metabolic crosstalk: molecular links between glycogen and lipid ... · 07-04-2014 · 1 metabolic...

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Metabolic Crosstalk: molecular links between glycogen and lipid metabolism in

obesity

Binbin Lu1, Dave Bridges

1, Yemen Yang

1, Kaleigh Fisher

1, Alan Cheng

1, Louise Chang

1,

Zhuoxian Meng1, Jiandie Lin

1, Michael Downes

2, Ruth T. Yu

2, Christopher Liddle

2,3,

Ronald M. Evans2, and Alan R. Saltiel

1

1Life Sciences Institute, University of Michigan, Ann Arbor, MI 48109, USA

2Salk Institute for Biological Sciences, La Jolla, CA 92037, USA

3Storr Liver Unit, Westmead Millennium Institute and University of Sydney, Westmead

Hospital, Westmead, NSW 2145, Australia

Address correspondence to:

A.R. Saltiel, Life Sciences Institute, University of Michigan, Ann Arbor, Michigan 48109,

USA. Phone: (734) 615-9787; Fax: (734) 763-6492; E-mail: [email protected].

Running title: PTG-Dependent Glycogen and Lipid Accumulation

Abstract word count: 185

Total word Count: 4450

Tables: 0

Figures: 8

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ABSTRACT

Glycogen and lipid are major storage forms of energy that are tightly regulated by

hormones and metabolic signals. Here, we demonstrate that feeding mice a high fat diet

(HFD) increased hepatic glycogen, due to increased expression of the glycogenic

scaffolding protein PTG/R5. PTG promoter activity was increased and glycogen levels

were augmented in mice and cells after activation of mechanistic target of rapamycin

complex 1 (mTORC1) and its downstream target sterol regulatory element binding

protein 1 (SREBP1). Deletion of the PTG gene in mice prevented HFD-induced hepatic

glycogen accumulation. Surprisingly, PTG deletion also blocked hepatic steatosis in

HFD-fed mice, and reduced the expression of numerous lipogenic genes. Additionally,

PTG deletion reduced fasting glucose and insulin levels in obese mice, while improving

insulin sensitivity, a result of reduced hepatic glucose output. This metabolic crosstalk

was due to decreased mTORC1 and SREBP activity in PTG knockout mice or

knockdown cells, suggesting a positive feedback loop in which once accumulated,

glycogen stimulates the mTORC1/SREBP1 pathway to shift energy storage to

lipogenesis. Together, these data reveal a previously unappreciated broad role for

glycogen in the control of energy homeostasis.

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INTRODUCTION

Glycogen represents the first choice for energy storage and utilization. Its metabolism is

tightly regulated by both hormones and nutritional status, which control the activities of

glycogen synthase (GS) and phosphorylase (GP) through a variety of pathways (1). GS

and GP activities are coordinated by glycogen-targeting subunits that serve as “molecular

scaffolds”, bringing together these enzymes with phosphatases and kinases in a

macromolecular complex, and in the process promoting activation of GS and inactivation

of GP. Six different proteins target protein phosphatase 1 (PP1) to glycogen (2). In liver,

PTG and GL are expressed at approximately equivalent levels (3), and together facilitate

the mobilization and storage of hepatic glycogen. PTG overexpression dramatically

increases glycogen content, due to a redistribution of PP1 and GS to glycogen particles,

and a corresponding marked increase in GS activity and glycogen synthesis (4–7).

Despite its profound effects, how PTG controls glycogen metabolism remains uncertain.

One potential mode of regulation may be transcriptional. Both noradrenaline and

adenosine up-regulate PTG mRNA levels in astrocytes and hepatocytes, concomitant

with increased glycogen synthesis (Allaman et al., 2000). The FoxA2 forkhead class

transcription factor directly binds to and transactivates the PTG promoter in vitro (10).

Moreover, two SREBP/USF binding units in the PTG promoter also suggest potential

transcriptional regulation by SREBP.

