Changes in hepatic glycogen cycling during a glucose load in healthy humans.

AIMS/HYPOTHESIS: Glycogen cycling, i.e. simultaneous glycogen synthesis and glycogenolysis, affects estimates of glucose fluxes using tracer techniques and may contribute to hyperglycaemia in diabetic conditions. This study presents a new method for quantifying hepatic glycogen cycling in the fed state. Glycogen is synthesised from glucose by the direct and indirect (gluconeogenic) pathways. Since glycogen is also synthesised from glycogen, i.e. glycogen-->glucose 1-phosphate-->glycogen, that synthesised through the direct and indirect pathways does not account for 100% of glycogen synthesis. The percentage contribution of glycogen cycling to glycogen synthesis then equals the difference between the sum of the percentage contributions of the direct and indirect pathways and 100. MATERIALS AND METHODS: The indirect and direct pathways were measured independently in nine healthy volunteers who had fasted overnight. They ingested (2)H(2)O (5 ml/kg body water) and were infused with [5-(3)H]glucose and acetaminophen (paracetamol; 1 g) during hyperglycaemic clamps (7.8 mmol/l) lasting 8 h. The percentage contribution of the indirect pathway was calculated from the ratio of (2)H enrichments at carbon 5 to that at carbon 2, and the contribution of the direct pathway was determined from the (3)H-specific activity, relative to plasma glucose, of the urinary glucuronide excreted between 2 and 4, 4 and 6, and 6 and 8 h. RESULTS: Glucose infusion rates increased (p<0.01) to approximately 50 mumol kg(-1) min(-1). Plasma insulin and the insulin : glucagon ratio rose approximately 3.6- and approximately 8.3-fold (p<0.001), respectively. From the difference between 100% and the sum of the direct (2-4 h, 54+/-6%; 4-6 h, 59+/-5%; 6-8 h, 63+/-4%) and indirect (32+/-3, 38+/-4, 36+/-3%) pathways, glycogen cycling was seen to be decreased (p<0.05) from 14+/-4% (2-4 h) to 4+/-3% (4-6 h) and 1+/-3% (6-8 h). CONCLUSIONS/INTERPRETATION: This method allows measurement of hepatic glycogen cycling in the fed state and demonstrates that glycogen cycling occurs most in the early hours after glucose loading subsequent to a fast.

Measurement of fractional whole-body gluconeogenesis in humans from blood samples using 2H nuclear magnetic resonance spectroscopy.

Several problems limit quantification of gluconeogenesis. We applied in vitro 2H-nuclear magnetic resonance (NMR) spectroscopy to simultaneously measure 2H in all glucose carbons for direct assessment of gluconeogenesis. This method was compared with 2H measurement in carbons 5 and 2 using gas chromatography-mass spectrometry (hexamethylenetetramine [HMT]) and with in vivo 13C magnetic resonance spectroscopy (MRS). After 14 h of fasting, and following 2H2O ingestion, blood was obtained from nine healthy and seven type 2 diabetic subjects. Glucose was purified, acetylated, and analyzed for 2H in carbons 1-6 with 2H-NMR. Using 5:2 ratios, gluconeogenesis increased (P < 0.05) over time and mean gluconeogenesis was lower in control subjects than in type 2 diabetic patients (63 +/- 3 vs. 75 +/- 2%, P < 0.01). 13C-MRS revealed higher hepatic glycogenolysis in control subjects (3.9 +/- 0.4 vs. 2.3 +/- 0.2 micromol.kg(-1).min(-1)) yielding mean contribution of gluconeogenesis of 65 +/- 3 and 77 +/- 2% (P < 0.005). Measurement of gluconeogenesis by 2H-NMR correlated linearly with 13C-MRS (r = 0.758, P = 0.0007) and HMT (r = 0.759, P = 0.0007). In an additional protocol, 2H enrichments demonstrated a fast decline of gluconeogenesis from approximately 100 to approximately 68% (P < 0.02) within 4 h of galactose infusion after 40-44 h of fasting. Thus, in vitro 2H-NMR offers an alternative approach to determine fractional gluconeogenesis in good agreement with standard methods and allows monitoring of rapid metabolic alterations.

Free fatty acids inhibit the glucose-stimulated increase of intramuscular glucose-6-phosphate concentration in humans.

