Dealing with stress
Authors: Neil Kaplowitz
Journal: Hepatology (2011)

I am honored to contribute to this outstanding Master's Perspective series and wish to thank the Editors for inviting me. The colleagues who have previously contributed to this section of HEPATOLOGY have all been role models, and their stories are inspirational for me even at this late stage of my career. I will tell you my story and hope that younger investigators and physician–scientists will gain some insights into the approach to a career in academic medicine from my experiences. AASLD, American Association for the Study of Liver Diseases; AP-1, activator protein 1; APAP, acetaminophen; ATF, activating transcription factor; Bcl2, B cell lymphoma 2; Bcl-XL, B cell lymphoma extra large; BSP, bromosulfopthalein; cFLIP, cellular FLICE-inhibitory protein; CHOP, CCAAT/enhancer-binding protein homologous protein; CREB, cyclic adenosine monophosphate response element-binding; CRTC, cyclic adenosine monophosphate response element-binding regulated transcription coactivator; CURE, Center for Ulcer Research and Education; DILI, drug-induced liver injury; ER, endoplasmic reticulum; ETC, electron transport chain; GSH, glutathione; GST, glutathione S-transferase; HLA, human leukocyte antigen; HSF, heat shock factor; IRE-1, inositol-requiring gene 1; JNK, c-Jun-N-terminal kinase; MAP, mitogen-activated protein; MAP2K, mitogen-activated protein kinase kinase; MAP3K, mitogen-activated protein kinase kinase kinase; MHC, major histocompatibility complex; MOMP, mitochondrial outer membrane permeability; MPT, mitochondrial permeability transition; NAPQI, N-acetyl-p-benzoquinone imine; NF-κB, nuclear factor kappa B; Nrf-2, nuclear erythroid 2 p45-related factor 2; PERK, protein kinase RNA-like endoplasmic reticulum kinase; PGC-1α, peroxisome proliferator-activated receptor γ coactivator 1α; P-JNK, phosphorylated c-Jun N-terminal kinase; ROS, reactive oxygen species; Sab, synthetic antigen binder; sXBP1, spliced X-box binding protein 1; TNF, tumor necrosis factor; UCLA, University of California Los Angeles; UPR, unfolded protein response; USC, University of Southern California. I was born in the Bronx in 1943. My father emigrated from Belarus in 1922, and my mother was born in New York, the child of German, Hungarian immigrants. They had no opportunity to receive higher education but very strongly encouraged me to do well in school and to read as much as possible. A turning point for me was the opportunity to attend Stuyvesant High School. This is a public school, and admission is determined competitively by entrance examination; the highest-scoring students were assigned to Stuyvesant or Bronx High School of Science, depending on the location of their residence. My class had approximately 700 students, every single one of whom went on to college; many of the students were brilliant, and the teachers were top notch and delighted to have such a motivated group. In high school, despite the emphasis on math and science, I developed an interest in art, particularly sculpting and drawing, and felt the joy of self-expression by using my imagination. After graduating, I attended NYU–University College in the Bronx, now an extinct campus. I comajored in French literature and Far Eastern history. My father wanted me to be an architect, but my mother convinced me to apply to medical school in my junior year just to see if I could get in. I reluctantly agreed, although torn by the possibility of spending my senior year abroad, but was swayed by the altruistic goal of helping to alleviate human suffering from disease. To my surprise, having only partially fulfilled the prerequisite science courses, I was accepted to NYU School of Medicine and was able to receive my bachelor's degree at the end of my first year of medical school. My acceptance was contingent upon taking organic chemistry in summer school. This course had a profound impact on me. I found the chemical reactions and syntheses, which are built logically on a set of facts, to be a thing of beauty. Medical school began with the basic sciences. This was challenging for me as a liberal arts major with minimal science background. However, two individuals made major impressions on me. First, there was Professor Severoa Ochoa, a Nobel Laureate and Chair of Biochemistry. He gave almost half of the lectures in our biochemistry course. These were presented as experiments rather than facts and pathways. He would identify a question or hypothesis and tell us how he or others designed experiments to test the hypothesis and so on and so on. Even our examinations were structured on how to design an experiment that would prove or disprove a biochemical fact. The course was truly inspirational and gave me insight into the creativity involved in the approach to medical science. I learned that the creative process in science and art are very similar and that a career in scientific discovery might fulfill my need to find self-expression. The