Enrichment of hepatic glycogen and plasma glucose from H218 O informs gluconeogenic and indirect pathway fluxes in naturally feeding mice.

Deuterated water (2 H2 O) is a widely used tracer of carbohydrate biosynthesis in both preclinical and clinical settings, but the significant kinetic isotope effects (KIE) of 2 H can distort metabolic information and mediate toxicity. 18 O-water (H2 18 O) has no significant KIE and is incorporated into specific carbohydrate oxygens via well-defined mechanisms, but to date it has not been evaluated in any animal model. Mice were given H2 18 O during overnight feeding and 18 O-enrichments of liver glycogen, triglyceride glycerol (TG), and blood glucose were quantified by 13 C NMR and mass spectrometry (MS). Enrichment of oxygens 5 and 6 relative to body water informed indirect pathway contributions from the Krebs cycle and triose phosphate sources. Compared with mice fed normal chow (NC), mice whose NC was supplemented with a fructose/glucose mix (i.e., a high sugar [HS] diet) had significantly higher indirect pathway contributions from triose phosphate sources, consistent with fructose glycogenesis. Blood glucose and liver TG 18 O-enrichments were quantified by MS. Blood glucose 18 O-enrichment was significantly higher for HS versus NC mice and was consistent with gluconeogenic fructose metabolism. TG 18 O-enrichment was extensive for both NC and HS mice, indicating a high turnover of liver triglyceride, independent of diet. Thus H2 18 O informs hepatic carbohydrate biosynthesis in similar detail to 2 H2 O but without KIE-associated risks.

Integration of Liver Glycogen and Triglyceride NMR Isotopomer Analyses Provides a Comprehensive Coverage of Hepatic Glucose and Fructose Metabolism.

Dietary glucose and fructose are both efficiently assimilated by the liver but a comprehensive measurement of this process starting from their conversion to sugar phosphates, involvement of the pentose phosphate pathway (PPP), and conversion to glycogen and lipid storage products, remains incomplete. Mice were fed a chow diet supplemented with 35 g/100 mL drinking water of a 55/45 fructose/glucose mixture for 18 weeks. On the final night, the sugar mixture was enriched with either [U-13C]glucose or [U-13C]fructose, and deuterated water (2H2O) was also administered. 13C-isotopomers representing newly synthesized hepatic glucose-6-phosphate (glucose-6-P), glycerol-3-phosphate, and lipogenic acetyl-CoA were quantified by 2H and 13C NMR analysis of post-mortem liver glycogen and triglyceride. These data were applied to a metabolic model covering glucose-6-P, PPP, triose-P, and de novo lipogenesis (DNL) fluxes. The glucose supplement was converted to glucose-6-P via the direct pathway, while the fructose supplement was metabolized by the liver to gluconeogenic triose-P via fructokinase-aldolase-triokinase. Glucose-6-P from all carbohydrate sources accounted for 40-60% of lipogenic acetyl-CoA and 10-12% was oxidized by the pentose phosphate pathway (PPP). The yield of NADPH from PPP flux accounted for a minority (~30%) of the total DNL requirement. In conclusion, this approach integrates measurements of glucose-6-P, PPP, and DNL fluxes to provide a holistic and informative assessment of hepatic glucose and fructose metabolism.

Insulin Pulse Characteristics and Insulin Action in Non-diabetic Humans.

OBJECTIVE: Pulsatile insulin secretion is impaired in diseases such as type 2 diabetes that are characterized by insulin resistance. This has led to the suggestion that changes in insulin pulsatility directly impair insulin signaling. We sought to examine the effects of pulse characteristics on insulin action in humans, hypothesizing that a decrease in pulse amplitude or frequency is associated with impaired hepatic insulin action. METHODS: We studied 29 nondiabetic subjects on two occasions. On 1 occasion, hepatic and peripheral insulin action was measured using a euglycemic clamp. The deuterated water method was used to estimate the contribution of gluconeogenesis to endogenous glucose production. On a separate study day, we utilized nonparametric stochastic deconvolution of frequently sampled peripheral C-peptide concentrations during fasting to reconstruct portal insulin secretion. In addition to measuring basal and pulsatile insulin secretion, we used approximate entropy to measure orderliness and Fourier transform to measure the average, and the dispersion of, insulin pulse frequencies. RESULTS: In univariate analysis, basal insulin secretion (R2 = 0.16) and insulin pulse amplitude (R2 = 0.09) correlated weakly with insulin-induced suppression of gluconeogenesis. However, after adjustment for age, sex, and weight, these associations were no longer significant. The other pulse characteristics also did not correlate with the ability of insulin to suppress endogenous glucose production (and gluconeogenesis) or to stimulate glucose disappearance. CONCLUSIONS: Overall, our data demonstrate that insulin pulse characteristics, considered independently of other factors, do not correlate with measures of hepatic and peripheral insulin sensitivity in nondiabetic humans.