These indications that PTG may be under transcriptional regulation prompted us to study

its expression during different dietary conditions, and assess its importance in energy

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homeostasis. We show here that HFD produces an increase in hepatic glycogen via a

process that involves induction of PTG mRNA and protein levels, which occurs

downstream of mTORC1 activation and SREBP1 induction. Surprisingly, PTG knockout

mice on HFD not only exhibited decreased glycogen levels in liver, but also improved

insulin sensitivity as well as decreased hepatic steatosis, both of which can be attributed

to reduced activation of mTORC1. Taken together, these data indicate that upon its

accumulation in liver, glycogen elicits a positive feedback loop involving mTORC1,

resulting in increased expression of lipogenic genes, and thus a coordinated synthesis of

glycogen and lipid.

RESEARCH DESIGN AND METHODS

Animals. Generation of PTG KO mice has been described previously (11). These mice

had been maintained on a mixed genetic background, but for these studies were crossed

ten times with C57BL/6J mice (Jackson Laboratories). Wild-type littermates were used as

control. Male mice between 8-10 weeks were given a high fat diet (45% fat, D12451;

Research Diets Inc.) or continued on a normal diet (4.5% fat, 5L0D; Lab Diet) for the

same duration. All mice were maintained in temperature and humidity controlled

conditions with a 12hr light/dark cycle, and had free access to food and water. All

protocols were approved by the University of Michigan Animal Care and Use Committee.

Antibodies. The following antibodies were used: GS, pGS (S641), S6K1, pS6K1 (T389),

S6, pS6 (S235/236) (all from Cell Signaling), SREBP1 (a gift from Dr. Jay Horton). PTG

was detected after AMPD using a rabbit polyclonal antibody raised against the murine

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PTG sequence as described previously (11). A mouse monoclonal antibody raised against

RalA (from BD sciences) and rabbit polyclonal antibody raised against CREB (from Cell

Signaling) was used as a loading control for whole cell lysate and nuclear extracts,

respectively. For detection, Western Lightning Enhanced Chemiluminescence kit (Perkin

Elmer Life Sciences) was used. Individual protein bands were quantified by Image J.

Glycogen Measurement and Glucose conversion into Glycogen Assay. Glycogen was

measured as previously published (12). Glucose conversion into glycogen assay was

performed as described in (13).

Triglyceride Measurement. Frozen tissues were homogenized in buffer containing 50

mM Tris, 5 mM EDTA, 30 mM Mannitol. Crude lysate was mixed with KOH and then

extracted with chloroform: methanol (2:1). After vigorous vortexing and short incubation,

samples were spun down at 13,000 rpm for 10 min. The bottom layer was dried and

resuspended in butanol mixture (3 mL butanol, 1.66 mL Triton-X114, 0.33 mL Methanol)

before being assayed for triglyceride content using a Triglyceride Assay Kit from Sigma-

Aldrich.

Quantitative Real-Time PCR. Total RNA was isolated using Ambion RNA isolation kit

(Life Technologies) and subjected to quantitative real-time PCR as described (14).

Primers are listed in Supplementary Table 1.

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Western blotting and Amylose Pull-down Assay. Whole cell lysates and nuclear

extracts were prepared as described (11,15). Amylose pull-down (AMPD) assays were

performed for analyzing PTG and glycogen-bound GS protein levels. Briefly, 1 mg of

total proteins from crude lysates were incubated with amylose resin (New England

Biolabs) for 2hrs at 4°C before spun down at 3,000 rpm for 2 min. Proteins bound to the

resin were washed and released by boiling in the presence of SDS sample buffer, and

subjected to western blotting.

Luciferase Constructs and Luciferase Reporter Assay. PTG promoter fragments were

generated as described previously (10). The -493 to +36 fragment (T3) was used as a

template to produce mutations in the putative SREBP binding sites. The following primer

sequences were used in site-directed mutagenesis: M1, 5′-

GATTGGTCGGAGGGACCGTGGCTTTGATAAGCTGCC -3′ and 5′-

GGCAGCTTATCAAAGCCACGGTCCCTCCGACCAATC-3′; M2, 5'

GATTGGTCGGAGGGACCGGTTGTCGTGATCTGGCTTTGATAAG-3'and 5'-

CTTATCAAAGCCAGATCACGACAACCGGTCCCTCCGACCAATC-3'; M3, 5'-

GTCGGAGGGACCGGTTGTCGACATCTGGCTTTGATAAGCTGC-3' and 5'-

GCAGCTTATCAAAGCCAGATGTCGACAACCGGTCCCTCCGAC-3'. HEK-293A

and TSC2 MEF cells were cultured in DMEM containing 10% FBS. TSC2 null MEF’s

were obtained from Dr. David Kwiatkowski (Brigham and Women’s Hospital) (16).