To test Randle's hypothesis we examined whether free fatty acids (FFAs) affect glucose-stimulated glucose transport/phosphorylation and allosteric mediators of muscle glucose metabolism under conditions of fasting peripheral insulinemia. Seven healthy men were studied during somatostatin-glucose-insulin clamp tests [plasma insulin, 50 pmol/L; plasma glucose, 5 mmol/L (0-180 min), 10 mmol/L (180-300 min)] in the presence of low (0.05 mmol/L) and increased (2.6 mmol/L) plasma FFA concentrations. (31)P and (1)H nuclear magnetic resonance spectroscopy was used to determine intracellular concentrations of glucose-6-phosphate (G6P), inorganic phosphate, phosphocreatine, ADP, pH, and intramyocellular lipids. Rates of glucose turnover were measured using D-[6,6-(2)H(2)]glucose. Plasma FFA elevation reduced rates of glucose uptake at the end of the euglycemic period (R(d 150-180 min): 8.6 +/- 0.5 vs. 12.6 +/- 1.6 micromol/kg.min, P < 0.05) and during hyperglycemia (R(d 270-300 min): 9.9 +/- 0.6 vs. 22.3 +/- 1.7 micromol/kg.min, P < 0.01). Similarly, intramuscular G6P was lower at the end of both euglycemic (G6P(167-180 min): -22 +/- 7 vs. +24 +/- 7 micromol/L, P < 0.05) and hyperglycemic periods (G6P(287-300 min): -7 +/- 9 vs. +28 +/- 7 micromol/L, P < 0.05). Changes in intracellular inorganic phosphate exhibited a similar pattern, whereas FFA did not affect phosphocreatine, ADP, pH, and intramyocellular lipid contents. In conclusion, the lack of an increase in muscular G6P along with reduction of whole body glucose clearance indicates that FFA might directly inhibit glucose transport/phosphorylation in skeletal muscle.

Effects of short-term improvement of insulin treatment and glycemia on hepatic glycogen metabolism in type 1 diabetes.

Insufficiently treated type 1 diabetic patients exhibit inappropriate postprandial hyperglycemia and reduction in liver glycogen stores. To examine the effect of acute improvement of metabolic control on hepatic glycogen metabolism, lean young type 1 diabetic (HbA1c 8.8 +/- 0.3%) and matched nondiabetic subjects (HbA1c 5.4 +/- 0.1%) were studied during the course of a day with three isocaloric mixed meals. Hepatic glycogen concentrations were determined noninvasively using in vivo 13C nuclear magnetic resonance spectroscopy. Rates of net glycogen synthesis and breakdown were calculated from linear regression of the glycogen concentration time curves from 7:30-10:30 P.M. and from 10:30 P.M. to 8:00 A.M., respectively. The mean plasma glucose concentration was approximately 2.4-fold higher in diabetic than in nondiabetic subjects (13.6 +/- 0.4 vs. 5.8 +/- 0.1 mmol/l, P < 0.001). Rates of net glycogen synthesis and net glycogen breakdown were reduced by approximately 74% (0.11 +/- 0.02 vs. 0.43 +/- 0.04 mmol/l liver/min, P < 0.001) and by approximately 47% (0.10 +/- 0.01 vs. 0.19 +/- 0.01 mmol/l liver/min, P < 0.001) in diabetic patients, respectively. During short-term (24-h) intensified insulin treatment, the mean plasma glucose level was not different between diabetic and nondiabetic subjects (6.4 +/- 0.1 mmol/l). Net glycogen synthesis and breakdown increased by approximately 92% (0.23 +/- 0.04 mmol/l liver/min, P = 0.017) and by approximately 40% (0.14 approximately 0.01 mmol/l liver/min, P = 0.011), respectively. In conclusion, poorly controlled type 1 diabetic patients present with marked reduction in both hepatic glycogen synthesis and breakdown. Both defects in glycogen metabolism are improved but not normalized by short-term restoration of insulinemia and glycemia.

Lipid-dependent control of hepatic glycogen stores in healthy humans.