Effects of Meal Fructose/Glucose Composition on Postprandial Glucose Appearance and Hepatic Glycogen Synthesis in Healthy Subjects.

Dietary fructose overshadows glucose in promoting metabolic complications. Intestinal fructose metabolism (IFM) protects against these effects in rodents, by favoring gluconeogenesis, but the extent of IFM in humans is not known. We therefore aimed to infer the extent of IFM by comparing the contribution of dietary fructose to systemic glucose and hepatic glycogen appearance postprandially. Twelve fasting healthy subjects ingested two protein meals in random order, one supplemented with 50 g 5/95 fructose/glucose (LF) and the other with 50 g 55/45 fructose/glucose (HF). Sources of postprandial plasma glucose appearance and hepatic glycogen synthesis were determined with deuterated water. Plasma glucose excursions, as well as pre- and post-meal insulin, c-peptide, and triglyceride levels were nearly identical for both meals. The total gluconeogenic contribution to plasma glucose appearance was significantly higher for HF versus LF (65 ± 2% vs. 34 ± 3%, p < 0.001). For HF, Krebs cycle anaplerosis accounted for two-thirds of total gluconeogenesis (43 ± 2%) with one-third from Triose-P sources (22 ± 1%). With LF, three-quarters of the total gluconeogenic contribution originated via Krebs cycle anaplerosis (26 ± 2%) with one-quarter from Triose-P sources (9 ± 2%). HF and LF gave similar direct and indirect pathway contributions to hepatic glycogen synthesis. Increasing the fructose/glucose ratio had significant effects on glucose appearance sources but no effects on hepatic glycogen synthesis sources, consistent with extensive IFM. The majority of fructose carbons were converted to glucose via the Krebs cycle.

Metabolic incorporation of H218 O into specific glucose-6-phosphate oxygens by red-blood-cell lysates as observed by 13 C isotope-shifted NMR signals.

Water enriched with oxygen-18 (H218 O) is a potential tracer for evaluating the sources of glucose and glycogen synthesis since it is incorporated into specific sites of glucose-6-phosphate via specific enzyme-mediated exchange/addition mechanisms. Unlike 2 H, 18 O does not experience significant isotope effects for any of these processes. Therefore, H218 O might provide more precise estimates of endogenous carbohydrate synthesis compared with deuterated water provided that positional 18 O enrichments of glucose can be measured. As a proof of concept, H218 O was incorporated into a well characterized hemolysate model of sugar phosphate metabolism and 13 C NMR was applied to quantify positional 18 O enrichment of glucose-6-phosphate oxygens. Human erythrocyte hemolysate preparations were incubated overnight at 37 °C with a buffer containing sugar phosphate precursors and 20% (n = 5) and 80% (n = 1) H218 O. Enrichment of glucose-6-phosphate was analyzed by 13 C NMR analysis of 18 O-shifted versus unshifted signals following derivatization to monoacetone glucose (MAG). 13 C NMR MAG spectra from hemolysate revealed resolved 18 O-shifted signals in Positions 1-5. Mean 18 O enrichments were 16.4 ± 1.6% (Position 1), 13.3 ± 1.3% (Position 2), 4.1 ± 1.1% (Position 3), 12.6 ± 0.8% (Position 4), 10.7 ± 1.4% (Position 5), and no detectable enrichment of Position 6. No 18 O-shifted glucose-6-phosphate signals were detected in preparations containing sugar phosphate precursors only. H218 O is incorporated into Positions 1-5 of glucose-6-phosphate in accordance with spontaneous aldose hydration and specific enzymatic reaction mechanisms. This provides a basis for its deployment as a tracer for glucose and glycogen biosynthesis.

Determining the contribution of a high-fructose corn syrup formulation to hepatic glycogen synthesis during ad-libitum feeding in mice.