Luciferase reporter assay was conducted as described (10).

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Chromatin Immunoprecipitation (ChIP) Assay. TSC2 MEF cells were treated with 1%

formaldehyde for 10 min and processed with a ChIP assay kit as described in (10). An

aliquot of DNA was used for quantitative real-time PCR using the following primers

corresponding to the mouse PTG genomic sequences. P1, 5’-

CTGGAGTTCCAGGCTGTGTA-3’ and 5’-GAGAACGGCTCAGAGGGTAG-3’; P2,

5’-GGGAAACACACACACACACA-3’ and 5’-GAGAAGTTGGCTGGCTCTTC-3’; P3,

5’-TCACGTGATCTGGCTTTGAT-3’ and 5’-CTGAGCAACTTCGCACTCAG-3’; P4,

5’-TTTGATTGGTCGGAGGGA-3’ and 5’-AACCGGGAGGCAGCTTAT-3’.

Blood Glucose and Insulin Measurements. Blood glucose was measured by OneTouch

Ultra Glucometer. Plasma insulin concentrations were measured by Ultra Sensitive

Mouse Insulin ELISA kit (Crystal Chem Inc.).

Glucose, Insulin and Pyruvate Tolerance Tests. For glucose tolerance test (GTT), mice

were fasted 6hrs before an intraperitoneal (IP) injection of 1.5g/kg of body weight of

glucose. For insulin tolerance tests (ITT), mice were fasted 3hrs before an IP injection of

0.75U/kg (ND) or 1.5 U/kg (HFD) of body weight of Humulin R (Eli Lilly). For pyruvate

tolerance test (PTT), mice were fasted overnight before an IP injection of 1.5g/kg of body

weight of sodium pyruvate. Blood glucose concentrations were measured before and after

the injection at the indicated time points using the One Touch Ultra glucometer

(Lifescan).

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shRNA Knockdown Experiments. Mouse pGIPZ lentiviral-shRNA clones targeting

SREBP1and PTG were provided by the University of Michigan Biomedical Research

Core Facilities. Lentiviral production was done as described (17).

RNA-seq and Data Analysis. Total RNA was isolated from livers of biological

triplicates using Trizol (Invitrogen) and the RNeasy mini kit (Qiagen). Libraries were

prepared from 100-500ng total RNA (TrueSeq v2, Illumina) and sequenced on the

Illumina HiSeq 2000 using bar-coded multiplexing and a 100bp read length. Data

analysis was accomplished according to (16) utilizing UCSC mm9 as a reference

sequence. Heatmaps were visualized using Java Treeview 1.1.6r2

(http://jtreeview.sourceforge.net). RNA-seq data are available from the Gene Expression

Omnibus (http://www.ncbi.nlm.nih.gov/geo/) as accession GSE45319.

RESULTS

High Fat Diet Increases Hepatic Glycogen in Fasted Mice.

To understand better the role of glycogen metabolism in the overall control of energy

disposition, C57BL/6J mice were put on high fat diet (HFD) for 12 weeks and became

obese, glucose intolerant and insulin resistant (Supplementary Figure 1). Mice were

subjected to 16hrs fasting followed by 6hrs refeeding before sacrifice. Hepatic glycogen

levels were almost completely depleted by fasting and replenished after refeeding (Figure

1A). Interestingly, while levels were similar in the refed state, HFD produced a 3-fold

increase in hepatic glycogen levels in fasted mice (Figure 1A). Hepatic PTG (Ppp1r3c)

mRNA levels were approximately 2-fold increased by HFD in both the fasted and refed

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states, concomitant with similar induction in SREBP1 (Srebf1) mRNA levels (Figure 1B).

