Bernard R. Landau

One Week’s Treatment With the Long-Acting Glucagon-Like Peptide 1 Derivative Liraglutide (NN2211) Markedly Improves 24-h Glycemia and α- and β-Cell Function and Reduces Endogenous Glucose Release in Patients with Type 2 Diabetes

Glucagon-like peptide 1 (GLP-1) is potentially a very attractive agent for treating type 2 diabetes. We explored the effect of short-term (1 week) treatment with a GLP-1 derivative, liraglutide (NN2211), on 24-h dynamics in glycemia and circulating free fatty acids, islet cell hormone profiles, and gastric emptying during meals using acetaminophen. Furthermore, fasting endogenous glucose release and gluconeogenesis (3-(3)H-glucose infusion and (2)H(2)O ingestion, respectively) were determined, and aspects of pancreatic islet cell function were elucidated on the subsequent day using homeostasis model assessment and first- and second-phase insulin response during a hyperglycemic clamp (plasma glucose approximately 16 mmol/l), and, finally, on top of hyperglycemia, an arginine stimulation test was performed. For accomplishing this, 13 patients with type 2 diabetes were examined in a double-blind, placebo-controlled crossover design. Liraglutide (6 micro g/kg) was administered subcutaneously once daily. Liraglutide significantly reduced the 24-h area under the curve for glucose (P = 0.01) and glucagon (P = 0.04), whereas the area under the curve for circulating free fatty acids was unaltered. Twenty-four-hour insulin secretion rates as assessed by deconvolution of serum C-peptide concentrations were unchanged, indicating a relative increase. Gastric emptying was not influenced at the dose of liraglutide used. Fasting endogenous glucose release was decreased (P = 0.04) as a result of a reduced glycogenolysis (P = 0.01), whereas gluconeogenesis was unaltered. First-phase insulin response and the insulin response to an arginine stimulation test with the presence of hyperglycemia were markedly increased (P < 0.001), whereas the proinsulin/insulin ratio fell (P = 0.001). The disposition index (peak insulin concentration after intravenous bolus of glucose multiplied by insulin sensitivity as assessed by homeostasis model assessment) almost doubled during liraglutide treatment (P < 0.01). Both during hyperglycemia per se and after arginine exposure, the glucagon responses were reduced during liraglutide administration (P < 0.01 and P = 0.01). Thus, 1 weeks treatment with a single daily dose of the GLP-1 derivative liraglutide, operating through several different mechanisms including an ameliorated pancreatic islet cell function in individuals with type 2 diabetes, improves glycemic control throughout 24 h of daily living, i.e., prandial and nocturnal periods. This study further emphasizes GLP-1 and its derivatives as a promising novel concept for treatment of type 2 diabetes.

Contributions of gluconeogenesis to glucose production in the fasted state.

Healthy subjects ingested 2H2O and after 14, 22, and 42 h of fasting the enrichments of deuterium in the hydrogens bound to carbons 2, 5, and 6 of blood glucose and in body water were determined. The hydrogens bound to the carbons were isolated in formaldehyde which was converted to hexamethylenetetramine for assay. Enrichment of the deuterium bound to carbon 5 of glucose to that in water or to carbon 2 directly equals the fraction of glucose formed by gluconeogenesis. The contribution of gluconeogenesis to glucose production was 47 +/- 49% after 14 h, 67 +/- 41% after 22 h, and 93 +/- 2% after 42 h of fasting. Glycerols conversion to glucose is included in estimates using the enrichment at carbon 5, but not carbon 6. Equilibrations with water of the hydrogens bound to carbon 3 of pyruvate that become those bound to carbon 6 of glucose and of the hydrogen at carbon 2 of glucose produced via glycogenolysis are estimated from the enrichments to be approximately 80% complete. Thus, rates of gluconeogenesis can be determined without corrections required in other tracer methodologies. After an overnight fast gluconeogenesis accounts for approximately 50% and after 42 h of fasting for almost all of glucose production in healthy subjects.

Use of 2H2O for estimating rates of gluconeogenesis. Application to the fasted state.

