Christopher D. Hardin

PGC-1α overexpression results in increased hepatic fatty acid oxidation with reduced triacylglycerol accumulation and secretion

Studies have shown that decreased mitochondrial content and function are associated with hepatic steatosis. We examined whether peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α) overexpression and a subsequent increase in mitochondrial content and function in rat primary hepatocytes (in vitro) and Sprague-Dawley rats (in vivo) would comprehensively alter mitochondrial lipid metabolism, including complete (CO(2)) and incomplete (acid-soluble metabolites) fatty acid oxidation (FAO), tricarboxylic acid cycle flux, and triacylglycerol (TAG) storage and export. PGC-1α overexpression in primary hepatocytes produced an increase in markers of mitochondrial content and function (citrate synthase, mitochondrial DNA, and electron transport system complex proteins) and an increase in FAO, which was accompanied by reduced TAG storage and TAG secretion compared with control. Also, the PGC-1α-overexpressing hepatocytes were protected from excess TAG accumulation following overnight lipid treatment. PGC-1α overexpression in hepatocytes lowered expression of genes critical to VLDL assembly and secretion (apolipoprotein B and microsomal triglyceride transfer protein). Adenoviral transduction of rats with PGC-1α resulted in a liver-specific increase in PGC-1α expression and produced an in vivo liver phenotype of increased FAO via increased mitochondrial function that also resulted in reduced hepatic TAG storage and decreased plasma TAG levels. In conclusion, overexpression of hepatic PGC-1α and subsequent increases in FAO through elevated mitochondrial content and/or function result in reduced TAG storage and secretion in the in vitro and in vivo milieu.

Overexpression of caveolin-1 results in increased plasma membrane targeting of glycolytic enzymes: the structural basis for a membrane associated metabolic compartment.

Although membrane‐associated glycolysis has been observed in a variety of cell types, the mechanism of localization of glycolytic enzymes to the plasma membrane is not known. We hypothesized that caveolin‐1 (CAV‐1) serves as a scaffolding protein for glycolytic enzymes and may play a role in the organization of cell metabolism. To test this hypothesis, we over‐expressed CAV‐1 in cultured A7r5 (rat aorta vascular smooth muscle; VSM) cells. Confocal immunofluorescence microscopy was used to study the distribution of phosphofructokinase (PFK) and CAV‐1 in the transfected cells. Areas of interest (AOI) were analyzed in a central Z‐plane across the cell transversing the perinuclear region. To quantify any shift in PFK localization resulting from CAV‐1 over‐expression, we calculated a periphery to center (PC) index by taking the average of the two outer AOIs from each membrane region and dividing by the central one or two AOIs. We found the PC index to be 1.92 ± 0.57 (mean ± SEM, N = 8) for transfected cells and 0.59 ± 0.05 (mean ± SEM, N = 11) for control cells. Colocalization analysis demonstrated that the percentage of PFK associated with CAV‐1 increased in transfected cells compared to control cells. The localization of aldolase (ALD) was also shifted towards the plasma membrane (and colocalized with PFK) in CAV‐1 over‐expressing cells. These results demonstrate that CAV‐1 creates binding sites for PFK and ALD that may be of higher affinity than those binding sites localized in the cytoplasm. We conclude that CAV‐1 functions as a scaffolding protein for PFK, ALD and perhaps other glycolytic enzymes, either through direct interaction or accessory proteins, thus contributing to compartmented metabolism in vascular smooth muscle. J. Cell. Biochem. 98: 861–871, 2006.

Expression of caveolin-1 in lymphocytes induces caveolae formation and recruitment of phosphofructokinase to the plasma membrane

