time PCR (qPCR). Glucose tolerance was also 29 periodically assessed. N, G and F mice had similar weight gains and glucose tolerance. Multivariate 30 analysis of NMR profiles indicated that F mice were separated from both N and G, with decreased 31 butyrate and glutamate and increased fructose, succinate, taurine, tyrosine and xylose. Compared to 32 N and G, F mice showed a shift in microbe populations from gram-positive Lactobacillus spp. to 33 gram-negative Enterobacteria species. Substitution of normal chow carbohydrate mixture by either 34 pure glucose or fructose for 10 weeks did not alter adiposity or glucose tolerance. However, F G and 35 N mice generated distinctive fecal metabolite signatures with incomplete fructose absorption as a 36 dominant feature of F mice. 37

Sources of hepatic glycogen synthesis in mice fed with glucose or fructose as the sole dietary carbohydrate

The positional analysis of hepatic glycogen enrichment from deuterated water (2H2O) by 2H NMR has been applied previously to resolve the contributions of glucose and fructose to glycogen synthesis in rodents fed a high sucrose diet. To further validate this method, this analysis was applied to mice fed with synthetic diets whose carbohydrate components consisted solely of either glucose or fructose.

Intestinal Microbial and Metabolic Profiling of Mice Fed with High-Glucose and High-Fructose Diets

Increased sugar intake is implicated in Type-2 diabetes and fatty liver disease; however, the mechanisms through which glucose and fructose promote these conditions are unclear. We hypothesize that alterations in intestinal metabolite and microbiota profiles specific to each monosaccharide are involved. Two groups of six adult C57BL/6 mice were fed for 10-weeks with diets with glucose (G) or fructose (F) as sole carbohydrates, and a third group was fed with a normal chow carbohydrate mixture (N). Fecal metabolites were profiled by nuclear magnetic resonance (NMR) and microbial composition by real-time polymerase chain reaction (qPCR). Although N, G and F mice exhibited similar weight gains (with slight slower gains for F) and glucose tolerance, multivariate analysis of NMR data indicated that F mice were separated from N and G, with decreased butyrate and glutamate and increased fructose, succinate, taurine, tyrosine, and xylose. The different sugar diets also resulted in distinct intestinal microbiota profiles. That associated with fructose seemed to hold more potential to induce host metabolic disturbances compared to glucose, mainly by promoting bile acid deconjugation and taurine release and compromising intestinal barrier integrity. This may reflect the noted nonquantitative intestinal fructose absorption hence increasing its availability for microbial metabolism, a subject for further investigation.