second individual was Norman Javitt. Norm gave a few lectures in our clinical pathology course in year 2, which focused on the clinical chemistry of the liver, particularly pyrrole (bilirubin, porphyrins) and sterol (cholesterol/bile acid) metabolism. This was pure organic chemistry—a beautiful, logical series of chemical reactions modifying organic substances to change their physical chemical properties and elimination. Research in hepatology 45 years ago was mainly focused at the interface of organic chemistry and biology. I charged up to him at the end of the lecture and asked if I could do research with him. That was the beginning of a relationship in medical school and later in fellowship as I followed him to Cornell University and Rockefeller University, where Attallah Kappas was my comentor. This led to a number of my early publications on porphyrin and bile acid metabolism.1-4 Norm was a great mentor for my personality. He is rigorous, very critical, and a brilliant individual. I have always been a very curious, fiercely independent person who prefers to figure things out on his own. We were perfect together. He would say to me “Why don't you figure out why coproporphyrin isomer distribution in bile is different than urine or are there bile acid binding proteins in liver?” Then I would go off and develop an experimental approach and return with the data, which he would critique, and I would return to the laboratory and so on. These were wonderful years for me, because I was given the opportunity to develop my skills and approach experiments under the tutelage of a mentor who asked the tough questions. At the same time, I learned to integrate existing knowledge and use my imagination to develop my own ideas. The completion of a manuscript for publication which described the hypothesis, experimental results, and their interpretation gave me the same sense of fulfillment as painting a picture or sculpting a statue. During the third year of my fellowship at Cornell, Ian Percy-Robb, a biochemist from Edinburgh, UK, came to Norm's laboratory at Cornell for sabbatical. We hit it off and worked closely together for a year. During that time, he helped me with my research project to discover bile acid binding proteins in the liver. This brings me to the story of the fraction collector. In the early 1970s when doing column chromatography, we would manually collect timed fractions eluting from the column, which was very tedious. However, the neighboring laboratory had just purchased an automated fraction collector. Percy-Robb had taught me to perform gel filtration column chromatography to size-fractionate liver cytosol proteins. The mission I accepted from Norm was to find bile acid binders, so I decided to start by identifying the known bromosulfophthalein (BSP) organic anion binding proteins, called Y (ligandin) and Z (fatty acid binder) as a frame of reference. I added BSP to rat liver cytosol and loaded the column in the cold room, turned on the automatic fraction collector, and went home. Had I done this manually, I would have stopped after the last of the protein eluted, leaving the remainder of the excess unbound BSP to drain into a waste receptacle. When I returned the next day, there were hundreds of tubes. I added a drop of NaOH to each tube. BSP turns visible purple, and one can measure its absorbance at 580 nm wavelength. After doing this on hundreds of samples, I graphed (manually of course) the data expecting to see 2 peaks of bound BSP (Y+Z) followed by a peak of unbound BSP after the protein had eluted from the column. But to my surprise, there were two peaks of unbound BSP. This was very strange, but I remembered that my mentor had previously shown that BSP is conjugated with glutathione (GSH), and this is mediated by enzymes in the liver cytosol known as GSH S-transferases (GSTs). I checked the two unbound peaks by thin-layer chromatography, and indeed one contained BSP and the other contained BSP-GSH. I then assayed GST enzyme activity and found that the Y peak contained the enzyme.5 The story eventually turned out to be a bit more complicated as some of the family of GSTs also exhibit a nonsubstrate organic anion binding site for ligands such as bilirubin. Nevertheless, this was an example of fortuitous discovery which I owe to the fraction collector and curiosity. This event propelled me into the fields of biochemistry and physiology of GSTs6-8 and GSH metabolism. The study of these vital components of antioxidant defense eventually lead me into the fields of toxicology, drug toxicity, and oxidative stress. In other words, after some exploration of porphyrin metabolism, I was preparing for a career in bile acid metabolism, following in my mentor's footsteps, when chance turned me in a completely unforeseen direction. After finishing fellowship training at Rockefeller University and Cornell University with Attallah Kappas and Norm Javitt, and 1 year on the faculty at Cornell, I spent 2 years (1973-1975) in research at the Oakland Naval Hospital under the Berry Plan. This was a great opportunity to set up my own lab and continue my research on the biochemistry of GSTs.6-8 There, I met John Kuhlenkamp, who has been my technician and laboratory manager to this day. Also, being close to University of California San Francisco gave me the opportunity to develop a friendship with Rudi Schmid, a charismatic figure and a giant in our field. His kindness and interest in me were very important and provided a certain level of mentorship for much of my career. In 1975, after completion of my military service, I took a position at University of California Los Angeles (UCLA) based in the Center for Ulcer Research and Education (CURE) at the Wadsworth Veterans Administration (VA) Hospital. John Kuhlenkamp accompanied me, and we set up a small laboratory with seed support and then spent the next 15 years building our research program. I was the only hepatology investigator at UCLA during that period, which suited my desire for independence. Nevertheless, I benefited greatly from the intellectual environment and outstanding scientists at CURE, such as Mort Grossman, Charles Code, Jon Isenberg, John Walsh, and George Sachs. The gastroenterology fellows during that era were amazing: Tachi Yamada, Ian Taylor, Andrew Soll, Steph Targan, Emeran Mayer, Fergus Shanahan, Andrew Stolz, Bob Gish, Laurie DeLeve, Stuart Sherman, and Shelly Lu, to name a few who have gone on to illustrious careers. I benefited tremendously from guidance and support of the CURE colleagues. Clearly, a young investigator's chances for success are greatly enhanced by working in an outstanding environment with role models, cutting edge science, and access to highly motivated trainees; I was fortunate that some of these talented gastroenterology fellows chose to work with me. Laurie DeLeve, Shelly Lu, and Andrew Stolz established their own successful independent research programs after joining me at the University of Southern California (USC) in Los Angeles. During this period, Murad Ookhtens, a Ph.D. in biomedical engineering, joined my group, which led to a lasting professional relationship and close friendship. Also, a series of postdoctoral fellows from Japan came to Los Angeles to work with me, and this connection with Japan continues. Together with some of my early postdoctoral fellows, Yuichi Sugiyama, Motonobu Sugimoto, Hajime Takikawa, and Andrew Stolz, we did return to explore and discover bile acid binders and their role in bile acid metabolism and transport.9-14 However, I was really captivated with GSH metabolism and its role in detoxification, and this occupied much of my focus from the early 1980s (Fig. 1). The following individuals were among the postdoctoral fellows who worked with me in the GSH field over the years: Tachi Yamada, David Eberle, Tak Yee Aw, Murad Ookhtens, Jose C. Fernandez-Checa, Laurie DeLeve, and Shelly Lu. These individuals were a remarkably talented group of trainees who took up the challenges with intelligence, dedication, and resourcefulness as we discovered that GSH is transported into bile and blood,15-23 that compartmentation of GSH in mitochondria is dysregulated in alcohol-fed animals,24-28 that mitochondrial GSH is vital for defense against physiological and pathological oxidative stress, that the synthesis and transport of hepatic GSH is regulated by various hormonal factors,29-31 and that sinusoidal endothelial GSH is vital for defense.32, 33 Nearly all of these postdoctoral fellows have gone on to very successful careers in academic medicine and/or science. They were all wonderful to work with. Being a mentor is a two-way street. The fun of scientific discovery is greatly enhanced by sharing it with others. Mentorship is more than simply telling trainees what to do; it is sharing one's curiosity and the joy of exploring uncharted territory. I try to teach two other important things. First, the novel or original observation one makes is a small, albeit crucial, part of the scientific process; much of the work has to do with considering alternative explanations for the findings and thus the design of as many experiments as possible to try to prove oneself wrong. These are the controls. The second is that a “failed” experiment, one in which the results are completely opposite to what is expected, represents a unique opportunity to gain a much deeper understanding of what you are studying. Remember the tale of the fraction collector. Falk meeting on GSH in Basel, Switzerland, in 1992. I will digress here to provide a little conceptual context about stress as we understand it today. Disease-causing agents promote subcellular organelle dysfunction (e.g., cytoplasm, endoplasmic reticulum [ER], mitochondria, or nucleus) to varying degrees, which set in motion the activation of transcription factors and signal transduction pathways that are designed to facilitate adaptation to the stress. However, if the cell is unable to