AIMS/HYPOTHESIS: Non-esterified fatty acids and glycerol could stimulate gluconeogenesis and also contribute to regulating hepatic glycogen stores. We examined their effect on liver glycogen breakdown in humans. METHODS: After an overnight fast healthy subjects participated in three protocols with lipid/heparin (plasma non-esterified fatty acids: 2.2 +/- 0.1 mol/l; plasma glycerol: 0.5 +/- 0.03 mol/l; n = 7), glycerol (0.4 +/- 0.1 mol/l; 1.5 +/- 0.2 mol/l; n = 5) and saline infusion (control; 0.5 +/- 0.1 mol/l; 0.2 +/- 0.02 mol/l; n = 7). Net rates of glycogen breakdown were calculated from the decrease of liver glycogen within 9 h using 13C nuclear magnetic resonance spectroscopy. Endogenous glucose production was measured with infusion of D-[6,6-2H2]glucose. RESULTS: Endogenous glucose production decreased by about 25 % during lipid and saline infusion (p < 0.005) but not during glycerol infusion (p < 0.001 vs lipid, saline). An increase of plasma non-esterified fatty acids or glycerol reduced the net glycogen breakdown by about 84 % to 0.6 +/- 0.3 micromol x kg(-1) x min(-1) (p < 0.001 vs saline: 3.7 +/- 0.5 micromol x kg(-1) x min(-1)) and by about 46 % to 2.0 +/- 0.4 micromol x kg(-1) x min(-1) (p < 0.01 vs saline and lipid), respectively. Rates of gluconeogenesis increased to 11.5 +/- 0.8 micromol x kg(-1) x min(-1) (p < 0.01) and 12.8 +/- 1.0 micromol x kg(-1) min(-1) (p < 0.01 vs saline: 8.2 +/- 0.7 micromol x l(-1) x min(-1)), respectively. CONCLUSION/INTERPRETATION: An increase of non-esterifled fatty acid leads to a pronounced inhibition of net hepatic glycogen breakdown and increases gluconeogenesis whereas glucose production does not differ from the control condition. We suggest that this effect is not due to increased availability of glycerol alone but rather to lipid-dependent control of hepatic glycogen stores.

Hormonal and metabolic counterregulation during and after high-dose insulin-induced hypoglycemia in diabetes mellitus type 2.

Non-obese type 2 diabetic subjects in good metabolic control (n=6, HbA1c 7.0 +/- 0.3%, mean diabetes duration: 5.7 +/- 1 years) and matched non-diabetic subjects (control; n = 6) were studied during hyperinsulinemic (approximately 3 nmol/l)-hypoglycemic (approximately 3.1 mmol/l) clamp tests (0-120 min) and the subsequent recovery period (120-240 min). Plasma glucagon rose gradually but not significantly, whereas norepinephrine and epinephrine similarly increased approximately 2 and approximately 25-fold in both groups. Islet amyloid polypeptide (IAPP) decreased to approximately 41% and approximately 24% of basal values during hypoglycemia and rapidly rose approximately 4.7-fold during the recovery period, while plasma C-peptide remained suppressed in both groups. Within 140 min, plasma free fatty acids similarly decreased to approximately 70 micromol/l (p < 0.05), but then rose to values being approximately 50% higher in diabetic than in control subjects (240 min: 907 +/- 93 vs. 602 +/- 90 micromol/l; p < 0.05). Glucose infusion rates were comparable during hypoglycemia, but approximately 40% lower during recovery in diabetic patients (1.88 +/- 0.27 vs. 3.44 +/- 0.27 mg x kg(-1) x min(-1), p < 0.001). These results demonstrate that (i) hypoglycemia induced by high-dose insulin largely abolishes the counterregulatory response of glucagon, but not of catecholamines in nondiabetic and well-controlled type 2 diabetic subjects, (ii) the rapid posthypoglycemic increase of plasma IAPP occurs independently of plasma insulin, and (iii) the superior rise in plasma free fatty acids may account at least in part for the posthypoglycemic insulin resistance of type 2 diabetic patients.

Effects of free fatty acid elevation on postabsorptive endogenous glucose production and gluconeogenesis in humans.

