Publications

Despite altered regulation of insulin signaling, Pten(+/-) heterodeficient standard diet-fed mice, approximately 4 months old, exhibit normal fasting glucose and insulin levels. We report here a stable isotope flux phenotyping study of this "silent" phenotype, in which tissue-specific insulin effects in whole-body Pten(+/-)-deficient mice were dissected in vivo. Flux phenotyping showed gain of function in Pten(+/-) mice, seen as increased peripheral glucose disposal, and compensation by a metabolic feedback mechanism that 1) decreases hepatic glucose recycling via suppression of glucokinase expression in the basal state to preserve hepatic glucose production and 2) increases hepatic responsiveness in the fasted-to-fed transition. In Pten(+/-) mice, hepatic gene expression of glucokinase was 10-fold less than wild-type (Pten(+/+)) mice in the fasted state and reached Pten(+/+) values in the fed state. Glucose-6-phosphatase expression was the same for Pten(+/-) and Pten(+/+) mice in the fasted state, and its expression for Pten(+/-) was 25% of Pten(+/+) in the fed state. This study demonstrates how intra- and interorgan flux compensations can preserve glucose homeostasis (despite a specific gene defect that accelerates glucose disposal) and how flux phenotyping can dissect these tissue-specific flux compensations in mice presenting with a "silent" phenotype.

Fatty liver is a common feature of both obesity and lipodystrophy, reflecting compromised adipose tissue function. The lipin-deficient fatty liver dystrophy (fld) mouse is an exception, as there is lipodystrophy without a fatty liver. Using a combination of indirect calorimetry and stable-isotope flux phenotyping, we determined that fld mice exhibit abnormal fuel utilization throughout the diurnal cycle, with increased glucose oxidation near the end of the fasting period and increased fatty acid oxidation during the feeding period. The mechanisms underlying these alterations include a twofold increase compared with wild-type mice in tissue glycogen storage during the fed state, a 40% reduction in hepatic glucose production in the fasted state, and a 27-fold increase in de novo fatty acid synthesis in liver during the fed state. Thus, the inability to store energy in adipose tissue in the fld mouse leads to a compensatory increase in glycogen storage for use during the fasting period and reliance upon hepatic fatty acid synthesis to provide fuel for peripheral tissues during the fed state. The increase in hepatic fatty acid synthesis and peripheral utilization provides a potential mechanism to ameliorate fatty liver in the fld that would otherwise occur as a consequence of adipose tissue dysfunction.

Our previous work led to the hypothesis that peroxisomal proliferator-activated receptor alpha (PPAR alpha) modulates insulin action in a compensatory fashion for hepatic glucose balance vs. peripheral glucose disposal. Therefore, we have examined the expression of insulin-dependent gluconeogenic/glycolytic/pentose cycle enzymes and compared these to insulin responsiveness for peripheral vs. hepatic substrate flux and futile cycling in the PPAR alpha knockout mouse. Hepatic gluconeogenic flux, glucose absorption, clearance and recycling, as well as in vivo glucose disposal were evaluated using new mass isotopomer methods. Insulin-dependent gluconeogenic/glycolytic/pentose cycle enzyme expression and glucose futile cycling were diminished; however, glucose disappearance was increased. This supports the hypothesis of hepatic insulin resistance and increased peripheral glucose uptake as compensatory events secondary to the decrease in fatty acid oxidation characteristic of the PPAR alpha knockout. We conclude that 1) the loss of PPAR alpha results in lower expression levels and diminished response to meal regulation for gluconeogenic/glycolytic enzyme expression; and 2) consequently, substrate/futile cycling of glucose is decreased when PPAR alpha is absent despite increased gluconeogenesis. The compensatory changes in liver and peripheral tissue substrate flux and the resultant adaptation for enzyme expression in the liver to have a diminished insulin dependence reflect the loosely linked correlation between phenotype and genotype in hepatic glucose metabolism.

