Overexpression of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase in mouse liver lowers blood glucose by suppressing hepatic glucose production

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126

Adiposity is commonly associated with adipose tissue dysfunction and many overnutrition-related metabolic diseases including type 2 diabetes. Much attention has been paid to reducing adiposity as a way to improve adipose tissue function and systemic insulin sensitivity. PFKFB3/iPFK2 is a master regulator of adipocyte nutrient metabolism. Using PFKFB3 In an in vitro system, knockdown of PFKFB3/iPFK2 in 3T3-L1 adipocytes caused a decrease in the rate of glucose incorporation into lipid but an increase in the production of reactive oxygen species. Furthermore, knockdown of PFKFB3/iPFK2 in 3T3-L1 adipocytes inappropriately altered the expression of adipokines, decreased insulin signaling, increased the phosphorylation states of JNK and NFB p65, and enhanced the production of proinflammatory cytokines. Together, these data suggest that PFKFB3/iPFK2, although contributing to adiposity, protects against diet-induced insulin resistance and adipose tissue inflammatory response.

Section: Methodsmentioning

confidence: 99%

“…Similarly, the amount of iPFK2 in the brown adipose and liver tissues was determined using Western blot. The levels of F26P 2 were measured using the 6PFK1 activation method (32).…”

Section: Methodsmentioning

confidence: 99%

Disruption of Inducible 6-Phosphofructo-2-kinase Ameliorates Diet-induced Adiposity but Exacerbates Systemic Insulin Resistance and Adipose Tissue Inflammatory Response

Huo

Guo

et al. 2010

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126

“…PFK-2 Expression Analysis-PFK-2 protein was analyzed by Western blot as reported earlier (19). In brief, PFK-2 was extracted from fresh hearts by homogenizing in buffer containing 20 mM TES, 1 mM dithiothreitol, 100 mM KCl, 5 mM EDTA, 5 mM EGTA (pH 7.8), 1.2 mM phenylmethylsulfonyl fluoride, 2.5 mg/liter leupeptin.…”

Section: Analysis Of Transgene Expressionmentioning

confidence: 99%

Cardiac Expression of Kinase-deficient 6-Phosphofructo-2-kinase/Fructose-2,6-bisphosphatase Inhibits Glycolysis, Promotes Hypertrophy, Impairs Myocyte Function, and Reduces Insulin Sensitivity

Donthi

et al. 2004

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Glycolysis is important to cardiac metabolism and reduced glycolysis may contribute to diabetic cardiomyopathy. To understand its role independent of diabetes or hypoxic injury, we modulated glycolysis by cardiacspecific overexpression of kinase-deficient 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (kd-PFK-2). PFK-2 controls the level of fructose 2,6-bisphosphate (Fru-2,6-P 2 ), an important regulator of glycolysis. Transgenic mice had over 2-fold reduced levels of Fru-2,6-P 2 . Heart weight/body weight ratio indicated mild hypertrophy. Sirius red staining for collagen was significantly increased. We observed a 2-fold elevation in glucose 6-phosphate and fructose 6-phosphate levels, whereas fructose 1,6-bisphosphate was reduced 2-fold. Pathways branching off of glycolysis above phosphofructokinase were activated as indicated by over 2-fold elevated UDP-N-acetylglucosamine and glycogen. The kd-PFK-2 transgene significantly inhibited glycolysis in perfused hearts. Insulin stimulation of metabolism and Akt phosphorylation were sharply reduced. In addition, contractility of isolated cardiomyocytes was impaired during basal and hypoxic incubations. The present study shows that cardiac overexpression of kinase-deficient PFK-2 reduces cardiac glycolysis that produced negative consequences to the heart including hypertrophy, fibrosis, and reduced cardiomyocyte function. In addition, metabolic and signaling responses to insulin were significantly decreased.

