Prime Minister Abe Shinzo made this comment when he convened the first meeting of the Council for Designing 100-Year Life Society on September 11, 2017.
Prime Minister Abe reshuffled his Cabinet on August 3, 2017 to halt the drop in his approval ratings due to the Kake Gakuen issue. The Prime Minister has identified the “revolution in human resources development” as the most pressing issue for the reshuffled Cabinet, and established the Council for Designing 100-Year Life Society to discuss specific measures.
The Second Abe administration has been characterized by its ability to secure high approval ratings from the public with the exception of the periods when he pushed through the Act on the Protection of Specially Designated Secrets, and the new security legislation (Fig. 1). Prime Minister Abe also has a certain number of achievements under his belt. In terms of diplomacy and security guarantees, he has exercised “diplomacy with a bird’s eye view of the globe,” visiting a total of 127 countries and regions. He has worked with the Obama administration to deepen the alliance between Japan and the United States, and he has also built stable relations with the Trump administration. He has succeeded in having the negotiation of the TPP 11 concluded in January 2018. He also has reached an agreement with the EU in the FTA negotiations in July 2018.
He has facilitated the right to collective defense under certain conditions, and established security legislation. On the economic side, he has realized a number of policies including resolving the deflationary recession, reducing corporate taxes, dismantling the policy of reducing acreage of rice cultivation (gentan), reforming corporate governance, and lifting the ban on private residences providing lodging services (minpaku).
Commenting on the stability of the Abe administration, the mass media now talk about Abe ikkyo (the Abe government’s political power) or kantei shudo (Prime Minister-led initiatives). One of the factors behind the stability is that compared to his first administration, Prime Minister Abe himself is now more skilled at controlling the administration.
But, more importantly, the power of the Japanese Prime Minister was systematically strengthened at the time of launching the second Abe Cabinet. In Japan, two major reforms—political reform and restructuring of ministries and agencies—were implemented between the 1990s and the early 2000s and prime ministerial power has been expanding ever since. The Abe government’s political power overlaps with this buildup. We also need to pay attention to the fact that the Prime Minister’s power base has grown even stronger under the second Abe administration.
But the growing power of the Prime Minister is also exposing cracks. This has been apparent in the past few months with fluctuating support for the Cabinet and the lowest Cabinet approval ratings and the highest disapproval rate since the launch of the administration.
Since February 2017, the Abe Cabinet has been criticized for selling state-owned land to the Moritomo Gakuen, for approving an application by the Kake Gakuen to open a veterinary school in a National Strategic Special Zone, and for concealing daily activity logs from the SDF troops posted to South Sudan on peace-keeping missions. The criticisms against the administration re-exasperated after it had become apparent in March this year that the Ministry of Finance had forged the official document, which was used to approve the selling of the state-owned land to the Moritomo Gakuen ater the approval.
Cabinet approval ratings have fallen sharply since May when the spotlight was turned on the Kake Gakuen issue. Even a survey by the Yomiuri Shimbun-sha, where one might generally expect high approval ratings for the Abe Cabinet, recorded a decline in the approval ratings from 61 percent in May to 49 percent in June, and 36 percent in July while the disapproval rating rose from 28 percent in May to 52 percent in July (Fig. 1). As a result, the LDP suffered an overwhelming defeat in the Tokyo Metropolitan Elections in July capturing a mere 23 seats in the 127-seat assembly.
The Abe Cabinet was roundly criticized during the phases of deliberating the Specially Designated Secrets Act and the security legislation. But at this time, Prime Minister Abe himself is facing unprecedented and strong criticism over the Moritomo Gakuen and Kake Gakuen issues. In the Yomiuri shimbun opinion poll, this is indicated point-blank by the sharp rise in the percentage of respondents who answered that they could not trust the Prime Minister—from 29 percent in May to 48 percent in June.
Why is the person of the Prime Minister criticized? To start with, the two issues have something in common. Firstly, there are the suspicions that a special relationship may exist between the Prime Minister and the operators of Moritomo Gakuen and Kake Gakuen. Secondly, the Prime Minister has invited suspicions of the two educational corporations affording preferential treatment. Rather than the right or wrong of any decisions involving Moritomo Gakuen and Kake Gakuen, another point is whether or not documentation exists that clarifies the relationship between the Prime Minister and public officials who were directly responsible for making a series of policy decisions related to Moritomo Gauen and Kake Gakuen.
So far, there is absolutely no evidence that the Prime Minister directly issued any instructions in the two affairs of the Moritomo Gakuen and the Kake Gakuen.
In the case of the Kake Gakuen issue, we have leaks of internal government documents. There are, however, misgivings concerning the testimony of the persons involved and suspicions that persons close to the Prime Minister may have exercised their influence.
There are only “suspicions” that persons close to the Prime Minister have exercised their influence in the policy decision process. There is no conclusive evidence of influence-peddling in the documentation and testimony at the center of attention. The internal MEXT documents that have been brought to light are no more than summaries of statements by Hagiuda Koichi, Deputy Chief Cabinet Secretary, and the Vice-Minister for Policy Coordination compiled by civil servants at the ministry. The Prime Minister has also denied any involvement during deliberations in the Diet, using the phrase “impression management” in his rebuttals (Committee on Audit and Oversight of Administration, June 5, 2017).
But the strengthening of prime ministerial powers over the past twenty years provides the context for the extent of the attention on the Moritomo Gakuen and Kake Gakuen affairs, and for the criticism of the person of the Prime Minister.
It is symbolic that all the politicians and bureaucrats who are the focus of attention where Moritomo Gakuen and Kake Gakuen are concerned are persons whose jobs bring them into the periphery of the Prime Minister. For example, did Hagiuda Koichi, the Deputy Chief Cabinet Secretary at the time, exert his influence on the process of selecting Kake Gakuen as a business operator in a special deregulation zone? Parliamentary officials noted for their competence, including Ozawa Ichiro in the Takeshita Cabinet and Yosano Kaoru in the second Hashimoto Ryutaro Cabinet, have held the post of Deputy Chief Cabinet Secretary in the past. But, they were both experienced Cabinet ministers, having clocked up seven election wins each. In contrast, Hagiuda, the previous Deputy Chief Cabinet Secretary, who had the record of having won four elections, had had no Cabinet minister experience.
In short, public opinion takes a stern view of the behavior of the Prime Minister because prime ministerial power has grown stronger, increasing the potential for strong criticism of any move that can be misrepresented. Although detailed testimony is an issue for the future, it has been pointed out that the broad coverage of the scandals involving lawmakers from the ruling parties in recent years has even influenced the Cabinet approval ratings (e.g., Tokyo shimbun dated July 19, 2017). The context here is, conceivably, the increasing power of the Prime Minister. In short, the Prime Minister now exerts more control within the administration, and as the head of the organization, he is perhaps held to account in a stricter manner than before.
To start with, I would like to discuss how the power of the Prime Minister is understood. I would also like to reflect on the gradual expansion of the power of the Prime Minister since the 1990s. In addition, I would like to demonstrate in detail how the power of the Prime Minister has further expanded since the launch of the second Abe Cabinet in 2012. Lastly, after addressing some anticipated objections to the arguments presented in this article, I would like to consider the present general election.
I would particularly like to focus the discussion on two matters. Firstly, the gradual expansion of prime ministerial power under previous administrations, which provides the context for the strong power exercised by the Prime Minister under the present Abe administration. Secondly, since the Prime Minister has obtained strong powers, he is in a position where he can introduce reforms to further expand his own powers.
Many researchers have already focused on the recent expansion of prime ministerial power including Kantei gaiko (Diplomacy Led by the Prime Minister) by Shinoda Tomohito, Nihon no tochi kozo (Political Structures in Japan) by Iio Jun, Koizumi seiken (The Koizumi Administration) by Uchiyama Yu, and Shusho shihai—Nihon seiji no henbo (Prime Ministerial Control—Transforming Politics in Japan) by Takenaka Harukata, among others. There has been a particularly strong focus on Prime Minister Koizumi Junichi, but recently the published research is looking at the changes since the Koizumi administration. For example, Abe Ikkyo no nazo (The Riddle of the Abe Government’s Power) by Makihara Izuru, Jiminto—Ikkyo no Jitsuzo (The LDP—The Reality of Government Power) by Nakakita Koji, and Shusho seiji no seido bunseki (An Institutional Analysis of Politics Led by the Prime Minister) by Machidori Satoshi. I would like to further develop the discussion based on this series of research.
The power of the Prime Minister is supported by the following three elements. Firstly, there is the authority of the Prime Minister. Secondly, the resources, such as the politicians, staff, and organizations, which the Prime Minister can turn to in drafting policy. Thirdly, public opinion support.
The Prime Minister’s power can be further divided into two parts: his power as the head of the ruling party, and his legal authority as prime minister.
In evaluating the power of the Prime Minister, we need to take into account the politicians, staff, and organizations assisting him as he would find it difficult to show strong leadership in the process of drafting policy were he not given these resources.
In this article, I will focus on the civil servants and the organizations that are recognized in law as resources available for the Prime Minister. Basically, I will treat the civil servants and organizations in the Cabinet Secretariat, and the civil servants and organizations in the Cabinet Office as the Prime Minister’s resources.
In addition, public support is important because of the current electoral system. Under the single-seat constituency system, the political parties fight the election race with the party leader as the face of the election whose popularity influences the results for the party. The popularity of the Prime Minister is important for the ruling party. Whether or not the Prime Minister is able to freely exercise his power depends on whether or not he has secured sufficient support among the public. Due to space limitations, I will only point out how public opinion acts on the power of the Prime Minister.
Let us start from the authority of the Prime Minister. The power of endorsing candidacy and the right to allocate political funds are important sources of the prime minister as the leader of the ruling party. These two types of powers have become politically important because of the political reform introduced in 1994.
Under the political reform of 1994, the electoral system for the House of Representatives was reorganized from the SNTV (Single Non-Transferable Voting System) to a system combining the first-past-the-post voting system with the proportional representation system. For a candidate considering a run in an election under the single-seat constituency system, party endorsement is vitally important to win in elections. A candidate’s ranking is also important when running under the proportional representation system. The controls over political funds have also been strengthened. As a result, it is no longer easy for individual politicians to acquire political funding. In the meantime, as public subsidies to the parties have been introduced.
The party leadership with the head of the party at the top controls party endorsement to candidates in the elections and has the power over how to distribute political funds within the parties. Therefore, under the present system, the head of the party can project a strong influence on the party politicians.
The impact of the political reforms is gradually penetrated into Japanese politics. At the time of the general elections in 1996 and 2000, the electoral districts were adjusted among some of the ruling party candidates with the final decisions frequently taken by the party leadership. As a result of the tighter restrictions on the collection of the political funds after 1994 reform, there has also been a steady decline in the ability of factions to obtain funds. As a result, factions can no longer wield as much power as they did prior to the reforms.
