A new global fibrinolysis capacity assay for the sensitive detection of hyperfibrinolysis and hypofibrinogenemia in trauma patients.

BACKGROUND: Conventional clotting tests are not expeditious enough to allow timely targeted interventions in trauma, and current point-of-care analyzers, such as rotational thromboelastometry (ROTEM), have limited sensitivity for hyperfibrinolysis and hypofibrinogenemia. OBJECTIVES: To evaluate the performance of a recently developed global fibrinolysis capacity (GFC) assay in identifying fibrinolysis and hypofibrinogenemia in trauma patients. METHODS: Exploratory analysis of a prospective cohort of adult trauma patients admitted to a single UK major trauma center and of commercially available healthy donor samples was performed. Lysis time (LT) was measured in plasma according to the GFC manufacturer's protocol, and a novel fibrinogen-related parameter (percentage reduction in GFC optical density from baseline at 1 minute) was derived from the GFC curve. Hyperfibrinolysis was defined as a tissue factor-activated ROTEM maximum lysis of >15% or LT of ≤30 minutes. RESULTS: Compared to healthy donors (n = 19), non-tranexamic acid-treated trauma patients (n = 82) showed shortened LT, indicative of hyperfibrinolysis (29 minutes [16-35] vs 43 minutes [40-47]; p < .001). Of the 63 patients without overt ROTEM-hyperfibrinolysis, 31 (49%) had LT of ≤30 minutes, with 26% (8 of 31) of them requiring major transfusions. LT showed increased accuracy compared to maximum lysis in predicting 28-day mortality (area under the receiver operating characteristic curve, 0.96 [0.92-1.00] vs 0.65 [0.49-0.81]; p = .001). Percentage reduction in GFC optical density from baseline at 1 minute showed comparable specificity (76% vs 79%) to ROTEM clot amplitude at 5 minutes from tissue factor-activated ROTEM with cytochalasin D in detecting hypofibrinogenemia but correctly reclassified >50% of the patients with false negative results, leading to higher sensitivity (90% vs 77%). CONCLUSION: Severe trauma patients are characterized by a hyperfibrinolytic profile upon admission to the emergency department. The GFC assay is more sensitive than ROTEM in capturing hyperfibrinolysis and hypofibrinogenemia but requires further development and automation.

Defective glycogenesis contributes toward the inability to suppress hepatic glucose production in response to hyperglycemia and hyperinsulinemia in zucker diabetic fatty rats.

OBJECTIVE: Examine whether normalizing net hepatic glycogenesis restores endogenous glucose production and hepatic glucose phosphorylation in response to diabetic levels of plasma glucose and insulin in Zucker diabetic fatty rats (ZDF). RESEARCH DESIGN AND METHODS: Hepatic glucose and intermediate fluxes (µmol · kg(-1) · min(-1)) were measured with and without a glycogen phosphorylase inhibitor (GPI) using [2-(3)H]glucose, [3-(3)H]glucose, and [U-(14)C]alanine in 20 h-fasted conscious ZDF and their lean littermates (ZCL) under clamp conditions designed to maintain diabetic levels of plasma glucose and insulin. RESULTS: With infusion of GPI into ZDF (ZDF-GPI+G), compared with vehicle infused ZDF (ZDF-V), high glycogen phosphorylase a activity was decreased and low synthase I activity was increased to that of ZCL. Low net glycogenesis from plasma glucose rose to 75% of ZCL levels (4 ± 1 in ZDF-V, 18 ± 1 in ZDF-GPI+G, and 24 ± 2 in ZCL) and phosphoenolpyruvate 260% (4 ± 2 in ZDF-V, 16 ± 1 in ZDF+GPI-G, and 6 ± 2 in ZCL). High endogenous glucose production was suppressed with GPI infusion but not to that of ZCL (46 ± 4 in ZDF-V, 18 ± 4 in ZDF-GPI+G, and -8 ± 3 in ZCL). This was accompanied by reduction of the higher glucose-6-phosphatase flux (75 ± 4 in ZDF-V, 41 ± 4 in ZDF-GPI+G, and 86 ± 12 in ZCL) and no change in low glucose phosphorylation or total gluconeogenesis. CONCLUSIONS: In the presence of hyperglycemic-hyperinsulinemia in ZDF, reduced glycogenic flux partially contributes to a lack of suppression of hepatic glucose production by failing to redirect glucose-6-phosphate flux from production of glucose to glycogen but is not responsible for a lower rate of glucose phosphorylation.

Impact of a glycogen phosphorylase inhibitor and metformin on basal and glucagon-stimulated hepatic glucose flux in conscious dogs.

