Role of pyruvate carboxylase in fatty acid synthesis: Alterations during preadipocyte differentiation

Transport of mitochondrial acetyl units to the cytoplasm for fatty acid synthesis via the citrate cleavage pathway requires replenishment of mitochondrial oxaloacetate. Pyruvate carboxylase is though to fulfill this role although compelling evidence has been lacking. During lipogenic differentiation of 3T3-L1 preadipocytes, pyruvate carboxylase activity rises 18-fold in close coordination with fat accumulation and the activity of ATP-citrate lyase, an established lipogenic enzyme. The activities of enzymes less directly related to lipogenesis rise only 3–5-fold while other unrelated enzymes do not increase significantly. These results indicate that pyruvate carboxylase is in fact a lipogenic enzyme.     

Conjugated linoleic acid and hepatic lipogenesis in mouse: role of the mitochondrial citrate carrier

Conjugated linoleic acid (CLA) is able to reduce adiposity by affecting lipid metabolism. In particular, CLA administration to mice reduces body fat mass with a concomitant lipid accumulation in the liver. We investigated the effects of CLA on the activity of the mitochondrial citrate carrier (CIC), which is implicated in hepatic lipogenesis. The transport activity of the CIC, measured both in intact mitochondria and in the proteoliposomes, progressively increased with the duration of CLA feeding. An increase in the CIC activity of approximately 1.7-fold was found in 16 week CLA-treated mice with respect to control animals. A kinetic analysis showed a 1.6-fold increase in the V(max) of citrate transport but no change in the K(m) value. Western blot experiments revealed an increase of approximately 1.7-fold in the expression of CIC after CLA treatment. A strict correlation between the increase in CIC activity and the stimulation of the cytosolic lipogenic enzymes was also found. These data indicate that the CIC may play a role in the onset of hepatic steatosis in CLA-fed mice by supplying the carbon source for de novo fatty acid synthesis.     

The citrate cleavage pathway and lipogenesis in rat adipose tissue: replenishment of oxaloacetate

Fatty acid synthesis via the citrate cleavage pathway requires the continual replenishment of oxaloacetate within the mitochondria, probably by carboxylation of pyruvate. Malic enzyme, although present in adipose tissue, is completely localized in the cytoplasm and has insufficient activity to support lipogenesis. Pyruvate carboxylase was found to be active in both the mitochondria and cytoplasm of epididymal adipose tissue cells; it was dependent on both ATP and biotin. Alteractions in dietary conditions induced no significant changes in mitochondrial pyruvate carboxylase activity, but the soluble activity was depressed in fat-fed animals. The possible importance of the soluble activity in lipogenesis lies in its participation in a soluble malate transhydrogenation cycle with NAD malate dehydrogenase and malic enzyme, whereby a continual supply of NADPH is produced. Consequently, the pyruvate carboxylase in adipose tissue both generates mitochondrial oxaloacetate for the citrate cleavage pathway and supplies soluble NADPH for the conversion of acetyl-CoA to fatty acid.     

Intracellular localization of enzymes in white-adipose-tissue fat-cells and permeability properties of fat-cell mitochondria. Transfer of acetyl units and reducing power between mitochondria and cytoplasm

