Geniposide Improves Glucose Homeostasis via Regulating FoxO1

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Bioactive Constituents, Metabolites, and Functions

Geniposide improves glucose homeostasis via regulating FoxO1/PDK4 in skeletal muscle Yan Li, Haiou Pan, Xuetong Zhang, Hui Wang, Shengnan Liu, Hui Zhang, Haifeng Qian, Li Wang, and Hao Ying J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00402 • Publication Date (Web): 30 Mar 2019 Downloaded from http://pubs.acs.org on April 2, 2019

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Journal of Agricultural and Food Chemistry

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Geniposide improves glucose homeostasis via regulating FoxO1/PDK4 in skeletal

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muscle

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Yan Li†,

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Zhang‡, Haifeng Qian‡, Li Wang†, ‡, *, Hao Ying§, *

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†

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Technology, Jiangnan University, Wuxi 214122, China

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‡

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Technology, Jiangnan University, Wuxi 214122, China

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§

‡, §, #,

Haiou Pan‡, #, Xuetong Zhang ∥ , #, Hui Wang§, Shengnan Liu§, Hui

Jiangsu Key Laboratory of Advanced Food Manufacturing Equipment &

State Key Laboratory of Food Science and Technology, School of Food Science and

CAS Key laboratory of nutrition, metabolism and food safety, Shanghai Institutes

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for Biological Sciences, University of Chinese Academy of Sciences, Chinese

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Academy of Sciences, Shanghai 200031, China

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∥

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University, Wuxi 214062, China

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*

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# These

Affiliated hospital of Jiangnan University (Wuxi No. 4 People’s Hospital), Jiangnan

Correspondence: [email protected]; [email protected] authors contribute equally to this work

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ACS Paragon Plus Environment


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Abstract

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It is well known that imbalance state of glucose metabolism triggers many metabolic

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diseases and glucose uptake in skeletal muscle accounts for 90% of body weight.

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Geniposide is one of the major natural bioactive constituents of gardenia fruit, and the

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regulation of geniposide on glucose metabolism in skeletal muscle has not been

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investigated yet. Here, based on microarray analysis, we discovered that geinposide

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decreased PDK4 expression in skeletal muscle of mice, and we subsequently found

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that geniposide inhibited the expressions of FoxO1, PDK4, P-PDH in vitro and in

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vivo. Moreover, geniposide promoted a switch of slow-to-fast myofiber type and

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glucose utilization, suggesting that geniposide improved glucose homeostasis. In

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addition, mechanistic studies revealed that geniposide played above roles by

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regulating FoxO1/PDK4, which controlled fuel selection via PDH. Meanwhile, effects

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of geniposide mentioned above could be reversed by FoxO1 overexpression.

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Together, these results establish that geniposide confers controls on fuel usage and

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glucose homeostasis through FoxO1/PDK4 in skeletal muscle.

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Key words: geniposide; skeletal muscle; glucose utilization; PDK4; FoxO1

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Introduction

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Nowadays, type 2 diabetes mellitus (T2DM) has become a research focus.

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Statistically, about 415 million people worldwide suffer from diabetes aged 20–79

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years.1 Insulin resistance is the major characteristic of T2DM, referring to the inability

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of cells to respond adequately to insulin, consequently leads to impaired glucose

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uptake.2 Skeletal muscle plays a vital role in the pathogenesis of diabetes, which is the

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main organ participating in the uptake, storage and metabolism of glucose provided

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by glucose.3 Skeletal muscle is composed of slow-twitch and fast-twitch

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fiber, which are defined by the presence of the myosin heavy chain (MyHC) subtypes

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(including MyHC-I, MyHC-IIa, MyHC-IIb, and MyHC-IIx).4 Slow-twitch myofiber

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(MyHC-I is representative) rich in mitochondria have high oxidative capacity whereas

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fast-twitch fiber (MyHC-IIb is representative) generate ATP primarily through

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glycolysis.5 The muscle-fiber specification impacts metabolic homeostasis, function