sufficiently adapt, the cell enters a fail-safe mode of self-destruction (Fig. 2). For any type of injury scenario, there are opposing forces battling to sustain or overcome the balance. This occurs at the interorgan, intraorgan, intercellular, cellular, and subcellular level. This concept of balance is now well recognized in the adaptive and innate immune systems, regenerative responses, and fibrogenesis. My particular research focus developed along the lines of organelle stress responses, and I will describe how and why this came about. Cell stress in defense and injury. Adaptive responses are activated by organelle dysfunction, leading to specific transcriptional programs designed to mitigate the stress. If balance cannot be successfully achieved, the cell moves into a default mode, promoting injury. CHOP,; Mitoch, mitochondria; ROS, reactive oxygen species. As I look back over the four decades of my academic career, starting with my first faculty position at Cornell in 1972, I can divide my career into two eras. The first 20-25 years was a period when scientists spent the bulk of their time developing reagents, purifying proteins, studying kinetics, and raising antibodies. The second period to the present, especially the last decade, has been the most exciting for physician–scientists. The study of disease pathogenesis, development of animal models, and the ability to gain deeper insights have been greatly facilitated by the availability of the reagents and tools at our fingertips, and therefore my laboratory has been able to focus most of its activity on the study of regulatory pathways in disease pathogenesis. This transformation that occurred in the 1990s brings me to the recent part of my career and the subject of stress, particularly in the context of hepatotoxicity from drugs and alcohol. The transition began as I moved my laboratory across town to USC in 1990 (Fig. 3). I have always preferred to ask questions for which the tools were available to generate meaningful answers, so the advent in the past 15-20 years of polymerase chain reaction, western blotting, microarrays, conditional knockouts, and so forth has enabled me to return to earlier studies in acetaminophen (APAP) toxicity and alcoholic liver disease with new vigor and begin to deepen my understanding of disease pathogenesis. Kaplowitz Lab just arriving at the University of Southern California in 1990. Standing from right to left, Neil Kaplowitz, Hilo Yoshida, Hideki Saijki, Shelly Lu, Jose Fernandez-Checa, Andrew Stolz, Carmen Garcia-Ruiz, John Kuhlenkamp, technician, Tony Koo, Laurie DeLeve, and Murad Ookhtens. Seated right to left, H. Zeng, Linda Hammond, Terry Maddatu. During most of my academic career, I have maintained a strong interest in drug-induced liver injury (DILI), as I realized that my interest in GSH metabolism was very relevant to DILI. Looking back at the development of the field of drug hepatotoxicity, Hy Zimmerman certainly was responsible for putting the spotlight on DILI, especially organizing the clinical and scientific knowledge into a working classification and conceptual approach. But a scientific milestone was the work of Mitchell and colleagues34 in the early 1970s, which appeared as I was first exploring GSTs as detoxifying enzymes and organic anion binding proteins. Mitchell's group showed that APAP toxicity depended on the metabolism to an electrophilic product, N-acetyl-p-quinoneimine I (NAPQI), which was detoxified by GSH; once GSH defense was exhausted, NAPQI was free to bind to protein thiols and this was believed to somehow cause necrosis, presumably by inhibiting the function of key proteins. This work was very exciting to me, because it showed the importance of GSH in defense against drug toxicity and the value of boosting or maintaining its levels with N-acetylcysteine (NAC). In addition, as the role of oxidative stress in many diseases began to emerge, this gave me great reassurance that studying GSH metabolism, cellular defense and redox biology was directly relevant to the physiology and pathophysiology of the liver. Approximately 10-12 years ago, my interest in GSH redox extended to gene regulation and cell death. I was interested in whether the GSH redox state affects susceptibility to tumor necrosis factor (TNF)-induced apoptosis. Indeed, we found that even modest GSH depletion sensitized hepatocytes to killing by TNF and this was due to impaired transcriptional activity of nuclear factor–κB (NF-κB). A number of antiapoptosis genes are regulated by NF-κB accounting for normal resistance of the liver to TNF-induced apoptosis; one aspect is that some of the up-regulated NF-κB–dependent genes dampen or inhibit c-Jun N-terminal kinase (JNK) so that its activation is only transient. When we depleted GSH, this feedback shut-off of JNK was blocked so TNF induced apoptosis could proceed, at least partly because JNK activation was sustained. In these experiments, one of the approaches to depleting GSH which we