1. A method is described for extracting separately mitochondrial and extramitochondrial enzymes from fat-cells prepared by collagenase digestion from rat epididymal fat-pads. The following distribution of enzymes has been observed (with the total activities of the enzymes as units/mg of fat-cell DNA at 25 degrees C given in parenthesis). Exclusively mitochondrial enzymes: glutamate dehydrogenase (1.8), NAD-isocitrate dehydrogenase (0.5), citrate synthase (5.2), pyruvate carboxylase (3.0); exclusively extramitochondrial enzymes: glucose 6-phosphate dehydrogenase (5.8), 6-phosphogluconate dehydrogenase (5.2), NADP-malate dehydrogenase (11.0), ATP-citrate lyase (5.1); enzymes present in both mitochondrial and extramitochondrial compartments: NADP-isocitrate dehydrogenase (3.7), NAD-malate dehydrogenase (330), aconitate hydratase (1.1), carnitine acetyltransferase (0.4), acetyl-CoA synthetase (1.0), aspartate aminotransferase (1.7), alanine aminotransferase (6.1). The mean DNA content of eight preparations of fat-cells was 109mug/g dry weight of cells. 2. Mitochondria showing respiratory control ratios of 3-6 with pyruvate, about 3 with succinate and P/O ratios of approaching 3 and 2 respectively have been isolated from fat-cells. From studies of rates of oxygen uptake and of swelling in iso-osmotic solutions of ammonium salts, it is concluded that fat-cell mitochondria are permeable to the monocarboxylic acids, pyruvate and acetate; that in the presence of phosphate they are permeable to malate and succinate and to a lesser extent oxaloacetate but not fumarate; and that in the presence of both malate and phosphate they are permeable to citrate, isocitrate and 2-oxoglutarate. In addition, isolated fat-cell mitochondria have been found to oxidize acetyl l-carnitine and, slowly, l-glycerol 3-phosphate. 3. It is concluded that the major means of transport of acetyl units into the cytoplasm for fatty acid synthesis is as citrate. Extensive transport as glutamate, 2-oxoglutarate and isocitrate, as acetate and as acetyl l-carnitine appears to be ruled out by the low activities of mitochondrial aconitate hydratase, mitochondrial acetyl-CoA hydrolyase and carnitine acetyltransferase respectively. Pathways whereby oxaloacetate generated in the cytoplasm during fatty acid synthesis by ATP-citrate lyase may be returned to mitochondria for further citrate synthesis are discussed. 4. It is also concluded that fat-cells contain pathways that will allow the excess of reducing power formed in the cytoplasm when adipose tissue is incubated in glucose and insulin to be transferred to mitochondria as l-glycerol 3-phosphate or malate. When adipose tissue is incubated in pyruvate alone, reducing power for fatty acid, l-glycerol 3-phosphate and lactate formation may be transferred to the cytoplasm as citrate and malate.     

Citrate carrier and lipogenic enzyme activities in lead nitrate-induced proliferative and apoptotic phase in rat liver.

After in vivo administration of lead nitrate, functional changes of the mitochondrial tricarboxylate carrier and of the cytosolic lipogenic enzymes acetyl-CoA carboxylase and fatty acid synthetase have been detected in rat liver. The rate of citrate transport was greatly reduced in rats during both the proliferative phase (3 days after the lead nitrate administration) and the involutive phase (5 days after the metal injection), which follows hepatic hyperplasia and corresponds to the peak of hepatocyte apoptosis. In both phases, a decrease of the lipogenic enzyme activities has been detected. In treated animals, an alteration of mitochondrial lipid composition has also been found. The modified lipid microenvironment could be responsible for the decreased carrier activity which, in turn, may account for the reduced activities of the lipogenic enzymes.     

Development of fatty acid-synthetic capacity in interscapular brown adipose tissue during suckling in genetically obese Zucker rats.

The development of the lipogenic capacity in brown adipose tissue was studied in suckling lean (Fa/fa) and obese (fa/fa) Zucker pups aged from 7 to 22 days. In both lean and obese pups, activities of the two key lipogenic enzymes, fatty acid synthetase and acetyl-CoA carboxylase, and of citrate cleavage enzyme rose from the early to the late suckling period. Compared with lean pups, 7-day-old fa/fa pups showed a 35% increase in fat accumulation in interscapular brown adipose tissue and a 25% increase in fatty acid synthetase activity. By 10 days of age, fat deposition, lipogenesis in vivo (assessed by the incorporation of 3H from 3H2O into fatty acids) and fatty acid synthetase activity were 1.5-2-fold higher in pre-obese than in lean pups. Compared with lean pups, the increased lipogenesis in vivo observed in brown adipose tissue of 10-day-old pre-obese pups could not entirely account for the difference in fat deposition observed in this tissue, suggesting that additional mechanisms are at play to explain the increased fat content of this tissue.     