Excessive sugar intake including high-fructose corn syrup (HFCS) is implicated in the rise of obesity, insulin resistance and non-alcoholic fatty liver disease. Liver glycogen synthesis is influenced by both fructose and insulin signaling. Therefore, the effect of HFCS on hepatic glycogenesis was evaluated in mice feeding ad-libitum. Using deuterated water: the fraction of glycogen derived from triose-P sources, Krebs cycle substrates, and direct pathway + cycling, was measured in 9 normal-chow fed mice (NC) and 12 mice fed normal chow plus a 55% fructose/45% glucose mix in the drinking water at 30% w/v (HFCS-55). This was enriched with [U-13C]fructose or [U-13C]glucose to determine the contribution of each to glycogenesis. For NC, direct pathway + cycling, Krebs cycle, and triose-P sources accounted for 66 ± 0.7%, 23 ± 0.8% and 11 ± 0.4% of glycogen synthesis, respectively. HFCS-55 mice had similar direct pathway + cycling (64 ± 1%) but lower Krebs cycle (12 ± 1%, p < 0.001) and higher triose-P contributions (24 ± 1%, p < 0.001). HFCS-55-fructose contributed 17 ± 1% via triose-P and 2 ± 0% via Krebs cycle. HFCS-55-glucose contributed 16 ± 3% via direct pathway and 1 ± 0% via Krebs cycle. In conclusion, HFCS-55 supplementation resulted in similar hepatic glycogen deposition rates. Indirect pathway contributions shifted from Krebs cycle to Triose-P sources reflecting HFCS-55-fructose utilization, while HFCS-55-glucose was incorporated almost exclusively by the direct pathway.

Monounsaturated fat rapidly induces hepatic gluconeogenesis and whole-body insulin resistance.

BACKGROUNDWhile saturated fat intake leads to insulin resistance and nonalcoholic fatty liver, Mediterranean-like diets enriched in monounsaturated fatty acids (MUFA) may have beneficial effects. This study examined effects of MUFA on tissue-specific insulin sensitivity and energy metabolism.METHODSA randomized placebo-controlled cross-over study enrolled 16 glucose-tolerant volunteers to receive either oil (OIL, ~1.18 g/kg), rich in MUFA, or vehicle (VCL, water) on 2 occasions. Insulin sensitivity was assessed during preclamp and hyperinsulinemic-euglycemic clamp conditions. Ingestion of 2H2O/acetaminophen was combined with [6,6-2H2]glucose infusion and in vivo 13C/31P/1H/ex vivo 2H-magnet resonance spectroscopy to quantify hepatic glucose and energy fluxes.RESULTSOIL increased plasma triglycerides and oleic acid concentrations by 44% and 66% compared with VCL. Upon OIL intervention, preclamp hepatic and whole-body insulin sensitivity markedly decreased by 28% and 27%, respectively, along with 61% higher rates of hepatic gluconeogenesis and 32% lower rates of net glycogenolysis, while hepatic triglyceride and ATP concentrations did not differ from VCL. During insulin stimulation hepatic and whole-body insulin sensitivity were reduced by 21% and 25%, respectively, after OIL ingestion compared with that in controls.CONCLUSIONA single MUFA-load suffices to induce insulin resistance but affects neither hepatic triglycerides nor energy-rich phosphates. These data indicate that amount of ingested fat, rather than its composition, primarily determines the development of acute insulin resistance.TRIAL REGISTRATIONClinicalTrials.gov NCT01736202.FUNDINGGerman Diabetes Center, German Federal Ministry of Health, Ministry of Culture and Science of the state of North Rhine-Westphalia, German Federal Ministry of Education and Research, German Diabetes Association, German Center for Diabetes Research, Portugal Foundation for Science and Technology, European Regional Development Fund, and Rede Nacional de Ressonancia Magnética Nuclear.

p-Aminobenzoic acid as an alternative chemical biopsy agent for human hepatic UDP-glucose.

p-Aminobenzoic acid (PABA) was evaluated for noninvasive sampling of UDP-glucose in the liver. Six healthy subjects ingested 550 mg PABA during a breakfast meal. Urine was collected 0-2 and 2-4 h after PABA ingestion. N-acetyl PABA glucuronide (NAPG) was identified with 522 ± 212 µmol recovered in the 2-4 h urines. One of the subjects ingested 2 g of 98% [U-2H7]glucose alongside PABA and the NAPG was analyzed for positional 2H-enrichment by 2H NMR following derivatization to 5-O-acetyl monoacetone glucuronolactone. In conclusion, PABA is an effective agent for the chemical biopsy of hepatic UDP-glucose in humans.

