Malonyl-CoA is utilised in fatty acid biosynthesis by the enzyme malonyl coenzyme A:acyl carrier protein transacylase (MCAT). MCAT serves to transfer malonate from malonyl-CoA to the terminal thiol of holo-acyl carrier protein (ACP).
Malonyl-CoA decarboxylase deficiency is a condition that prevents the body from converting certain fats to energy. The signs and symptoms of this disorder typically appear in early childhood. Almost all affected children have delayed development. Additional signs and symptoms can include weak muscle tone (hypotonia), seizures, diarrhea, vomiting, and low blood sugar (hypoglycemia). A heart condition called cardiomyopathy, which weakens and enlarges the heart muscle, is another common feature of malonyl-CoA decarboxylase deficiency.
Mutations in the MLYCD gene cause malonyl-CoA decarboxylase deficiency. The MLYCD gene provides instructions for making an enzyme called malonyl-CoA decarboxylase. Within cells, this enzyme helps regulate the formation and breakdown of a group of fats called fatty acids. Many tissues, including the heart muscle, use fatty acids as a major source of energy.
Mutations in the MLYCD gene reduce or eliminate the function of malonyl-CoA decarboxylase. A shortage of this enzyme disrupts the normal balance of fatty acid formation and breakdown in the body. As a result, fatty acids cannot be converted to energy, which can lead to characteristic features of this disorder including low blood sugar and cardiomyopathy. Byproducts of fatty acid processing build up in tissues, which also contributes to the signs and symptoms of malonyl-CoA decarboxylase deficiency.
The malonyl-CoA/long-chain acyl-CoA (LC-CoA) model of glucose-induced insulin secretion (GIIS) predicts that malonyl-CoA derived from glucose metabolism inhibits fatty acid oxidation, thereby increasing the availability of LC-CoA for lipid signaling to cellular processes involved in exocytosis. For directly testing the model, INSr3 cell clones overexpressing malonyl-CoA decarboxylase in the cytosol (MCDc) in a tetracycline regulatable manner were generated, and INS(832/13) and rat islets were infected with MCDc-expressing adenoviruses. MCD activity was increased more than fivefold, and the malonyl-CoA content was markedly diminished. This was associated with enhanced fat oxidation at high glucose, a suppression of the glucose-induced increase in cellular free fatty acid (FFA) content, and reduced partitioning at elevated glucose of exogenous palmitate into lipid esterification products. MCDc overexpression, in the presence of exogenous FFAs but not in their absence, reduced GIIS in all beta-cell lines and in rat islets. It also markedly curtailed the stimulation of insulin secretion by other fuel and nonfuel secretagogues. In the absence of MCDc overexpression, the secretory responses to all types of secretagogues were amplified by the provision of exogenous fatty acids. In the presence of exogenous FFAs, the fatty acyl-CoA synthetase inhibitor triacsin C reduced secretion in response to glucose and nonfuel stimuli. The data show the existence of important links between the metabolic coupling factor malonyl-CoA, the partitioning of fatty acids, and the stimulation of insulin secretion to both fuel and nonfuel stimuli.
In the catabolic state with no food intake, the liver generates ketones by breaking down fatty acids. During the nocturnal fast or longer starvation periods, this protects the brain, which cannot oxidize fatty acids. In 1977, we published a study in the JCI noting the surprising realization that malonyl-CoA, the substrate of fatty acid synthesis, was also an inhibitor of fatty acid oxidation. Subsequent experiments have borne out this finding and furthered our understanding of molecular metabolism.
We knew that glucagon was the primary on signal for hepatic ketogenesis (7). Once initiated, the rate of ketone production is dependent on the level of long-chain fatty acids reaching the liver. Glucagon signaling triggers the phosphorylation and activation of AMPK. In turn, AMPK phosphorylates the two acetyl-CoA carboxylases, thereby blocking synthesis of malonyl-CoA. It simultaneously enhances destruction of malonyl-CoA by activating malonyl-CoA decarboxylase (Figure 1). The fall in malonyl-CoA stops fatty acid synthesis and activates CPT1 and ketogenesis (8). We also showed that the malonyl-CoA system functions in skeletal and cardiac muscle, although these tissues do not make ketones (9).
Interestingly, we subsequently discovered that the interaction of malonyl-CoA and carnitine with CPT1 are different in liver and muscle. Inhibition of liver CPT1 requires ten times the concentration of malonyl-CoA as does the inhibition of CPT1 in the muscle and heart. Conversely, the Km for carnitine is much lower in liver than in muscle. These differences became important when we cloned and sequenced the liver and muscle enzymes.
The decrease in malonyl-CoA concentration is life saving during the overnight fast and, more importantly, during prolonged fasting or starvation (1, 2). However, it can also be deadly in uncontrolled type 1 diabetes, where markedly increased concentrations of long-chain fatty acids move the chemical state from modest ketosis to full-blown ketoacidosis if not treated (10).
A more serious problem than transient lowering of malonyl-CoA occurs in individuals who harbor genetic deficiencies in the enzymes that control carnitine levels and fat oxidation. Systemic carnitine deficiency due to a mutation in the carnitine transporter OCTN2 was the first identified cause of the syndrome of hypoketotic hypoglycemia, which can lead to sudden infant death (11). Carnitine deficiency also causes liver failure, high ammonia, cerebral edema, cardiac arrhythmias, cardiomyopathy, and muscle weakness with rhabdomyolysis.