A method is introduced for estimating the contribution of gluconeogenesis to glucose production. 2H2O is administered orally to achieve 0.5% deuterium enrichment in body water. Enrichments are determined in the hydrogens bound to carbons 2 and 6 of blood glucose and in urinary water. Enrichment at carbon 6 of glucose is assayed in hexamethylenetetramine, formed from formaldehyde produced by periodate oxidation of the glucose. Enrichment at carbon 2 is assayed in lactate formed by enzymatic transfer of the hydrogen from glucose via sorbitol to pyruvate. The fraction gluconeogenesis contributes to glucose production equals the ratio of the enrichment at carbon 6 to that at carbon 2 or in urinary water. Applying the method, the contribution of gluconeogenesis in healthy subjects was 23-42% after fasting 14 h, increasing to 59-84% after fasting 42 h. Enrichment at carbon 2 to that in urinary water was 1.12 +/- 0.13. Therefore, the assumption that hydrogen equilibrated during hexose-6-P isomerization was fulfilled. The 3H/14C ratio in glucose formed from [3-3H,3-14C]lactate given to healthy subjects was 0.1 to 0.2 of that in the lactate. Therefore equilibration during gluconeogenesis of the hydrogen bound to carbon 6 with that in body water was 80-90% complete, so that gluconeogenesis is underestimated by 10-20%. Glycerols contribution to gluconeogenesis is not included in these estimates. The method is applicable to studies in humans of gluconeogenesis at safe doses of 2H2O.

Quantifying the carboxylation of pyruvate in pancreatic islets.

Pyruvate has been estimated to enter the citric acid cycle in islets by carboxylation to the same extent or more than by decarboxylation. Those estimates were made assuming the dimethyl esters of [1,4-14C]succinate and [2-3-14C]succinate, incubated with islets at a concentration of 10 mM, gave the same ratio of 14CO yields as if [1-14C]acetate and [2-14C]acetate had been incubated. The labeled succinates, at 10 mM, but not 1 mM, are now shown to give ratios higher than the labeled acetates at those concentrations and therefore higher estimates when related to yields from [2-14C]glucose and [6-14C]glucose. Using the labeled acetate ratios in paired incubations, the rate of pyruvate carboxylation is still estimated to be about two-thirds the rate of pyruvate decarboxylation. Participation of the malic enzyme-catalyzed reaction explains the greater ratio of yields of 14CO from the succinates at 10 mM than 1 mM and increases in those ratios on glucose addition and can account for the removal from the citric acid cycle of oxaloacetate carbon formed in the carboxylation.

Contributions of net hepatic glycogenolysis and gluconeogenesis to glucose production in cirrhosis

Net hepatic glycogenolysis and gluconeogenesis were examined in normal ( n = 4) and cirrhotic ( n = 8) subjects using two independent methods [13C nuclear magnetic resonance spectroscopy (NMR) and a2H2O method]. Rates of net hepatic glycogenolysis were calculated by the change in hepatic glycogen content before (∼11:00 PM) and after (∼7:00 AM) an overnight fast using13C NMR and magnetic resonance imaging. Gluconeogenesis was calculated as the difference between the rates of glucose production determined with an infusion of [6,6-2H2]glucose and net hepatic glycogenolysis. In addition, the contribution of gluconeogenesis to glucose production was determined by the2H enrichment in C-5/C-2 of blood glucose after intake of2H2O (5 ml/kg body water). Plasma levels of total and free insulin-like growth factor I (IGF-I) and IGF-I binding proteins-1 and -3 were significantly decreased in the cirrhotic subjects ( P < 0.01 vs. controls). Postprandial hepatic glycogen concentrations were 34% lower in the cirrhotic subjects ( P = 0.007). Rates of glucose production were similar between the cirrhotic and healthy subjects [9.0 ± 0.9 and 10.0 ± 0.8 μmol ⋅ kg body wt-1 ⋅ min-1, respectively]. Net hepatic glycogenolysis was 3.5-fold lower in the cirrhotic subjects ( P = 0.01) and accounted for only 13 ± 6% of glucose production compared with 40 ± 10% ( P = 0.03) in the control subjects. Gluconeogenesis was markedly increased in the cirrhotic subjects and accounted for 87 ± 6% of glucose production vs. controls: 60 ± 10% ( P = 0.03). Gluconeogenesis in the cirrhotic subjects, as determined from the2H enrichment in glucose C-5/C-2, was also increased and accounted for 68 ± 3% of glucose production compared with 54 ± 2% ( P = 0.02) in the control subjects. In conclusion, cirrhotic subjects have increased rates of gluconeogenesis and decreased rates of net hepatic glycogenolysis compared with control subjects. These alterations are likely important contributing factors to their altered carbohydrate metabolism.

