Shaji K. Chacko

Methionine transmethylation and transsulfuration in the piglet gastrointestinal tract

Methionine is an indispensable sulfur amino acid that functions as a key precursor for the synthesis of homocysteine and cysteine. Studies in adult humans suggest that splanchnic tissues convert dietary methionine to homocysteine and cysteine by means of transmethylation and transsulfuration, respectively. Studies in piglets show that significant metabolism of dietary indispensable amino acids occurs in the gastrointestinal tissues (GIT), yet the metabolic fate of methionine in GIT is unknown. We show here that 20% of the dietary methionine intake is metabolized by the GIT in piglets implanted with portal and arterial catheters and fed milk formula. Based on analyses from intraduodenal and intravenous infusions of [1-13C]methionine and [2H3]methionine, we found that the whole-body methionine transmethylation and remethylation rates were significantly higher during duodenal than intravenous tracer infusion. First-pass splanchnic metabolism accounted for 18% and 43% of the whole-body transmethylation and remethylation, respectively. Significant transmethylation and transsulfuration was demonstrated in the GIT, representing ≈27% and ≈23% of whole-body fluxes, respectively. The methionine used by the GIT was metabolized into homocysteine (31%), CO2 (40%), or tissue protein (29%). Cystathionine β-synthase mRNA and activity was present in multiple GITs, including intestinal epithelial cells, but was significantly lower than liver. We conclude that the GIT consumes 20% of the dietary methionine and is a significant site of net homocysteine production. Moreover, the GITs represent a significant site of whole-body transmethylation and transsulfuration, and these two pathways account for a majority of methionine used by the GITs.

Sulfur amino acid deficiency upregulates intestinal methionine cycle activity and suppresses epithelial growth in neonatal pigs

We recently showed that the developing gut is a significant site of methionine transmethylation to homocysteine and transsulfuration to cysteine. We hypothesized that sulfur amino acid (SAA) deficiency would preferentially reduce mucosal growth and antioxidant function in neonatal pigs. Neonatal pigs were enterally fed a control or an SAA-free diet for 7 days, and then whole body methionine and cysteine kinetics were measured using an intravenous infusion of [1-(13)C;methyl-(2)H(3)]methionine and [(15)N]cysteine. Body weight gain and plasma methionine, cysteine, homocysteine, and taurine and total erythrocyte glutathione concentrations were markedly decreased (-46% to -85%) in SAA-free compared with control pigs. Whole body methionine and cysteine fluxes were reduced, yet methionine utilization for protein synthesis and methionine remethylation were relatively preserved at the expense of methionine transsulfuration, in response to SAA deficiency. Intestinal tissue concentrations of methionine and cysteine were markedly reduced and hepatic levels were maintained in SAA-free compared with control pigs. SAA deficiency increased the activity of methionine metabolic enzymes, i.e., methionine adenosyltransferase, methionine synthase, and cystathionine beta-synthase, and S-adenosylmethionine concentration in the jejunum, whereas methionine synthase activity increased and S-adenosylmethionine level decreased in the liver. Small intestine weight and protein and DNA mass were lower, whereas liver weight and DNA mass were unchanged, in SAA-free compared with control pigs. Dietary SAA deficiency induced small intestinal villus atrophy, lower goblet cell numbers, and Ki-67-positive proliferative crypt cells in association with lower tissue glutathione, especially in the jejunum. We conclude that SAA deficiency upregulates intestinal methionine cycle activity and suppresses epithelial growth in neonatal pigs.

New generation lipid emulsions prevent PNALD in chronic parenterally fed preterm pigs