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

PURPOSE: The positional analysis of hepatic glycogen enrichment from deuterated water (2 H2 O) by 2 H 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. METHODS: Eight glucose-fed and 12 fructose-fed mice were given 2 H2 O followed by ad libitum feeding overnight. Mice were then euthanized, hepatic glycogen was isolated and derivatized to monoacetone glucose, and 2 H-enrichment of positions 2, 5, and 6S were measured by 2 H NMR. From these data, the fraction of overnight glycogen appearance from the direct pathway and/or glycogen cycling and indirect pathway were estimated. Indirect pathway fractions were resolved into Krebs cycle and triose-phosphate sources-the latter including contributions from fructose metabolism. RESULTS: After overnight feeding, the fraction of overnight glycogen appearance derived from direct pathway and/or glycogen cycling in glucose-fed-mice was 63 ± 1%. For the indirect pathway, Krebs cycle and triose-phosphate sources contributed 22 ± 1% and 15 ± 1%, respectively. For fructose-fed-mice, glycogen appearance was dominated by triose-phosphate sources (60 ± 2%) with lesser contributions from Krebs cycle (14 ± 1%) and direct and/or glycogen cycling (26 ± 2%). CONCLUSIONS: 2 H NMR analysis of hepatic glycogen 2 H enrichment from 2 H2 O provides realistic profiles of dietary glucose and fructose contributions to hepatic glycogen synthesis in mice fed with diets containing 1 or the other sugar as the sole carbohydrate source.

Acute dietary fat intake initiates alterations in energy metabolism and insulin resistance.

BACKGROUND: Dietary intake of saturated fat is a likely contributor to nonalcoholic fatty liver disease (NAFLD) and insulin resistance, but the mechanisms that initiate these abnormalities in humans remain unclear. We examined the effects of a single oral saturated fat load on insulin sensitivity, hepatic glucose metabolism, and lipid metabolism in humans. Similarly, initiating mechanisms were examined after an equivalent challenge in mice. METHODS: Fourteen lean, healthy individuals randomly received either palm oil (PO) or vehicle (VCL). Hepatic metabolism was analyzed using in vivo 13C/31P/1H and ex vivo 2H magnetic resonance spectroscopy before and during hyperinsulinemic-euglycemic clamps with isotope dilution. Mice underwent identical clamp procedures and hepatic transcriptome analyses. RESULTS: PO administration decreased whole-body, hepatic, and adipose tissue insulin sensitivity by 25%, 15%, and 34%, respectively. Hepatic triglyceride and ATP content rose by 35% and 16%, respectively. Hepatic gluconeogenesis increased by 70%, and net glycogenolysis declined by 20%. Mouse transcriptomics revealed that PO differentially regulates predicted upstream regulators and pathways, including LPS, members of the TLR and PPAR families, NF-κB, and TNF-related weak inducer of apoptosis (TWEAK). CONCLUSION: Saturated fat ingestion rapidly increases hepatic lipid storage, energy metabolism, and insulin resistance. This is accompanied by regulation of hepatic gene expression and signaling that may contribute to development of NAFLD.REGISTRATION. ClinicalTrials.gov NCT01736202. FUNDING: Germany: Ministry of Innovation, Science, and Research North Rhine-Westfalia, German Federal Ministry of Health, Federal Ministry of Education and Research, German Center for Diabetes Research, German Research Foundation, and German Diabetes Association. Portugal: Portuguese Foundation for Science and Technology, FEDER - European Regional Development Fund, Portuguese Foundation for Science and Technology, and Rede Nacional de Ressonância Magnética Nuclear.

Mechanisms Underlying the Pathogenesis of Isolated Impaired Glucose Tolerance in Humans.

