ATP produced by substrate level phosphorylation. Reduced co-enzyme. ATP produced by oxidative phosphorylation. Oxidation of pyruvic acid. Total: Glycolysis occurs exactly as it does in aerobic respiration, but in anaerobic respiration, pyruvate is reduced and NAD is regenerated. This prevents the cell from exhausting its supply of NAD that is necessary for aerobic respiration. The pyruvate then undergoes fermentation. There are 2 types of fermentation.
Alcoholic fermentation: occurs in plants, yeast and bacteria. Pyruvate is converted to ethanol. Pyruvate loses CO 2 and is converted to the 2-carbon compound acetaldehyde. NADH is oxidized and acetaldehyde id reduced to ethanol.
Lactic acid fermentation: occurs in animal cells. Pyruvate is converted to lactic acid. Used to make cheese and yogurt and in human muscle cells when oxygen is scarce. NADH is oxidized and pyruvate is converted to lactic acid. Aerobic respiration. Uses glycolysis to oxidize glucose to form pyruvate and produce 2 ATP. NADH reduces pyruvate.
Electrons released are not used to make ATP. Electrons carried by NADH are used to power oxidative phosphorylation. Pyruvate is the final electron acceptor. Oxygen is the final electron acceptor. Amount of ATP produced. Requires oxygen. Breaks down pyruvate into CO 2 3. Krebs Cycle The Krebs cycle completes the oxidation of organic molecules.
Isocitric acid loses CO 2 leaving a 5 carbon molecule b. The 5 carbon compound is oxidized and NAD is reduced 4. Catalyzed by multiple enzymes: a.
CO 2 is removed from the 5 carbon molecule b. Remaining 4 carbon molecule is oxidized and NAD is reduced 5.
To balance the oxidation half reaction, we need to add 6 water molecules to add enough oxygen atoms to make all of the carbon dioxide molecules. There will also be 24 protons as products. Now consider the reduction of molecular oxygen 0 oxidation state to water -II oxidiation state. Each oxygen atom requires 2 electrons, for a total of 4. To balance the reaction, we can add 4 protons to the reactant side of the equation.
To get the net reaction, we multiply one of the equations by some factor so that the electrons produced equals the electrons used. The oxidation of glucose to carbon dioxide is the same as above. The Cl V in the chlorate ion is reduced to Cl -I in the chloride anion for a reduction of 6 electrons. Multiplying the reduction half reaction by 4, to use 24 electrons, and combining the oxidation and reduction half reactions gives us the net reaction.
The mitochondrial oxidation of pyruvate derived from glucose glucose oxidation is a major source of acetyl CoA for the tricarboxylic acid TCA cycle and reducing equivalents for adenosine triphosphate ATP production in the heart. Insulin plays a crucial role in cardiac energy metabolism by orchestrating the contribution of oxidative substrates, including glucose and fatty acids, to cardiac ATP production.
Insulin causes a switch in cardiac energy substrate preference, by stimulating glucose oxidation and inhibiting fatty acid oxidation [ 1 , 2 , 3 , 4 , 5 , 6 ]. Insulin indirectly stimulates glucose oxidation via increasing glucose uptake and subsequent glycolysis that increases pyruvate supply for mitochondrial glucose oxidation by the pyruvate dehydrogenase PDH complex, the rate-limiting enzyme of glucose oxidation. In the heart, glucose uptake is mainly mediated by the role glucose transporter 1 and 4 GLUT1 and GLUT4 , which behave differently following insulin stimulation [ 7 ].
GLUT4 the insulin sensitive glucose transporter translocates from the endosome stores to the sarcolemmal membrane for glucose assimilation following insulin stimulation, while GLUT1 is more localized in the cytosol and less dominant in the sarcolemma [ 8 , 9 , 10 ]. Increased glucose uptake drives glycolysis to convert glucose to pyruvate. The activity of PDH is largely regulated by its phosphorylation status and it is active when it is dephosphorylated.
The PDH complex can be phosphorylated and inhibited by pyruvate dehydrogenase kinase PDK , while it can be dephosphorylated and activated by pyruvate dehydrogenase phosphatase PDP. It has also been shown that insulin can directly stimulate glucose oxidation independent of enhancing glucose uptake or glycolysis [ 11 , 12 , 13 , 14 ]. The hallmark of this direct stimulation of glucose oxidation by insulin is the dephosphorylation and activation of the mitochondrial PDH complex [ 11 , 12 , 13 , 14 ].