Compartmentation of carbohydrate metabolism has been shown in a wide range of tissues including reports of one compartment of glycolysis associated with the plasma membrane of cells. However, only in the erythrocyte has the physical basis for plasma membrane‐associated glycolytic pathway been established. We have previously found that phosphofructokinase (PFK) appeared to colocalize with the fairly ubiquitous plasma membrane protein caveolin‐1 (CAV‐1), consistent with a role for CAV‐1 as an anchor for glycolysis to the plasma membrane. To test the hypothesis that CAV‐1 functions as a scaffolding protein for PFK, we transfected human lymphocytes (a cell without CAV‐1 expression) with human CAV‐1 cDNA. We demonstrate that expression of CAV‐1 in lymphocytes results in the formation of caveolae at the plasma membrane and affects the subcellular localization of PFK by recruiting PFK to the plasma membrane. Targeting of PFK by CAV‐1 also was validated by the significant colocalization between the proteins after transfection, which resulted in a correlation of 0.97 ± 0.004 between the two fluorophores. This finding is significant in as much as it illustrates the CAV‐1 feasibility of generating binding sites for glycolytic enzymes on the plasma membrane. We therefore conclude that CAV‐1 functions as a scaffolding protein for PFK and that this may contribute to the elucidation of the basis for carbohydrate compartmentation to the plasma membrane in a wide variety of cell types.

Glycolytic flux in permeabilized freshly isolated vascular smooth muscle cells

To determine whether channeling of glycolytic intermediates can occur in vascular smooth muscle (VSM), we permeabilized freshly isolated VSM cells from hog carotid arteries with dextran sulfate. The dextran sulfate-treated cells did not exclude trypan blue, a dye with molecular weight of approximately 1,000. If glycolytic intermediates freely diffuse, plasmalemmal permeabilization would allow intermediates to exit the cell and glycolytic flux should cease. We incubated permeabilized and nonpermeabilized cells with 5 mM [1-13C]glucose at 37 degrees C for 3 h. 13C nuclear magnetic resonance (NMR) was used to determine relative [3-13C]lactate production and to identify any 13C-labeled glycolytic intermediates that exited from the permeabilized cells. [3-13C]lactate production from [1-13C]glucose was decreased by an average of 32% (n = 6) in permeabilized cells compared with intact cells. No 13C-labeled glycolytic intermediates were observed in the bathing solution of permeabilized cells. We conclude that channeling of glycolytic intermediates can occur in VSM cells.To determine whether channeling of glycolytic intermediates can occur in vascular smooth muscle (VSM), we permeabilized freshly isolated VSM cells from hog carotid arteries with dextran sulfate. The dextran sulfate-treated cells did not exclude trypan blue, a dye with molecular weight of ∼1,000. If glycolytic intermediates freely diffuse, plasmalemmal permeabilization would allow intermediates to exit the cell and glycolytic flux should cease. We incubated permeabilized and nonpermeabilized cells with 5 mM [1-13C]glucose at 37°C for 3 h. 13C nuclear magnetic resonance (NMR) was used to determine relative [3-13C]lactate production and to identify any13C-labeled glycolytic intermediates that exited from the permeabilized cells. [3-13C]lactate production from [1-13C]glucose was decreased by an average of 32% ( n = 6) in permeabilized cells compared with intact cells. No13C-labeled glycolytic intermediates were observed in the bathing solution of permeabilized cells. We conclude that channeling of glycolytic intermediates can occur in VSM cells.

Fructose-1,6-bisphosphate as a metabolic substrate in hog ileum smooth muscle during hypoxial

Exogenously applied fructose-1,6-bisphosphate has been reported to be effective in preventing some damage to the small intestine during ischemia. To determine whether exogenously applied fructose-1,6-bisphosphate protects ileum smooth muscle from damage from hypoxia and from reoxygenation, we examined the effect of fructose-1,6-bisphosphate on the ability of hog ileum smooth muscle to maintain isometric force during hypoxia and to generate isometric force after reoxygenation in the presence of 5 mM glucose. After 180 min of hypoxia, tissues incubated with 20 mM fructose-1,6-bisphosphate maintained significantly greater levels of isometric force than tissues incubated in the absence of exogenous substrate (23% of pre-hypoxia force compared to 16%). During the first contraction following reoxygenation there was a significantly greater force generation in tissues incubated with 20 mM fructose-1,6-bisphosphate during the hypoxia period compared to tissues with no exogenous substrate included during the hypoxia period (29% of pre-hypoxia force compared to 19%). However, glucose always was a better metabolic substrate compared to fructose-1,6-bisphosphate under all experimental conditions. The presence of fructose-1,6-bisphosphate during hypoxia likely improved tissue function by fructose-1,6-bisphosphate entering the cells and acting as a glycolytic intermediate, since during a 120 min period of hypoxia, unmounted ileum smooth muscle metabolized 1,6-13C-fructose-1,6-bisphosphate to 3-13C-lactate. This conversion of 1,6-13C-fructose-1,6-bisphosphate to 3-13C-lactate was inhibited by the addition of 1 mM iodoacetic acid, a glycolytic inhibitor. We conclude that exogenously provided fructose-1,6-bisphosphate does provide modest protection of ileum smooth muscle from hypoxic damage by functioning as a glycolytic intermediate and improving the cellular energy state.