In retrospect, the discovery of the malonyl-CoA regulatory system has had an impact far beyond the issue of ketogenesis. The system is active in the hypothalamus, where it contributes to the regulation of food intake, in the heart, where fatty acid oxidation influences the outcome of myocardial infarction, and in the liver, where nonalcoholic steatosis may be diminished by increased fatty acid oxidation, and it is relevant in obesity, where increased mitochondrial function may cause weight loss.
Previous studies showed that i.p. administration of C75, a potent inhibitor of fatty acid synthase (FAS), blocked fasting-induced up-regulation of orexigenic neuropeptides and down-regulation of anorexigenic neuropeptides in the hypothalami of mice. As a result, food intake and body weight were drastically reduced. Here we provide evidence supporting the hypothesis that hypothalamic malonyl-CoA, a substrate of FAS, is an indicator of global energy status and mediates the feeding behavior of mice. We use a sensitive recycling assay to quantify malonyl-CoA to show that the hypothalamic malonyl-CoA level is low in fasted mice and rapidly (< or = 2 h) increases (approximately 5-fold) on refeeding. Intracerebroventricular (i.c.v.) administration of C75 to fasted mice rapidly (< or = 2 h) increased (by 4-fold) hypothalamic malonyl-CoA and blocked feeding when the mice were presented with food. Moreover, prior i.c.v. administration of an acetyl-CoA carboxylase inhibitor, 5-(tetradecyloxy)-2-furoic acid, rapidly (although only partially) prevented the C75-induced rise of hypothalamic malonyl-CoA and prevented the C75-induced decrease of food intake. These effects correlated closely with the rapid (< or = 2 h) and reciprocal effects of i.c.v. C75 on the expression of hypothalamic orexigenic (NPY and AgRP) and anorexigenic (proopiomelanocortin) neuropeptide mRNAs. Previous results showing that C75 administered i.c.v. rapidly activates hypothalamic neurons of the arcuate and paraventricular nuclei are consistent with the results reported in this paper. Together these findings suggest that level of hypothalamic malonyl-CoA, which depends on the relative activities of acetyl-CoA carboxylase and FAS, is an indicator of energy status and mediates feeding behavior.
Acetyl-CoA, derived from glucose metabolism, is converted to malonyl-CoA by acetyl-CoA carboxylase (ACC). Malonyl-CoA is incorporated by fatty acid synthase (FAS) into long-chain fatty acid such as palmitic acid, or is degraded to acetyl-CoA by malonyl-CoA decarboxylase (MCD).
Malonyl-CoA in the hypothalamus is proposed as an anorectic mediator in the CNS control of food intake. Leptin inhibits AMP-activated kinase (AMPK) that inhibits ACC. This leads to activation of ACC resulting in increase in malonyl-CoA level. Glucose activates ACC by inhibiting AMPK, and increases availability of acetyl-CoA. Both effects result in increases in malonyl-CoA level. FAS inhibitors such as C75 and cerulenin, deletion of FAS protein, and tamoxifen that downregulates FAS level, increase malonyl-CoA levels. MCD inhibitor (MCDi) reduces MCD activity resulting in increase in malonyl-CoA level. The increases in malonyl-CoA level lead to decrease in food intake. Overexpression of MCD reduces malonyl-CoA level, which leads to increase in food intake. Antisense oligonucleotide reduces ACC protein level decreasing malonyl-CoA level, which increases food intake.
In the Arc nucleus, CPT-1c and ceramide de novo biosynthesis mediates downstream effect of malonyl-CoA action on feeding. Malonyl-CoA may inhibit CPT-1c function, and CPT-1c regulates ceramide level, possibly by enhancing de novo biosynthesis. Leptin and cerulenin that increase the levels of malonyl-CoA would inhibit CPT-1c, which leads to decreases in ceramide level. The reduction of ceramide level is involved in the decrease in food intake. In contrast, MCD overexpression reducing the malonyl-CoA level would activate CPT-1c, which leads to increase in ceramide level. The upregulation of ceramide level contributes to the increase in food intake.
The formation of fusion protein in biosynthetic pathways usually improves metabolic efficiency either channeling intermediates and/or colocalizing enzymes. In the metabolic engineering of biochemical pathways, generating unnatural protein fusions between sequential biosynthetic enzymes is a useful method to increase system efficiency and product yield. Here, we reported a special case. The malonyl-CoA reductase (MCR) of Chloroflexus aurantiacus catalyzes the conversion of malonyl-CoA to 3-hydroxypropionate (3HP), and is a key enzyme in microbial production of 3HP, an important platform chemical. Functional domain analysis revealed that the N-terminal region of MCR (MCR-N; amino acids 1-549) and the C-terminal region of MCR (MCR-C; amino acids 550-1219) were functionally distinct. The malonyl-CoA was reduced into free intermediate malonate semialdehyde with NADPH by MCR-C fragment, and further reduced to 3HP by MCR-N fragment. In this process, the initial reduction of malonyl-CoA was rate limiting. Site-directed mutagenesis demonstrated that the TGXXXG(A)X(1-2)G and YXXXK motifs were important for enzyme activities of both MCR-N and MCR-C fragments. Moreover, the enzyme activity increased when MCR was separated into two individual fragments. Kinetic analysis showed that MCR-C fragment had higher affinity for malonyl-CoA and 4-time higher Kcat/Km value than MCR. Dissecting MCR into MCR-N and MCR-C fragments also had a positive effect on the 3HP production in a recombinant Escherichia coli strain. Our study showed the feasibility of protein dissection as a new strategy in biosynthetic systems. 2b1af7f3a8