Phosphorylation of the mTORC1 downstream target S6K and its substrate S6 were

dramatically increased by HFD in fasted mice (Figure 1C). Fasting reduced SREBP1

processing; however, we still detected an increase in the active, mature form of SREBP1

protein after HFD (Figure 1C), consistent with previous reports (17,18). PTG protein

levels corresponded well with the HFD-dependent increase in mTORC1 activity and

SREBP1 induction (Figure 1C), suggesting that the mTORC1/SREBP1 pathway may

contribute to the elevated glycogenesis observed in obesity in the fasted state.

Glycogen phosphorylase (GP) activity did not change, ruling out the possibility that

increased glycogen content in obesity results from reduced GP activity (Figure 1D).

While total GS protein levels decreased during refeeding, the mRNA levels of the

predominant GS isoform GYS2 in liver were not changed, suggesting a post-

transcriptional modification of GS in response to refeeding (Figure 1B). Since the GS

antibody might not efficiently recognize the dephosphorylated form of the enzyme, we

measured GS activity (-/+ G6P ratio) in liver lysate, and observed increased GS activity

in response to refeeding in liver from ND but not HFD mice (Figure 1E).

The PTG Promoter is Regulated by SREBP1 and mTORC1

The coordinated increases in hepatic SREBP1 and PTG protein and transcript levels in

response to nutritional status, and the correlation of these findings with increased

glycogen levels, suggested an SREBP-dependent mechanism for the regulation of PTG

transcription. Therefore, we generated mutations of SREBP binding sites on the PTG

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promoter (10) and assessed their activity by a luciferase-based promoter assay.

Expression of active SREBP-1c led to an approximately 2-fold increase in the activity of

the wild-type PTG promoter, an effect significantly blunted in the mutants (Figure 2A),

suggesting that SREBP1 modulates PTG promoter activity through SRE elements

proximal to the transcriptional start site.

To determine whether mTORC1 can regulate PTG transcript levels, we examined TSC2-/-

murine embryo fibroblasts (MEFs) that have high mTORC1 activity (19). PTG promoter

activity was elevated in TSC2-/-

cells, and was reduced by rapamycin treatment (Figure

2B). However, this activation was not seen in the PTG promoter mutant M1, in which the

only available SRE was deleted. Chromatin Immunoprecipitation (ChIP) assay confirmed

the direct interaction of SREBP1 with the PTG promoter, and this interaction was

enhanced at the SREBP1 binding site by constitutive activation of mTORC1 (Figure 2C).

Similarly, overexpression of the constitutively active form of Rheb (20) activated the

PTG promoter in a rapamycin-sensitive manner, and this effect was abolished by deletion

of the proximal SRE in the PTG promoter (Figure 2D).

Transcriptional Regulation of PTG expression by SREBP1 is Required for the

Increased Glycogen Produced by mTORC1 Activation

TSC2-/-

MEFs have increased protein levels of PTG and SREBP1, and higher steady state

levels of glycogen and a greater rate of glucose conversion into glycogen compared to

wild type MEFs (Figure 3A, C and D, control), suggesting that mTORC1 activation is

sufficient for increased glycogen content in vitro. Because PTG binds to and localizes

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PP1 and its substrate GS to glycogen (4), we performed amylose pull-down (AMPD)

assays to measure PTG and GS bound to a glycogen-like matrix. Amounts of glycogen-

bound GS increased with PTG (Figure 3A, control), explaining the elevated glycogen

content in TSC2-/-

cells. These data reinforce the notion that PTG plays a dual

scaffolding/dephosphorylation role by recruiting GS to the glycogen pellet and promoting

its dephosphorylation.

In order to test if the stimulation of glycogen synthesis by mTORC1 is dependent on

SREBP1, we performed lentiviral-mediated shRNA knockdown of SREBP1. The levels

of PTG protein and mRNA were reduced by SREBP1 knockdown, as was the recruitment

of GS to the amylose precipitate (Figure 3A and B). Glycogen levels (Figure 3C) and the

conversion of glucose into glycogen (Figure 3D) were similarly reduced. Collectively,

these data suggest a role for mTORC1 in the regulation of PTG gene expression, via

SREBP binding to an SRE in the PTG promoter.