Total parenteral nutrition (TPN) is associated with the development of parenteral nutrition-associated liver disease (PNALD) in infants. Fish oil-based lipid emulsions can reverse PNALD, yet it is unknown if they can prevent PNALD. We studied preterm pigs administered TPN for 14 days with either 100% soybean oil (IL), 100% fish oil (OV), or a mixture of soybean oil, medium chain triglycerides (MCTs), olive oil, and fish oil (SL); a group was fed formula enterally (ENT). In TPN-fed pigs, serum direct bilirubin, gamma glutamyl transferase (GGT), and plasma bile acids increased after the 14 day treatment but were highest in IL pigs. All TPN pigs had suppressed hepatic expression of farnesoid X receptor (FXR), cholesterol 7-hydroxylase (CYP7A1), and plasma 7α-hydroxy-4-cholesten-3-one (C4) concentrations, yet hepatic CYP7A1 protein abundance was increased only in the IL versus ENT group. Organic solute transporter alpha (OSTα) gene expression was the highest in the IL group and paralleled plasma bile acid levels. In cultured hepatocytes, bile acid-induced bile salt export pump (BSEP) expression was inhibited by phytosterol treatment. We show that TPN-fed pigs given soybean oil developed cholestasis and steatosis that was prevented with both OV and SL emulsions. Due to the presence of phytosterols in the SL emulsion, the differences in cholestasis and liver injury among lipid emulsion groups in vivo were weakly correlated with plasma and hepatic phytosterol content.

Gluconeogenesis is Not Regulated by Either Glucose or Insulin in Extremely Low Birth Weight Infants Receiving Total Parenteral Nutrition

OBJECTIVE To determine potential factors regulating gluconeogenesis (GNG) in extremely low birth weight infants receiving total parenteral nutrition. STUDY DESIGN Seven infants (birth weight, 0.824 ± 0.068 kg; gestational age, 25.4 ± 0.5 weeks; postnatal age, 3.3 ± 0.2 days) were studied for 11 hours, with parenteral lipid and amino acid therapy continued at prestudy rates. Glucose was supplied at prestudy rates for the first 5 hours (period 1) and was then reduced to 6 mg/kg·min for 1 hour and further to ~3 mg/kg·min for 5 hours (period 2). A total of 2.5 mg/kg·min of the glucose was replaced by [U-(13)C]glucose throughout the study for measurements of glucose production and GNG. Concentrations of glucose, insulin, glucagons, and cortisol were determined. RESULTS GNG and glucose production remained unchanged (2.12 ± 0.23 vs. 1.84 ± 0.25 mg/kg·min [P = NS] and 2.44 ± 0.27 vs. 2.51 ± 0.31 mg/kg·min [P = NS], respectively), despite a 60% reduction of the glucose infusion rate and subsequent 30% (124.7 ± 10.8 to 82.6 ± 8.9 mg/dL; P = .009) and 70% (26.9 ± 4.7 to 6.6 ± 0.4 μU/mL; P = .002) decreases in glucose and insulin concentrations, respectively. Cortisol and glucagon concentrations remained unchanged. CONCLUSION In extremely low birth weight infants receiving total parenteral nutrition, GNG is a continuous process that is not affected by infusion rates of glucose or concentrations of glucose or insulin.

Sulforaphane reduces hepatic glucose production and improves glucose control in patients with type 2 diabetes

Sulforaphane, a natural compound identified by drug repurposing, reduces hepatic glucose production and improves glucose control in type 2 diabetes. Another reason to eat your broccoli Type 2 diabetes is becoming increasingly common worldwide, and not all patients can be successfully treated with the existing drugs. Axelsson et al. analyzed the pattern of gene expression associated with type 2 diabetes and compared it to the gene signatures for thousands of drug candidates to find compounds that could counteract the effects of diabetes. The leading candidate from this analysis was sulforaphane, a natural compound found in broccoli and other vegetables. The authors showed that sulforaphane inhibits glucose production in cultured cells and improves glucose tolerance in rodents on high-fat or high-fructose diets. Moreover, in a clinical trial, sulforaphane-containing broccoli sprout extract was well tolerated and improved fasting glucose in human patients with obesity and dysregulated type 2 diabetes. A potentially useful approach for drug discovery is to connect gene expression profiles of disease-affected tissues (“disease signatures”) to drug signatures, but it remains to be shown whether it can be used to identify clinically relevant treatment options. We analyzed coexpression networks and genetic data to identify a disease signature for type 2 diabetes in liver tissue. By interrogating a library of 3800 drug signatures, we identified sulforaphane as a compound that may reverse the disease signature. Sulforaphane suppressed glucose production from hepatic cells by nuclear translocation of nuclear factor erythroid 2–related factor 2 (NRF2) and decreased expression of key enzymes in gluconeogenesis. Moreover, sulforaphane reversed the disease signature in the livers from diabetic animals and attenuated exaggerated glucose production and glucose intolerance by a magnitude similar to that of metformin. Finally, sulforaphane, provided as concentrated broccoli sprout extract, reduced fasting blood glucose and glycated hemoglobin (HbA1c) in obese patients with dysregulated type 2 diabetes.