Critical role of the peroxisomal protein PEX16 in white adipocyte development and lipid homeostasis

The importance of peroxisomes for adipocyte function is poorly understood. Herein, we provide insights into the critical role of peroxin 16 (PEX16)-mediated peroxisome biogenesis in adipocyte development and lipid metabolism. Pex16 is highly expressed in adipose tissues and upregulated during adipogenesis of murine and human cells. We demonstrate that Pex16 is a target gene of the adipogenesis “master-regulator” PPARγ. Stable silencing of Pex16 in 3T3-L1 cells strongly reduced the number of peroxisomes while mitochondrial number was unaffected. Concomitantly, peroxisomal fatty acid (FA) oxidation was reduced, thereby causing accumulation of long- and very long-chain (polyunsaturated) FAs and reduction of odd-chain FAs. Further, Pex16-silencing decreased cellular oxygen consumption and increased FA release. Additionally, silencing of Pex16 impaired adipocyte differentiation, lipogenic and adipogenic marker gene expression, and cellular triglyceride stores. Addition of PPARγ agonist rosiglitazone and peroxisome-related lipid species to Pex16-silenced 3T3-L1 cells rescued adipogenesis. These data provide evidence that PEX16 is required for peroxisome biogenesis and highlights the relevance of peroxisomes for adipogenesis and adipocyte lipid metabolism.     

Metabolism of pyruvate and malate by isolated fat-cell mitochondria

1. Metabolism of pyruvate and malate by isolated fat-cell mitochondria incubated in the presence of ADP and phosphate has been studied by measuring rates of pyruvate uptake, malate utilization or production, citrate production and oxygen consumption. From these measurements calculations of the flow rates through pyruvate carboxylase, pyruvate dehydrogenase and citrate cycle have been made under various conditions. 2. In the presence of bicarbonate, pyruvate was largely converted into citrate and malate and only about 10% was oxidized by the citrate cycle; citrate and malate outputs were linear after lag periods of 6-9min and 3min respectively, and no other end products of pyruvate metabolism were detected. On the further addition of malate or hydroxymalonate, the lag in the rate of citrate output was less marked but no net malate disappearance was detected. If, however, bicarbonate was omitted then net malate uptake was observed. Addition of butyl malonate was found to greatly inhibit the metabolism of pyruvate to citrate and malate in the presence of bicarbonate. 3. These results are in agreement with earlier conclusions that in adipose tissue acetyl units for fatty acid synthesis are transferred to the cytoplasm as citrate and that this transfer requires malate presumably for counter transport. They also support the view that oxaloacetate for citrate synthesis is preferentially formed from pyruvate through pyruvate carboxylase rather than malate through malate dehydrogenase and that the mitochondrial metabolism of citrate in fat-cells is restricted. The possible consequences of these conclusions are discussed. 4. Studies on the effects of additions of adenine nucleotides to pyruvate metabolism by isolated fat-cell mitochondria are consistent with inhibition of pyruvate carboxylase in the presence of ADP and pyruvate dehydrogenase in the presence of ATP.     

Silybin exerts antioxidant effects and induces mitochondrial biogenesis in liver of rat with secondary biliary cirrhosis

The accumulation of toxic hydrophobic bile acids in hepatocytes, observed during chronic cholestasis, induces substantial modification in the redox state and in mitochondrial functions. Recent reports have suggested a significant role of impaired lipid metabolism in the progression of chronic cholestasis. In this work we report that changes observed in the expression of the lipogenic enzymes acetyl-CoA carboxylase and fatty acid synthase were associated with a decrease in the activity of citrate carrier (CIC), a protein of the inner mitochondrial membrane closely related to hepatic lipogenesis. We also verified that the impairment of citrate transport was dependent on modification of the phospholipid composition of the mitochondrial membrane and on cardiolipin oxidation. Silybin, an extract of silymarin with antioxidant and anti-inflammatory properties, prevented mitochondrial reactive oxygen species (ROS) production, cardiolipin oxidation, and CIC failure in cirrhotic livers but did not affect the expression of lipogenic enzymes. Moreover, supplementation of silybin was also associated with mitochondrial biogenesis. In conclusion, we demonstrate that chronic cholestasis induces cardiolipin oxidation that in turn impairs mitochondrial function and further promotes ROS production. The capacity of silybin to limit mitochondrial failure is part of its hepatoprotective property.     