The increasing power of the Prime Minister is vividly demonstrated in the way Cabinet posts are filled. Traditionally, the Prime Minister would take account of the influence of the factions when allocating posts in his Cabinet. He would also respect the recommendations of the factions when appointing the cabinet ministers. The Prime Minister had limited discretion to select Cabinet ministers. However, when Prime Minister Obuchi Keizo assumed office in July 1998, he was able to appoint about half of the Cabinet ministers at his discretion.
The reorganization of central government ministries and agencies in 2001 is important when considering the legal authority of the Prime Minister. Oddly enough, until that time, the Prime Minister of Japan had no clear formal authority to propose policy. In the Cabinet, it was the ministers who had the prerogatives to draft policy. Of course, the Prime Minister could take the initiative on substantial policy proposals. In initiating policies, he had to rely on informal power deriving from the position of the prime minister.
In addition, Article 12-2 of the Cabinet Act established that one of the tasks of the Cabinet Secretariat was the “planning, drafting, and overall coordination concerning the basic principles involving important policies of the Cabinet.” In short, providing assistance to the Prime Minister when he was formulating important policies was added to the tasks of the Cabinet Secretariat.
Prior to the reorganization of ministries and agencies, it was recognized that the Cabinet Secretariat retained the power to coordinate policy among the ministries and agencies and it was possible to rely on this power to draft policy. For example, the Cabinet Secretariat formulated the PKO bill, which involved several ministries and agencies including the Ministry of Foreign Affairs and the Japan Defense Agency. However, this was an exception and it was rare for the Cabinet Secretariat to take initiatives in formulating policy.
The Cabinet Office was established as a body to assist the Prime Minister with the formulation of policy in addition to the Cabinet Secretariat. The Council on Economic and Fiscal Policy for drafting economic and fiscal policies, and the Council for Science and Technology Policy for discussing science and technology policy (currently, the Council for Science, Technology and Innovation) were set up under the Cabinet Office.
Prime Minister Koizumi Junichiro took office in April 2001 amid the expansion of the authority of the Prime Minister and the enhancement of the supporting organizations.
Prime Minister Koizumi largely ignored the intentions of the faction in appointing ministers to his Cabinet. He also implemented one policy after another that would have been unthinkable under a conventional LDP administration including cuts in public construction expenditure, the privatization of the Japan Highway Public Corporation, and the privatization of the postal services.
Prime Minister Koizumi used the Cabinet Secretariat and the Cabinet Office when designing the policies that he wanted to focus on. In the process of formulating economic and fiscal policies, in particular, he made maximum use of the clout of the Council on Economic and Fiscal Policy, essentially using the Council as the forum where he made decisions on key policies such as reducing public works expenditure, writing off non-performing loans, and privatizing the postal services. Takenaka Heizo, Prime Minister Koizumi’s appointee to the post of Minister of State for Economic and Fiscal Policy, was put in charge of managing the Council from April 2001 to October 2005.
Prime Minister Koizumi also made use of the Cabinet Secretariat with the formulation of important legislation. In terms of economic policies, for example, the Cabinet Secretariat prepared the bill related to the privatization of the postal services to implement the privatization, and the bill establishing the Promotion Committee for the Privatization of the Four Highway-Related Public Corporations to discuss the privatization of the Japan Highway Public Corporation. As security policies, the Cabinet Secretariat formulated the bill on anti-terrorism special measures to facilitate cooperation with the war on terror after 9/11, and the bill on special measures for humanitarian relief and reconstruction to dispatch Self-Defense Forces to Iraq after the Iraq War.
Prime ministerial powers have steadily expanded since the Koizumi Cabinet. Nakakita Koji, one of the authors listed above, has carried out a detailed verification of the expanding authority of recent Prime Ministers as leaders of the ruling party. Nakakita focuses on the percentage of politicians with no Cabinet experience among those who have been elected to LDP seats in at least six elections as an indicator of the Prime Minister’s enhanced authority over personnel issues. Calculating the percentages immediately before general elections from the 1980s to the early 2010s, he points out that although the percentage used to be below 15 percent, it has leapt to the upper 20 percent level under the second Abe Cabinet.
This indicator is significant because of substantial decline in the importance of factions in recent years. Conventionally, faction-related indicators were frequently used to scrutinize the strength of the Prime Minister’s power in providing patronage, but it is now highly doubtful that these indicators have the ability to measure such strength.
The fading importance of factions is also indicated by the increase in Diet members who do not belong to a faction. At present, the number of members of the House of Representatives who are not aligned with any faction has risen to sixty, which is on the same scale as the number of members in the largest faction, the Hosoda faction.
The appointments to the posts of Chief Cabinet Secretary and Deputy Cabinet Secretary under the second Abe administration also symbolize the dwindling presence of the factions. Traditionally, with LDP administrations, the Prime Minister has appointed members of his own faction to the posts of Chief Cabinet Secretary and Deputy Cabinet Secretary. There are some precedents, including the appointment of Chief Cabinet Secretary Gotoda Masaharu (Tanaka faction) under the Nakasone Yasuhiro Cabinet, but, ultimately, these were exceptions. The second Abe Cabinet appointed Suga Yoshihide as Chief Cabinet Secretary, and Kato Katsunobu and Seko Hiroshige as Deputy Cabinet Secretaries, but Seko is the only one who is a member of the Prime Minister’s original faction (currently, Abe is not aligned with any faction).
Consequently, the percentage of Diet members who have been elected at least six times without joining the Cabinet is a significant indicator replacing the faction-related indicator. The customary practice in the LDP of focusing on the number of election wins is thought to have begun at the time of the Sato Eisaku Cabinet. There is no doubt that the purpose was to guarantee the loyalty of LDP backbenchers to the prime minister and to restrain rebellions. If the power of the Prime Minister increases, it should be possible to step away from these practices and to appoint the right person to the right position as he wishes.
I have worked out the figures for each Cabinet from the second Mori Yoshiro Cabinet formed in July 2000 to the third reshuffle of the third Abe Cabinet. My conclusions are essentially the same as those in Nakakita’s study. The trend has remained unchanged since the 2014 general election when the percentage of Diet members who had been elected at least six times without joining the Cabinet leapt to 38 percent under the third Abe Cabinet. The level after the third Cabinet reshuffle was 26 percent, which is still high compared to the past (Fig. 2, based on the Kokkai Binran).
Next, I will consider changes in the Prime Minister’s authority and support system, both of which are aspects where prime ministerial power is expanding.
To start with, staff numbers at the Cabinet Secretariat, which is the core of the Prime Minister’s support organization, are rising rapidly. In fiscal 2000, prior to the reorganization of the ministries and agencies, there were no more than 377 regular staff at the Cabinet Secretariat, but by fiscal 2016 the number had risen to 1,119. With the number of persons holding concurrent posts also rising, the total number of regular staff and concurrent posts was 822 in fiscal 2000. By fiscal 2016, the number had more than tripled to 2,779 (Fig. 3, based on the Cabinet Secretariat website, and other sources).
Within the Cabinet Secretariat, the number of divisions under the Cabinet Affairs Office and the Assistant Chief Cabinet Secretary, thought to participate closely in drafting policy, have also risen rapidly. In brief, under the reshuffled second Mori Cabinet immediately after the reorganization of the ministries and agencies, there were only ten divisions. In contrast, the number increased to 39 after the second reshuffle of the third Abe Cabinet (Fig. 4, based on the Kokkai Binran, and other sources).
The Cabinet Office organization is also expanding. There were 2,210 regular staff at the time of the reorganization of the ministries and agencies in fiscal 2001, but the number had risen to 2,324 by fiscal 2016 (Fig. 5, based on the Cabinet Secretariat website, and other sources). In addition, as outlined in the Act for the Establishment of the Cabinet Office, the Cabinet Office was responsible for 76 administrative tasks in 2001, but by the end of 2016, the number had reached 100. The number of councils under the Cabinet Office has also increased from 24 in 2001 to 39 in 2016.
There are three trends in the expansion of the Prime Minister’s support system.
Firstly, the Cabinet Secretariat, the Cabinet Office, and the number of Cabinet Councils have been expanding continuously over the long term. This trend was no different under the administrations of the Democratic Party of Japan when policymaking was unstable.
Secondly, in terms of the structure of the Cabinet Secretariat, there was a period between 2009 and 2012 when the number of internal groups specifically created to take charge of policy decreased briefly due to changes in the political administration (Fig. 4). This suggests a link with policy reviews when administrations change.
Thirdly, the Cabinet Secretariat structure and the Cabinet Councils have expanded rapidly since the advent of the second Abe Cabinet. At the end of 2012, shortly after the inauguration of the Cabinet, the Cabinet Secretariat had 807 regular staff, but by the end of 2016, the number had risen to 1,119. When concurrent posts are included, the numbers have expanded from 2,331 to 2,779 posts (Fig. 3). Directly after the inauguration of the second Abe Cabinet, there were 28 policy organizations, but by the third Abe Cabinet, the number had risen to 39 (Fig. 4).
As a result, the Prime Minister, the Cabinet Secretariat, and the Cabinet Office are now directly responsible for an increasing number of policy domains. The current Prime Minister is, of course, in charge of foreign diplomacy, security guarantees, and the economy, but his brief also ranges the whole gamut of policymaking from regional development to social security, and from the oceans to space.
I would also like to take a close look at how prime ministerial powers have changed under the second and third Abe Cabinets.
There are two distinct features. Firstly, institutional reforms have further broadened the legal authority of the prime minister. Secondly, as I have already described, the Prime Minister’s supporting institutions have been expanded.
The second Abe Cabinet has reformed the civil service based on the Basic Act on Reform of National Public Officers’ Systems enacted by the Fukuda Yasuo Cabinet. In July 2013, an office for civil service reform was set up under the Headquarters for the Promotion of Administrative Reform at the Cabinet Secretariat to formulate a bill to deliver the reforms.
In November the same year, the bill related to the civil service reform was approved by Cabinet decision and the legislation was enacted in April 2014. The legislation has two key dimensions. Firstly, to increase the Prime Minister’s authority over the civil service and its executive leadership. The Prime Minister has taken charge of the civil service system, traditionally the responsibility of the Ministry of Internal Affairs and Communications. The Prime Minister has also acquired more political power in making the list which put the candidates for high ranking officials at the grade of Deputy Director-General and higher at each ministry and agency. Reasonably, the Prime Minister has entrusted the use of this authority to the Chief Cabinet Secretary.
Those who have the authority to manage personnel affairs, including each minister, must choose among the candidates listed in this list when making appointments to leadership posts. They also have to consult with the Prime Minister and the Chief Cabinet Secretary.