Effects of free fatty acids (FFAs) on endogenous glucose production (EGP) and gluconeogenesis (GNG) were examined in healthy subjects (n = 6) during stepwise increased Intralipid/heparin infusion (plasma FFAs 0.8+/-0.1, 1.8+/-0.2, and 2.8+/-0.3 mmol/l) and during glycerol infusion (plasma FFAs approximately 0.5 mmol/l). Rates of EGP were determined with D-[6,6-2H2]glucose and contributions of GNG from 2H enrichments in carbons 2 and 5 of blood glucose after 2H2O ingestion. Plasma glucose concentrations decreased by approximately 10% (P < 0.01), whereas plasma insulin increased by approximately 47% (P = 0.02) after 9 h of lipid infusion. EGP declined from 9.3+/-0.5 (lipid) and 9.0+/-0.8 pmol x kg(-1) x min(-1) (glycerol) to 8.4+/-0.5 and 8.2+/-0.7 micromol x kg(-1) x min(-1), respectively (P < 0.01). Contribution of GNG similarly rose (P < 0.01) from 46+/-4 and 52+/-3% to 65+/-8 and 78+/-7%. To exclude interaction of FFAs with insulin secretion, the study was repeated at fasting plasma insulin (approximately 35 pmol/l) and glucagon (approximately 90 ng/ml) concentrations using somatostatin-insulin-glucagon clamps. Plasma glucose increased by approximately 50% (P < 0.005) during lipid but decreased by approximately 12% during glycerol infusion (P < 0.005). EGP remained unchanged over the 9-h period (9.9+/-1.2 vs. 9.0+/-1.1 micromol x kg(-1) x min(-1)). GNG accounted for 62+/-5 (lipid) and 60+/-6% (glycerol) of EGP at time 0 and rose to 74+/-3% during lipid infusion only (P < 0.05 vs. glycerol: 64+/-4%). In conclusion, high plasma FFA concentrations increase the percent contribution of GNG to EGP and may contribute to increased rates of GNG in patients with type 2 diabetes.

Prevention of in vitro lipolysis by tetrahydrolipstatin.

BACKGROUND: Metabolic effects of free fatty acids (FFAs) frequently are tested using combined infusion of triglycerides and heparin, which stimulates lipolysis in vivo. Ongoing in vitro lipolysis, however, probably produces falsely high plasma FFA concentrations under these conditions. Therefore, this study aims to assess the efficacy of tetrahydrolipstatin (THL) in inhibiting plasma lipolytic activity and to improve plasma FFA determination. METHODS: Plasma concentrations of FFAs and glycerol were measured in five healthy subjects in the presence and absence of THL. Blood was drawn at baseline, during infusion of a triglyceride emulsion (1.5 mL/min), and during infusion of triglycerides plus heparin (0.2 IU. kg(-1). min(-1)). In addition, the effects of storage temperature of the samples were analyzed. RESULTS: In samples frozen immediately after collection, plasma FFAs were 28% lower in the presence of THL than in its absence (P = 0.008). When THL-free plasma was incubated for 3 h on ice or at room temperature, plasma FFAs were 22% (P = 0.02) and 91% (P = 0.0004) higher, respectively, than in samples frozen immediately. The addition of THL blunted temperature-dependent in vitro lipolysis by 88% (P<0.01) and 89% (P <0.001) after incubation on ice and at room temperature, respectively. Changes in plasma glycerol concentrations exhibited similar behavior. CONCLUSIONS: THL, which is safe and easy to handle, is a potent inhibitor of in vitro lipolysis and could, therefore, be added to blood samples drawn during triglyceride/heparin infusions to allow more accurate determination of plasma FFA concentrations.

Elevated serum free fatty acid concentrations inhibit T lymphocyte signaling.

Unbound cis-unsaturated free (i.e., nonesterified) fatty acids (FFA) inhibit T lymphocyte activation in vitro and therefore may exert immunosuppressive effects. However, in blood serum the major proportion of FFA is tightly bound to albumin, whereas unbound FFA are hardly detectable. Since serum FFA elevation occurs under pathological conditions like insulin resistance or cancer, which are often associated with a disturbed immune response, we addressed the question of whether increased serum FFA concentrations could affect T lymphocyte activation under in vivo conditions. Our studies revealed that 1) addition of pure long-chain cis-unsaturated FFA in the absence of albumin inhibited calcium response in cultured Jurkat T cells. 2) In healthy volunteers, serum FFA elevation by a lipid/heparin infusion, including predominantly unsaturated fatty acids, decreased calcium response of cultured T cells in contrast to studies without heparin. 3) Most notably, stepwise increasing serum FFA by lipid/heparin infusion also inhibited calcium response of simultaneously isolated autologous peripheral blood T lymphocytes as well as their CD4(+) and CD8(+) subsets. In conclusion, our data emphasize that serum FFA elevation is able to exert immunosuppressive effects in vivo.