Metabolic pathways involved in lipogenesis from lactate and acetate in bovine adipose tissue: Effects of metabolic inhibitors

Metabolic inhibitors were used in vitro in an attempt to elucidate the biochemical pathways by which lactate is converted to fatty acids by bovine adipose tissue. Subcutaneous adipose tissue samples were obtained by biopsy techniques from steers fed a high-energy ration. Kynurenate (α-2-diamino-γ-oxabenzenebutanoic acid) (5–10 mm), an inhibitor of acetyl-CoA carboxylase, and cerulenin (2,3-epoxy-4-oxo-7,10-dodecadienamide) (20–100 μg/ml), an inhibitor of the fatty acid synthetase enzyme complex, inhibited fatty acid synthesis from both acetate and lactate. The hydrogen acceptor, N-methylphenazonium methosulfate (10 μm) inhibited acetate but not lactate incorporation into fatty acids. α-Cyanohydroxycinnamate (5 mm) and phenylpyruvate (10 mm), which inhibit pyruvate entry into the mitochondria and pyruvate carboxylase, respectively, decreased lipogenesis from both acetate and lactate. The effects of phenylpyruvate on lipogenesis from acetate were greater in the presence of glucose plus insulin. Agaric acid (2-hydroxy-1,2,3-nonadecanetricarboxylic acid) (0.2 and 1.0 mm), which inhibits citrate efflux from the mitochondria also decreased lipogenesis from both acetate and lactate. Fluoroacetate (2.5 mm), an inhibitor of aconitate hydratase, had no effect on lipogenesis from acetate; but, in the presence of glucose or pyruvate, decreased lactate incorporation into fatty acids. n-Butylmalonate (5 mm), which blocks malate transport across the mitochondrial membrane, decreased lipogenesis from lactate but not acetate. Malate transport during lipogenesis is not associated with an operative malate:asparate shuttle in bovine adipose tissue, as indicated by the lack of effect of either 0.2 or 1.0 mm aminooxyacetate, a transaminase inhibitor, on lipogenesis from acetate or lactate. The results suggest a functional ATP-citrate lyase:NADP-malate dehydrogenase pathway in bovine subcutaneous adipose tissue and that this pathway may be involved in lipogenesis from acetate as well as lactate.     

Pathway of carbon flow during fatty acid synthesis from lactate and pyruvate in rat adipose tissue

The metabolism of pyruvate and lactate by rat adipose tissue was studied. Pyruvate and lactate conversion to fatty acids is strongly concentration-dependent. Lactate can be used to an appreciable extent only by adipose tissue from fasted-refed rats. A number of compounds, including glucose, pyruvate, aspartate, propionate, and butyrate, stimulated lactate conversion to fatty acids. Based on studies of incorporation of lactate-2-(3)H and lactate-2-(14)C into fatty acids it was suggested that the transhydrogenation sequence of the "citrate-malate cycle"(1) was not providing all of the NADPH required for fatty acid synthesis from lactate. An alternative pathway for NADPH formation involving the conversion of isocitrate to alpha-ketoglutarate via cytosolic isocitrate dehydrogenase was proposed. Indirect support for this proposal was provided by the rapid labeling of glutamate from lactate-2-(14)C by adipose tissue incubated in vitro, as well as the demonstration that glutamate can be readily metabolized by adipose tissue via reactions localized largely in the cytosol. Furthermore, isolated adipose tissue mitochondria convert alpha-ketoglutarate to malate, or in the presence of added pyruvate, to citrate. Glutamate itself can not be metabolized by these mitochondria, a finding in keeping with the demonstration of negligible levels of NAD-glutamate dehydrogenase activity in adipose tissue mitochondria. Pyruvate stimulated alpha-ketoglutarate and malate conversion to citrate and reduced their oxidation to CO(2). It is proposed that under conditions of excess generation of NADH malate may act as a shuttle carrying reducing equivalents across the mitochondrial membrane. Malate at low concentrations increased pyruvate conversion $$Word$$ citrate and markedly decreased the formation of CO(2) by isolated adipose tissue mitochondria. Malate also stimulated citrate and isocitrate metabolism by these mitochondria, an effect that could be blocked by 2-n-butylmalonate. This potentially important role of malate in the regulation of carbon flow during lipogenesis is underlined by the observation that 2-n-butylmalonate inhibited fatty acid synthesis from pyruvate, but not from glucose and acetate, and decreased the stimulatory effect of pyruvate on acetate conversion to fatty acids.     