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of muscle and eventually whole-body physiology. Some studies have shown that the

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promotion of slow-twitch myofiber in skeletal muscle increasing oxidative capacity

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may be a key factor regulating salutary effects of lifestyle interventions on

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hyperglycemia and insulin resistance.6-8 In contrast, data showed that an increase of

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type IIb fiber is sufficient to maintain glucose homeostasis and reverse the decline in

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metabolic function in middle-aged individuals.9 Therefore, we need to further

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investigate the fiber-type switch in skeletal muscle, which may provide new

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therapeutic strategies on metabolic disease.

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muscle

It has been found that some natural compounds derived from edible resources



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have certain effects on skeletal muscle. For instance, dihydromyricetin has protective

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effect on mitochondrial function and muscle autophagy, lauric acid accelerates

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glycolytic fiber formation, Epigallocatechin gallate reduces the formation of

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slow-twitch muscle fiber, fermented rice germ extract increases the strength and

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weight of gastrocnemius muscle and myofiber size and so on.10-13 Gardenia fruit is a

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kind of Chinese herbal and can also be used as edible resource and geniposide is one

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of the major natural bioactive components in gardenia fruit which can be used in soft

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drink, pastry and instant noodles.14 Studies of the beneficial effects of geniposide have

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been of interest to researchers for decades, and according to our previous study, it is

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proved that geniposide could attenuate fibrotic progress in injured skeletal muscle.15

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However, the effects of geniposide on muscle fiber-type conversion, glucose

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metabolism and the underlying mechanism have not been performed yet.

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Metabolic flexibility defines the ability for an organism to respond and adapt to

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conditional changes (fasted state, exercise and obesity) in metabolic demand, such as

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fuel selection between glucose and fatty acids. The normal, healthy transition from

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fasting to feeding involves shifts in fuel selection from fatty acid oxidation to more

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glucose oxidation in skeletal muscle, highlighting the metabolic inflexibility of T2DM

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and obesity.16 Pyruvate dehydrogenase (PDH) is the critical regulator of metabolic

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flexibility and the key rate-limiting enzyme for glucose oxidation, which catalyzes

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pyruvate to form acetyl-CoA, links glycolysis to the tricarboxylic acid (TCA) cycle

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and ATP production and is inactivated after phosphorylation (P-PDH). P-PDH is

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under the regulation of pyruvate dehydrogenase kinase (PDK) isoenzymes to respond


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to physiologic conditions. Four PDK isoenzymes (including PDK1, 2, 3 and 4) are

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known to be expressed in a tissue-specific manner in mammals, and among these

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isoenzymes, PDK2 and PDK4 have attracted the most interest due to the finding that

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their expressions are increased during starvation and diabetic state.17 Particularly, the

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expression of PDK4 is higher than that of PDK2 in skeletal muscle of mice.18 In

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addition, it is verified that forkhead box O1 (FoxO1) is a critical transcription factor

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in muscle energy homeostasis controlling PDH activity through directly regulating

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PDK4.19-21 Previous studies showed that down-regulation of FoxO1 and activation of

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protein kinase B (Akt/PKB) in skeletal muscle could induce slow-to-fast myofiber

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transition and this improved glucose metabolism.21,

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akt/FoxO1/PDK4/PDH axis in metabolic flexibility has been suggested.

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The regulatory role of the

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In this study, we started from the expression of PDK4 in myocytes and mice

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treated with geniposide at mRNA and protein levels. The in vitro and in vivo results

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displayed that geniposide significantly inhibited PDK4 expression. We accordingly

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speculated that the regulation of geniposide was via FoxO1/PDK4/PDH signaling

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pathway in C2C12 myotubes and this speculation was evaluated. Then it was

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confirmed that geniposide promoted fast-twitch muscle phenotype formation and

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induced a slow-to-fast muscle fiber conversion. Additionally, further studies revealed

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that geniposide affected enzyme activity of PDH in muscle and modulated glucose

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utilization, thereby improving glucose homeostasis. Meanwhile, we continued to

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identify that the effects of geniposide on myocytes mentioned above could be

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reversed by FoxO1 overexpression. Taken together, our data demonstrated that



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geniposide played a pivotal role in muscle fiber-type switch and glucose homeostasis,

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thus constituting a therapeutic strategy for metabolic diseases.