The Cabinet Bureau of Personnel Affairs was set up in May 2014 to manage preparation of the list of the candidates for high ranking positions and administer the civil service as a whole.
Prior to the reforms, the Prime Minister and the Chief Cabinet Secretary exercised a certain degree of influence on the appointment of senior public officers at the ministries and agencies. In May 1997, the second Hashimoto Cabinet agreed to set up an advisory panel on reviewing Cabinet personnel affairs, which meant that all senior positions at the grade of Director and higher had to be reviewed in advance by the Chief Cabinet Secretary and the Deputy Cabinet Secretary. On such occasions, the opinion of the Prime Minister would, of course, be taken into consideration. But, it must be remembered that it was the relevant Minister who had the right to appoint and dismiss bureaucrats and the authority to independently manage personnel affairs. The Prime Minister and the Chief Cabinet Secretary may very well have rejected a proposed appointment, but it is difficult to find any examples where they would have endorsed a specific candidate for a post at a ministry or agency.
In this regard, Chief Cabinet Secretary Suga has made use of the new system to actively promote certain bureaucrats to senior positions in ministries and agencies. Specific examples include the vice-ministers at the Ministry of Agriculture, Forestry, and Fisheries, and at the Ministry of Defense, as well as the Commandant of the Japan Coast Guard. In an interview with the Asahi shimbun, the Chief Cabinet Secretary has personally acknowledged that he has used his influence (Asahi shimbun, online edition, February 27, 2017).
Another important institutional change was implemented at the same time as the civil service reforms in 2014. In brief, the Prime Minister gained the authority to set up new organizations and to make staffing decisions at the ministries and agencies (Cabinet Act Article 12-2, items 13 and 14). Seeing that the Prime Minister considers policy for the whole of Japan, it is only natural that he has the authority to make decisions about organization and staffing at each ministry. However, in the past, these tasks have been under the jurisdiction of the Ministry of Internal Affairs and Communications. In this respect as well, the Prime Minister’s influence over each ministry seems to be growing stronger.
In December 2013, the Abe Cabinet reorganized the Security Council, which designed such policies as principles of Japan’s defense policy and National Defense Program Guidelines, into the National Security Council (NSC). In January 2014, the National Security Secretariat was established as the secretariat for the Council.
The main purpose of the Security Council had been to discuss important matters related to the fundamental principles of defense policy as well as the National Defense Program Guidelines. In contrast, the National Security Council also discusses the principles for foreign and defense policies related Japanese security in addition to the policies discussed by the Security Council. Specifically, in principal, members of the 4-Minister meeting, which discusses foreign and defense policies are limited to the Prime Minister, the Chief Cabinet Secretary, the Minister of Foreign Affairs, and the Minister of Defense. The establishment of this meeting was a major reform.
The Cabinet Secretariat prepared the bill to revise the law to create a National Security Council and the bill was enacted in November 2013. Since the establishment of the National Security Council and the National Security Secretariat, the Prime Minister’s ability to formulate security policies has increased significantly. The 4-Minister Meeting is convened regularly. Since the Assistant Chief Cabinet Secretaries, respectively seconded from the Ministry of Foreign Affairs and the Ministry of Defense, serve as Deputy Secretaries General of the National Security Secretariat, it has become possible to coordinate foreign and defense policy much more effectively on a constant basis.
Within the Cabinet Secretariat, there are three Assistant Chief Cabinet Secretaries who coordinate policies involving various ministries. The way they coordinate security policies have drastically changed since the establishment of the Security Secretariat.
Their predecessor organizations are the Cabinet Office for Internal Affairs (COIA), the Cabinet Office for External Affairs (COEA), and the Cabinet Office for Security Affairs (COSA), which were set up by the Nakasone Cabinet in July 1986. The posts have often been held by officials seconded from the Ministry of Finance, the Ministry of Foreign Affairs, and the Ministry of Defense. The COEA was responsible for coordinating matters related international relations, while the COSA coordinated policies related to national security. Prior to the revisions, Article 4 of the Order for the Organization of the Cabinet Secretariat clearly specified that matters under the jurisdiction of the COSA were “excluded” from the jurisdiction of the COEA. It is necessary to coordinate foreign policy and defense policy to design security policies, but since COEA and COSA were separated, the Cabinet Secretariat could not make effective coordination of security policies. (In April 1998, the COSA also assumed responsibility for crisis management and the name was changed to the Cabinet Office for Security Affairs and Crisis Management).
The supporting institutions for the Prime Minister have expanded in an area which is not often noticed. In May 2013, the Abe Cabinet amended the Cabinet Act to establish the post of Deputy Chief Cabinet Secretary for Information Technology Policy with responsibility for coordinating issues involving information and communication technologies. The Deputy Chief Cabinet Secretary for Information Technology Policy works on the integration of information systems at each ministry.
In addition to the series of institutional reforms, the decision-making process within the Cabinet Secretariat has also changed since the launch of the Second Abe Cabinet. The Prime Minister, the Chief Cabinet Secretary, the three Assistant Chief Cabinet Secretaries, and the Secretary to the Prime Minister has come to meet regularly to facilitate communications among them. It is perhaps surprising that there were no regular meetings of the Prime Minister, the Chief Cabinet Secretary, and the Assistant Chief Cabinet Secretaries under previous Cabinets.
Under the Second Abe Administration, the Cabinet Secretariat plays a central role in numerous policy formulation processes. I would like to consider this from the perspective of economic policy.
As soon as Prime Minister Abe assumed office, he made it clear that he intended to press forward with the three arrows of Abenomics—bold monetary policy, flexible fiscal policy, growth strategy to stimulate private investment—as his fundamental economic policy. To formulate the growth strategy, the Prime Minister established the Headquarters for Japan’s Economic Revitalization and created the post of Minister in charge of Economic Revitalization. Under the auspices of the Headquarters, he also set up the Industrial Competitiveness Council, which worked on the Japan Revitalization Strategy, an important Cabinet policy, on an annual basis until 2016. (In September 2016, the Industrial Competitiveness Council was reorganized as the Council on Investments for the Future. In 2017, the Council started coordinating the Investments for the Future Strategy in place of the Japan Revitalization Strategy.) To coordinate revitalization strategies, the General Secretariat for Japan’s Economic Revitalization was set up as the secretariat for the Headquarters and for the Competitiveness Council.
Specifically, in 2013 and 2014, the revitalization strategies included, for example, corporate governance reform, the reform of the Government Pension Investment Fund (GPIF), reduction of corporate taxes, and establishing National Strategic Special Zones. Each policy was delivered. Prime Minister Koizumi made maximum use of the Council on Economic and Fiscal Policy to formulate economic policy. In contrast, Prime Minister Abe has used the Cabinet Secretariat to formulate micro-economic policies.
After the general election in the fall of 2015, Prime Minister Abe launched the “Promotion of the Dynamic Engagement of All Citizens” as an important economic policy for the Cabinet, adding “Work-Style Reform” after reshuffling the Cabinet in August 2016. He created the posts of Minister in Charge of Promoting Dynamic Engagement of All Citizens, and Minister in Charge of Work Style Reform. He also established the Secretariat for Promoting the Dynamic Engagement of All Citizens and the Secretariat for Promoting the Realization of Work Style Reform at the Cabinet Secretariat as the organizations with responsibility for each policy. By March 2017, the Third Abe Cabinet designed the policy for limiting overtime and levying penalties on firms found to be non-compliant.
Prime Minister Abe has also relied on the Cabinet Secretariat for the Trans-Pacific Partnership (TPP) negotiations. In March 2013, the Prime Minister announced that he would participate in the TPP negotiations and in April and set up the Governmental Headquarters for the TPP in the Cabinet Secretariat. He appointed Economy Minister Amari Akira the director of the Headquarters. He also created the positions of Chief Domestic Coordinator to make coordination within the government and Chief Negotiator to lead negotiations. The Headquarters formulated basic policies for the TPP negotiations and handled domestic coordination. A general agreement was reached in October 2015. More than two and a half years passed since the Abe cabinet had announced that Japan would participate in the negotiations.
Next, I would like to take a look at security policies. As soon as Prime Minister Abe assumed office, he indicated that he was considering a review of the right of exercising collective defense. In February 2013, he also reconvened the Advisory Panel on Reconstruction of the Legal Basis for Security, which was established under the First Abe Cabinet, to review the policies vital for the security of Japan including the right of collective self-defense. The Cabinet Secretariat served as the secretariat for the panel. In May 2014, the Advisory Panel compiled a report which recommended that the government should allow exercise of collective self-defense under certain conditions and that it should provide more flexibly rear support for other countries in times of international crisis such as the Gulf War.
Subsequently, Prime Minister Abe had the cabinet make a cabinet decision to revise the Constitutional interpretation of the right of collective self-defense outlined and permit the government to exercise the right collective self-defense under certain conditions. The Cabinet Secretariat did the groundwork for the cabinet decision. Based on the decision, Prime Minister Abe went ahead with preparations for a security-related bill. In May 2015, the Abe Cabinet adopted the bill by Cabinet decision and the bill was enacted in September. The National Security Secretariat at the Cabinet Secretariat prepared and drafted the bill.
This is how Prime Minister Abe has developed, expanded, and made full use of his own supporting organizations to formulate important bills.
Prime Minister Abe has not only expanded his supporting organizations, but he has also carried out an institutional reform.
The reform was based on a recommendation by the Liberal Democratic Party (LDP). The LDP considered that organizations of the Cabinet Secretariat and the Cabinet Office became excessive and they asked for the reduction of the respective institutions. In January 2014, the LDP started to design complete plans for reductions and submitted a proposal to the government in November.
Based on the proposal, the Abe Cabinet examined ways to slim down the Cabinet Secretariat and the Cabinet Office. The plan became official by a cabinet decision in in January 2015. In September, the Cabinet succeeded in having the Diet pass the bill to implement the plan.
The plan consisted from two pillars. First was to eliminate some organizations in the Cabinet Secretariat and to transfer some of them to the Cabinet Office, while moving some of the organizations from the Cabinet Office to other ministries and agencies. Many of the abolished or transferred organizations were introduced prior to the formation of the Second Abe Cabinet. It is possible to interpret that Prime Minister Abe has reorganized the two institutions in such a way that they truly serve his own policy goals.
