高原低氧重塑能量代谢的研究进展※

白振忠综述，格日力审校

(1.青海大学医学院青海-犹他高原医学联合重点实验室；2.青海大学高原医学研究中心)

Introduction

With the ringing bell of the 2019 Nobel prize，HIF-1α has stood in the center of the stage for the researching frontiers of oxygen sensing and hypoxia activation[1，2].Hemostasis of oxygen allows the survival of the cell in the most end of our body and populations standing in the highest sites of our planate[3].From sensing oxygen in cellularly to modulating in globally，it makes the fundamental requirements of the regular functions in human life，energy metabolisms[4].Considering the HIF-1α has orchestrated the rhythm of the symphony for both cancer[5，6]and high-altitude medicine research，together a broad genes involved metabolism has been modulated by HIF-1α，from enhanced lactate dehydratase LDH for lactate producing to increased glycolysis，and blocked pyruvate into Krebs Cycle as well as β-oxidation in lipids by shunt pyruvate dehydratase PDH，to inhibit the electric chain reactions ETCs for mitochondrial respiration.Metabolic reshaping is the major adaptation approaches in cellular strategy for hypoxia，a lot of papers have been decently reviewed for cancer cell，the current review has been focused on the published studies on metabolic remodeling for high altitudes most of them from our lab to provided macroenvironment effects both on injury and adaptions at the population and organism level at adipose tissues.

Over 400 million population globally living above 2 000 m altitudes，taken together with the millions of travelers and construction workers going up to high-altitude for climbing and recreation as well as railroad and road constructions.They are specifically evolved the mechanisms for successful survival and dwelling in hostile and extreme high-altitudes.High-altitude provides the most intangible and approaching arena for researching the macroenvironmental changes caused the diseases and adaptations.Although abundant literature on the hypoxia effects of regulations on metabolism for tumorigenesis and carcinoma metastasis process，there is little understood for hypoxia impacting globally on metabolism，especially on lower oxygen partial pressure due to high-altitude exposure(macroenvironmental).

Currently，multiple aspects have been identified for hypoxia-inducible factors pathways involved in metabolisms modulations and regulations，such as mobilizing and utilizations for macronutrients like glucose，fat and protein，as well as micronutrients such as iron and copper and zine.In addition，the mitochondrial activity and respirations have also been clarified，which can be impacted by high-altitude hypoxia.In the current review，we have focused on the high-altitude hypoxia effects on the metabolism in acute，chronic exposure and genetically adaptive population like Tibetans and Sherpa in Qinghai-Tibetan Plateau and Himalaya mountains ranges.What it is unclarified is that whether the environmental factors rather than genetic differences influence the metabolic manifestations in an individual[7].The purpose of the present review is to discuss the most recent published studies from our lab to summarize the potential influence of high altitudes on adjustment in metabolism，as well as high-altitude shaping the various phenotypes in adipose tissues，as well as gut microbiota，in order to have review the evidences to clarify the answers and challenges on the light destinations of the research for high altitudes remodeling metabolism.

1 High-altitude exposure in population

Rapid ascending to high altitudes mostly for mountain climbers as an example，hypoxia caused stress is the first and overall effects on the human body[8].A mountain lines of evidence indicate that on men and women at altitude showed the fuel preference mostly dependent on carbohydrate-derived fuel energy sources(glycogen，glucose，and lactate)，slightly on lipids，only in working muscle utilizes if the ample sources of diet available[9].Which can be highlighted on lactate，based on the demanding of oxygen but less supplement，glycolysis has been boosted，a large amount of lactate can be produced，after most of lactate can be utilized as the gluconeogenesis precursor to stabilize the glycemia，even some of disparities for lactate paradox implied as well for reverse effects[10].For long-term and sustained high-altitude exposure，after expelled the chronic mountain sickness patients，the metabolism remodeled approached have been zoomed in to the specific organs such as heart，muscle and liver respectively.Based on Murry et al.，high altitude both moderate and extreme high-altitude can diminish the numbers of mitochondrial as well as the suppressed fatty acids oxidation in skeletal muscle and heart.However，the high altitude has the fixed protections in Tibetan populations from obesity and metabolic syndromes like Type 2 Diabetes，and NAFLD in living place above 2 500 m，if worse after move down to sea levels and much easier to suffer from obese and diabetic disorders，which can be explained that the prolonged impacts from activations of the hypoxia-inducible factors pathways in Tibetans.