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Methods

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Animal study

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All procedures and protocols of animal experiments were approved by the

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Animal Care and Use Committee of the School of Food Science and Technology,

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Jiangnan University (No. JN 2013-4). Male mice aged 8-12 weeks were used in this

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study. Mice were maintained as described previously.21 After adapting to new

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environment, mice were treated with 25 mg geniposide (Sigma Aldrich, SML0153) or

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control vehicle (dimethyl sulfoxide, DMSO, Sigma) per kilogram of body weight per

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day through intraperitoneal injection (i.p.). For intraperitoneal glucose tolerance test

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(IPGTT), glucose was injected through i.p. (1 g/kg body weight) after fast overnight

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for more than 16 h. For oral glucose tolerance test (OGTT), glucose was treated

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through intragastric administration (1.5 g/kg body weight) after fast overnight for

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more than 16 h. Blood glucose levels were detected at the indicated intervals.

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C57BL/6 wild-type mice (8-week-old) were fed a high-fat diet (HFD, 45% calories

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from fat) for 8 weeks and were kept on HFD diet and received geniposide (25 mg/kg

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body weight) every day for 1 week before conducting IPGTT assay. For evaluating

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fuel

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lab-animal-monitoring system (CLAMS, Columbus Instruments) for 24 h to make the

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mice adapt to experimental condition, and then the volume of O2 consumption and

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CO2 production were continuously recorded during the next 24 hours as described

selection

in

vivo,

mice

were

maintained


in

a

comprehensive

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before.21 Tissues including gastrocnemius (GAS) and soleus (SOL) were harvested

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and frozen in lipid nitrogen and stored at -80℃ for future analysis.

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Microarray analysis

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Microarray analysis was performed in Shanghai Majorbio Bio-pharm

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Technology Limited Company. Brief procedures were as follows. At first, total RNA

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was extracted from skeletal muscle and quantified. Next, according to the Illumina’s

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TruSeq RNA sample preparation Kit (San Diego, CA), 5 μg RNA was used to prepare

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RNA-seq transcriptome library. After quantified by TBS380, paired-end RNA-seq

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sequencing library was sequenced with the Illumina HiSeq X Ten (2 × 150 bp read

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length).23 The raw paired end reads were trimmed and quality controlled by SeqPrep

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and Sickle with default parameters. Then clean reads were separately aligned to

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reference genome with orientation mode using TopHat software.24 To identify

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differential expression genes between two different groups, the expression level of

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each transcript was calculated according to the fragments per kilobase of exon per

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million mapped reads method. RSEM software was used to quantify gene

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abundances.25 The differential expression analysis between two groups was performed

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by DEGseq software on the free online platform of Majorbio I-Sanger Cloud Platform

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(www.i-sanger.com).

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Cell culture, transfection and luciferase assay

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Mouse C2C12 myoblasts were cultured in Dulbecco’s modified Eagle medium

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(DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), and 100

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units/mL penicillin (Gibco), and 100 mg/mL streptomycin (Thermo Fisher Scientific).