Increased glucose cycling between glucose and glucose-6-phosphate is characteristic of insulin resistance and hyperglycemia seen with Type II diabetes. Traditionally, glucose cycling is determined by the difference between hepatic glucose output measured with separate [2-3H]glucose and [6-3H]glucose infusions. We demonstrate a novel method for determining hepatic glucose recycling from an intraperitoneal glucose tolerance test (IPGTT). A single tracer, [1, 2-13C(2)]glucose (a M2 glucose isotopomer), was administered at 1mg/g body weight to 4-month-old C57BL/6 mice. Hepatic glucose recycling was monitored by the appearance of a plasma M1 isotopomer of glucose, which is produced by the action of the pentose cycle on the M2 glucose isotopomer in the liver. The initial M2 enrichment was 56% and decreased to 13% at the end of 3 h, and the M1 enrichment peaked at 2 h. The ratio of plasma M1/M2 glucose increased linearly with time to approximately 25%, and the regression of the M1/M2 ratio against time gives a slope, termed the in vivo glucose-dependent futile recycling rate constant k(HR). k(HR) estimates glucose/glucose-6-phosphate futile cycling, along with glucose recycling through the pentose cycle. These observations demonstrate complex substrate cycling during an IPGTT using a single stable isotope tracer.

The hypoglycemia seen in the fasting PPARalpha null mouse is thought to be due to impaired liver fatty acid beta-oxidation. The etiology of hypoglycemia in the PPARalpha null mouse was determined via stable isotope studies. Glucose, lactate, and glycerol flux was assessed in the fasted and fed states in 4-month-old PPARalpha null mice and in C57BL/6 WT maintained on standard chow using a new protocol for flux assessment in the fasted and fed states. Hepatic glucose production (HGP) and glucose carbon recycling were estimated using [U-(13)C(6)]glucose, and HGP, lactate, and glycerol turnover was estimated utilizing either [U-(13)C(3)]lactate or [2-(13)C]glycerol infused subcutaneously via Alza miniosmotic pumps. At the end of a 17-h fast, HGP was higher in the PPARalpha null mice than in WT by 37% (p < 0.01). However, recycling of glucose carbon from lactate back to glucose was lower in the PPARalpha null than in WT (39% versus 51%, p < 0.02). The lack of conversion of lactate to glucose was confirmed using an [U-(13)C(3)]lactate infusion. In the fasted state, HGP from lactate and lactate production were decreased by 65 and 55%, respectively (p < 0.05) in PPARalpha null mice. In contrast, when [2-(13)C]glycerol was infused, glycerol production and HGP from glycerol increased by 80 and 250%, respectively (p < 0.01), in the fasted state of PPARalpha null mice. The increased HGP from glycerol was not suppressed in the fed state. While little change was evident for phosphoenolpyruvate carboxykinase (PEPCK) expression, pyruvate kinase expression was decreased 16-fold in fasted PPARalpha null mice as compared with the wild-type control. The fasted and fed insulin levels were comparable, but blood glucose levels were lower in the PPARalpha null mice than in controls. In conclusion, PPARalpha receptor function creates a setpoint for a metabolic network that regulates the rate and route of HGP in the fasted and fed states, in part, by controlling the flux of glycerol and lactate between the triose-phosphate and pyruvate/lactate pools.

Liver and skeletal muscle isoforms of 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF2K/Fru-2,6-P(2)ase) isoenzymes are products of alternatively spliced first exons of the same gene, with common kinase and bisphosphatase domains. The muscle-specific exon-1 encodes nine unique amino acids, that lack the cAMP-dependent protein kinase (PK-A) phosphorylation site, and differ in sequence from those encoded by the liver-specific exon-1 (32 amino acids), contributing to its much lower affinity for fructose 6-phosphate (Fru-6-P). PK-A phosphorylation of the liver isoform at Ser(32) reduces the affinity of the kinase for Fru-6-P, and stimulates the bisphosphatase V(max). In the present study, we have defined the locus of interaction of the N-terminal residues with the N-terminal kinase and C-terminal domains by successive N- and C-terminal deletions. This study shows that: (1) residues Gly(5)-Glu(6)-Leu(7) of the liver isoform are responsible for increasing the affinity of 6PF2K for Fru-6-P, maintaining the inhibition of Fru-2,6-P(2)ase activity, and mediating the effects of PK-A phosphorylation on the two activities; (2) the loss of Fru-6-P inhibition of the bisphosphatase and the enhancement of its V(max), rather than the inhibition of the kinase, may be responsible for the behaviour of the muscle isoform primarily as a bisphosphatase; (3) the composition of residues 24-32 of the liver form appears to confer the enhanced kinase catalytic rate of this form over that of the muscle isoform. It is concluded that specific regions of the N-terminus of liver and skeletal muscle 6PF2K/Fru-2,6-P(2)ase have a role in adapting the two activities to work in the physiological range of pH and substrate concentrations found in each particular tissue.