The lipogenic enzyme acetoacetyl-CoA synthetase and ketone body utilization for denovo lipid synthesis,a review

Acetoacetyl-CoA synthetase (AACS) is the key enzyme in the anabolic utilization of ketone bodies (KBs) for denovo lipid synthesis, a process that bypasses citrate and ATP citrate lyase. This review shows that AACS is a highly regulated, cytosolic, and lipogenic enzyme and that many tissues can readily use KBs for denovo lipid synthesis. AACS has a low micromolar K_m for acetoacetate, and supply of acetoacetate should not limit its activity in the fed state. In many tissues, AACS appears to be regulated in conjunction with the need for cholesterol, but in adipose tissue, it seems tied to fatty acid synthesis. KBs are readily utilized as substrates for lipid synthesis in lipogenic tissues, including liver, adipose tissue, lactating mammary gland, skin, intestinal mucosa, adrenals, and developing brain. In numerous studied cases, KBs served several-fold better than glucose as substrates for lipid synthesis, and when present, KBs suppressed the utilization of glucose for lipid synthesis. Here, it is hypothesized that a physiological role for the utilization of KBs for lipid synthesis is a metabolic process of lipid interconversion. Fatty acids are converted to KBs in liver, and then, the KBs are utilized to synthesize cholesterol and other long-chain fatty acids in liver and nonhepatic tissues. The conversion of fatty acids to cholesterol via the KBs may be a particularly important example of lipid interconversion. Utilizing KBs for lipid synthesis is glucose sparing and probably is important with low carbohydrate diets. Metabolic situations and tissues where this pathway may be important are discussed.     

Quantifying reductive carboxylation flux of glutamine to lipid in a brown adipocyte cell line

We previously reported that glutamine was a major source of carbon for de novo fatty acid synthesis in a brown adipocyte cell line. The pathway for fatty acid synthesis from glutamine may follow either of two distinct pathways after it enters the citric acid cycle. The glutaminolysis pathway follows the citric acid cycle, whereas the reductive carboxylation pathway travels in reverse of the citric acid cycle from alpha-ketoglutarate to citrate. To quantify fluxes in these pathways we incubated brown adipocyte cells in [U-(13)C]glutamine or [5-(13)C]glutamine and analyzed the mass isotopomer distribution of key metabolites using models that fit the isotopomer distribution to fluxes. We also investigated inhibitors of NADP-dependent isocitrate dehydrogenase and mitochondrial citrate export. The results indicated that one third of glutamine entering the citric acid cycle travels to citrate via reductive carboxylation while the remainder is oxidized through succinate. The reductive carboxylation flux accounted for 90% of all flux of glutamine to lipid. The inhibitor studies were compatible with reductive carboxylation flux through mitochondrial isocitrate dehydrogenase. Total cell citrate and alpha-ketoglutarate were near isotopic equilibrium as expected if rapid cycling exists between these compounds involving the mitochondrial membrane NAD/NADP transhydrogenase. Taken together, these studies demonstrate a new role for glutamine as a lipogenic precursor and propose an alternative to the glutaminolysis pathway where flux of glutamine to lipogenic acetyl-CoA occurs via reductive carboxylation. These findings were enabled by a new modeling tool and software implementation (Metran) for global flux estimation.     

Hepatic oleate regulates adipose tissue lipogenesis and fatty acid oxidation

Hepatic steatosis is associated with detrimental metabolic phenotypes including enhanced risk for diabetes. Stearoyl-CoA desaturases (SCDs) catalyze the synthesis of MUFAs. In mice, genetic ablation of SCDs reduces hepatic de novo lipogenesis (DNL) and protects against diet-induced hepatic steatosis and adiposity. To understand the mechanism by which hepatic MUFA production influences adipose tissue stores, we created two liver-specific transgenic mouse models in the SCD1 knockout that express either human SCD5 or mouse SCD3, that synthesize oleate and palmitoleate, respectively. We demonstrate that hepatic de novo synthesized oleate, but not palmitoleate, stimulate hepatic lipid accumulation and adiposity, reversing the protective effect of the global SCD1 knockout under lipogenic conditions. Unexpectedly, the accumulation of hepatic lipid occurred without induction of the hepatic DNL program. Changes in hepatic lipid composition were reflected in plasma and in adipose tissue. Importantly, endogenously synthesized hepatic oleate was associated with suppressed DNL and fatty acid oxidation in white adipose tissue. Regression analysis revealed a strong correlation between adipose tissue lipid fuel utilization and hepatic and adipose tissue lipid storage. These data suggest an extrahepatic mechanism where endogenous hepatic oleate regulates lipid homeostasis in adipose tissues.     