CONTEXT: Prediabetes is a heterogeneous disorder classified on the basis of fasting glucose concentrations and 2-hour glucose tolerance. OBJECTIVE: We sought to determine the relative contributions of insulin secretion and action to the pathogenesis of isolated impaired glucose tolerance (IGT). DESIGN: The study consisted of an oral glucose tolerance test and a euglycemic clamp performed in two cohorts matched for anthropometric characteristics and fasting glucose but discordant for glucose tolerance. SETTING: An inpatient clinical research unit at an academic medical center. PATIENTS OR OTHER PARTICIPANTS: Twenty-five subjects who had normal fasting glucose (NFG) and normal glucose tolerance (NGT) and 19 NFG/IGT subjects participated in this study. INTERVENTION(S): Subjects underwent a seven-sample oral glucose tolerance test and a 4-hour euglycemic, hyperinsulinemic clamp on separate occasions. Glucose turnover during the clamp was measured using tracers, and endogenous hormone secretion was inhibited by somatostatin. MAIN OUTCOME MEASURES: We sought to determine whether hepatic glucose metabolism, specifically the contribution of gluconeogenesis to endogenous glucose production, differed between subjects with NFG/NGT and those with NFG/IGT. RESULTS: Endogenous glucose production did not differ between groups before or during the clamp. Insulin-stimulated glucose disappearance was lower in NFG/IGT (24.6 ± 2.2 vs 35.0 ± 3.6 µmol/kg/min; P = .03). The disposition index was decreased in NFG/IGT (681 ± 102 vs 2231 ± 413 × 10-14 dL/kg/min2 per pmol/L; P < .001). CONCLUSIONS: We conclude that innate defects in the regulation of glycogenolysis and gluconeogenesis do not contribute to NFG/IGT. However, insulin-stimulated glucose disposal is impaired, exacerbating defects in ß-cell function.

Diabetes-Associated Variation in TCF7L2 Is Not Associated With Hepatic or Extrahepatic Insulin Resistance.

A common genetic variation in TCF7L2 is associated with type 2 diabetes. However, the mechanism by which this occurs remains elusive. In addition to affecting insulin secretion, genetic variation at the TCF7L2 locus may alter insulin action or directly modify hepatic glucose metabolism. We sought to determine whether the diabetes-associated variant in this locus (the T allele of rs7903146) increases fasting endogenous glucose production (EGP), and impairs insulin-induced suppression of EGP and insulin-stimulated glucose disappearance. To address this, we studied individuals who were either homozygous for the diabetes-associated allele (TT) at rs7903146 or were homozygous for the protective allele (CC). Subjects were matched for other anthropometric characteristics and were studied using a euglycemic clamp. EGP and glucose uptake were measured using the tracer dilution technique, and the relative contribution of gluconeogenesis to EGP was quantitated using deuterated water corrected for transaldolase exchange. We report that the diabetes-associated variation in TCF7L2 did not associate with fasting EGP, insulin-induced suppression of EGP, and insulin-induced stimulation of glucose uptake. There was no association with the contribution of gluconeogenesis and glycogenolysis to EGP. These data indicate that genetic variation at TCF7L2 does not predispose an individual to type 2 diabetes by altering either hepatic or extrahepatic insulin action.

Demonstration of glucose-6-phosphate hydrogen 5 enrichment from deuterated water by transaldolase-mediated exchange alone.

PURPOSE: Enrichment of glucose position 5 (H5) from deuterated water ((2)H2O) is widely used for quantifying gluconeogenesis. Exchanges of hexose and triose phosphates mediated by transaldolase have been postulated to enrich H5 independently of gluconeogenesis, but to date this mechanism has not been proven. We determined the enrichment of glucose-6-phosphate (G6P), the immediate precursor of endogenously produced glucose, from (2)H2O in erythrocyte hemolysate preparations. Here, transaldolase exchange is active but gluconeogenesis is absent. METHODS: Hemolysates were prepared from human erythrocytes and incubated with a buffer containing 5% [U-(13)C]G6P, unlabeled fructose 1,6-bisphosphate, and 10% (2)H2O. G6P (2)H-enrichment and (13)C-isotopomer distributions were analyzed by (2)H and (13)C NMR following derivatization to monoacetone glucose. RESULTS: (2)H NMR analysis revealed high (2)H-enrichment of G6P hydrogens 2, 4, and 5; low enrichment of hydrogen 3, and residual enrichments of hydrogens 1, 6R, and 6S. (13)C NMR isotopomer analysis revealed that [U-(13)C]G6P was converted to [1,2,3-(13)C3]G6P, a predicted product of transaldolase-mediated exchange, as well as [1,2-(13)C2]G6P and [3-(13)C]G6P, predicted products of combined transaldolase and transketolase exchanges. CONCLUSION: Hydrogen 5 of G6P was enriched from (2)H2O through exchanges mediated by transaldolase. These studies prove that G6P can be enriched in hydrogen 5 by (2)H2O independently of gluconeogenesis.