PTG Levels Control Glycogen Synthesis and Contribute to Feedback Regulation of

mTORC1 and SREBP1.

Lentiviral-mediated shRNA knockdown of PTG produced a 60-70% reduction in both

protein and transcript levels of PTG in TSC2-/-

cells (Figure 4A and B). PTG knockdown

did not affect other glycogen-targeting subunits such as GL, GM and the catalytic subunit

PP1 (Supplementary Figure 2A and B and Figure 4A). Interestingly, PTG knockdown

also reduced SREBP1 protein and mRNA levels, suggesting a positive feedback

mechanism in which PTG regulates SREBP1.

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PTG knockdown produced a 60-70% reduction in both steady state glycogen content and

the rate of glycogen synthesis in TSC-/-

but not TSC+/+

cells (Figure 4C and D), consistent

with the knockdown efficiency of PTG in these cells. The reduced glycogen synthesis

was not due to reduced glucose transport, as glucose uptake was not affected by PTG

knockdown (Figure 4E).

We next examined regulation of mTORC1 activity and glycogen levels by PTG in

HEK293A cells in which we stably overexpressed Flag-GFP-tagged PTG. Glycogen

levels in these cells were 50 fold higher compared to control cells (Figure 4F) due to the

constitutive activation of PTG proteins (Figure 4G). Moreover, phosphorylation of S6K

was 2-fold increased in cells overexpressing PTG (Figure 4G), suggesting that the

scaffolding protein also regulates mTORC1 signaling through a positive feedback

mechanism. Taken together, these results provide evidence that regulation of PTG

expression is directly responsible for the increased glycogen in the context of activated

mTORC1.

In HEK293A cells and the liver cell line Hepa1c, we also observed similar regulation of

glycogen synthesis through PTG. Transient overexpression of WT PTG in these cells

resulted in a 2-3 fold increase in glycogen content. More importantly, these effects were

blocked by overexpression of PTG mutants that are incompetent to bind to GS or GP,

suggesting that PTG is critical in the recruitment of GS and GP to glycogen particles

(Supplementary Figure 2C).

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Homozygous Deletion of PTG Reduces Glycogen and Lipid Levels in vivo.

Our previous studies on a PTG heterozygous KO mouse suggested that PTG acts as a

PP1 scaffolding protein and plays a vital role in glycogen metabolism (11). These mice

had been maintained on a mixed background in which homozygous deletion of the PTG

gene was embryonic lethal. Subsequent backcrosses of these mice into a C57BL/6J

background produced viable homozygous PTG KO mice (Supplementary Figure 3A).

The genotyping of more than 200 female and male homozygous PTG KO pups derived

from heterozygous breeding pairs coming from the first backcross accounted for only ~10%

of the total births, a number significantly lower than the expected Mendelian inheritance

ratio of 25% (Supplementary Figure 3B). However, sequential backcross of the

heterozygous PTG KO mice with C57BL/6J mice produced both female and male

homozygous PTG KO mice born at the expected frequency (Supplementary Figure 3B).

While the mechanism underlying the effect of genetic background on survival of PTG

KO mice remains unknown, all the experiments described in this study were performed

on male PTG KO mice derived from heterozygous breeding pairs backcrossed ten times

with C57BL/6J mice, and littermates without PTG gene deletion (WT) were used as

controls.

To study the role of PTG in glucose homeostasis in vivo, we fed PTG KO mice HFD. No

obvious differences in weight gain or food intake were observed between WT and KO,

fed either ND or HFD, demonstrating that the PTG KO mouse exhibits a normal growth

rate and eating behavior (Supplementary Figure 3C and D).