Inhibition of Fructose 1,6-Bisphosphatase Reduces Excessive Endogenous Glucose Production and Attenuates Hyperglycemia in Zucker Diabetic Fatty Rats

Gluconeogenesis is increased in type 2 diabetes and contributes significantly to fasting and postprandial hyperglycemia. We recently reported the discovery of the first potent and selective inhibitors of fructose 1,6-bisphosphatase (FBPase), a rate-controlling enzyme of gluconeogenesis. Herein we describe acute and chronic effects of the lead inhibitor, MB06322 (CS-917), in rodent models of type 2 diabetes. In fasting male ZDF rats with overt diabetes, a single dose of MB06322 inhibited gluconeogenesis by 70% and overall endogenous glucose production by 46%, leading to a reduction in blood glucose of >200 mg/dl. Chronic treatment of freely feeding 6-week-old male Zucker diabetic fatty (ZDF) rats delayed the development of hyperglycemia and preserved pancreatic function. Elevation of lactate (∼1.5-fold) occurred after 4 weeks of treatment, as did the apparent shunting of precursors into triglycerides. Profound glucose lowering (∼44%) and similar metabolic ramifications were associated with 2-week intervention therapy of 10-week-old male ZDF rats. In high-fat diet–fed female ZDF rats, MB06322 treatment for 2 weeks fully attenuated hyperglycemia without evidence of metabolic perturbation other than a modest reduction in glycogen stores (∼20%). The studies confirm that excessive gluconeogenesis plays an integral role in the pathophysiology of type 2 diabetes and suggest that FBPase inhibitors may provide a future treatment option.

Glucose Cycling in Islets From Healthy and Diabetic Rats

Pancreatic islets from healthy (control) and neonatally streptozocin-induced diabetic (STZ-D) rats, a model for non-insulin-dependent diabetes mellitus, were incubated with 3H2O and 5.5 or 16.7 m M glucose. At 5.5 mM glucose, no detectable [3H]glucose was formed. At 16.7 mM, 2.2 patom · islet−1 · h−1 of 3H was incorporated into glucose by the control islets and 5.4 · patom · islet−1 · h−1 by STZ-D islets. About 75% of the 3H was bound to carbon-2 of the glucose. Glucose utilization was 35.3 pmol · islet−1 · h−1 by the control and 19.0 pmol · islet−1 · h−1 by the STZ-D islets· Therefore, 4.5% of the glucose-6-phosphate formed by the control islets and 15.7% by the STZ-D islets was dephosphorylated. This presumably occurred in the β-cells of the islets catalyzed by glucose-6-phosphatase. An increased glucose cycling, i.e., glucose → glucose-6-phosphate → glucose, in islets of STZ-D rats may contribute to the decreased insulin secretion found in these animals.

Quantitation of pathways of ethanol metabolism

A method has been developed for estimating the sum of the contributions to ethanol oxidation by the microsomal ethanol-oxidizing system (MEOS) and catalase in the intact liver cell. It depends upon a comparison of the fate of the R hydrogen of ethanol and the hydrogen bound to carbon-2 of sorbitol under identical conditions. Limitations of the approach, particularly as regards isotopic effects, are defined. Under the condition of incubation of liver slices from rat and monkey at a concentration of ethanol of 3 mg/ml and from rat at 1 mg/ml, alcohol dehydrogenase catalysis is concluded to account, on the average, for 89% or more of the initial metabolism of ethanol. As by-products of this study, the stereospecificity of the sorbitol dehydrogenase-catalyzed reaction is shown to be of the A type in the rat, and evidence is obtained for the irreversibility of sorbitol oxidation in the intact liver cell.

A study on the equivalence of metabolic patterns in rat adipose tissue: Insulin versus glucose concentration

Abstract The rat epididymal fat pad has been incubated with increasing concentrations of glucose. The metabolic patterns obtained in the absence and presence of insulin are quantitatively different in several respects. In particular, insulin appears to stimulate glycogen synthesis, the oxidation of carbon 1 of glucose to CO 2 and fatty acid synthesis, while increasing glucose concentrations favor the oxidation of carbon 6 of glucose to CO 2 and the synthesis of glycerol from glucose. The mechanism of action of insulin in adipose tissue is considered in the light of these observations. It is suggested that insulin action on glucose permeability or activation alone does not adequately explain the patterns observed.

Adrenal Steroids and Carbohydrate Metabolism

Publisher Summary This chapter describes adrenal steroids and carbohydrate metabolism. It has generally been concluded that adrenal steroids increase carbohydrate content when administered to an animal. In the strictest sense carbohydrate content of the entire carcass of the animal would have to be determined to justify this conclusion. In some tissues, content has been estimated as glucose, following preliminary treatment of homogenates to hydrolyse polysaccharides and phosphorylated sugars. Usually carbohydrate content has meant glucose and glycogen content as measured by more or less specific analytical procedures. Increased carbohydrate content after steroid administration to intact or adrenalectomized animals appears to be well established, but the mechanism by which it occurs remains in doubt. Increased content can be consequent to either increased glucose synthesis (i.e., increased gluconeogenesis) or to decreased utilization, or both. Evidence considered to support all three of these possibilities has accumulated. Data on glucose production and utilization following adrenal steroid administration are inadequate. In the dog during the first hour after cortisol infusion, hepatic glucose output appears to be decreased.