However, the exact mechanism through which the insulin signal is transmitted from the cell membrane to the mitochondria to activate the PDH complex has not been previously elucidated. The mouse heart has high glycolytic rates that are almost maximized even in the absence of insulin [ 15 , 16 ]. Because of this, insulin does not have dramatic effects on glucose uptake and glycolysis in the mouse heart [ 15 , 16 ]. Despite this, insulin still directly stimulates cardiac glucose oxidation, independent of any change in glucose uptake or glycolysis rates [ 3 , 4 , 5 , 15 , 16 ].
How insulin directly stimulates glucose oxidation in the heart is presently not known. However, because of the high insulin-independent glycolytic rates, the mouse heart provides a valuable tool to examine how insulin stimulates glucose oxidation in the heart independent of pyruvate supply to the mitochondria. Interestingly, it has been shown that these kinases can be translocated to the mitochondria following insulin stimulation [ 17 , 18 , 19 , 20 , 21 ].
The translocation of these kinases has also been linked to modulating mitochondrial oxidative phosphorylation. For example, it has been reported that Akt can rapidly be translocated to the mitochondria following insulin stimulation in a PI3K-dependent manner in SH-SY5Y human neuroblastoma cells [ 17 ].
Taken together, it seems plausible to propose that these kinases are potential candidates to transduce insulin signal from the cell membrane to the mitochondria to directly stimulate the PDH complex and glucose oxidation in the heart. We also hypothesized that manipulating the activity of these kinases in the mitochondria will influence the direct stimulatory effect of insulin on glucose oxidation independent of any change in glucose uptake or glycolysis rates.
Mice were housed at the University of Alberta Health Sciences Lab Animal Services facility in a temperature- and humidity-controlled room with a 12 h light dark cycle. This study used isolated working mouse hearts. The mouse heart has high glycolytic rates that are maximally stimulated in the absence of insulin. In Study 1, male and female mice hearts were randomized to be perfused for 30 min with or without insulin Fig. In Study 2 Fig. In both cohorts, hearts were perfused with Krebs—Henseleit solution The aorta was cannulated with an gauge plastic cannula, while the left atrium was connected to the preload reservoir oxygenator by cannulating the pulmonary vein with a gauge steel cannula.
The preload line and perfusate reservoir was wrapped with a water jacket and heated to When the heart was switched from the Langendorff to the working mode, the left atrium was perfused at a preload pressure of 15 mmHg; the left ventricle worked against a hydrostatic column set at a height equivalent to a pressure of 50 mmHg.
Insulin stimulation of glucose oxidation rates is independent of glycolysis in the mouse heart. Hearts were perfused in an isolated working heart mode for 30 min with either vehicle or insulin throughout the perfusion protocol.
Arrows indicate the time of adding the vehicle of insulin to the perfusate. Data were analyzed using an unpaired student t-test. Glycolysis, glucose oxidation and palmitate oxidation rates were measured by simultaneously sampling 14 CO 2 and 3 H 2 O produced from the metabolism of [5- 3 H]glucose, [U- 14 C] glucose and [9, 3 H] palmitate [ 4 , 16 ]. For ATP production, the rates of glycolysis, glucose oxidation and fatty acid oxidation were multiplied by the number of ATP molecules produced from each process i.
To directly measure cardiac oxygen consumption rates, we cannulated the pulmonary artery and oxygen concentrations difference in the buffer were measured between the left atria and the pulmonary artery using in-line oxygen probes Microelectrodes, USA. The effluent was sampled in triplicates every 10 min during the perfusion protocol and these samples were processed then analyzed and the values for each time point were averaged.
To calculate cardiac efficiency throughout the perfusion protocol, we divided the cardiac work by the oxygen consumption at each time point. This approach allows us to correct for any changes in oxygen consumption that are secondary to changes in cardiac function and not due to alterations in cardiac energy metabolism.
At the end of the perfusions the hearts were homogenized using a modified protocol described previously [ 22 ]. The hearts were then chopped into small pieces with small scissors and homogenized with a teflon pestle in a glass tube. For subsequent investigations, mitochondrial and cytosolic fractions were used. The probed membrane was incubated with the correspondent secondary antibody for 1 h then protein bands were visualized using the Amersham enhanced chemiluminescence kit Cell Signaling Technologies, Danvers, Massachusetts, USA.
Protein bands intensity analysis was performed in a blind fashion using ImageJ program 1. An unpaired student t-test was used for comparison between two experimental groups and one-way or two-way analysis of variance ANOVA followed by Bonferroni post-hoc test was used for multiple comparison.