Whereas many reports substantiated the suitability of using [2-(13)C]glycerol and Mass Isotoper Distribution Analysis for gluconeogenesis, the use of [(13)C]glycerol had been shown to give lower estimates of gluconeogenesis (GNG). The reason for the underestimation has been attributed to asymmetric isotope incorporation during gluconeogenesis as well as zonation of gluconeogenic enzymes and a [(13)C]glycerol gradient across the liver. Since the cycling of glycerol carbons through the pentose cycle pathways can introduce asymmetry in glucose labeling pattern and tracer dilution, we present here a study of the role of the pentose cycle in gluconeogenesis in Fao cells. The metabolic regulation of glucose release and gluconeogenesis by insulin was also studied. Serum-starved cells were incubated for 24 h in Dulbecco's modified Eagle's media containing 1.5 mm [U-(13)C]glycerol. Mass isotopomers of whole glucose from medium or glycogen and those of the C-1-C-4 fragment were highly asymmetrical, typical of that resulting from the cycling of glucose carbon through the pentose cycle. Substantial exchange of tracer between hexose and pentose intermediates was observed. Our results offer an alternative mechanism for the asymmetrical labeling of glucose carbon from triose phosphate. The scrambling of (13)C in hexose phosphate via the pentose phosphate cycle prior to glucose release into the medium is indistinguishable from dilution of labeled glucose by glycogen using MIDA and probably accounts for the underestimation of GNG using (13)C tracer methods.

The hepatic bifunctional enzyme, 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (6PF-2-K/Fru-2,6-P2ase), E.C. 2.7-1-105/E.C. 3-1-3-46, is one member of a family of unique bifunctional proteins that catalyze the synthesis and degradation of the regulatory metabolite fructose-2,6-bisphosphate (Fru-2,6-P2). Fru-2,6-P2 is a potent activator of the glycolytic enzyme 6-phosphofructo-1-kinase and an inhibitor of the gluconeogenic enzyme fructose-1,6-bisphosphatase, and provides a switching mechanism between these two opposing pathways of hepatic carbohydrate metabolism. The activities of the hepatic 6PF-2-K/Fru-2,6-P2ase isoform are reciprocally regulated by a cyclic AMP-dependent protein kinase (cAPK)-catalyzed phosphorylation at a single NH2-terminal residue, Ser-32. Phosphorylation at Ser-32 inhibits the kinase and activates the bisphosphatase, in part through an electrostatic mechanism. Substitution of Asp for Ser-32 mimics the effects of cAPK-catalyzed phosphorylation. In the dephosphorylated homodimer, the NH2- and COOH-terminal tail regions also have an interaction with their respective active sites on the same subunit to produce an autoregulatory inhibition of the bisphosphatase and activation of the kinase. In support of this hypothesis, deletion of either the NH2- or COOH-terminal tail region, or both regions, leads to a disruption of these interactions with a maximal activation of the bisphosphatase. Inhibition of the kinase is observed with the NH2-truncated forms, in which there is also a diminution of cAPK phosphorylation to decrease the Km for Fru-6-P. Phosphorylation of the bifunctional enzyme by cAPK disrupts these autoregulatory interactions, resulting in inhibition of the kinase and activation of the bisphosphatase. Therefore, effects of cyclic AMP-dependent phosphorylation are mediated by a combination of electrostatic and autoregulatory control mechanisms.

The relationship between the in vivo insulin secretory responsiveness of the pancreatic beta-cell to glucose and the flux of glucose through the enzyme glucokinase was investigated in six subjects with heterozygous glucokinase mutations and in six matched control subjects. This was done by combining data published previously on the in vivo dose-response relationships between glucose and insulin secretion and on the in vitro enzymatic properties of wild-type and mutant forms of glucokinase. The flux of glucose through glucokinase (GK flux) in these subjects was estimated using a model based on the approximate Michaelis-Menten kinetics of wild-type and mutant forms of the enzyme. In two subjects with glucokinase mutations, which resulted in only a small reduction in enzymatic activity, the decrease in insulin secretion was directly proportional to the decrease in GK flux predicted using a Michaelis-Menten model for both mutant and wild-type glucokinase. However, in four subjects with glucokinase mutations, which resulted in severe reductions in enzymatic activity, insulin secretion was reduced compared with control subjects but less than predicted. This latter result implies the existence of a compensatory change in the beta-cells of such subjects, which results in a relative increase in insulin secretory response. We propose modifications to the simple model relating glucose concentration and GK flux, including glucose-induced overexpression of the normal allele and a role of glucokinase regulatory protein. The modifications take into account the possibility that the degree of compensation may be directly related to the severity of the mutation.