The second pillar was to amend the National Government Organization Act and to set up a mechanism whereby the Prime Minister by a cabinet decision can assign each minister of state with the responsibilities of “comprehensive” coordination between ministries and agencies concerning important cabinet policies. Until this reform the Cabinet Secretariat and the Cabinet Office were the only organizations which had power to make comprehensive coordination among the ministries. Therefore, the Prime Minister basically had no choice but to rely on the Cabinet Secretariat or the Cabinet Office when formulating policies that involved multiple ministries and agencies. With the reform it has become possible for the Prime Minister to turn to each minister of state, as well as the organizations below the minister for comprehensive coordination. For example, in January 2016, Prime Minister Abe instructed Foreign Minister Kishida Fumio to be in charge of making comprehensive policy coordination within the government for the EU-Japan Economic Partnership Agreement negotiations.
So far, I have argued that the powers of the Japan’s Prime Minister’s have continued to expand both in terms of his authority as well as supporting institutions. Here I anticipate that objections might be raised against the proposition of the continuous expansion of prime ministerial powers. The period between 2006 and 2012 was characterized by a series of short-lived governments. In fact, the Prime Minister changed every year during this period. Even the longest serving cabinet during this period, the Noda Yoshihiko Cabinet, lasted only for one year and four months.
In brief, if the prime ministerial power is so strong, the objection would be to question why it was impossible for successive prime ministers to sustain their administrations for a long period of time.
Certainly, it would be difficult to claim that the Prime Ministers exercised strong power during the period between the first Abe Cabinet and the Noda Yoshihiko Cabinet.
The First Abe Cabinet and the Hatoyama Yukio Cabinet were short-lived because the Prime Ministers poorly managed their administrations. A major reason for the short duration of the Cabinets of Prime Ministers Fukuda, Aso, Kan, and Noda is that the ruling parties were unable to secure a majority in the House of Councilors. The two chambers of the Diet were controlled by different parties, causing the policy process to stagnate.
However, even during this time, the Prime Ministers used their own support institutions to formulate many important policies. Basically, the function of formulating government policy was concentrated in the hands of the Prime Minister and those around him. The seeds for the expanding authority of the Prime Minister under the current Abe Cabinet were sown in this period.
Under the three LDP Cabinets after Koizumi, the number of councils, where the Prime Minister was the principal minister, increased, and the Prime Minister made use of the Cabinet Secretariat for formulating some important policies. For example, in 2006, Prime Minister Abe set up a Council on Strengthening the functions of the Prime Minister’s Office on National Security, with its secretariat in the Cabinet Secretariat, to examine a possible establishment of the National Security Council. This council recommended the establishment of the National Security Council. Prime Minister Abe then set up a task force at the Cabinet Secretariat to prepare for the establishment of the National Security Council, asking them to draft a legislation to amend the Act for the Establishment of the Security Council of Japan. (However, the amendments were not delivered at the time.) He also set up the Advisory Panel on Reconstruction of the Legal Basis for Security to examine possible exercise of the right of collective defense and had the Cabinet Secretariat serve as the secretariat for the panel.
The Fukuda Yasuo Cabinet asked the Cabinet Secretariat to prepare the bill for the Basic Act on Civil Service Reform. A pillar of this bill was to set up the Cabinet Agency for Personnel Affairs to centralize the management of personnel policy of the civil service at the Cabinet. In the end, Prime Minister Fukuda did not set up the agency. Instead, he accepted a proposal from the Democratic Party of Japan to ask the Chief Cabinet Secretary to prepare the register of candidates for senior ranking officials for the ministries and agencies and he decided to introduce the Cabinet Bureau of Personnel Affairs at the Cabinet Secretariat. The bill was enacted in June 2008.
When Prime Minister Aso set up the Council for the Realization of a Reassuring Society to re-examine the nature of social security in Japan, he also put the secretariat for the council at the Cabinet Secretariat. This council proposed the expansion of social security for the working generation of people in employment, which clearly marked a departure from the social security policies which have been pursued so far. Under the Aso Cabinet, the Cabinet Secretariat also formulated the anti-piracy bill which would allow the MSDF to guard private vessels from the piracy off the coast of Somalia.
The DPJ administrations were unable to deliver on the plans for Centralization of policy formulation to the Cabinet and the National Policy Unit included in their manifesto for the 2009 general election, but even so, each Prime Minister used his supporting institutions to formulate several important policies.
Prime Minister Hatoyama Yukio established a National Policy Office instead of the National Policy Unit. The National Policy Office formulated two important policies under the Hatoyama and Kan Cabinets. Firstly, it set the targets for improving of fiscal conditions of Japan, which have been carried over to the Second and Third Abe Cabinets. In July 2010, the office also compiled the New Growth Strategy as the key economic policy of the Kan Cabinet. The strategy incorporated infrastructure exports, expanding exports of agricultural products, building airport facilities, and the introduction of other concessions to social capital. These policies have continued under the Second Abe administration.
The Kan Cabinet was in power in March 2011 when the Great East Japan Earthquake and the Fukushima nuclear disaster occurred. The Kan Cabinet has been criticized for its response, but the Cabinet Secretariat and the Cabinet Office played the central role in designing policies in response to the earthquake and nuclear disaster.
The Kan and Noda Cabinets also pushed for the Comprehensive Social Security and Tax Reform with an increase in the consumption tax as an important pillar of the reform. Prime Minister Kan appointed Yosano Kaoru as Minister for Social Security and Tax Reform while Prime Minister Noda appointed Okada Katsuya Minister for Social Security and Tax Reform.
The Headquarters of the Government and Ruling Parties for Social Security Reform reviewed the reform proposals and the Office for Social Security Reform in the Cabinet Secretariat served as the secretariat for the Headquarters.
As discussed at the outset, since the formation of the Second Abe Cabinet, the Prime Minister’s strength which has become often apparent in the policy formulation process of the current administrations often referred to as Abe Ikkyo (the Dominance of Prime Minister Abe). Prime Minister Abe has, in actual fact, acquired more power than previous prime ministers. In addition to the expansion of the Prime Minister’s legal authority, his supporting institutions centered on the Cabinet Secretariat and the Cabinet Office have also been strengthened.
The institutional strengthening of the prime ministerial powers has allowed Prime Minister Abe to manage the administration in a stable manner and to leave the record of steady policy performance until the first half of 2017. The Prime Minister used the Cabinet Secretariat as the forum for formulating security policies including changes to the interpretation of the right of collective self-defense. He also used the Cabinet Secretariat to formulate a Growth Strategy, Work-Style Reform, and other economic and social policies.
It should be noted that Prime Minister Abe has obtained stronger powers as a steady extension of the increase in prime ministerial powers since the 1994 political reforms. The Prime Minister’s support system, in particular, has continuously expanded since the Koizumi Cabinet.
When you look at the institution of the Prime Minister rather than the individuals in the post, it is interesting to note that the Prime Minister can use his supporting organizations to implement institutional reforms to secure even stronger powers. The establishment of the Cabinet Bureau of Personnel Affairs and the National Security Council are examples of the Prime Minister using the Cabinet Secretariat to expand his own powers even further. The beginnings of the Civil Service Reform, which brought in the Cabinet Bureau of Personnel Affairs, can be traced back to the First Abe Cabinet, or from even a longer perspective, to the Second Hashimoto Cabinet (Final Report of the Administrative Reform Council, 1997). Likewise, the discussions about the National Security Council started during the First Abe Cabinet.
The political process under the Second Abe Cabinet also points to the risks of expanding his supporting institutions. The public has started to take a critical view of the Prime Minister as a result of the expansion of his powers. The problems involving the Moritomo Gakuen and Kake Gakuen have shown that any action that can be interpreted as the Prime Minister’s use of power to favor himself or those close to him is likely to shake the foundations of the administration. The emergence of documents suggesting the involvement of politicians and bureaucrats in the Cabinet Secretariat and the Cabinet Office in the Kake Gakuen issue also suggests problems with management at the continually expanding Cabinet Secretariat.
If the Cabinet Secretariat becomes bloated, it is likely that the Prime Minister and the Chief Cabinet Secretary will be less scrupulous as regard to the management of the Cabinet Secretariat. If there is insufficient oversight, there is a growing risk that the politicians and bureaucrats in the Cabinet Secretariat will instruct the ministries of the alleged intentions of the Prime Minister in the same way that Mito Komon (anonym of the second daimyo of Mito Domain [currently Ibaraki Pref.], Mito Mitsukuni, 1628–1701) brandishes his inro seal in the television drama.
The potential problems caused by huge supporting structure for the Prime Minster were recognized when the Hashimoto Cabinet reorganized the ministries and agencies. It had already been pointed out that if a single organization is given the responsibility for the supporting functions, the issue of how to manage such an organization emerges. Therefore, when the ministries and agencies were reorganized in 2001, the original idea was to divide the supporting functions for the Prime Minister among the Cabinet Secretariat and the Cabinet Office. Cabinet Office would handle implementation and daily coordination, while the Cabinet Secretariat, as a small-scale organization, would only plan and formulate important policies.
There is no doubt that the British system of government has been taken into account as a point of reference when implementing the political system reform in Japan since the 1990s. If we compare the Prime Ministers of Japan and Britain, the powers of the Japanese Prime Minister fall far short of those of the British Prime Minister due to the authority of the Diet committees and the restrictions imposed by the bicameral system. There is still room for improvements in the Diet system. However, the concentration of powers into the hands of the Prime Minister with regard to formulation of policies and drafting of legislation has progressed to a significant degree. In terms of the appropriate scale of an organization managed by a small group of politicians comprising the Prime Minister, the Chief Cabinet Secretary, and the Deputy Chief Cabinet Secretaries, it is doubtful that the Cabinet Secretariat will be able to continue to expand its personnel as it has done in the past.
Prime Minister Abe recovered his support ratings when he reshuffled the Cabinet on August 3, 2017. According to a Yomiuri shimbun survey, support for the Cabinet rose to 50 percent in September. The Prime Minister dissolved the House of Representatives on September 28 and general elections were held on November 22. There are undoubtedly two ulterior motives behind the sudden dissolution of the House. Firstly, there is the concern that the Diet will pursue the Kake Gakuen issue and that the support ratings will once again fall if the matter is discussed at an extraordinary session of the Diet. Secondly, there is the ongoing excitement generated by the Democratic Party. It would be more advantageous to contrive a general election before the new party of Koike Yuriko, the Governor of Tokyo, has completed preparations for entering the national political scene.
Although the Prime Minister’s powers have been enhanced, it is up to the nation to decide whether the administration survives or not. The fact that the Prime Minister retains strong powers clearly indicates the seat of responsibility for national policy. Regardless of the Prime Minister’s intentions, in this general election, there is a sense that a new wind is blowing with Governor Koike Yuriko’s new party (the party of hope) and that it will “reset Japan.” In this context, we are asked to make an overall assessment of whether or not to support an administration where the Prime Minister has accumulated certain achievements due to his enhanced power base, and where the management of the administration has started to unravel as a result of those enhanced powers.