Pathogenesis of prediabetes: role of the liver in isolated fasting hyperglycemia and combined fasting and postprandial hyperglycemia.

CONTEXT: People with prediabetes are at high risk of developing diabetes. OBJECTIVE: The objective of this study was to determine the pathogenesis of fasting and postprandial hyperglycemia in prediabetes. DESIGN: Glucose production, gluconeogenesis, glycogenolysis, and glucose disappearance were measured before and during a hyperinsulinemic clamp using [6,6-(2)H2]glucose and the deuterated water method corrected for transaldolase exchange. SETTING: The study was conducted at the Mayo Clinic Clinical Research Unit. PARTICIPANTS: Subjects with impaired fasting glucose (IFG)/normal glucose tolerance (NGT) (n = 14), IFG/impaired glucose tolerance (IGT) (n = 18), and normal fasting glucose (NFG)/NGT (n = 16) were studied. INTERVENTION: A hyperinsulinemic clamp was used. OUTCOME MEASURES: Glucose production, glucose disappearance, gluconeogenesis, and glycogenolysis were measured. RESULTS: Fasting glucose production was higher (P < .0001) in subjects with IFG/NGT than in those with NFG/NGT because of increased rates of gluconeogenesis (P = .003). On the other hand, insulin-induced suppression of glucose production, gluconeogenesis, glycogenolysis, and stimulation of glucose disappearance all were normal. Although fasting glucose production also was increased (P = .0002) in subjects with IFG/IGT, insulin-induced suppression of glucose production, gluconeogenesis, and glycogenolysis and stimulation of glucose disappearance were impaired (P = .005). CONCLUSIONS: Fasting hyperglycemia is due to excessive glucose production in people with either IFG/NGT or IFG/IGT. Both insulin action and postprandial glucose concentrations are normal in IFG/NGT but abnormal in IFG/IGT. This finding suggests that hepatic and extrahepatic insulin resistance causes or exacerbates postprandial glucose intolerance in IFG/IGT. Elevated gluconeogenesis in the fasting state in IFG/NGT and impaired insulin-induced suppression of both gluconeogenesis and glycogenolysis in IFG/IGT suggest that alteration in the regulation of these pathways occurs early in the evolution of type 2 diabetes.

Acetaminophen glucuronide and plasma glucose report identical estimates of gluconeogenesis and glycogenolysis for healthy and prediabetic subjects using the deuterated water method.

Plasma glucose (2) H-enrichment in positions 5 ((2) H5) and 2 ((2) H2) from deuterated water ((2) H2 O) provides a measure of the gluconeogenic contribution to endogenous glucose production. Urinary glucuronide analysis can circumvent blood sampling but it is not known if glucuronide and glucose enrichments are equal. Thirteen subjects with impaired fasting glucose/impaired glucose tolerance and 11 subjects with normal fasting glucose and normal glucose tolerance ingested (2) H2 O to ∼0.5% body water and acetaminophen. Glucose and glucuronide (2) H5 and (2) H2 were measured by (2) H NMR spectroscopy of monoacetone glucose. For normal fasting glucose/normal glucose tolerance, (2) H5 was 0.23 ± 0.02% and 0.25 ± 0.02% for glucose and glucuronide, respectively, whereas (2) H2 was 0.47 ± 0.01% and 0.49 ± 0.02%, respectively. For impaired fasting glucose/impaired glucose tolerance, (2) H5 was 0.22 ± 0.01% and 0.26 ± 0.02% for glucose and glucuronide, respectively, whereas (2) H2 was 0.46 ± 0.01% and 0.49 ± 0.02%, respectively. The gluconeogenic contribution to endogenous glucose production measured from glucose and glucuronide were identical for both normal fasting glucose/normal glucose tolerance (48 ± 4 vs. 51 ± 3%) and impaired fasting glucose/impaired glucose tolerance (48 ± 2 vs. 53 ± 3%).

Effect of cyclosporine A on hepatic carbohydrate metabolism and hepatic gene expression in rat.