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Both hepatic glycogen and triglyceride levels were greatly reduced in PTG KO mice fed

HFD for 12 weeks (Supplementary Figure 3E and F). Consistent with the increased

hepatic glycogen levels observed in HFD WT mice, a 2-fold increase in PTG and

SREBP1 mRNA was detected (Supplementary Figure 3G). The mRNA levels of the

glycogen targeting subunit GL were also increased by about 2-fold under HFD conditions

(Supplementary Figure 3G). However, glucose and insulin tolerance tests were

indistinguishable between PTG WT and KO mice, either on ND or HFD (Supplementary

Figure 3H and I). Interestingly, neither GS nor GP activity was changed in the liver

lysates from KO mice (Supplementary Figure 4), reinforcing the hypothesis that PTG is a

critical scaffold to recruit GS or GP to the glycogen particles. In this regard, it is possible

that the phosphorylation status or activity of non-glycogen targeted GS or GP (i.e. GS or

GP in whole cell lysate) is largely irrelevant to glycogen accumulation.

We next explored glycogen synthesis more broadly by directly examining glucose

conversion into glycogen in vitro in isolated primary hepatocytes (Figure 5 A-C) or in

vivo during a hyperinsulinemic-euglycemic clamp study (Figure 5D and E). In primary

hepatocytes isolated from ND mice, we observed a 60% reduction in 14C-glucose

incorporation into glycogen in PTG KO, but no obvious difference in lipid or CO2

incorporation, consistent with the mild reduction in TG observed in ND PTG KO mice.

Both 3H-glucose incorporation into glycogen and lipids were 40-50% reduced in PTG

KO mice fed HFD for 8-10 weeks, as measured in a hyperinsulinemic-euglycemic clamp

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study, suggesting that both glycogen and lipid synthesis in HFD mice are reduced when

PTG is depleted.

PTG Ablation Reduces Hepatic Steatosis after Long-term HFD.

Because of the profound effects of PTG deletion on lipid accumulation in liver, we also

studied mice fed HFD for 24 weeks. Similar to what was observed with shorter-term

HFD feeding, the 24-week HFD-induced elevation in glycogen was prevented in PTG

KO mice (Figure 6A). Triglyceride content was 40% decreased in the liver from PTG KO

mice fed HFD compared to WT littermates, corresponding to decreased glycogen levels

(Figure 6B). Moreover, HFD-induced liver steatosis was significantly attenuated by PTG

ablation, consistent with the marked reduction in triglycerides (Figure 6C).

To further investigate alleviation of the hepatosteatotic phenotype, we measured the

expression of key metabolic and inflammatory genes in the livers. PTG KO mice on HFD

had lower expression of Srebf1 and its downstream target gene fatty acid synthase (Fasn)

(Figure 6D). Gluconeogenic gene expression was modestly higher in HFD mice and was

not different with PTG ablation (Figure 6E). However, glycolytic genes such as pyruvate

kinase isozymes R/L (Pklr) and glucokinase (Gck), which were more highly expressed in

HFD relative to ND mice, were expressed at lower levels in PTG KO mice (Figure 6E).

In addition, HFD-induced expression of several inflammatory genes and markers of

macrophage infiltration, including F4/80 (Emr1), CD11C (Itgax), Rantes (Ccl5), and

MCP-1 (Ccl2), were reduced in livers from PTG KO mice (Figure 6F).

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PTG Ablation Improves Glucose Tolerance and Insulin Sensitivity after Long-term

HFD.

These changes in glycogen and lipid metabolism prompted us to investigate the role of

PTG in the overall control of carbohydrate metabolism. After 24 weeks of HFD, both

hyperglycemia and hyperinsulinemia were attenuated in PTG KO mice (Figure 7A). We

also performed oral glucose and intraperitoneal (IP) insulin and pyruvate tolerance tests.

PTG ablation did not affect insulin sensitivity or glucose tolerance in ND mice. However,

PTG KO mice displayed much improved glucose tolerance (Figure 7B), as well as

improved sensitivity to IP injection of insulin (Figure 7C) and pyruvate (Figure 7D).

AMPD assays from liver lysates revealed that levels of glycogen-bound GS protein were

increased by HFD, consistent with the HFD-induced elevation in PTG protein levels

(Figure 7E). Moreover, the increase in glycogen-bound GS protein was suppressed in

PTG KO mice, likely due to the lack of recruitment of GS to glycogen in the absence of

PTG. These data demonstrate that the recruitment of GS to glycogen is elevated in HFD

mice due at least in part to increased PTG protein, causing increased hepatic glycogen

content. Interestingly, both mTORC1 activity and SREBP1 levels were greatly

attenuated in PTG KO mice, confirming in vivo the feedback regulation of the

mTORC1/SREBP1 pathway by PTG and glycogen.