Born in Tokyo in 1971. He joined the Ministry of Finance after graduating from the Faculty of Law at the University of Tokyo. Earned his Ph.D. in political studies at Stanford University. Professor Takenaka was Associate Professor at the National Graduate Institute for Policy Studies before being appointed to his current position in 2010. His publications include Shusho shihai—Nihon seiji no henbo (Prime Ministerial Control—Transforming Politics in Japan), Sangiin toha nanika (Defining the House of Councilors). Awarded the Osaragi Jiro Prize.
Prime Minister Abe Shinzo received a report the Report on the National Council on Social Security System Reform from the Chairman Seike Atsushi (President of Keio University) and said that he would like to decide on the outline for a bill that includes the format and schedule of the reform by August 21. (Source: Prime Minister of Japan and His Cabinet) Prime Minister Abe Shinzo attended a ceremony of raising a signboard for the National Security Secretariat, Cabinet Secretariat. August 2 “We will work with increased speed to give shape to the policies to ensure that everyone throughout Japan fully experiences the economic recovery firsthand. I would appreciate your cooperation in this effort,” said Prime Minister Abe Shinzo at the Council on Economic and Fiscal Policy. (Source: Prime Minister of Japan and His Cabinet) Prime Minister Abe Shinzo attended the ASEAN-Japan Ministerial Policy Meeting on Cyber Security Cooperation held in Tokyo. First and Second Day of the Prime Minister’s Visit to the United States Prime Minister Abe Shinzo visited the United States and held a Japan-France Summit Meeting on 24. On the following day, the Prime Minister held talks with Mr. William (Bill) H. Gates, and then participated in the Special Event towards achieving the Millennium Development Goals (MDGs). Later, the Prime Minister held talks with Mr. Ban Ki-moon, Secretary-General of the United Nations, and received a courtesy call from Bono of U2. After that, the Prime Minister attended a side event related to the post-2015 development agenda and health. In the afternoon, the Prime Minister delivered an address at an award ceremony held at the Hudson Institute, where he received the Herman Kahn Award. The Prime Minister then visited the New York Stock Exchange and delivered a speech. Later, the Prime Minister visited Ground Zero and offered flowers.


Although autophagy is classically viewed as a non-selective degradation system, recent studies have revealed that various forms of selective autophagy also play crucial physiological roles. However, the induction of selective autophagy is not well understood. In this study, we established a forced selective autophagy system using a fusion of an autophagy adaptor and a substrate-binding protein. In both mammalian cells and fertilized mouse embryos, efficient forced lipophagy was induced by expression of a fusion of p62 (Sqstm1) and a lipid droplet (LD)-binding domain. In mouse embryos, induction of forced lipophagy caused a reduction in LD size and number, and decreased the triglyceride level throughout embryonic development, resulting in developmental retardation. Furthermore, lipophagy-induced embryos could eliminate excess LDs and were tolerant of lipotoxicity. Thus, by inducing forced lipophagy, expression of the p62 fusion protein generated LD-depleted cells, revealing an unexpected role of LD during preimplantation development.
Macroautophagy (hereafter referred to as autophagy) is an evolutionarily conserved system of ‘self-eating’, in which cytoplasmic content is engulfed by the autophagosome and delivered to the lysosome for degradation (Mizushima, 2007). Although autophagy was originally characterized as a non-selective bulk degradation pathway, recent studies revealed that autophagy plays crucial roles in selective degradation of cellular components, including organelles (Okamoto, 2014; Stolz et al., 2014). Autophagy adaptors, which act as cargo receptors for selective degradation, play important roles in determining selectivity for target organelles. The most well-characterized adaptor is p62 (also known as Sqstm1), which contains an LC3-recognition sequence (LRS)/LC3-interacting region (LIR), a ubiquitin-associated (UBA) domain, and a Phox and Bem1p (PB1) domain, which are responsible for dimerization and multimerization of the protein (Katsuragi et al., 2015). To activate selective autophagy, surface proteins on damaged organelles (e.g. mitochondria) or microbes are first ubiquitylated by ubiquitin ligase. Then, autophagy adaptors, such as p62, are recruited onto the damaged organelle via interactions with the ubiquitylated proteins. The adaptors on the damaged organelle are recognized by LC3 (Map1lc3b), and the target is then engulfed by the autophagosome (Anding and Baehrecke, 2017; Stolz et al., 2014). Therefore, the crucial steps in selective autophagy are recruitment of an adaptor to the substrate and the interaction of the adaptor with LC3.
Lipid droplets (LDs) are unique organelles that store lipids essential for cellular energy (metabolism) and membrane production (Thiam et al., 2013; Walther and Farese, 2012). LDs consist of a neutral lipid core, mainly made up of triglyceride (TG) and cholesterol esters, and are coated by a phospholipid monolayer and various proteins such as the perilipins (Plins) [Plin1, ADRP (Plin2; also known as adipophilin), TIP47 (Plin3), Plin4 (also known as S3-12) and Plin5 (also known as OXPAT)]. The Plins share a conserved sequence, the PAT domain, primarily at the N terminus, which is essential for the LD interaction (Bickel et al., 2009). In mammals, several Plins are widely expressed (e.g. ADRP and TIP47), whereas others are expressed in a tissue-specific manner (e.g. Plin1 in adipocytes and steroidogenic cells) (Bickel et al., 2009). LDs are present in all tissues, and serve various functions in addition to lipid storage (Fujimoto and Parton, 2011).
Lysosomes contain both proteases and acidic lipases. Indeed, LDs are also targets of a form of selective autophagy termed lipophagy. During this process in mammals, yeast and Caenorhabditis elegans, LDs are transported to the lysosomes (O'Rourke and Ruvkun, 2013; Singh et al., 2009; Wang et al., 2014), where they are broken down by acidic lipases. This suggests that regulation of lipophagy might provide a therapeutic approach to preventing metabolic syndrome. However, the molecular machinery responsible for lipophagy, including its specific adaptors, remains uncharacterized.
In the mammalian oocyte, a considerable number of LDs accumulate during oogenesis, although the amount differs across species (Sturmey et al., 2009). For example, in comparison with mouse, porcine oocytes contain massive amounts of LDs (Leese, 2012), which interferes with embryonic manipulation, especially in the context of cryopreservation (Nagashima et al., 1995). The physiological function of LDs stored in the oocyte/embryo is species specific, but, in general, LDs are required for energy (i.e. ATP) production during preimplantation embryonic development (Dunning et al., 2014). LDs stored in the oocyte dynamically change their morphology during oocyte maturation and subsequent embryonic development. In unfertilized (MII) oocytes (before fertilization), LDs are mostly detected as clustered structures, which disperse into smaller droplets soon after fertilization (Bradley et al., 2016; Watanabe et al., 2010). Interestingly, when MII oocytes are cultured without pyruvate and glutamine (i.e. energy-depleted conditions), the clustered LDs are dispersed (Bradley et al., 2016). Thus, LD morphology changes in response to cellular energy level.
In this study, we developed a system of forced lipophagy using a fusion protein of p62 and TIP47, an LD-binding domain. Using this system, we demonstrated that the amount of LDs was decreased upon induction of forced lipophagy in cultured mammalian cells expressing TIP47-p62. Our results demonstrate that maintenance of the proper amount of LDs is important for early embryonic development.
We hypothesized that expression of an artificial linker protein that connects substrates to the autophagosomal membrane would induce selective autophagy. p62 is a well-characterized adaptor that binds to LC3 via the LIR motif (Ichimura et al., 2008; Pankiv et al., 2007). Given that overexpression of p62 results in Nrf2 (Nfe2l2) activation through Keap1 binding, we used the p62T352A mutant, which lacks the Keap1 interaction (Komatsu et al., 2010). The PAT domain of TIP47 has a high binding affinity for LD surfaces, and efficiently competes with other proteins from LD surface (Bulankina et al., 2009; Kory et al., 2015). Therefore, we fused the PAT domain with p62T352A and the red fluorescent protein mCherry, yielding TIP47-mCherry-p62T352A (Fig. 1A). To determine whether this fusion protein could induce lipophagy, we expressed TIP47-mCherry-p62T352A in HeLa cells and visualized LDs using the fluorescent neutral lipid dye BODIPY 493/503. HeLa cells in regular media contain few LDs, and no significant change in the number of LDs was detected in cells expressing TIP47-mCherry-p62T352A (data not shown). Therefore, to induce high levels of LDs, we treated the cells with oleic acid (OA). In the treated cells, the amount of LDs was markedly decreased by expression of TIP47-mCherry-p62T352A in comparison with TIP47-mCherry (Fig. 1B). To determine whether the reduction of LDs was dependent on autophagic degradation activity, we expressed TIP47-mCherry-p62T352A in Atg14 knockout (KO) cells, which are defective in autophagosome formation (Itakura et al., 2008). The Atg14 KO cells did not exhibit any reduction in the amount of LDs upon expression of TIP47-mCherry-p62T352A (Fig. 1B), suggesting that TIP47-mCherry-p62T352A induced degradation of LDs, but did not inhibit LD synthesis or recruitment of endogenous LD-associated proteins to LDs. Analysis of the fluorescence intensity of BODIPY 493/503 by flow cytometry confirmed that the amount of LDs per cell was lower in OA-treated cells expressing TIP47-mCherry-p62T352A than in mCherry-negative cells (Fig. 1C), indicating that expression of TIP47-mCherry-p62T352A in cells accelerates breakdown of LDs via forced lipophagy.
Forced lipophagy by expression of TIP47-mCherry-p62T352A in cultured mammalian cells. (A) Schematic of forced lipophagy. The LD-binding domain from TIP47 was fused with p62T352A and mCherry. TIP47-mCherry-p62T352A interacts with LC3 on the autophagosomal membrane via the LD-binding domain, and is then incorporated into autophagosomes via the p62–LC3 interaction. TIP47-mCherry-p62T352A-coated LDs were broken down by lysosomal lipases. (B) Wild-type and Atg14 KO HeLa cells expressing TIP47-mCherry or TIP47-mCherry-p62T352A were cultured with OA for 24 h. LDs were stained with BODIPY 493/503 and analyzed by immunofluorescence microscopy. Red and blue dashed lines outline cells expressing or not expressing mCherry-fusion protein, respectively. Bottom panels show higher magnification images of the boxed regions. Scale bars: 10 µm (top panels); 1 µm (bottom panels). (C) Cells expressing TIP47-mCherry or TIP47-mCherry-p62T352A were cultured with OA for 24 h. Then, LDs were stained with BODIPY 493/503, and the total amount of LD per cell was determined by flow cytometry. The relative intensity of BODIPY 493/503 fluorescence in mCherry-positive cells is expressed as a percentage relative to that in mCherry-negative cells. Data represent mean±s.e.m. *P<0.05. Data are representative of three independent experiments.