Caveolae and the organization of carbohydrate metabolism in vascular smooth muscle

We have previously found that glycolysis and gluconeogenesis occur in separate “compartments” of the VSM cell. These compartments may result from spatial separation of glycolytic and gluconeogenic enzymes (Lloyd and Hardin [ 1999 ] Am J Physiol Cell Physiol. 277:C1250‐C1262). We have also found that an intact plasma membrane is essential for compartmentation to exist (Lloyd and Hardin [ 2000 ] Am J Physiol Cell Physiol. 278:C803‐C811), suggesting that glycolysis and gluconeogenesis may be associated with distinct plasma membrane microdomains. Caveolae are one such microdomain, in which proteins of related function colocalize. Thus, we hypothesized that membrane‐associated glycolysis occurs in association with caveolae, while gluconeogenesis is localized to non‐caveolae domains. To test this hypothesis, we disrupted caveolae in vascular smooth muscle (VSM) of pig cerebral microvessels (PCMV) with β methyl‐cyclodextrin (CD) and examined the metabolism of [2‐13C]glucose (a glycolytic substrate) and [1‐13C]fructose 1,6‐bisphosphate (FBP, a gluconeogenic substrate in PCMV) using 13C nuclear magnetic resonance spectroscopy. Caveolar disruption reduced flux of [2‐13C]glucose to [2‐13C]lactate, suggesting that caveolar disruption partially disrupted the glycolytic pathway. Caveolae disruption may also have resulted in a breakdown of compartmentation, since conversion of [1‐13C]FBP to [3‐13C]lactate was increased by CD treatment. Alternatively, the increased [3‐13C]lactate production may reflect changes in FBP uptake, since conversion of [1‐13C]FBP to [3‐13C]glucose was also elevated in CD‐treated cells. Thus, a link between caveolar organization and metabolic organization may exist. J. Cell. Biochem. 82:399–408, 2001.

Tension responses of sheep aorta to simultaneous decreases in phosphocreatine, inorganic phosphate and ATP.

1. Tension responses of sheep aortae were investigated when different substrates were included in the superfusing medium. The magnitude of tension development was similar whether or not 5 mM glucose was present in the medium. However, the rate of tension development was greater in the absence of glucose. 2. When 5 mM 2‐deoxyglucose (2DG) was present in the medium, the magnitude of tension generation was 1.6 times that in the absence of exogenous substrate. A second sequential contraction with 2DG generated tension 1.25 times that in the absence of exogenous substrate. The rate of tension development during the first contraction in the presence of 2DG was similar to that in the absence of substrate. However, the second contraction in the presence of 2DG had a substantially increased rate of tension development. 3. 31P nuclear magnetic resonance (NMR) spectroscopy revealed that, at resting tone, in the presence of 2DG, inorganic phosphate (P(i)) and phosphocreatine (PCr) simultaneously decrease while 2‐deoxyglucose‐6‐phosphate accumulates. During contraction‐relaxation cycles, in the presence of 2DG, P(i) and PCr become undetectable while ATP declines to approximately 50% of control values as determined by NMR. During the second contraction in the presence of 2DG, the area of the ADP resonance was similar to that of the alpha‐ATP resonance. 4. The increase in the magnitude of tension generation, during 2DG administration, correlated with a decrease in P(i) levels. The rate of relaxation from a contraction, in the presence of 2DG, was slower than in the presence of glucose or in the absence of exogenous substrate. These results are consistent with the role of P(i) in the release of the proposed ‘latch‐bridge’ state of maintained contraction at low energy demand. 5. The increase in isometric tension generation during contraction in the presence of 2DG appears to be related to the decreased levels of P(i). In the presence of 2DG, the reduction of PCr and of ATP occur to a similar extent to that during hypoxia, yet no inhibition of force takes place. The low levels of ATP and PCr reported with 2DG administration in these studies do not energetically limit the contractile apparatus.