Gluconeogenesis continues in premature infants receiving total parenteral nutrition

Objective To determine the contribution of total gluconeogenesis to glucose production in preterm infants receiving total parenteral nutrition (TPN) providing glucose exceeding normal infant glucose turnover rates. Study Design Eight infants (0.955±0.066 kg, 26.5±0.5 weeks, 4±1 days) were studied while receiving routine TPN. The glucose appearance rate (the sum of rates of glucose infusion and residual glucose production) and gluconeogenesis were measured by stable isotope–gas chromatography–mass spectrometry techniques using deuterated water and applying the Chacko and Landau methods. Results Blood glucose ranged from 5.2 to 14.3 mmol/l (94–257 mg/dl) and the glucose infusion rate from 7.4 to 11.4 mg/kg per min, thus exceeding the normal glucose production rates (GPR) of newborn infants in most of the babies. The glucose appearance rate was 12.4±0.6 and GPR 2.1±0.3 mg/kg per min. Gluconeogenesis as a fraction of the glucose appearance rate was 11.2±1.1% (Chacko) and 10.5±1.2% (Landau) (NS) and the rate of gluconeogenesis was 1.35±0.15 mg/kg per min (Chacko) and 1.29±0.14 mg/kg per min (Landau) (NS). Gluconeogenesis accounted for 73±11% and 68±10 (NS) of the GPR for the two methods, respectively. Gluconeogenesis and glycogenolysis were not affected by the total glucose infusion rate, glucose concentration, gestational age or birth weight. Glucose concentration correlated with the total glucose infusion rate and gestational age (combined R2=0.79, p=0.02). Conclusions Gluconeogenesis is sustained in preterm infants receiving routine TPN providing glucose at rates exceeding normal infant glucose turnover rates and accounts for the major part of residual glucose production. Gluconeogenesis is not affected by the glucose infusion rate or blood glucose concentration.

Effect of ghrelin on glucose regulation in mice

Improvement of glucose metabolism after bariatric surgery appears to be from the composite effect of the alterations in multiple circulating gut hormone concentrations. However, their individual effect on glucose metabolism during different conditions is not clear. The objective of this study was to determine whether ghrelin has an impact on glycogenolysis, gluconeogenesis, and insulin sensitivity (using a mice model). Rate of appearance of glucose, glycogenolysis, and gluconeogenesis were measured in wild-type (WT), ghrelin knockout (ghrelin(-/-)), and growth hormone secretagogue receptor knockout (Ghsr(-/-)) mice in the postabsorptive state. The physiological nature of the fasting condition was ascertained by a short-term fast commenced immediately at the end of the dark cycle. Concentrations of glucose and insulin were measured, and insulin resistance and hepatic insulin sensitivity were calculated. Glucose concentrations were not different among the groups during the food-deprived period. However, plasma insulin concentrations were lower in the ghrelin(-/-) and Ghsr(-/-) than WT mice. The rates of gluconeogenesis, glycogenolysis, and indexes of insulin sensitivity were higher in the ghrelin(-/-) and Ghsr(-/-) than WT mice during the postabsorptive state. Insulin receptor substrate 1 and glucose transporter 2 gene expressions in hepatic tissues of the ghrelin(-/-) and Ghsr(-/-) were higher compared with that in WT mice. This study demonstrates that gluconeogenesis and glycogenolysis are increased and insulin sensitivity is improved by the ablation of the ghrelin or growth hormone secretagogue receptor in mice.

Gluconeogenesis during endurance exercise in cyclists habituated to a long‐term low carbohydrate high‐fat diet

Blood glucose is an important fuel for endurance exercise. It can be derived from ingested carbohydrate, stored liver glycogen and newly synthesized glucose (gluconeogenesis). We hypothesized that athletes habitually following a low carbohydrate high fat (LCHF) diet would have higher rates of gluconeogenesis during exercise compared to those who follow a mixed macronutrient diet. We used stable isotope tracers to study glucose production kinetics during a 2 h ride in cyclists habituated to either a LCHF or mixed macronutrient diet. The LCHF cyclists had lower rates of total glucose production and hepatic glycogenolysis but similar rates of gluconeogenesis compared to those on the mixed diet. The LCHF cyclists did not compensate for reduced dietary carbohydrate availability by increasing glucose synthesis during exercise but rather adapted by altering whole body substrate utilization.