This finding prompted us to perform a functional analysis of cells in which LDs were depleted by forced lipophagy. For this purpose, we focused on mouse early embryonic development, for several reasons. First, because LDs accumulate during oogenesis, oocytes contain considerable numbers of LDs. Second, autophagy is highly activated after fertilization, and is essential for early embryonic development (Tsukamoto et al., 2008). Third, the physiological function of LDs during embryonic development remains largely unknown. To induce forced lipophagy after fertilization, we microinjected mRNAs encoding either TIP47-mCherry (control) or TIP47-mCherry-p62T352A (lipophagy-induced) into the cytoplasm of one-cell embryos (Fig. 2A). The embryos were collected 4-5 h after in vitro fertilization (IVF), the time at which autophagy is highly activated (Tsukamoto et al., 2008). Induction of lipophagy resulted in formation of LD clusters, which were subsequently delivered to the cell periphery during embryonic development. No such clustering and movement of LDs was observed in control embryos (Fig. 2B, Fig. S1A, Movie 1). We confirmed that the clustered LDs observed in lipophagy-induced embryos were specifically labeled with BODIPY 493/503 (Fig. 2C). Similar changes in LD morphology after lipophagy induction occurred in embryos collected from another mouse strain, C3H (Fig. S1B). By contrast, in control embryos, LDs were distributed uniformly, as reported previously (Bradley et al., 2016; Watanabe et al., 2010), although TIP47-mCherry signals failed to localize to the LD surface and were diffused throughout the cytoplasm, probably owing to the abundance of ADRP protein in mouse oocytes and embryos, which could block access of TIP47-mCherry to LDs (see also Fig. S1C,D and Discussion).
Forced lipophagy in fertilized mouse embryos. (A) Experimental design for analysis of forced lipophagy during preimplantation embryonic development. To induce lipophagy, TIP47-mCherry-p62T352A mRNA was microinjected into the cytoplasm at the one-cell stage, and the embryos were analyzed. (B) Morphology of LDs in two-cell embryos expressing either TIP47-mCherry or TIP47-mCherry-p62T352A. (C) Embryos microinjected with or without TIP47-mCherry-p62T352A mRNA were stained with BODIPY 493/503 at the indicated stages, and observed by laser confocal fluorescence microscopy. DIC, differential interference contrast. Insets show higher-magnification images. (D) Triglyceride (TG) levels at the eight-cell stage in embryos microinjected with either TIP47-mCherry (control) or TIP47-mCherry-p62T352A mRNA. Data are representative of five independent experiments with 40 embryos per experiment. (E) Electron microscopic analysis of embryos not injected (non-injected control) or injected with TIP47-mCherry-p62T352A mRNA (lipophagy-induced) at the four-cell stage. Right panels: quantification of LD size and number per blastomere in non-injected control (15 blastomeres selected randomly from five embryos) and lipophagy-induced (11 blastomeres selected randomly from four embryos) embryos. Error bars represent s.e.m.; *P<0.05 (Student's t-test). Scale bars: 10 µm (B,C); 5 µm (E).
In lipophagy-induced embryos, the content of TG, a major component of LDs, decreased approximately 50% at the eight-cell stage in comparison with control embryos (Fig. 2D). Furthermore, quantitative analysis of electron micrographs of the four-cell stage revealed that induction of lipophagy significantly decreased LD number and size (Fig. 2E). Thus, forced lipophagy caused not only morphological changes of LDs, but also a reduction in intracellular lipid storage.
Although we used the p62T352A mutant to prevent secondary effects of p62, we were still concerned that p62 overexpression alone might induce lipophagy, largely because p62 plays multiple roles in cell signaling, including lipid metabolism (Rodriguez et al., 2006). To eliminate this concern, we synthesized mRNA encoding DsRed-p62WT (fusion protein of DsRed and wild-type p62) and microinjected it into one-cell embryos. DsRed-p62WT was detected as cytoplasmic puncta that stained with an anti-ubiquitin antibody that recognizes poly-ubiquitylated proteins, representing protein aggregates, indicating that DsRed-p62WT was functional (Fig. 3A). We also confirmed that these p62-positive foci partially overlapped with LC3 (autophagosome marker) and Lamp1 (lysosome-associated membrane protein 1, a lysosome marker), indicating that, like LC3, overexpressed p62 also functions as an autophagy adaptor (Fig. 3B). Importantly, DsRed-p62WT expression induced neither LD clustering nor a close interaction between DsRed-p62WT and LDs throughout embryonic development (Fig. 3C). Therefore, we concluded that the clustering and movement of LDs observed in lipophagy-induced embryos was unlikely to be due to p62 overexpression, and was instead dependent on p62 localization on the LD surface.
p62 overexpression does not alter LD morphology. (A) Embryos microinjected with DsRed-p62WT mRNA were cultured to the two-cell stage, stained with anti-ubiquitin antibody and observed by laser confocal fluorescence microscopy. (B) Embryos microinjected with DsRed-p62WT mRNA were cultured to the four-cell stage, stained with anti-LC3 or Lamp1 antibodies, and observed by laser confocal fluorescence microscopy. (C) Embryos microinjected with DsRed-p62WT mRNA were cultured to the indicated stages, stained with BODIPY 493/503, and observed by laser confocal fluorescence microscopy. DIC, differential interference contrast. Insets show higher-magnification images. Scale bars: 10 µm.
To determine whether forced lipophagy was autophagy dependent, we first analyzed the localization of clustered LDs relative to autophagosomes and lysosomes. Immunofluorescence analysis revealed that both LC3 (autophagosome) and Lamp1 (lysosome) were in close proximity to the clustered LDs in lipophagy-induced embryos, but not in non-injected control embryos (Fig. 4A,B). In the presence of wortmannin [a phosphoinositide 3-kinase (PI3K) inhibitor, which blocks the initial step of autophagy], this close localization was lost, although the clustered LDs were still present. Furthermore, treatment with chloroquine, an inhibitor of lysosomal hydrolases, not only inhibited LD clustering and association with autophagosomes and lysosomes, but also changed the lysosome positioning: specifically, the predominantly peripheral localization of lysosomes was abolished (Fig. 4A,B).
Forced lipophagy depends on autophagy. (A,B) Immunofluorescence imaging of LD localization with LC3 (autophagosomes) or Lamp1 (lysosomes) in two-cell embryos injected with TIP47-mCherry-p62T352A mRNA (lipophagy-induced) or not injected (non-injected control), and then co-cultured in the presence or absence of wortmannin or chloroquine for 24 h. (C,D) Immunofluorescence analysis of LD localization with LC3 or Lamp1 in two-cell embryos simultaneously injected with siRNA targeting Atg14 and either TIP47-mCherry (control) or TIP47-mCherry-p62T352A mRNA (lipophagy-induced). (E) Electron microscopy images of lipophagy-induced embryos at the four-cell stage. Right three panels show higher-magnification images of the representative boxed areas (1, 2 and 3) in the left panel. Red arrows and blue arrowheads indicate lysosome and autolysosome, respectively. AP, autophagosome; LD, lipid droplet; MI, mitochondria. (F) Autophagic flux assay of embryos microinjected with TIP47-mCherry or TIP47-mCherry-p62T352A mRNA. Embryos were co-cultured in the absence or presence of bafilomycin A1 (BafA1), and total cell extracts (70 embryos per lane) were subjected to immunoblot analysis using anti-LC3 antibody. The membrane was re-probed with anti-tubulin antibody as a loading control. (G) Unfertilized (MII) oocytes microinjected with or without TIP47-mCherry-p62T352A mRNA were cultured for 2 days in vitro, stained with BODIPY 493/503, and observed by laser confocal fluorescence microscopy. DIC, differential interference contrast. Insets show higher-magnification images. Scale bars: 10 µm (A-D,G); 5 µm (E, left panel); 0.5 µm (E, right three panels).
To further assess the specificity of LD clustering upon autophagy, we microinjected siRNA targeting Atg14 along with mRNAs encoding either TIP47-mCherry or TIP47-mCherry-p62T352A. In these embryos, LC3 dots (representing autophagosomes) were almost absent (Fig. 4C), and embryonic development was impaired, as a result of efficient inhibition of autophagy (Fig. S2A,B). Additionally, LD clustering was diminished, and lysosomal association of LDs was weaker in siRNA-treated lipophagy-induced embryos than in control lipophagy-induced embryos (Fig. 4D, Fig. S2C).
Consistent with our immunofluorescence results, electron microscopic analysis of lipophagy-induced embryos confirmed the abundance of lysosomes and autolysosomes, which were localized proximally to the clustered LDs at the cell periphery (Fig. 4E).
We next investigated whether lipophagy induction would alter endogenous autophagic activity. To this end, we cultured embryos microinjected with either TIP47-mCherry or TIP47-mCherry-p62T352A mRNA in the presence or absence of bafilomycin A1 (BafA1, a lysosomal inhibitor), and then performed immunoblotting with anti-LC3 antibody. The autophagic flux did not differ significantly between control and lipophagy-induced embryos (Fig. 4F), indicating that endogenous autophagic activity was not altered after induction of lipophagy.
To further assess whether lipophagy induction was autophagy specific, we expressed TIP47-mCherry-p62T352A in unfertilized (MII) oocytes, in which autophagic activity is low, and cultured them for 2 days in vitro. Under these conditions, LD clustering occurred, whereas LD translocation did not (Fig. 4G), indicating that p62 expression at the LD surface is responsible for LD clustering even under low (or absent) autophagic activity, whereas high autophagic activity is required for LD movement to the periphery. Taken together, these results suggested that forced lipophagy occurs in an autophagy-dependent manner.
Based on these results, we hypothesized that lipophagy-induced embryos have higher developmental potential as a result of their more efficient utilization of intracellular LDs in comparison with normal embryos. To investigate this possibility, we cultured embryos microinjected with either TIP47-mCherry or TIP47-mCherry-p62T352A mRNA for 4-5 days in vitro. Unexpectedly, the proportion of embryos reaching the late blastocyst stage within this time frame was lower in lipophagy-induced embryos (30.7%) than in control embryos (56.7%) (Fig. 5A,B). Because the mouse embryo is particularly reliant on exogenous nutrients (Leese, 2012), we then cultured control and lipophagy-induced embryos without amino acids. The blastocyst formation rate of the lipophagy-induced embryos was not influenced by the presence or absence of amino acids, whereas amino acid deficiency significantly decreased the rate of development in control embryos (Fig. 5A,B). These results imply that utilization of intracellular LDs is important for early embryogenesis, particularly in the absence of extracellular energy sources.