Transport and metabolism of exogenous fumarate and 3-phosphoglycerate in vascular smooth muscle

The keto (linear) form of exogenous fructose 1,6-bisphosphate, a highly charged glycolytic intermediate, may utilize a dicarboxylate transporter to cross the cell membrane, support glycolysis, and produce ATP anaerobically. We tested the hypothesis that fumarate, a dicarboxylate, and 3-phosphoglycerate (3-PG), an intermediate structurally similar to a dicarboxylate, could support contraction in vascular smooth muscle during hypoxia. To assess ATP production during hypoxia we measured isometric force maintenance in hog carotid arteries during hypoxia in the presence or absence of 20 mM fumarate or 3-PG. 3-PG improved maintenance of force (p < 0.05) during the 30-80 min period of hypoxia. Fumarate decreased peak isometric force development by 9.5% (p = 0.008) but modestly improved maintenance of force (p < 0.05) throughout the first 80 min of hypoxia. 13C-NMR on tissue extracts and superfusates revealed 1,2,3,4-13C-fumarate (5 mM) metabolism to 1,2,3,4-13C-malate under oxygenated and hypoxic conditions suggesting uptake and metabolism of fumarate. In conclusion, exogenous fumarate and 3-PG readily enter vascular smooth muscle cells, presumably by a dicarboxylate transporter, and support energetically important pathways.

Role of microtubules in the regulation of metabolism in isolated cerebral microvessels

We used13C-labeled substrates and nuclear magnetic resonance spectroscopy to examine carbohydrate metabolism in vascular smooth muscle of freshly isolated pig cerebral microvessels (PCMV). PCMV utilized [2-13C]glucose mainly for glycolysis, producing [2-13C]lactate. Simultaneously, PCMV utilized the glycolytic intermediate [1-13C]fructose 1,6-bisphosphate (FBP) mainly for gluconeogenesis, producing [1-13C]glucose with only minor [3-13C]lactate production. The dissimilarity in metabolism of [2-13C]FBP derived from [2-13C]glucose breakdown and metabolism of exogenous [1-13C]FBP demonstrates that carbohydrate metabolism is compartmented in PCMV. Because glycolytic enzymes interact with microtubules, we disrupted microtubules with vinblastine. Vinblastine treatment significantly decreased [2-13C]lactate peak intensity (87.8 ± 3.7% of control). The microtubule-stabilizing agent taxol also reduced [2-13C]lactate peak intensity (90.0 ± 2.4% of control). Treatment with both agents further decreased [2-13C]lactate production (73.3 ± 4.0% of control). Neither vinblastine, taxol, or the combined drugs affected [1-13C]glucose peak intensity (gluconeogenesis) or disrupted the compartmentation of carbohydrate metabolism. The similar effects of taxol and vinblastine, drugs that have opposite effects on microtubule assembly, suggest that they produce their effects on glycolytic rate by competing with glycolytic enzymes for binding, not by affecting the overall assembly state of the microtubule network. Glycolysis, but not gluconeogenesis, may be regulated in part by glycolytic enzyme-microtubule interactions.

Pattern of substrate utilization in vascular smooth muscle using 13C isotopomer analysis of glutamate

Although vascular smooth muscle (VSM) derives the majority of its energy from oxidative phosphorylation, controversy exists concerning which substrates are utilized by the tricarboxylic acid (TCA) cycle. We used 13C isotopomer analysis of glutamate to directly measure the entry of exogenous [13C]glucose and acetate and unlabeled endogenous sources into the TCA cycle via acetyl-CoA. Hog carotid artery segments denuded of endothelium were superfused with 5 mM [1-13C]glucose and 0-5 mM [1,2-13C]acetate at 37°C for 3-12 h. We found that both resting and contracting VSM preferentially utilize [1,2-13C]acetate compared with [1-13C]glucose and unlabeled substrates. The entry of glucose into the TCA cycle (30-60% of total entry via acetyl-CoA) exhibited little change despite alterations in contractile state or acetate concentrations ranging from 0 to 5 mM. We conclude that glucose and nonglucose substrates are important oxidative substrates for resting and contracting VSM. These are the first direct measurements of relative substrate entry into the TCA cycle of VSM during activation and may provide a useful method to measure alterations in VSM metabolism under physiological and pathophysiological conditions.