OBJECTIVE: Cyclosporine A (CsA)-based therapy has been implicated in the development of diabetes. Hence, its effects on hepatic carbohydrate metabolism and gene expression will be investigated. METHODS: Sprague-Dawley rats given 15 mg/kg body weight/day of CsA for 20 days, as well as healthy untreated animals, received a glucose load enriched with [U-(13)C]glucose and deuterated water to resolve load and endogenous contributions to plasma glucose. Blood glucose and plasma insulin levels were assayed and at 60-min post-load, plasma glucose (13)C and (2)H-enrichments were analyzed by nuclear magnetic resonance spectroscopy and liver tissue analyzed for hepatic gene expression. RESULTS: CsA-treated rats were glucose intolerant relative to controls (AUC(glucose) = 21,297 ± 857 versus 14,183 ± 1094, p < 0.01). Contributions from endogenous glucose production (EGP) were significantly elevated in CsA-treated rats (179 ± 16 versus 123 ± 13 mg/dl, p < 0.05). The increased endogenous contributions were attributable to glycogenolysis or glucose-G6P cycling and not to gluconeogenesis. Significantly higher expressions of fatty acid synthase and acetyl-CoA carboxylase 1 and 2 genes were observed in CsA-treated rats. CONCLUSIONS: CsA-altered glucose metabolism and gene expression could reflect increased hepatic insulin resistance. In the liver of CsA-treated animals, EGP suppression is impaired whereas hepatic de novo lipogenesis is enhanced contributing to dysregulated glucose and lipid metabolism.

Resolving the sources of plasma glucose excursions following a glucose tolerance test in the rat with deuterated water and [U-13C]glucose.

Sources of plasma glucose excursions (PGE) following a glucose tolerance test enriched with [U-(13)C]glucose and deuterated water were directly resolved by (13)C and (2)H Nuclear Magnetic Resonance spectroscopy analysis of plasma glucose and water enrichments in rat. Plasma water (2)H-enrichment attained isotopic steady-state within 2-4 minutes following the load. The fraction of PGE derived from endogenous sources was determined from the ratio of plasma glucose position 2 and plasma water (2)H-enrichments. The fractional gluconeogenic contributions to PGE were obtained from plasma glucose positions 2 and 5 (2)H-positional enrichment ratios and load contributions were estimated from plasma [U-(13)C]glucose enrichments. At 15 minutes, the load contributed 26±5% of PGE while 14±2% originated from gluconeogenesis in healthy control rats. Between 15 and 120 minutes, the load contribution fell whereas the gluconeogenic contribution remained constant. High-fat fed animals had significant higher 120-minute blood glucose (173±6 mg/dL vs. 139±10 mg/dL, p<0.05) and gluconeogenic contributions to PGE (59±5 mg/dL vs. 38±3 mg/dL, p<0.01) relative to standard chow-fed controls. In summary, the endogenous and load components of PGE can be resolved during a glucose tolerance test and these measurements revealed that plasma glucose synthesis via gluconeogenesis remained active during the period immediately following a glucose load. In rats that were placed on high-fat diet, the development of glucose intolerance was associated with a significantly higher gluconeogenic contribution to plasma glucose levels after the load.

Sources of hepatic glycogen synthesis following a milk-containing breakfast meal in healthy subjects.

During feeding, dietary galactose is a potential source of hepatic glycogen synthesis; but its contribution has not been measured to date. In the presence of deuterated water ((2)H(2)O), uridine diphosphate (UDP)-glucose derived from galactose is not enriched, whereas the remainder derived from glucose-6-phosphate (G6P) is enriched in position 2 to the same level as body water, assuming complete G6P-fructose-6-phosphate (F6P) exchange. Hence, the difference between UDP-glucose position 2 and body water enrichments reflects the contribution of galactose to glycogen synthesis relative to all other sources. In study 1, G6P-F6P exchange in 6 healthy subjects was quantified by supplementing a milk-containing breakfast meal with 10 g of [U-(2)H(7)]glucose and quantifying the depletion of position 2 enrichment in urinary menthol glucuronide. In study 2, another 6 subjects ingested (2)H(2)O and acetaminophen followed by an identical breakfast meal with 10 g of [1-(13)C]glucose to resolve direct/indirect pathways and galactose contributions to glycogen synthesis. Metabolite enrichments were determined by (2)H and (13)C nuclear magnetic resonance. In study 1, G6P-F6P exchange approached completion; therefore, the difference between position 2 and body water enrichments in study 2 (0.20% ± 0.03% vs 0.27% ± 0.03%, P < .005) was attributed to galactose glycogenesis. Dietary galactose contributed 19% ± 3% to glycogen synthesis. Of the remainder, 58% ± 5% was derived from the direct pathway and 22% ± 4% via the indirect pathway. The contribution of galactose to hepatic glycogen synthesis was resolved from that of direct and indirect pathways using a combination of (2)H(2)O and [1-(13)C]glucose tracers.