Taken together, these results suggest that in short term HFD-fed PTG KO mice, the

reduction in hepatic glycogen likely contributes to lower glucose output, in the process

mildly reducing serum glucose and insulin levels. Over time, this modulation of

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metabolic activity also leads to dramatically improved insulin sensitivity in the KO mice,

perhaps through reductions in mTORC1 activity known to negatively regulate insulin

receptor signaling (21,22).

A PTG-SREBP1/mTORC1 Feedback Mechanism Regulates Lipogenesis.

Although ablation of PTG did not have a major impact on systemic glucose tolerance and

insulin sensitivity after short term HFD, there were dramatic effects on hepatic lipid

metabolism (Figure 5F), which might reflect a crosstalk between pools of glycogen and

upstream nutrient-sensing pathways. In an effort to determine the mechanism, we

conducted quantitative real-time PCR for Srebf1 and its target genes Fasn, Ldlr, and

Hmgcr. We observed a trend toward decreases in the mRNA levels of these genes in the

PTG KO mice on HFD, although these changes were not statistically significant (Figure

8A).

To thoroughly examine the entire transcriptional landscape of PTG WT and KO livers,

we performed unbiased RNA-Seq and gene set enrichment analysis. We found that sterol,

steroid, cholesterol, and lipid biosynthetic/metabolic processes were differentially

regulated by PTG ablation in HFD mice (Figure 8B). More specifically, the transcript

levels of Srebf1 and its hepatic target genes such as Fasn, Ldlr, Mvk, Hmgcr were

significantly reduced in the PTG KO (Figure 8C and Supplementary Table 2), consistent

with quantitative real-time PCR data. Collectively, these data reveal a novel crosstalk

mechanism in which the mTORC1/SREBP pathway can sense elevated levels of

glycogen, and then initiate the transcriptional changes that increase lipid synthesis.

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DISCUSSION

Despite its well-established role as the first choice for energy storage and mobilization,

the coordination of glycogen metabolism with that of lipid synthesis and breakdown

remains poorly understood, particularly in the liver. Data presented here reveals that upon

reaching threshold levels during nutrient excess, glycogen may trigger increased

expression of lipogenic genes, thus shifting energy storage from glycogen to lipid. This

shift in the mode of energy storage may result from a positive feedback loop involving

mTORC1 and its transcriptional factor target SREBP1.

The activation of glycogen synthase by insulin requires the localization of PP1 to

glycogen particles through glycogen-targeting subunits, which act as molecular scaffolds

to unite PP1 with GS and GP for regulation (23). Studies reported here confirm the

crucial role of PTG in controlling glycogen synthesis (4,5). PTG KO mice have markedly

reduced glycogen levels in liver. Moreover, biochemical evaluation of GS localization in

PTG KO mice demonstrates conclusively that PTG is essential to direct GS to glycogen

particles in liver.

Experiments described here also reveal the surprising finding that the regulation of

glycogen synthesis by fasting/refeeding and HFD can be largely attributed to changes in

the expression of PTG. The increase in glycogen levels found in liver from HFD mice are

mirrored by increased expression of PTG, but attenuated in PTG KO mice. Both in vivo

and in vitro studies revealed an important role for mTORC1 in PTG expression,

consistent with findings by ourselves and others that glycogen synthesis is sensitive to

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inhibition by rapamycin (24–26). We propose that mTORC1 may regulate glycogen

metabolism through a transcriptional mTORC1-SREBP-PTG pathway, which in turn

modifies the phosphorylation status of GS in the glycogen compartment (Figure 8D).

This idea is supported by studies in both embryonic fibroblasts and mice, in which there

was a clear causal relationship between mTORC1 function, SREBP and PTG levels and

glycogen accumulation. The regulation of SREBP processing by mTORC1 has been

widely studied in the context of lipid homeostasis (27,28). Our data suggest that dietary

modulations in mTORC1 activity may affect not only lipid storage, but also carbohydrate

storage through regulation of glycogen synthesis.