The effects of a glycogen phosphorylase inhibitor (GPI) and metformin (MT) on hepatic glucose fluxes (µmol · kg(-1) · min(-1)) in the presence of basal and 4-fold basal levels of plasma glucagon were investigated in 18-h fasted conscious dogs. Compared with the vehicle treatment, GPI infusion suppressed net hepatic glucose output (NHGO) completely (-3.8 ± 1.3 versus 9.9 ± 2.8) despite increased glucose 6-phosphate (G-6-P) neogenesis from gluconeogenic precursors (8.1 ± 1.1 versus 5.5 ± 1.1). MT infusion did not alter those parameters. In response to a 4-fold rise in plasma glucagon levels, in the vehicle group, plasma glucose levels were increased 2-fold, and NHGO was increased (43.9 ± 5.7 at 10 min and 22.7 ± 3.4 at steady state) without altering G-6-P neogenesis (3.7 ± 1.5 and 5.5 ± 0.5, respectively). In the GPI group, there was no increase in NHGO due to decreased glucose-6-phosphatase flux associated with reduced G-6-P concentration. A lower G-6-P concentration was the result of increased net glycogenesis without altering G-6-P neogenesis. In the MT group, the increment in NHGO (22.2 ± 4.4 at 10 min and 12.1 ± 3.6 at steady state) was approximately half of that of the vehicle group. The lesser NHGO was associated with reduced glucose-6-phosphatase flux but a rise in G-6-P concentration and only a small incorporation of plasma glucose into glycogen. In conclusion, the inhibition of glycogen phosphorylase a activity decreases basal and glucagon-induced NHGO via redirecting glucose 6-phosphate flux from glucose toward glycogen, and MT decreases glucagon-induced NHGO by inhibiting glucose-6-phosphatase flux and thereby reducing glycogen breakdown.

Restoration of hepatic glucokinase expression corrects hepatic glucose flux and normalizes plasma glucose in zucker diabetic fatty rats.

OBJECTIVE: We examined in 20-week-old Zucker diabetic fatty (ZDF) rats whether restoration of hepatic glucokinase (GK) expression would alter hepatic glucose flux and improve hyperglycemia. RESEARCH DESIGN AND METHODS: ZDF rats were treated at various doses with an adenovirus that directs the expression of rat liver GK (AdvCMV-GKL) dose dependently, and various metabolic parameters were compared with those of nondiabetic lean littermates (ZCL rats) before and during a hyperglycemic clamp. Viral infection per se did not affect hepatic GK activity, since expression of a catalytically inactive form of GK did not alter endogenous hepatic GK activity. RESULTS: ZDF rats compared with ZCL rats have lower hepatic GK activity (11.6 +/- 1.9 vs. 32.5 +/- 3.2 mU/mg protein), marked hyperglycemia (23.9 +/- 1.2 vs. 7.4 +/- 0.3 mmol/l), higher endogenous glucose production (80 +/- 3 vs. 38 +/- 3 micromol x kg(-1) x min(-1)), increased glucose-6-phosphatase flux (150 +/- 11 vs. 58 +/- 8 micromol x kg(-1) x min(-1)), and during a hyperglycemic clamp, a failure to suppress endogenous glucose production (80 +/- 7 vs. -7 +/- 4 micromol x kg(-1) x min(-1)) and promote glucose incorporation into glycogen (15 +/- 5 vs. 43 +/- 3 micromol/g liver). Treatment of ZDF rats with different doses of AdvCMV-GKL, which restored hepatic GK activity to one to two times that of ZCL rats, normalized plasma glucose levels and endogenous glucose production. During a hyperglycemic clamp, glucose production was suppressed and glucose incorporation into glycogen was normal. CONCLUSIONS: Alteration of hepatic GK activity in ZDF rats has profound effects on plasma glucose and hepatic glucose flux.

Hepatic overexpression of glycerol-sn-3-phosphate acyltransferase 1 in rats causes insulin resistance.