employed was to expose primary mouse hepatocytes (PMH) to APAP. Inhibition of JNK blocked the TNF-induced apoptosis. As a control, we used APAP alone (no TNF) which, as expected, caused dose-dependent necrosis. However, to our great surprise, the JNK inhibitor protected against APAP-induced necrosis. This was a minor aspect of the article we published in HEPATOLOGY35 and other publications from our laboratory which dealt with the role of oxidative stress and GSH redox status in sensitization to TNF-induced apoptosis36-38 which in itself was of potential importance. However, I could not let go of the unexpected protection against APAP by the JNK inhibitor and suggested that postdoctoral fellows Basuki Gunawan and Derick Han explore this further. Indeed, either a small molecule inhibitor of JNK or antisense silencing of JNK1 and 2 markedly protected against APAP toxicity in vivo.39 We then showed that glycogen synthase kinase 3β (GSK3β) was upstream and that translocation of these kinases to mitochondria was occurring during the early phase of toxicity.40 Mitochondria have emerged as a critical organelle in generating ROS, which then play a key role in activating signal transduction pathways. Mitochondria were known to be a critical target of NAPQI. In the toxicology field, APAP-induced necrosis had been assumed to directly result from a combination of covalent binding and GSH depletion, particularly in mitochondria, leading to reactive oxygen species (ROS) and reactive nitrogen species (RNS) which induced mitochondrial membrane permeability pore transition (MPT) leading to a bioenergetic catastrophe and necrotic cell death. We found that activation and participation of signal transduction pathways were required in order for mitochondrial function to collapse and these pathways were downstream of covalent binding and GSH depletion. Indeed, this could be viewed as a unique type of programmed cell necrosis. One fascinating aspect of this work was that these signaling molecules (e.g., JNK) translocated to the mitochondria, and the binding of activated JNK appeared to worsen mitochondrial function leading to MPT, but only if mitochondria were made vulnerable by prior APAP treatment.41 With this hint that activated JNK binds to mitochondria, I asked another postdoctoral fellow, Sanda Win, to address the target of JNK binding in mitochondria and to determine if this interaction was necessary for toxicity to occur. We identified Sab,42 a protein described a decade ago to bind Bruton's tyrosine kinase in lymphocytes and P-JNK in fibroblast cell lines.43 First, we identified Sab exclusively in the outer membrane of liver mitochondria and then showed that activated JNK induced by APAP coimmunoprecipitated with Sab. We then knocked-down the expression of hepatic Sab in vivo and found that this markedly inhibited both APAP-induced necrosis and TNF/galactosamine-induced apoptosis. Silencing Sab prevented sustained JNK activation in both models.42 Another laboratory has recently generated complementary information in cell lines treated with anisomycin (JNK activator), demonstrating that isolated mitochondria from these cells are susceptible to activated JNK induced inhibition of the electron transport chain (ETC) which was prevented by silencing Sab.43 Our current working model is that Sab serves as a platform for assembly and activation of mitogen-activated protein (MAP) kinases and that this interaction promotes sustained ROS by mediating an inhibitory effect on electron transport; the sustained ROS then promotes sustained MAP kinase activation in a self-amplifying cycle (Fig. 4). During the past 10-12 years, the work in my laboratory on the pathogenesis of hepatotoxicity has been conducted by a sequence of outstanding postdoctoral fellows starting with Guopeng Feng, Hide Nagai, Katsu Matsumaru, Basuki Gunawan, Naoko Hanawa, and Mie Shinohara, who each spent 2 years with me and Cheng Ji, Derick Han, Huan Lou, Zhang-Xu Liu, Tin Aung Than, and Sanda Win, who have remained in the research group after postdoctoral training, and the current postdoctoral fellows, Lily Dara and Behnam Saberi (Fig. 5). This talented, dedicated group of young investigators make it fun for me to continue to pursue the science of hepatotoxicity as we explore new and often unexpected directions. As I noted above, the joy of scientific exploration is an important part of an academician's career. However, sharing this with trainees and young colleagues and helping them move to independent careers is equally satisfying. Model of JNK/Sab in hepatotoxicity. NAPQI or TNF in the presence of inhibition of NF-κB (galactosamine or actinomycin D) inhibit electron transport (ETC), promoting release of ROS from mitochondria. This leads to activation of MAP kinase kinase kinase (MAP3K), e.g., ASK-1, which in turn activates MAP2K (e.g., MKK4) which activates JNK (thin black arrows). P-JNK then binds to Sab which leads to further impairment of ETC and enhanced ROS production