Developmental potential of lipophagy-induced embryos. (A) Representative images of embryos microinjected with TIP47-mCherry (control) or TIP47-mCherry-p62T352A mRNA (lipophagy-induced) and cultured with or without amino acids (AA) until the blastocyst stage (96 h after IVF). (B) Percentage of control or lipophagy-induced embryos cultured with or without amino acids (AA) that reached the late blastocyst stage. (C) Representative images of embryos expressing TIP47-mCherry (control) or TIP47-mCherry-p62T352A (lipophagy-induced) after 120 h of culture under the indicated conditions. (D) Percentage of control or lipophagy-induced embryos that reached the late blastocyst stage under the indicated culture conditions. (E) Representative images of embryos expressing TIP47-mCherry (control) or TIP47-mCherry-p62T352A (lipophagy-induced) after 120 h of culture in the presence of PA. (F) Percentage of control or lipophagy-induced embryos that reached the late blastocyst stage when co-cultured with PA. (G) Intracellular level of apoptosis in control and lipophagy-induced embryos. Embryos expressing TIP47-mCherry or TIP47-mCherry-p62T352A, cultured with PA to the blastocyst stage, were stained with Annexin V-EGFP, and total cellular fluorescence was measured by CCM. Error bars represent s.e.m.; numbers in parentheses indicate total number of embryos analyzed from three independent experiments; *P<0.05 (Student's t-test). Scale bars: 100 µm.
The intracellular distribution of LDs may be linked to energy balance, e.g. the close association of LDs with mitochondria facilitates ATP production via β-oxidation (Herms et al., 2015). Accordingly, we analyzed the ATP content in lipophagy-induced embryos. Throughout embryonic development, ATP content did not differ significantly between lipophagy-induced and control embryos (Fig. S3A). Consistent with this, clustered LDs were not localized in close proximity to mitochondria in lipophagy-induced embryos (Fig. S3B). In addition, we observed no significant difference in cellular levels of reactive oxygen species (ROS) between control and lipophagy-induced embryos (Fig. S3C). These data indicate that lipophagy induction is not directly involved in increasing cellular ATP, even when mitochondrial function is not impaired.
We further assessed the contribution of lipolysis to lipophagy-induced embryos. To this end, we cultured either control or lipophagy-induced embryos with OA, to stimulate TG synthesis, in the presence or absence of the lipase inhibitor diethylumbelliferyl phosphate (DEUP). OA supplementation had a negative influence on embryonic development in control embryos, but not in lipophagy-induced embryos (Fig. 5A-D, Fig. S4). Moreover, in the presence of DEUP, almost all control embryos were developmentally retarded, probably owing to excess accumulation of LDs, whereas 12.7% of lipophagy-induced embryos survived even to the late blastocyst stage (Fig. 5C,D, Fig. S4).
Palmitic acid (PA, a saturated fatty acid) is more lipotoxic than OA (unsaturated fatty acid) in a variety of cell lines (Listenberger et al., 2003). To assess tolerance of lipotoxicity, we cultured control or lipophagy-induced embryos with PA to the blastocyst stage. PA treatment substantially decreased the developmental rate (i.e. the rate at which the embryos formed blastocysts) in both groups (Fig. 5A,B,E,F), probably as a result of lipotoxicity. However, the level of apoptosis, indicative of lipotoxicity, was lower in the lipophagy-induced embryos than in control embryos (Fig. 5G).
Taken together, these data indicate that forced lipophagy plays an important role in avoiding excess LD accumulation, and can confer resistance to lipotoxicity.
In this study, we developed a system for inducing forced selective autophagy of LDs, termed forced lipophagy, using a fusion protein of p62 and an LD-binding domain. p62 on the surface of LDs can be recognized as an autophagic cargo, facilitating the interaction with LC3 through its LC3-interacting region (LIR). LC3 stably binds to the autophagosomal membrane, forming a bridge between LDs and the membrane. The autophagosomal membrane then elongates to form the autophagosome, which encloses a portion of LDs, and subsequently fuses with the lysosome for degradation (see our proposed model, Fig. S5). LDs are also metabolized (i.e. degraded) by cytosolic neutral lipases such as adipose triglyceride lipase (ATGL; PNPLA2) and hormone-sensitive lipase (HSL; LIPE), both of which contain LC3-binding motifs required for their lipolytic activities (Martinez-Lopez et al., 2016). In addition, lipolysis is tightly coupled with chaperone-mediated autophagy (CMA) (Kaushik and Cuervo, 2015). Although additional unknown factor(s) and mechanism(s) such as self-oligomerization of p62 via the PB1 domain (which may promote LD clustering) and degradation of LD coat proteins (Plins) by the proteasome (which may stabilize the association of p62 on the LD surface) might contribute to this process, our results strongly suggest that expression of p62 on the LD surface is sufficient to induce selective LD degradation in mammalian cells and fertilized mouse embryos.
We observed that induction of lipophagy in mouse embryos caused LD clustering and translocation to the cell periphery. This clustering also occurred in cultured mammalian cells, whereas translocation did not (data not shown), suggesting that the latter process is mediated by an embryo-specific mechanism. Although the mechanism underlying this process is not fully understood, lipophagic activity in embryos might be required for subsequent translocation of clustered LDs to the cell periphery. Consistent with this idea, in unfertilized oocytes (in which autophagic activity is low), expression of TIP47-mCherry-p62T352A caused LD clustering but not subsequent movement. Similar results were obtained when lipophagy-induced embryos were cultured with wortmannin, an inhibitor of autophagy, and injected with siRNA targeting Atg14. In contrast, lysosomal inhibition by chloroquine treatment shifted lysosome localization, but caused neither LD clustering nor translocation in lipophagy-induced embryos, indicating that lysosomal positioning influences the association between clustered LDs and lysosomes.
Unexpectedly, we found that forced lipophagy induction decreased embryonic viability (Fig. 5A,B), suggesting that LDs consumed by forced lipophagy at an early stage of embryonic development might be necessary at a later stage. Thus, preimplantation embryonic development requires not only endogenous lipolysis, but also the proper amount of LDs. On the other hand, the developmental potential of lipophagy-induced embryos was not significantly influenced by the presence of amino acids. At present, we know little about the molecular machinery responsible for this phenomenon, but speculate that a signaling pathway that regulates autophagy [e.g. mammalian target of rapamycin (mTOR), which negatively regulates autophagy] or cellular energy balance [e.g. AMP-activated protein kinase (AMPK), which is activated by ATP depletion] could be involved, facilitating the recycling of fatty acids (FAs) generated by forced lipophagy.
By contrast, some lipophagy-induced embryos survived under conditions in which excess LDs accumulated (i.e. in the presence of OA and a lipase inhibitor) or lipotoxic species were generated (i.e. under PA supplementation). This result indicates that forced lipophagy attenuates lipotoxicity by eliminating excess LDs. Although the precise mechanism underlying tolerance of lipotoxicity remains unknown, it is possible that unsaturated FAs generated by forced lipophagy protect the cells against lipotoxic FAs, such as PA, as previously demonstrated in cultured mammalian cells (Listenberger et al., 2003). Further analysis will be required to determine whether tolerance of lipotoxicity is related to apoptosis.
An unresolved issue, based on our observations in mouse early embryos, is that TIP47-mCherry, used as an LD marker, did not work as expected. In contrast to the lipophagy-induced embryos (microinjected with TIP47-mCherry-p62T352A), in which fluorescence was specifically detected at the LD surface, TIP47-mCherry-injected embryos exhibited diffuse fluorescence (Fig. S1C). Because post-translational modification of Plins, including phosphorylation and ubiquitylation, are involved in stabilizing the LD localization of these proteins (Orlicky et al., 2008; Xu et al., 2005), we speculated that some modification occurs at the C terminus of TIP47, which might affect its LD localization. Therefore, we created mCherry-TIP47 (C-terminally mCherry-tagged TIP47) and repeatedly microinjected it into one-cell embryos. However, although the new fusion protein was expressed, fluorescence signals were not detected at the LD surface, but instead remained diffusely distributed in the cytoplasm (Fig. S1D). On the other hand, mRNAs encoding either TIP47-mCherry-p62T352A or mCherry-TIP47-p62T352A could induce lipophagy, with the mCherry signals localized specifically at clustered LDs (Fig. S1C,D), indicating that p62 expression at the LD surface might stabilize TIP47 or its interaction with LDs. Because a previous study in cultured mammalian cells showed that TIP47 fails to associate with LD when full-length ADRP is simultaneously overexpressed, but not when an N-terminally modified (mutant or GFP-tagged) ADRP is overexpressed (Orlicky et al., 2008), we speculate that access of TIP47 to LDs is dependent on the level of ADRP. Consistent with this idea, ADRP is abundant in mouse oocytes and embryos (T.T. and S.T., unpublished). Our ongoing research using ADRP KO mice should be able to determine why TIP47 protein fused to either the N or C terminus of mCherry does not function as an LD marker in fertilized mouse embryos.
Cellular lipid content varies between animal species, probably because of differences in ATP requirements between oocyte maturation and subsequent embryonic development (Dunning et al., 2014). For example, in comparison with mouse and human, porcine oocytes contain massive amounts of LDs [37.3 ng fat/nl volume of oocyte in pig, versus 6.25 ng fat/nl in mouse (Leese, 2012)], which can negatively influence embryonic manipulation, especially in the case of cryopreservation (Nagashima et al., 1995). Because autophagy is highly conserved across animal species, it would be interesting to investigate the consequences of forced lipophagy in porcine embryos.
In summary, we achieved forced selective autophagy by expressing a p62 fusion protein, thereby generating cells depleted of a specific organelle. Using this system, we revealed that the correct amount of LDs is important for early embryonic development. The same strategy could be applied to generate other organelle-depleted cells to remove unwanted organelles and thus analyze organelle functions. Therefore, this method represents a valuable tool for elucidating the roles of specific organelles.
To generate pCW TIP47-mCherry and pCW TIP47-mCherry-p62T352A, TIP47 (1-221 amino acids) and human p62T352A cDNA were cloned into pCW57.1 (Addgene plasmid #41393, deposited by David Root) along with mCherry. The plasmid DsRed-p62WT was a kind gift from Dr Masaaki Komatsu (Niigata University, Japan).
HeLa cells were cultured in Dulbecco's Modified Eagle's medium (DMEM, Nacalai Tesque) supplemented with 10% fetal bovine serum (FBS, Biosera), 50 mg/ml penicillin, and streptomycin (regular medium) in a 5% CO2 incubator. Atg14 KO HeLa cells were generated by clustered regularly interspaced short palindromic repeat (CRISPR) using pX459 GFP carrying the sgRNA expression cassette to target Atg14 (CAGAGGCATAATCGCAAACT) as previously described (Takayama et al., 2017). TIP47-mCherry and TIP47-mCherry-p62T352A cells were generated by lentiviral transduction with pCW, psPAX2 (Addgene plasmid #12260, deposited by Didier Trono) and pCMV VSV-G (Addgene plasmid #8454, deposited by Bob Weinberg). The stable cell lines were cultured in regular media containing 1 mg/ml doxycycline. For drug treatment, cells were incubated for the indicated times in medium containing 0.5 mM OA (Sigma-Aldrich). For LD staining, cells were cultured for 30 min in the presence of 1 µg/ml BODIPY 493/503 (Thermo Fisher Scientific).