“…For example, Kuwajima et al (10) reported continual production of liver glycogen in sucrose-fed rats despite high levels of Fru-2,6-P 2 , and Hue and Bartrons (11) observed stimulated glucose production by glucagon in isolated hepatocytes regardless of the levels of Fru-2,6-P 2 . Levels of Fru-2,6-P 2 have also been manipulated by recombinant adenovirus overexpression of the bifunctional enzyme phosphofructo-2-kinase:fructose-2,6-bisphosphatase (the enzyme that catalyzes both synthesis and degradation of Fru-2,6-P 2 ) in mice and rats in vivo (12)(13)(14). Here, increased hepatic Fru-2,6-P 2 in vivo actually resulted in increased glycogen synthesis from [1- 13 C]glucose via the indirect pathway (14), thereby suggesting that activation of glycolysis by Fru-2,6-P 2 is more important than inhibition of glyconeogenesis in vivo.…”

mentioning

confidence: 99%

Increased Hepatic Fructose 2,6-Bisphosphate after an Oral Glucose Load Does Not Affect Gluconeogenesis

Jin

Uyeda

Kawaguchi

et al. 2003

The generally accepted metabolic concept that fructose 2,6-bisphosphate (Fru-2,6-P 2 ) inhibits gluconeogenesis by directly inhibiting fructose 1,6-bisphosphatase is based entirely on in vitro observations. To establish whether gluconeogenesis is indeed inhibited by Fru-2,6-P 2 in intact animals, a novel NMR method was developed using [U-13 C]glucose and 2 H 2 O as tracers. The method was used to estimate the sources of plasma glucose from gastric absorption of oral [U-13 C]glucose, from gluconeogenesis, and from glycogen in 24-h fasted rats. Liver Fru-2,6-P 2 increased ϳ10-fold shortly after the glucose load, reached a maximum at 60 min, and then dropped to base-line levels by 150 min. The gastric contribution to plasma glucose reached ϳ50% at 30 min after the glucose load and gradually decreased thereafter. Although the contribution of glycogen to plasma glucose was small, glucose formed from gluconeogenesis was substantial throughout the study period even when liver Fru-2,6-P 2 was high. Liver glycogen repletion was also brisk throughout the study period, reaching ϳ30 mol/g at 3 h. These data demonstrate that Fru-2,6-P 2 does not inhibit gluconeogenesis significantly in vivo.Plasma glucose is preserved by gluconeogenesis after exhaustion of glycogen stores during a moderate fast. Following an oral glucose load, gluconeogenesis is thought to be modulated by allosteric regulation of fructose-1,6-bisphosphatase (1). This is an eminently satisfying model because a key regulatory site in glycolysis and gluconeogenesis occurs at level of fructose-6-P and fructose-1,6-P 2 (Fig. 1). Phosphofructokinase-1, the glycolytic enzyme, is potently activated by fructose-2,6-P 2 , whereas fructose-1,6-bisphosphatase, the gluconeogenic enzyme, is thought to be inhibited by this same effector molecule (2, 3). Thus, by regulating the activities of phosphofructo-1-kinase and fructose-1,6-bisphosphatase in a reciprocal manner, Fru-2,6-P 2 is thought to serve as an elegant regulator of glucose usage/production by the liver after an oral glucose load. This generally accepted model is based on kinetic analysis of fructose-1,6-bisphosphatase in vitro, which shows that Fru-2,6-P 2 competes with Fru-1,6-P 2 for the active site of fructose-1,6-bisphosphatase and that both molecules have similar affinity constants, 1-5 M (4 -6). However, the in vivo concentrations of Fru-1,6-P 2 in fasted and fed livers are 20 and 35 M, respectively, whereas those of Fru-2,6-P 2 are 1 and 8 M, respectively (7-9). This suggests that it would be difficult for Fru-2,6-P 2 to have a significant direct effect on fructose-1,6-bisphosphatase activity in vivo based simply upon concentration differences. Some evidence has been presented that suggests Fru-2,6-P 2 is not a potent inhibitor of gluconeogenesis in intact animals. For example, Kuwajima et al. (10) reported continual production of liver glycogen in sucrose-fed rats despite high levels of Fru-2,6-P 2 , and Hue and Bartrons (11) observed stimulated glucose production by glucagon in isolated hepatocytes reg...