There was also no significant difference in cardiac glycolytic rates in the presence and absence of insulin Fig. However, insulin did result in a significant increase in cardiac glucose oxidation rates Fig. In contrast, insulin inhibited fatty acid oxidation rates Fig.
Although insulin stimulation enhances glucose contribution to cardiac ATP production at the expense of fatty acid oxidation Fig. There were no significant differences between male and female mice in term of glycolysis, glucose oxidation or fatty acid oxidation data not shown. Therefore, we combined the data from male and female mice. At the end of the perfusion protocol, the heart was homogenized to isolate the cytosolic and mitochondrial fractions. Hearts were homogenized and fractionated using differential centrifugation to isolate mitochondrial and cytosolic portions.
All hearts were perfused for 30 min in the absence of insulin, following which insulin was then added to the perfusate and the hearts were perfused for an additional 30 min Fig. We found that none of the pharmacological inhibitors had any significant effect on cardiodynamics Additional file 1 : Table S2.
In addition, none of the pharmacological inhibitors caused any significant change in glycolysis rates or its contribution to the total cardiac ATP production in the presence and absence of insulin compared to the vehicle-treated hearts Fig.
Similar to Study 1, insulin caused a significant increase in cardiac glucose oxidation rates Fig. Akt inhibition was also accompanied by a significant reduction in the inhibitory effect of insulin on cardiac fatty acid oxidation rates Fig.
This could possibly be a compensatory response to the reduction in glucose oxidation contribution to acetyl CoA for the TCA cycle through the Randle cycle [ 26 ]. Inhibition of Akt abrogates the direct insulin stimulation of glucose oxidation. The metabolic profile of the heart is characterized by measuring b glycolysis, c glucose oxidation and d palmitate fatty acid oxidation along with e their contribution to cardiac ATP production.
Interestingly, there was no further increase in glucose oxidation rates following insulin stimulation Fig. We also found that the inhibition of insulin stimulation of glucose oxidation with Akt inhibitors was associated with abrogation of the phosphorylation of mitochondrial Akt Ser Fig. This abrogation of mitochondrial Akt was also accompanied by inhibition of the insulin-induced activation of the PDH complex Fig. Inhibition of mitochondrial Akt abrogates insulin stimulation of PDH.
Hearts from the second series of perfusions with the pharmacological inhibitors as shown in Fig. Interestingly, the presence of insulin in the Bisindolylmaleimide-treated hearts did not cause a further increase in PDH complex activity Fig.
Interestingly, the presence of insulin did not cause any further enhancement of mitochondrial Akt activity Fig. These findings further emphasize that mitochondrial Akt is prerequisite for mediating the direct insulin stimulation of glucose oxidation.
This study revealed, for the first time, a number of novel and important findings. Second, mitochondrial Akt is essential for the direct stimulation of insulin to cardiac glucose oxidation, independent of glucose uptake and glycolysis. Third, inhibition of mitochondrial Akt increases myocardial oxygen consumption and compromises cardiac efficiency. Moreover, insulin stimulates glucose uptake that increases glycolytic rates and subsequently mitochondrial glucose oxidation rates.
The hallmark of this direct stimulation of glucose oxidation by insulin is the dephosphorylation and activation of mitochondrial PDH complex [ 11 , 12 , 13 , 14 ]. However, the exact mechanism through which insulin signal is transmitted form the cell membrane to the mitochondria to activate PDH complex has not been previously elucidated. We then asked the question whether any of these kinases plays an indispensable role in transducing insulin signal to the PDH complex.
Therefore, we investigated how inhibiting the phosphorylation of each one of these kinases in the mitochondrial will influence insulin-stimulated PDH complex using pharmacological modulators for these kinases. Akt is an important component of the insulin signaling pathway and it mediates the majority of the metabolic actions of insulin.
It enhances glucose uptake by triggering the translocation of insulin-dependent glucose transporter-4 GLUT4 to the cell membrane. Impaired activity of Akt is positively correlated with reduced glucose oxidation [ 3 , 4 ]. It has also been shown that Akt can be translocated to the mitochondrial following insulin stimulation, an effect which is associated with modulating mitochondrial bioenergetics [ 17 , 36 ]. However, how mitochondrial translocation of Akt influences mitochondrial oxidative phosphorylation is not known.
In this study, we found that insulin-stimulated PDH complex is associated with enhanced phosphorylation of mitochondrial Akt.
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