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To induce differentiation of myoblasts, 2% horse serum (HS, Gibco) was used to

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replace FBS in culture medium. After 48 h of differentiation, the myotubes were

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treated with geniposide or control vehicle (DMSO) as indicated. The treatment

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method of geniposide is concentration gradient and time gradient. Cells were cultured

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in a humidified incubator at 37 °C and 5% CO2 condition. FoxO1 plasmids were

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gifted by Dr. Ying’s lab. FoxO1 or control vector transfection was performed by

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using Lipofectamine 2000 (Invitrogen). The luciferase reporter activities were

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determined by using the Dual-Luciferase Reporter Assay System (Promega)

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following the manufacturer’s instructions and by using a luminometer (Berthold

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Technologies). For primary myoblasts isolation, the protocol used was described

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previously.26 Briefly, the muscle tissues were collected. Washed, trimmed, minced,

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and then incubated in 0.1% pronase (Sigma) in DMEM at 37 °C for 1 h shaking. After

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centrifugation, removal of supernatant, the precipitate was resuspended, filtrated

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through a 40 μm nylon mesh cell strainer (Falcon 2350), pre-plated in a noncoated

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dish for 1 h and then cells were transferred to Matrigel-coated plates (BD

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Biosciences).

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RT-PCR and Western blot

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RT-PCR was performed on an ABI7900 Real Time PCR system (Applied

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Biosystems). The primer sequences for RT-PCR will be found in Table S1. Western

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blot analysis includes following antibodies against PDK4 (ProteinTech), FoxO1 (Cell

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Signaling), P-PDH (Millipore) and GAPDH (Sigma Aldrich).

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Measurement of glucose oxidation, glycolysis and PDH enzyme activity


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The level of glucose oxidation in C2C12 myotubes was determined as described

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before.27 Briefly, differentiated myotubes were treated with geniposide or control

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vehicle (DMSO) for 12 h, then the myotubes were cultured in serum-free medium for

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2 h at 37 °C, then [U-14C] D-glucose was added into each well for 4 h. Insulin

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treatment was used as a positive control. Each well was covered with a piece of

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Whatman paper with 3M NaOH.

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perchloric acid, then the collection filter paper were counted in liquid scintillation

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fluid (Perkin-Elmer, MA, USA) to measure glucose conversion to CO2.

14CO

2

was released by the injection of 70%

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Seahorse Bioscience XF24 instrument was applied to determine the levels of

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glycolysis and glycolysis capacity. C2C12 myotubes were incubated in 24-well XF24

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microplate. Glycolysis test kit was obtained from Seahorse Bioscience and the

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detection of extracellular acidification rate (ECAR) followed the manufacturer's

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instructions. The levels of glycolysis and glycolysis capacity were calculated as

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described before.28

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PDH activity was analyzed by using a pyruvate dehydrogenase enzyme activity

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microplate assay kit (Abcam, ab109902). Isolated mitochondria from muscle

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specimens and whole cell extracts were added into immunocaptured plate, then the

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PDH complex activities were measured at OD450, which were normalized to protein

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concentration.

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Metabolic profile determination

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The levels of pyruvate and acetyl-CoA in skeletal muscle or C2C12 myotubes

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were determined by using commercial available kits (Acetyl-CoA, Sigma Aldrich;



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pyruvate, Nanjing Jiancheng bio-company) following the manufacturer’s instructions.

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Immunostaining and histological analysis

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C2C12 muscle cells were incubated into 24-well plate and 2% HS was used to

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replace FBS in culture medium inducing myocytes into myotubes. After 24 h, the

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myotubes were treated with geniposide and control vehicle (DMSO) for 12 h.

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Anti-Myosin-slow (Sigma Aldrich) and anti-Myosin-fast (Sigma Aldrich) were used

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for immunofluorescence detection. Muscle tissues from geniposide-treated or control

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(DMSO) mice were frozen and fixed in isopentane. Muscle fiber types were measured

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by the Adenosine Triphosphatase (ATPase) staining and Succinate dehydrogenase

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(SDH) staining, and pathological states of muscle were observed using hematoxylin

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and eosin (HE) staining.

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Statistical analysis

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GraphPad Prism 5.0 software was applied to statistical analysis. All experiments

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were repeated at least three times and representative data are shown. Data are

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presented as mean ± standard deviation (SD). Student’s t test was performed to assess

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whether there is significant difference between means of two groups (p

Geniposide Improves Glucose Homeostasis via Regulating FoxO1

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