2 High-altitude genetically adaptive populations

HIFs pathway plays an important role in the adaptations of adipose manifested metabolic alterations in high-altitude.Back in 2010，our group have identified genetic evidence of the metabolic phenotypes of Tibetan populations，mostly in HIF pathway signals，which normally rooted for hematological traits such as lower of hemoglobin and erythrocytes counts，have also involved in the influence of the fuel substrates such as fat and glucose[11，12].There are three of the most prominent genes included EGLN1，EPAS1，and PPARA，have shown the special haplotypes among Tibetan populations for hypoxia sensing and transferring as well as regulations[13].EGLN1 the coding gene of proline hydrolases 2，has exhibited the gain-of-function mutations in Tibetan，which can degrade the HIF-1α in the ample of oxygen levels，but activated at the hypoxia[14].However，the mutated EGLN1 haplotypes can activate and increase the oxygen affinity with shown genetically blunted hypoxia sensitivity in Tibetan highlanders[15].Based on the results of the genotype and phenotypes associations studies，we also have found that the selective haplotypes of EGLN1 have related to the increased anaerobic metabolism on lactating productions.at the same time，the PPARA related to the accumulated free fatty acids concentrations in serum for decreased lipids oxidation capacities，the overall fuels substrates preferences shifts to carbohydrates rather than lipids[16].Metabolic adaptation in high-altitude hypoxia have more highlighted on the oxygen utilization rather than delivery[17].PPARA is one of the members of the upstream and downstream target of HIF-1α.Given the positive selective gene also present on the PPARA haplotype in the Tibetan population，a few studies have been investigated for identifying the PPARA roles played in the reprogramming of metabolism when exposed in high-altitude hypoxia.Murry et al.，have found that decreased PPARA-CPT1 signals as well as activities of β-oxidation enzymes for more efficiently consumed intermediates with long-chain fatty acids in mitochondria of muscle tissues to suppress fatty acids oxidation capacities for adaptation of Sherpa populations on high-altitude hypoxia.

In 2017，our team have collaborated with Dr.Zhao in Qinghai University Affiliated Hospital，we have identified the Tibetan populations have their increased tissue iron storages levels indicated by serum ferritin levels in healthy subjects，totally different from the sea level population for high iron stored in tissues have associated with metabolic syndrome，unlikely less prevalence for metabolic disorders and obesity in Tibetan populations living in higher altitudes.There were several possible mechanisms for explanation on the phenomenon，it could be mostly for HIF pathway alterations，together with the close relationship of the iron metabolisms with the hypoxia signal pathway.

3 Metabolic alterations in native species from Qinghai-Tibetan Plateau

Plateau pika as the typically native species dwelling above 3 500 m in Qinghai-Tibetan Plateau.Hinted from the conversions between the WAT and BAT，as cold and stimulators drugs given to WAT.we hypothesis that which might also have been occurred after high-altitude hypoxia exposure.Therefore，we have taken plateau pika to manipulate them into more cold and hypoxia conditions both intermittently and chronically.In 2015，we have found that visceral adipose can be browning around the main energy starving organs such as heart，liver，after cold exposure[18].More importantly，we also have found that cold exposure to pika can stimulate less inflammation and boosted the mitochondrial and vascular densities.More interestingly，it also can repetitive in the non-adaptive rodents in mice and rat the results，from 2018 unpublished data.

To further take the test for chronic cold effects，we next compared the seasonal alterations of adipose phenotypes in winter and summer from plateau pika from 4 300 m[19].Finally，the results indicated that enhanced the browning of the WAT to induced more ther mogenesis for the winter group than the summer group.From finding the results we have until now，just not sure only，one of the reasons which can be caused by high-altitude hypoxia，might be coldness.It also can be explained that plateau pika has evolved in genetically or others.This year，we have compared the mitochondrial activities in electric transport chain ETC capacities as well as OXPHS abilities in gradient altitudes habitant plateau pika，we have found that decreased the OXPHS and ETC leaking values in skeleton muscle and liver tissues but higher of total coupling ratios in 4 300 m living pika，in addition，more depend on the glucose rather than on lipid on 4 300 m for fuel substrates，which have compared with 2 900 m plateau pika[20].

Changed mitochondrial activities also have contributed to adaptation mechanisms for high-altitude hypoxia.With measuring fatty acid oxidative phosphorylation capacity and gauged the oxygen flux in muscle fibers，Sherpa has the lower fatty acid oxidation capacities than sea-level populations in FAO and OXPHO as well as LEAK(ADP absence)capacities，but greater coupling efficiency for ascending the altitudes.Correspondingly，we also have measured and compared the mitochondrial activity in muscle fibers from plateau pika of 2 900 m and 4 300 m living groups，there were lower FAO and OXPHO capacities for 4 300 m group than 2 900 m.From the results，we can confirm that high-altitude exposure actually suppressed the mitochondrial respiration activities even the ample fat substrate available for muscle，which would be a good explanation for lower prevalence in diabetes risks.