sustaining a high level of JNK activation in a self-amplifying cycle (thick red arrows). In the case of APAP, profound GSH depletion renders the mitochondria more susceptible to ROS leading to mitochondrial permeability transition (MPT) pore opening which induces a bioenergetic catastrophe causing the necrotic cell death characteristic of APAP toxicity. In contrast, in the case of TNF/galactosamine or TNF/actinomycin the sustained JNK activation promotes a proapoptotic combination of events mediated by the Bcl-2 family: Bax and Bim translocation to mitochondria, inhibition of Bcl-XL, t-Bid formation and translocation as a consequence of proteosomal degradation of cFLIP (caspase-8 inhibitor), all of which favors mitochondrial outer membrane permeability (MOMP). Adapted from Win et al.42 Current research group. From left to right: Maria Ybanez, Michelle MacViegh-Aloni, Behnam Saberi, Zhang-Xu Liu, Lily Dara, Derick Han, Neil Kaplowitz, Cheng Ji, Sanda Win, John Kuhlenkamp, Tin Aung Than, Murad Ookhtens (missing, Huan Lou). My longstanding interest in the pathogenesis of liver injury extended over the years to alcohol as a causative agent. This began back in the mid-1980s, when Jose Fernandez-Checa joined me as a postdoctoral fellow. We explored GSH metabolism in alcohol-fed rats and found that there was an impairment in the uptake of GSH by mitochondria.24-28 After 7 years working together at UCLA and USC, he moved back to Spain and has had a very successful career. We have continued to collaborate over the years on aspects of the role of mitochondrial GSH in alcoholic liver disease.44-51 Also, Hide Tsukamoto has had a very large impact on our work. He is an outstanding colleague who established an Animal Model Core at USC which has allowed us to perform many studies in the intragastric alcohol-fed mouse model. Then, Cheng Ji joined me as a postdoctoral fellow more than a decade ago. Cheng is an outstanding molecular biologist, so we decided to fish for new clues for pathogenesis by examining gene expression profiles in intragastric alcohol and pair-fed mice. To our surprise, a set of genes reflecting a response to ER stress stood out.52 Very little was known about ER stress response at the time but so this was a new finding and seemed important. Although we focused on alcohol, many others joined this field studying the role of ER stress in the metabolic syndrome and NAFLD/NASH. Cheng Ji has now successfully achieved independence, but we continue to collaborate as we try to understand how alcohol induces ER stress, particularly with respect to the role of one carbon metabolism and homocysteine, and how ER stress contributes to liver disease with respect to steatosis, cell death, fibrosis, and liver cancer.53-57 The latter work has been greatly facilitated by collaborations with our colleague Amy Lee and the creation of the liver-specific GRP78 knockout mouse model which spontaneously develops steatohepatitis and is very susceptible to alcohol, overfeeding, and drug/toxin-induced liver disease.56 ER stress response is an example in our view of a protective, adaptive response (unfolded protein response, UPR), which if overwhelmed or impaired, progresses to a fail-safe lethal outcome. However, along the way it plays a key role in dysregulating lipid metabolism and promoting insulin resistance. So you can see that for many years we have been attempting to understand the role of various types of stress responses in the protection or worsening of liver injury. Our early efforts focused on oxidative stress and GSH defense, then addressed stress responses in the cytoplasm (e.g., MAP kinases and other yet-to-be-published signaling pathways) and ER. Having extensively explored the ER stress response, I turned to another talented postdoctoral fellow, Tin Aung Than, approximately 5 years ago, and asked him to consider whether something analogous to the UPR in response to ER stress could exist in mitochondria in response to mitochondrial stress. Because an aspect of UPR is the activation of a transcriptional program which promotes the production of more ER as well as chaperones to compensate for demand, we decided to focus on mitochondrial biogenesis in response to mitochondrial specific stress induced by a toxin, rotenone, which inhibits complex I of the ETC. It is known that the induction of peroxisome proliferator-activated receptor γ coactivator-1α (PGC-1α) serves as a master activator/regulator of mitochondrial biogenesis. Thus, we set up a model in PMH and HepG2 cells and showed that rotenone at nonlethal doses induced mitochondrial biogenesis and this was blocked by PGC-1α silencing. Thus, the question is what is the retrograde signal from mitochondria which activates the expression of the nuclear PGC-1α gene. PGC-1α is regulated by cAMP-response element-binding protein (CREB), a cyclic AMP responsive transcription factor. cAMP and other signals are known to promote the dephosphorylation and nuclear translocation of CREB-regulated