Keap1-Knockdown Decreases Fasting-Induced Fatty Liver via Altered Lipid Metabolism and Decreased Fatty Acid Mobilization from Adipose Tissue

Aims

The purpose of this study was to determine whether Nrf2 activation, via Keap1-knockdown (Keap1-KD), regulates lipid metabolism and mobilization induced by food deprivation (e.g. fasting).

Methods and Results

Male C57BL/6 (WT) and Keap1-KD mice were either fed ad libitum or food deprived for 24 hours. After fasting, WT mice exhibited a marked increase in hepatic lipid accumulation, but Keap1-KD mice had an attenuated increase of lipid accumulation, along with reduced expression of lipogenic genes (acetyl-coA carboxylase, stearoyl-CoA desaturase-1, and fatty acid synthase) and reduced expression of genes related to fatty acid transport, such as fatty acid translocase/CD36 (CD36) and Fatty acid transport protein (FATP) 2, which may attribute to the reduced induction of Peroxisome proliferator-activated receptor (Ppar) α signaling in the liver. Additionally, enhanced Nrf2 activity by Keap1-KD increased AMP-activated protein kinase (AMPK) phosphorylation in liver. In white adipose tissue, enhanced Nrf2 activity did not change the lipolysis rate by fasting, but reduced expression of fatty acid transporters — CD36 and FATP1, via a PPARα-dependent mechanism, which impaired fatty acid transport from white adipose tissue to periphery circulation system, and resulted in increased white adipose tissue fatty acid content. Moreover, enhanced Nrf2 activity increased glucose tolerance and Akt phosphorylation levels upon insulin administration, suggesting Nrf2 signaling pathway plays a key role in regulating insulin signaling and enhanced insulin sensitivity in skeletal muscle.

Conclusion

Enhanced Nrf2 activity via Keap1-KD decreased fasting-induced steatosis, pointing to an important function of Nrf2 on lipid metabolism under the condition of nutrient deprivation.     

Covariance of tricarboxylate carrier activity and lipogenesis in liver of polyunsaturated fatty acid (n-6) fed rats.

The mitochondrial tricarboxylate (citrate) carrier plays an important role in hepatic intermediary metabolism because, among other functions, it supplies the cytosol with acetyl units for fatty-acid synthesis. In this study, the effect of polyunsaturated fatty acids (PUFA, n-6) on the function of this mitochondrial transporter and on lipogenic enzyme activities was investigated by feeding rats for 4 weeks with a 15%-fat diet composed of high linoleic safflower oil. Citrate transport was strongly reduced in liver mitochondria isolated from PUFA-treated rats. A reduced transport activity was also observed when solubilized mitochondrial citrate carrier from PUFA-treated rats was reconstituted into liposomes. In the same animals, a decrease of cytosolic lipogenic enzyme activities was observed. These results indicate a coordinated modulation of citrate carrier and of lipogenic enzyme activities by PUFA feeding. Kinetic analysis of the carrier activity showed that only V(max) decreased, whereas K(m) was almost virtually unaffected. The PUFA-mediated effect is most likely due to the reduced mRNA level and lower content of the citrate carrier protein observed in the safflower oil-fed rats.     