4 High-altitude hypoxia and adipose tissues

Over consumptions of high-fat diet might lead to obesity in heterogeneously and excessively accumulations as well as dysfunctions of fat tissues.However，a few of study have reported that high-altitude might offer the protections from obesity after HFD1，through changing the adipose tissue phenotypes.We have explored adaptation mechanisms of adipose tissues，which have been offered by high-altitude hypoxia，as included the unique phenotype changes in development and differentiation in the size，nuclear density，and mitochondrial density as well as the oxidation phosphorylation capacities in the brown adipose tissues.We have taken a broader view of the metabolic adaption mechanisms as the perspective of adipose tissues.

Recently，adipose-derived exosome(ADE)has been identified as the major mechanisms for adipose tissues locally impact the global metabolism3，mostly depend on the exosomal containing miRNAs4 to allow the cross-talk between adipose with far away liver5 and macrophages6，7.Adipose，as the major secretion site of exosomes in mammals，and the adipose-derived exosome(ADE)allowed the cross-talk communications as the fat-liver and fat-brain axis.Genetically modified rodents for adipose-dicer KO mice have shown the insulin resistance，obesity and diabetes，which can be recovered after injected the exogenous exosomes from healthy mice.Given the multiple factors can change the exosomal contains8，we have found that high-altitude hypoxia can change the ADE containing miRNAs as the expression levels and types.To identify the potential effects of high-altitude hypoxia on phenotypes of adipose tissues，we have raised mice in 4 300 m and 50 m with normal chew and HFD，respectively.After，8 weeks，we have found that the increased browning for increased mitochondrial numbers and vascularized in white adipose tissues in 4 300 m groups than 50 m group，after HFD were treated，shown in Figure.The unbiased transcriptomes study has indicated that rats with HFD in hypoxia exposure compared with HFD control group，shown significantly decreased free blood glucose，body weight and there was glucose，lipids metabolisms，lipids trafficking and angiogenesis pathways genes were different in adipose tissues2.

In 2017，we also investigated the exosomal differences between the small groups of population，who habitant above 4 100 m and below 2 250 m respectively，the results have displayed the significantly different exosomalmiRNAs profiles in serum，in addition，we also have found that some of the exosomalmiRNAs were strongly associated with brown adipose tissue activities in high-altitude adaptive populations(unpublished data).Next，we demonstrated that high-altitude hypoxia has altered the expression profiles of exosomalmiRNAs，through comparisons from the two groups of mice，which have been raised in 4 300 m and 50 m，after we have confirmed that the differently expressed miRNAs were originated from adipose tissues.However，it needs more emphasis on clarifying the details for modulations of the exosomalmiRNAs on the target genes and signals in future research.

Figure 1 Phenotypical alterations of adipocytes to adapt the high-altitude hypoxia exposure and seasonal changes in plateau pika

5 High altitudes and gut microbiota

Gut microbiota is the most prominent microbiota community in the human body.With the co-evolution of the host-microbiota interactions in the long-term periods on adaptions in high-altitude plateau.Ma et al.，from our group，also have investigated the alterations of the gut-microbiota recently，from adaptive herbivores Tibetan antelope to populations as Tibetan populations，there are several of groups of Tibetans residing at higher altitudes have higher levels of bacteria from Firmicutes，Clostridiales，and Ruminococcus in their gut microbiota than Tibetans from lower altitudes，and Ruminococcaceae is one of the three bacterial families most frequently identified in the gut microbiota of Tibetans[21].The potential mechanisms would be the Ruminococcus might secret a large amount of short-chain fatty acids(SCFA)，which contribute to adjusting the metabolic fuel substrates in the gut as well as liver and muscles for controlling the gut membrane integrity，metabolic control appetite regulations and immune function.

However，which species are specifically manipulating the gut ecosystem to allow the glucose is the first choice to fuel rather than lipids，with depending on glycolysis or Krebs cycle，what kind of substances were secreted to influences the gastric tract intestinal absorptions and excretions，a lot of unanswered questions need to be resolved，and first of all，to find the leaf in the big forest and identify the bacteria in species and strains in the huge oceans of gut microbiota will be the first step，and eventually，we have a long way to go.

By the courtesy，figure was artistically illustrated by Ph.D.student Yifan Zhang from our Lab.

Acknowledgment

Chinese Academy of Science“Western light” Talent for 2019；Applied and Basic Research Program from Department of Qinghai Science and Technology for 2020(2020-ZJ-721)supported grants for Dr.Bai.We are grateful for suggestions in Chinese writings from Dr.Liu Shou.