Postprandial and fasting hepatic glucose fluxes in long-standing type 1 diabetes.

OBJECTIVE: Intravenous insulin infusion partly improves liver glucose fluxes in type 1 diabetes (T1D). This study tests the hypothesis that continuous subcutaneous insulin infusion (CSII) normalizes hepatic glycogen metabolism. RESEARCH DESIGN AND METHODS: T1D with poor glycemic control (T1Dp; HbA(1c): 8.5 ± 0.4%), T1D with improved glycemic control on CSII (T1Di; 7.0 ± 0.3%), and healthy humans (control subjects [CON]; 5.2 ± 0.4%) were studied. Net hepatic glycogen synthesis and glycogenolysis were measured with in vivo (13)C magnetic resonance spectroscopy. Endogenous glucose production (EGP) and gluconeogenesis (GNG) were assessed with [6,6-(2)H(2)]glucose, glycogen phosphorylase (GP) flux, and gluconeogenic fluxes with (2)H(2)O/paracetamol. RESULTS: When compared with CON, net glycogen synthesis was 70% lower in T1Dp (P = 0.038) but not different in T1Di. During fasting, T1Dp had 25 and 42% higher EGP than T1Di (P = 0.004) and CON (P < 0.001; T1Di vs. CON: P = NS). GNG was 74 and 67% higher in T1Dp than in T1Di (P = 0.002) and CON (P = 0.001). In T1Dp, GP flux (7.0 ± 1.6 µmol ⋅ kg(-1) ⋅ min(-1)) was twofold higher than net glycogenolysis, but comparable in T1Di and CON (3.7 ± 0.8 and 4.9 ± 1.0 µmol ⋅ kg(-1) ⋅ min(-1)). Thus T1Dp exhibited glycogen cycling (3.5 ± 2.0 µmol ⋅ kg(-1) ⋅ min(-1)), which accounted for 47% of GP flux. CONCLUSIONS: Poorly controlled T1D not only exhibits augmented fasting gluconeogenesis but also increased glycogen cycling. Intensified subcutaneous insulin treatment restores these abnormalities, indicating that hepatic glucose metabolism is not irreversibly altered even in long-standing T1D.

Transaldolase exchange and its effects on measurements of gluconeogenesis in humans.

The deuterated water method is used extensively to measure gluconeogenesis in humans. This method assumes negligible exchange of the lower three carbons of fructose 6-phsophate via transaldolase exchange since this exchange will result in enrichment of carbon 5 of glucose in the absence of net gluconeogenesis. The present studies tested this assumption. ²H2O and acetaminophen were ingested and [1-¹³C]acetate infused in 11 nondiabetic subjects after a 16-h fast. Plasma and urinary glucuronide enrichments were measured using nuclear magnetic resonance spectroscopy before and during a 0.35 mU·kg FFM⁻¹·min⁻¹ insulin infusion. Rates of endogenous glucose production measured with [3-³H]- and [6,6-²H2]glucose did not differ either before (14.0 ± 0.7 vs. 13.8 ± 0.7 µmol·kg⁻¹·min⁻¹) or during the clamp (10.4 ± 0.9 vs. 10.9 ± 0.7 µmol·kg⁻¹·min⁻¹), consistent with equilibration and quantitative removal of tritium during triose isomerase exchange. Plasma [3-¹³C] glucose-to-[4-¹³C]glucose and urinary [3-¹³C] glucuronide-to-[4-¹³C]glucuronide ratios were <1.0 (P < 0.001) in all subjects both before (0.66 ± 0.04 and 0.60 ± 0.04) and during (059 ± 0.05 and 0.56 ± 0.06) the insulin infusion, respectively, indicating that ∼35-45% of the labeling of the 5th carbon of glucose by deuterium was due to transaldolase exchange rather than gluconeogenesis. When corrected for transaldolase exchange, rates of gluconeogenesis were lower (P < 0.001) and glycogenolysis higher (P < 0.001) than uncorrected rates both before and during the insulin infusion. In conclusion, assuming negligible dilution by glycerol and near-complete triose isomerase equilibration, these data provide strong experimental evidence that transaldolase exchange occurs in humans, resulting in an overestimate of gluconeogenesis and an underestimate of glycogenolysis when measured with the ²H2O method. Use of appropriate ¹³C tracers provides a means of correcting for transaldolase exchange.