Mechanisms to conserve glucose in lactating women during a 42-h fast

Little is known about how lactating women accommodate for their increased glucose demands during fasting to avoid maternal hypoglycemia. The objective of this study was to determine whether lactating women conserve plasma glucose by reducing maternal glucose utilization by increasing utilization of FFA and ketone bodies and/or increasing gluconeogenesis and mammary gland hexoneogenesis. Six healthy exclusively breastfeeding women and six nonlactating controls were studied during 42 h of fasting and 6 h of refeeding. Glucose and protein kinetic parameters were measured using stable isotopes and GCMS and energy expenditure and substrate oxidation using indirect calorimetry. After 42 h of fasting, milk production decreased by 16% but remained within normal range. Glucose, insulin, and C-peptide concentrations decreased with the duration of fasting in both groups but were lower (P < 0.05) in lactating women. Glucagon, FFA, and beta-hydroxybutyrate concentrations increased with fasting time (P < 0.001) and were higher (P < 0.0001) in lactating women during both fasting and refeeding. During 42 h of fasting, gluconeogenesis was higher in lactating women compared with nonlactating controls (7.7 +/- 0.4 vs. 6.5 +/- 0.2 micromol kg(-1) min(-1), P < 0.05), whereas glycogenolysis was suppressed to similar values (0.4 +/- 0.1 vs. 0.9 +/- 0.2 micromol kg(-1) min(-1), respectively). Mammary hexoneogenesis did not increase with the duration of fasting. Carbohydrate oxidation was lower and fat and protein oxidations higher (P < 0.05) in lactating women. In summary, lactating women are at risk for hypoglycemia if fasting is extended beyond 30 h. The extra glucose demands of extended fasting during lactation appear to be compensated by increasing gluconeogenesis associated with ketosis, decreasing carbohydrate oxidation, and increasing protein and FFA oxidations.

Reducing glucose infusion safely prevents hyperglycemia in post-surgical children.

BACKGROUND & AIMS To investigate the effects of two different glucose infusions on glucose homeostasis and amino acid metabolism in post-surgical children. METHODS This randomized crossover study evaluated glucose and amino acid metabolism in eight children (age 9.8 ± 1.9 months, weight 9.5 ± 1.1 kg) admitted to a pediatric intensive care unit in a tertiary university hospital after surgical correction for non-syndromal craniosynostosis. Patients were randomized to receive low (LG; 2.5 mg kg(-1) min(-1)) and standard (SG; 5.0 mg kg(-1) min(-1)) glucose infusion in a crossover setting. After a bolus (4 g kg(-1)) of deuterium oxide, we conducted a primed, constant, 8 h tracer infusion with [6,6-²H₂]Glucose, [1-¹³C]Leucine, [ring-²H₅]Phenylalanine and [3,3-²H₂]Tyrosine. RESULTS SG resulted in hyperglycemia (defined as > 6.1 mmol L(-1)), while during LG plasma glucose levels were normoglycemic (5.9 ± 0.6 vs. 7.5 ± 1.7 mmol L(-1); LG vs. SG respectively, p = 0.02). Hypoglycemia did not occur during LG infusion. Endogenous glucose production was not fully suppressed during the hyperglycemic state under SG and increased with reduced glucose infusion (2.6 ± 1.5 vs. 1.1 ± 1.4 mg kg(-1) min(-1); LG vs. SG; p = 0.05). Whole body protein balance derived from leucine and phenylalanine kinetics was slightly negative but not further affected with a decrease in glucose infusion. CONCLUSIONS The current recommended glucose infusion induces hyperglycemia in post-surgical children. A reduced glucose infusion safely reduced high glucose levels, while children were capable to sustain normoglycemia with increased endogenous glucose production. The reduced glucose infusion did not exacerbate the mild catabolic state in which the patients were.