Trypsinized cells were incubated with 1 µg/ml BODIPY 493/503 (Thermo Fisher Scientific) in PBS for 30 min at 4°C, and then sedimented by centrifugation at 5000 rpm (2500 g) for 2 min. The pelleted cells were washed with PBS and then re-suspended in PBS containing 10% FBS and 1 μg/ml 4′,6-diamidino-2-phenylindole (DAPI), passed through a 70-μm cell strainer, and subjected to flow cytometry on a CytoFLEX S flow cytometer equipped with NUV 375 nm (for DAPI), 488 nm (for GFP) and 561 nm (for mCherry) lasers (Beckman Coulter). Dead cells were detected by DAPI staining.
All mouse experiments were performed in accordance with the relevant guidelines and were approved by the Animal Care and Use Committee of the National Institute of Quantum and Radiological Science and Technology. Mice used in this study were 8- to 12-week-old C57BL/6J mice (Japan SLC), except for C3H mice (Japan SLC) used for the experiment described in Fig. S1B. Mice were housed under specific pathogen-free conditions. Metaphase II (MII) oocyte collection from superovulated females, embryo manipulation, in vitro fertilization, microinjection, and live-cell imaging were performed based on previously described methods (Kaizuka et al., 2016; Tsukamoto et al., 2014, 2013). For assessment of blastocysts, one-cell embryos were cultured for 4-5 days in vitro, and blastocyst formation was evaluated based on expansion of the blastocoel. We confirmed that 70% of control embryos (70.1±6.2%; from three independent experiments) injected with TIP47-mCherry mRNA developed to early and growing blastocysts with a cavity volume greater than half the volume of the embryo. We considered developing embryos with cavities that completely filled the embryo as late blastocysts. When necessary, embryos were cultured with KSOM lacking amino acids (Eagle's essential and non-essential amino acids), and glutamine. In some experiments, one-cell embryos were cultured with the following chemicals: 200 nM BafA1 (WAKO Pure Chemical Industries), 100 µM DEUP (Sigma-Aldrich), 0.1 mM OA, 0.1 mM PA (Sigma-Aldrich), 10 µM wortmannin (Sigma-Aldrich) or 50 µM chloroquine (WAKO Pure Chemical Industries).
For RNAi experiments, Stealth RNAi duplex oligonucleotides (siRNAs) were designed to target Atg3 (MSS228710, Invitrogen) or Atg14 (MSS211913, Invitrogen). Stealth RNAi Negative Control Duplexes (Invitrogen) were used as negative controls. Knockdown efficiency was calculated based on conversion of LC3-I to LC3-II, as determined by immunoblot analysis (Fig. S2B), as well as by immunofluorescence analysis of LC3 (Fig. 4C). In addition, we assessed whether embryos injected with siRNAs were developmentally compromised (Fig. S2A), based on our previous observation that autophagy-deficient embryos (derived from oocyte-specific Atg5 knockout females) die before implantation (Tsukamoto et al., 2008). Based on these analyses, we chose siRNA targeting Atg14, which inhibited development more efficiently than siRNA against Atg3, for further study.
In vitro transcription and microinjection were performed as described (Tsukamoto et al., 2014). Briefly, plasmids encoding the indicated mRNAs were linearized, and capped mRNAs with poly (A) tailing were synthesized using the mMESSAGE mMACHINE Ultra Kit (Ambion). The purified mRNAs were diluted to 100 ng/µl in TE buffer, filtered, and microinjected into the cytoplasm of oocytes or embryos. For the RNAi experiment, 10 µM siRNA was microinjected simultaneously with either TIP47-mCherry or TIP47-mCherry-p62T352A mRNA into the cytoplasm of fertilized embryos, as described above, except that these mixtures were not filtered before microinjection.
Cells expressing fluorescently tagged protein were grown on coverslips and fixed in 3.7% formaldehyde in PBS for 15 min. For LD staining, the fixed cells were incubated with 1 µg/ml BODIPY 493/503 in PBS for 30 min and observed under a confocal laser microscope (FV1000 IX81, Tokyo, Japan) using a 60× oil-immersion objective lens with a numerical aperture (NA) of 1.42.
Immunofluorescence of mouse embryos was performed as previously described (Tsukamoto et al., 2014). Briefly, fixed embryos were blocked and permeabilized overnight at 4°C with 0.4% Triton X-100 in PBS containing 10% goat serum and 3% bovine serum albumin (BSA). The permeabilized embryos were then incubated with the following primary antibodies, diluted in PBS containing 3% BSA (PBS/BSA), overnight at 4°C: anti-LC3B (Cosmo Bio; CAC-CTB-LC3-2-IC; 1:200), anti-Lamp1 (Abcam; ab25245; 1:200), anti-ubiquitin (Medial & Biological Laboratories; D058-3; 1:200) and anti-Atp5a (Abcam; ab14748; 1:200). The embryos were washed in PBS/BSA at least four times for 15 min each, and then incubated with the appropriate secondary antibodies (1:800) for 1 h at room temperature or overnight at 4°C. Embryos were then washed once more, and transferred to PBS/BSA in a glass-bottomed dish (Matsunami Glass) covered with mineral oil. Fluorescence images were acquired with a SP8 laser scanning confocal system (Leica, TCS, SP8) with a 63× Plan APO 1.4 NA oil immersion objective.
For labeling of LDs, fixed oocytes or embryos were stained with BODIPY 493/503 solution (1 µg/ml, diluted in PBS/BSA) in the dark for 10-15 min at room temperature, washed several times with PBS/BSA, and observed by laser confocal microscopy as described above. Fluorescence measurements were performed using ImageJ software (NIH). Images were adjusted in Photoshop and Illustrator (Adobe).
To measure ROS production, embryos were incubated with 10 µM aminophenyl fluorescein (APF, GORYO Chemical) for 30 min in PBS containing polyvinylpyrrolidone (PVP, Sigma-Aldrich) instead of BSA to avoid interference with fluorescence measurements. Fluorescence was imaged on a real-time cultured cell monitoring system (CCM, ASTEC Co.) with a 10× objective lens (Plan, NA0.25, Nikon), and analyzed using ImageJ software as described before.
To monitor cellular apoptosis, embryos were stained using the Annexin V-EGFP Apoptosis Kit (BioVision, #K104-25), which includes Annexin V-EGFP and propidium iodide. Fluorescence was analyzed by CCM, and fluorescence measurements were performed as described above.
Immunoblotting was performed based on previously described methods (Tsukamoto et al., 2014). The following antibodies were used: anti-LC3B (Cell Signaling Technology; #12741; 1:400) and anti-α/β-tubulin (Cell Signaling Technology; #2148; 1:500). Horseradish peroxidase (HRP)-conjugated anti-mouse and anti-rabbit IgG antibodies were purchased from Cell Signaling Technology (#7076 and #7074, respectively). For the autophagic flux assay, immunoblots were performed according to previously reported guidelines for monitoring autophagy (Klionsky et al., 2016). Briefly, 70 control or lipophagy-induced embryos were cultured in the absence or presence of BafA1 and lysed for immunoblotting. After SDS-PAGE, proteins were blotted onto PVDF membrane using a Trans-Blot Turbo Transfer System (Bio-Rad). The blots were blocked with 3% BSA in TBS-T (Bio-Rad) for 1 h, and then incubated with primary antibodies at 4°C overnight. Subsequently, the membranes were washed three times for 10 min each with TBS-T (Bio-Rad), and then incubated with the appropriate HRP-conjugated secondary antibodies as described above. The signals were visualized using a SuperSignal West Femto Chemiluminescent Substrate (Thermo Fisher) and imaged on a ChemiDoc system (UVP).
Electron microscopy was performed as described previously (Tsukamoto et al., 2013). LD number and size were quantified using the ImageJ segmentation tool (NIH).
For TG measurements, 40 embryos per experiment were transferred into 10 µl of water (Nippon Gene) and stored at −80°C until use. TG levels were assayed using the Picoprobe Triglyceride Fluorometric Assay kit (BioVision, #K614-100). In this assay, TG is converted to glycerol and FA, and the resultant glycerol is oxidized to generate a product that reacts with the picoprobe and developer (included in the kit) to generate fluorescence. Because the resultant fluorescence is directly proportional to the amount of TG, TG content was quantitated by fluorometry. The fluorescence was measured on a QFX Fluorometer (DeNovix), and TG level was calculated using a standard curve.
Intracellular ATP levels were determined using the IntraCellular ATP assay kit (TOYO B-Net Co.). In brief, embryos cultured to the indicated stage were individually transferred into 100 µl of water, and then mixed with 100 µl of the kit reaction buffer, followed by incubation for 10 min at room temperature. ATP was measured immediately using a GENELIGHT (Microtec Co., #GL-220A). ATP content was calculated using a standard curve, prepared using the ATP provided with the kit.
Data were compared using unpaired Student's t-test in Prism 6 (GraphPad). Statistical significance is represented as *P<0.05, unless otherwise stated.
We thank Dr Noboru Mizushima (The University of Tokyo) for his mentorship and encouragement. We also thank Masaaki Komatsu (Niigata University) for providing the DsRed-p62WT plasmid, and Ayako Wada and Tomomi Hatakeyama for technical assistance.
Conceptualization: T.T., E.I., S.T.; Methodology: T.T., K.T., S.I., E.I., S.T.; Investigation: T.T., K.T., S.I., A.Y., E.I., S.T.; Data curation: T.T., S.I., A.Y., T.H., N. Minami, N. Miyasaka, T.K., A.M., E.I., S.T.; Writing - original draft: T.T., E.I., S.T.; Writing - review & editing: T.H., N. Minami, N. Miyasaka, T.K., A.M., E.I., S.T.; Supervision: E.I., S.T.; Project administration: E.I., S.T.; Funding acquisition: E.I., S.T.
This work was supported by Japan Society for the Promotion of Science (KAKENHI Program; 16H06167 and 16H01194 to E.I.; 25111001 and 15H05637 to S.T.), Japan Agency for Medical Research and Development (16gk0110015h0001 to T.T. and S.T.), the Naito Foundation (to E.I.), the Senri Life Science Foundation (to E.I.) and the Takeda Science Foundation (to E.I. and S.T.).
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