Rapid impairment of skeletal muscle glucose transport/phosphorylation by free fatty acids in humans.

The initial effects of free fatty acids (FFAs) on glucose transport/phosphorylation were studied in seven healthy men in the presence of elevated (1.44 +/- 0.16 mmol/l), basal (0.35 +/- 0.06 mmol/l), and low (<0.01 mmol/l; control) plasma FFA concentrations (P < 0.05 between all groups) during euglycemic-hyperinsulinemic clamps. Concentrations of glucose-6-phosphate (G-6-P), inorganic phosphate (Pi), phosphocreatine, ADP, and pH in calf muscle were measured every 3.2 min for 180 min by using 31P nuclear magnetic resonance spectroscopy. Rates of whole-body glucose uptake increased similarly until 140 min but thereafter declined by approximately 20% in the presence of basal and high FFAs (42.8 +/- 3.6 and 41.6 +/- 3.3 vs. control: 52.7 +/- 3.3 micromol x kg(-1) x min(-1), P < 0.05). The rise of intramuscular G-6-P concentrations was already blunted at 45 min of high FFA exposure (184 +/- 17 vs. control: 238 +/- 17 micromol/l, P = 0.008). At 180 min, G-6-P was lower in the presence of both high and basal FFAs (197 +/- 21 and 213 +/- 18 vs. control: 286 +/- 19 micromol/l, P < 0.05). Intramuscular pH decreased by -0.013 +/- 0.001 (P < 0.005) during control but increased by +0.008 +/- 0.002 (P < 0.05) during high FFA exposure, while Pi rose by approximately 0.39 mmol/l (P < 0.005) within 70 min and then slowly decreased in all studies. In conclusion, the lack of an initial peak and the early decline of muscle G-6-P concentrations suggest that even at physiological concentrations, FFAs primarily inhibit glucose transport/phosphorylation, preceding the reduction of whole-body glucose disposal by up to 120 min in humans.

Acute troglitazone action in isolated perfused rat liver.

1. The thiazolidinedione compound, troglitazone, enhances insulin action and reduces plasma glucose concentrations when administered chronically to type 2 diabetic patients. 2. To analyse to what extent thiazolidinediones interfere with liver function, we examined the acute actions of troglitazone (0.61 and 3.15 microM) on hepatic glucose and lactate fluxes, bile secretion, and portal pressure under basal, insulin- and/or glucagon-stimulated conditions in isolated perfused rat livers. 3. During BSA-free perfusion, high dose troglitazone increased basal (P < 0.01), but inhibited glucagon-stimulated incremental glucose production by approximately 75% (10.0 +/- 2.5 vs control: 40.0 +/- 7.2 micromol g liver(-1), P < 0.01). In parallel, incremental lactate release rose approximately 6 fold (13.1 +/- 5.9 vs control: 2.2 +/- 0.8 mmol g liver(-1), P < 0.05), while bile secretion declined by approximately 67% [0.23 +/- 0.02 vs control: 0.70 +/- 0.05 mg g liver(-1) min(-1)), P < 0.001]. Low dose troglitazone infusion did not enhance the inhibitory effect of insulin on glucagon-stimulated glucose production, but rapidly increased lactate release (P < 0.0005) and portal venous pressure (+0.17 +/- 0.07 vs +0.54 +/- 0.07 cm buffer height, P < 0.0001). 4. These results indicate that troglitazone exerts both insulin-like and non-insulin-like hepatic effects, which are blunted by addition of albumin, possibly due to troglitazone binding.

[Fixation disparity in peripheral and central binocular vision under conditions of prismatic heterophoria (author's transl)]. / Fixationdisparität bei peripherem und zentralem Binokularsehen unter phorischer Belastung

A critical review of the literature shows that there are controversal interpretations of thephenomenon fixation disparity. The former findings of Ogle and coworkers are ascertained byinvertigator to close the central gap in the fusional patter. Formerly the gap was essentialdue to polarisation techiques. 1. Without central gap fixation disparity decreases, but remainssignificantly different from zero. 2. Diminution or closing of the gap leads to more preciseresults, since the position of the eyes is better controlled. 3. Straining the binocular vision with high prism power cortical tolerances increase when fusion was aided by central stimuli. Fixation disparity could be extended up to 20' (minutes of are) which is three times the well accepted size of the Panum area.