1. Hepatic insulin resistance: insulin resistance (see below) in the liver<br />Postprandial glucose tolerance: postrandial is the rise in glucose after eating a meal, normally this rise in glucose will lead to an increase I insulin to return blood sugar levels to normal range; tolerance is a condition associated with the continual elevation of glucose following eating due to abnormal insulin regulation and glucose metabolism.<br />Systemic dyslipidemia: abnormal amounts or types of lipids and glyolipids in tissues throughout the body<br />Nonalcoholic fatty liver disease: accumulation of fatty acids in the liver of people that consume little to no alcohol; causes inflammation and scarring and can lead to liver failure.<br />Type 2 Diabetes: (also known as adult-onset or noninsulin-dependent diabetes) chronic condition that affects glucose and lipid metabolism because insulin is unable to regulate glucose transport into cells. The body does not produce enough insulin to maintain a normal level of glucose.<br />Hyperinsulinemia: refers to high levels of insulin in the blood, this is not diabetes but can be associated with Type 2 diabetes and can be caused by insulin resistance.<br />Insulin resistance: a condition in which the body is resistant to the effects of insulin and the pancreas continues to make insulin to compensate for continual high levels of glucose in the blood. Type 2 diabetes can results with the pancreas is no longer able to secrete the amount of insulin required to keep the levels of blood sugar ina normal range.<br />Complex V: ATP synthetase is sometimes referred to as Complex V<br />Mitochondrial fission: Like bacteria mitochondrial undergo division when they become too large, thereby dividing into two mitochondria<br />Mitochondrial fusion: merging of two mitochondria to form a single compartment<br />QUESTIONS:<br />1. How do the authors measure mitochondrial number and size in control and DKO liver tissue? How does the number and size of mitochondria differ in control and DKO mice?<br />Electron microscopy of liver sections from DKO and control mice was used to measure the sizes and numbers of the tissue. DKO mice revealed larger and lucent mitochondria that were distinctly different from the compact and electron-dense mitochndria in control liver. The DKO liver contained twofold larger and 50% fewer fitochondria per liver cell than control liver cells.<br />2. What is Foxo1 and why is its regulation by insulin important for normal mitochondrial function? <br />Foxol is a transcription factor that regulates genes controlling many hepatic functions, including glucose production and lipid metabolism. Insulin inactivates Foxo1 by Akt-stimulated phosphorylation. Since Foxo1 was poorly phosphorylated in the liver of DKO mice, many hepatic genes were dysregulated in DKO livers, including several genes known to control mitochondrial function, biogenesis and dynamics.<br />3. In what way do concentrations of fission related proteins in mitochondria change in DKO mice? How is this related to Foxo1 activity in DKO mice? How to Cheng et al. demonstrate the link between Foxo1 activity and abnormal fission protein expression? What technique is used to measure the levels of these proteins?<br />The concentrations of proteins that regulate mitochondrial fission and fusion, changed markedly in DKO, which was consistent with the accumulation of large and deformed mitochondria.<br />An ablation of Foxo1 ameliorates the metabolic syndrome in DKO mice. Expression of the dysregulated genes in TKO liver was restored to the normal range. Moreover, mitochondrial number and morphology and the expression of fusion and fission proteins were normalized in TKO mice and in mice with Foxo1 deletion.<br />Immunoblotting, detection of expression of mitochondria fusion and fission proteins in control, DKO, TKO liver.<br />4. What is RCR? What does this term measure? What are RCR levels compared to control mice?<br />RCR stands for ‘respiratory control ratio.’ Mitochondrial respiration couples O2 consumption with ATP production, which is measured by RCR – the ratio of the oxygen consumption rate in the presence of ADP to that in the absense of ADP. The RCR was lower in DKO mitochondria, showing that the coupled mitochondrial respiration requires the Irs1 or Irs2 branch of the insulin signaling cascade. The RCR levels show that the ratio of oxygen consumption in the presence of ADP to that in the absence of ADP is much lower than in the controls. This can lead to mitochondrial dysfunciton because mice with lower RCRs were found to have a lower mitochondrial ATP production and a lower cellular ATP concentration.<br />5. How does ATP production differ in DKO and control mice? <br />The ATP production rate (APR) was lower in DKO mice.<br />6. How is electron transport chain activity measured in this study? How is ETC activity affected in DKO mice?<br />The ETC activity is measured by NADH oxidation. The ETC activity decreased 32% in DKO liver and about 50% in db/db and ob/ob liver compared to the controls.<br />7. What is Ppargc-1a? Ppargc-1a levels are increased in DKO mice but its function appears to be inhibited in this study. How do the authors explain this finding? What epigenetic modification is thought to be taking to inhibit Ppargc-1a function and what evidence is provided by the authors to support this idea?<br />Ppargc-1a induces the expression of nuclear respiration factor-1 (Nrf-1) and mitochondrial transcription factor A (Tfam)14,15; it plays a key role in mitochondrial biogenesis and function.<br />Although the authors discovered that Ppargc-1a levels were up to 14-times higher in DKO mice, its function was inhibited because Ppargc-1a was strongly acetylated. Acetylation inhibits Ppargc-1a function.<br />Deacetylation of Ppargc-1a is thought to be mediated by sirtuin-1, which is activated by NAD+. Because the DKO mice have a low NAD+/NADH ratio, sirtuin-1 was not activated, and Ppargc-1a was not deacetylized.<br />The authors tested this by infecting DKO mice with an adenovirus encoding wild-type Ppargc-1a or a mutant (R13- Ppargc-1a) that prevent acetylation and thus inactivate Ppargc-1a. The abundance of acetylated Ppargc-1a was much lower upon infection with R13- Ppargc-1a. Mitochondrial biogenesis and morphology were restored by R13- Ppargc-1a but not by wild-type Ppargc-1a, which shows that acetylation contributed to mitochondrial dysregulation in DKO mice.<br />8. What was the purpose of the targeting Hmox1 with siRNA to eliminate its function? How does Hmox1 affect electron transport chain function?<br />The researchers found the Hmox1 was directly related to the lower heme concentration in mitochondria. Since they levels were elevated, they attacked the Hmox1 with siRNA to reduce the numbers. Upon doing this, they found that the heme concentration returned to normal as well as ETC function became regular again along with a normalized NAD+/NADH ratio<br />9. Resveratrol restored mitochondrial function is this study. Why?<br />Ppargc-1a is strongly acetylated and unable to promote the mitochondrial biogenesis and function that are crucial for normal oxidative phosphorylation and hepatic nutrient homeostasis. Resveratrol (which can compensate for reduced NAD concentration by lowering the Michaelis constant for sirtuin-1 for NAD+) decreased the abundance of acetylated Ppargc-1a and restored mitochondrial function and biogenesis in the primary hepatocytes.<br />10. According to the authors, how does deletion of Foxo1 affect beta oxidation of fatty acids in DKO mice? <br />Deletion of Foxo1 from DKO liver normalized fatty acid oxidation and hepatic content, concurrent with the restoration of mitochondrial function. Thus, our results show how dysregulated insulin signaling can cause mitochondrial dysfunction that leads to metabolic disease.<br />11. Consequently, how strong of a case is made by Cheng et al, that reduction of hepatic Foxo1 is a good target for deducing both mitochondrial dysfunction and accumulation of fatty acids in the liver of people with Type 2 diabetes?<br />