Fatty liver is commonly associated with insulin resistance and type 2 diabetes, but it is unclear whether triacylglycerol accumulation or an excess flux of lipid intermediates in the pathway of triacyglycerol synthesis are sufficient to cause insulin resistance in the absence of genetic or diet-induced obesity. To determine whether increased glycerolipid flux can, by itself, cause hepatic insulin resistance, we used an adenoviral construct to overexpress glycerol-sn-3-phosphate acyltransferase-1 (Ad-GPAT1), the committed step in de novo triacylglycerol synthesis. After 5-7 days, food intake, body weight, and fat pad weight did not differ between Ad-GPAT1 and Ad-enhanced green fluorescent protein control rats, but the chow-fed Ad-GPAT1 rats developed fatty liver, hyperlipidemia, and insulin resistance. Liver was the predominant site of insulin resistance; Ad-GPAT1 rats had 2.5-fold higher hepatic glucose output than controls during a hyperinsulinemic-euglycemic clamp. Hepatic diacylglycerol and lysophosphatidate were elevated in Ad-GPAT1 rats, suggesting a role for these lipid metabolites in the development of hepatic insulin resistance, and hepatic protein kinase Cepsilon was activated, providing a potential mechanism for insulin resistance. Ad-GPAT1-treated rats had 50% lower hepatic NF-kappaB activity and no difference in expression of tumor necrosis factor-alpha and interleukin-beta, consistent with hepatic insulin resistance in the absence of increased hepatic inflammation. Glycogen synthesis and uptake of 2-deoxyglucose were reduced in skeletal muscle, suggesting mild peripheral insulin resistance associated with a higher content of skeletal muscle triacylglycerol. These results indicate that increased flux through the pathway of hepatic de novo triacylglycerol synthesis can cause hepatic and systemic insulin resistance in the absence of obesity or a lipogenic diet.

A defect in glucose-induced dissociation of glucokinase from the regulatory protein in Zucker diabetic fatty rats in the early stage of diabetes.

Effect of stimulation of glucokinase (GK) export from the nucleus by small amounts of sorbitol on hepatic glucose flux in response to elevated plasma glucose was examined in 6-h fasted Zucker diabetic fatty rats at 10 wk of age. Under basal conditions, plasma glucose, insulin, and glucagon were approximately 8 mM, 2,000 pmol/l, and 60 ng/l, respectively. Endogenous glucose production (EGP) was 44 +/- 4 micromol x kg(-1) x min(-1). When plasma glucose was raised to approximately 17 mM, GK was still predominantly localized with its inhibitory protein in the nucleus. EGP was not suppressed. When sorbitol was infused at 5.6 and 16.7 micromol x kg(-1) x min(-1), along with the increase in plasma glucose, GK was exported to the cytoplasm. EGP (23 +/- 19 and 12 +/- 5 micromol x kg(-1) x min(-1)) was suppressed without a decrease in glucose 6-phosphatase flux (145 +/- 23 and 126 +/- 16 vs. 122 +/- 10 micromol x kg(-1) x min(-1) without sorbitol) but increased in glucose phosphorylation as indicated by increases in glucose recycling (122 +/- 17 and 114 +/- 19 vs. 71 +/- 11 microl x kg(-1) x min(-1)), glucose-6-phosphate content (254 +/- 32 and 260 +/- 35 vs. 188 +/- 20 nmol/g liver), fractional contribution of plasma glucose to uridine 5'-diphosphate-glucose flux (43 +/- 8 and 42 +/- 8 vs. 27 +/- 6%), and glycogen synthesis from plasma glucose (20 +/- 4 and 22 +/- 5 vs. 9 +/- 4 mumol glucose/g liver). The decreased glucose effectiveness to suppress EGP and stimulate hepatic glucose uptake may result from failure of the sugar to activate GK by stimulating the translocation of the enzyme.

Glucose toxicity is responsible for the development of impaired regulation of endogenous glucose production and hepatic glucokinase in Zucker diabetic fatty rats.

The effect of restoration of normoglycemia by a novel sodium-dependent glucose transporter inhibitor (T-1095) on impaired hepatic glucose uptake was examined in 14-week-old Zucker diabetic fatty (ZDF) rats. The nontreated group exhibited persistent endogenous glucose production (EGP) despite marked hyperglycemia. Gluconeogenesis and glucose cycling (GC) were responsible for 46 and 51% of glucose-6-phosphatase (G6Pase) flux, respectively. Net incorporation of plasma glucose into hepatic glycogen was negligible. Glucokinase (GK) and its inhibitory protein, GK regulatory protein (GKRP), were colocalized in the cytoplasm of hepatocytes. At day 7 of drug administration, EGP was slightly reduced, but G6Pase flux and GC were markedly lower compared with the nontreated group. In this case, GK and GKRP were colocalized in the nuclei of hepatocytes. When plasma glucose and insulin levels were raised during a clamp, EGP was completely suppressed and GC, glycogen synthesis from plasma glucose, and the fractional contribution of plasma glucose to uridine diphosphoglucose flux were markedly increased. GK, but not GKRP, was translocated from the nucleus to the cytoplasm. Glucotoxicity may result in the blunted response of hepatic glucose flux to elevated plasma glucose and/or insulin associated with impaired regulation of GK by GKRP in ZDF rats.