Ensemble Modeling of Hepatic Fatty Acid Metabolism with a Synthetic Glyoxylate Shunt

The liver plays a central role in maintaining whole body metabolic and energy homeostasis by consuming and producing glucose and fatty acids. Glucose and fatty acids compete for hepatic substrate oxidation with regulation ensuring glucose is oxidized preferentially. Increasing fatty acid oxidation is expected to decrease lipid storage in the liver and avoid lipid-induced insulin-resistance. To increase hepatic lipid oxidation in the presence of glucose, we previously engineered a synthetic glyoxylate shunt into human hepatocyte cultures and a mouse model and showed that this synthetic pathway increases free fatty acid β-oxidation and confers resistance to diet-induced obesity in the mouse model. Here we used ensemble modeling to decipher the effects of perturbations to the hepatic metabolic network on fatty acid oxidation and glucose uptake. Despite sampling of kinetic parameters using the most fundamental elementary reaction models, the models based on current metabolic regulation did not readily describe the phenotype generated by glyoxylate shunt expression. Although not conclusive, this initial negative result prompted us to probe unknown regulations, and malate was identified as inhibitor of hexokinase 2 expression either through direct or indirect actions. This regulation allows the explanation of observed phenotypes (increased fatty acid degradation and decreased glucose consumption). Moreover, the result is a function of pyruvate-carboxylase, mitochondrial pyruvate transporter, citrate transporter protein, and citrate synthase activities. Some subsets of these flux ratios predict increases in fatty acid and decreases in glucose uptake after glyoxylate expression, whereas others predict no change. Altogether, this work defines the possible biochemical space where the synthetic shunt will produce the desired phenotype and demonstrates the efficacy of ensemble modeling for synthetic pathway design.     

An established preadipose cell line and its differentiation in culture. II. Factors affecting the adipose conversion.

When cells of the established preadipose line 3T3-L1 enter a resting state, they accumulate triglyceride and convert to adipose cells. The adipose conversion is brought about by a large increase in the rate of triglyceride synthesis, as measured by the incorporation rate of labeled palmitate, acetate, and glucose. In a resting 3T3 subline which dose not undergo the adipose conversion, the rate of triglyceride synthesis from these precursors is very low, and similar to that of growing 3T3-L1 cells, before their adipose conversion begins. If 3T3-L1 cells incorporate bromodeoxyuridine during growth, triglyceride synthesis does not increase when the cells reach a stationary state, and triglycerides do not accumulate. As would be expected from their known actions on tissue adipose cells, lipogenic and lipolytic hormones and drugs affect the rate of synthesis and accumulation of triglyceride by 3T3-L1 cells, but in contrast to bromodeoxyuridine, these modulating agents do not seem to affect the proportion of cells which undergoes the adipose conversion. Insulin markedly increases the rate of synthesis and accumulation of triglyceride by fatty 3T3-L1 cells, and produces a related increase in cell protein content. Of 20 randomly selected clones isolated from the original 3T3 stock, 19 are able to convert to adipose cells. The probability of such a conversion varies greatly among the different clones, in most cases being much lower than for 3T3-L1; but once the conversion takes place, the adipose cells produced from all of the 19 clones appear similar. The adipose conversion would seem to depend on an on-off switch, which is on with a different probability in different clones. This probability is quasistably inherited by the clonal progeny.     

Increased activity of glycerol 3-phosphate dehydrogenase and other lipogenic enzymes in human bladder cancer.

Common molecular changes in cancer cells are high carbon flux through the glycolytic pathway and overexpression of fatty acid synthase, a key lipogenic enzyme. Since glycerol 3-phosphate dehydrogenase creates a link between carbohydrates and the lipid metabolism, we have investigated the activity of glycerol 3-phosphate dehydrogenase and various lipogenic enzymes in human bladder cancer. The data presented in this paper indicate that glycerol 3-phosphate dehydrogenase activity in human bladder cancer is significantly higher compared to adjacent non-neoplastic tissue, serving as normal control bladder tissue. Increased glycerol 3-phosphate dehydrogenase activity is accompanied by increased enzyme activity, either directly (fatty acid synthase) or indirectly (through ATP-citrate lyase, glucose 6-phosphate dehydrogenase, 6-phosphogluconate dehydrogenase and citrate synthase) involved in fatty acid synthesis. Coordinated upregulation of glycerol 3-phosphate dehydrogenase and lipogenic enzymes activities in human bladder cancer suggests that glycerol 3-phosphate dehydrogenase supplies glycerol 3-phosphate for lipid biosynthesis.