Also surprising was the effect of PTG ablation on glucose metabolism. PTG mice on the

original mixed background were homozygous lethal, so we studied the heterozygous

knockouts and reported that they have decreased hepatic glycogen levels and a worsened

metabolic phenotype after 10 months or longer with ND feeding (11). However, we were

surprised to find after several years that the homozygous PTG KO mice were viable by

backcrossing heterozygotes against C57BL6/J mice. After short term HFD, the new

homozygous PTG KO mice showed reduced hepatic glucose output compared to WT

littermates, but little effect on systemic insulin sensitivity, likely reflecting reduced

glycogen that serves as a source of glucose. However, after longer term HFD, PTG KO

mice exhibited profoundly reduced fasting glucose and insulin levels, along with

improved glucose, pyruvate and insulin tolerance, reflecting increased systemic insulin

sensitivity, and accompanied by changes in expression of genes involved in lipogenesis,

glycolysis, inflammation and macrophage infiltration. Thus, although GS is increased

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while GP is decreased by insulin action, and both enzymes have been considered as

targets for the treatment of type 2 diabetes, reducing glycogen synthesis may

paradoxically improve insulin resistance in obesity.

The effect of PTG deletion on hepatic steatosis was particularly intriguing. Because PTG

does not interact with lipid-metabolizing enzymes, it is likely that this observation

reflects crosstalk between pools of glycogen and upstream nutrient-sensing pathways.

These ideas are consistent with observations using a glycogen phosphorylase inhibitor, in

which administration of the drug resulted in a short-term reduction in blood sugar by

prevention of hepatic glycogen breakdown, but was accompanied by steatosis after longer

term treatment due to increased lipid synthesis (29). Glycogen levels are increased in the

liver of HFD mice under fasting conditions, while the mTORC1 pathway is constitutively

elevated, along with increased levels of PTG, suggesting that diet-induced activation of

mTORC1 may increase glycogen stores in the fasted state by increasing the levels of

PTG. This occurs even in the face of reduced insulin signaling in HFD, and is similar to

what is observed for obesity-induced hepatic triglyceride accumulation (reviewed in (30)).

Conversely, PTG KO mice, which have reduced levels of hepatic glycogen, have reduced

hepatic lipids and reduced mTORC1/SREBP. Our transcriptional landscape analysis by

RNA-seq revealed a significant down-regulation of SREBP1 and numerous target genes

involved in lipogenesis. Although we cannot rule out other mechanisms explaining

reduced hepatic lipid levels, such as reduced glucose and insulin, these transcriptional

changes most likely underlie the alterations in lipid levels in the PTG KO livers, which

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perhaps reflect an interesting feedback regulation of PTG on mTORC1/SREBP and

lipogenesis to mediate crosstalk between pools of glycogen and lipids.

How might mTORC1 sense levels of glycogen? We propose a molecular sensor

upstream of the mTORC1 complex, similar to what might be involved in the sensing of

amino acids (31). One possible candidate is AMP-activated kinase, a negative regulator

of mTORC1 that is thought to be directly attenuated by the binding of glycogen to its β

subunit (32–34). Although we did not observe a decrease in AMPK or ACC

phosphorylation in liver from PTG mice on short term HFD, there were reductions in

muscle tissue (data not shown), suggesting that this enzyme may contribute to the overall

regulation of mTORC1. However, it also remains possible that there are other glycogen

sensors in the mTOR pathway. Future studies will focus on the regulation of this

pathway by glycogen, with the hope of developing new therapeutic targets for the

treatment of insulin resistance and nonalcoholic fatty liver disease.

ACKNOWLEDGEMENTS

We thank the University of Michigan Vector Core and the Comprehensive Cancer Center

Tissue Core for shRNA clones and histology service. We thank Dr. Jay Horton for

SREBP1 antibodies and Dr. David Kwiatkowski for the TSC2 knockout fibroblasts. We

also thank Dr. Ken Inoki and the members of the Saltiel laboratory for helpful

discussions.

FUNDING

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This work was supported by NIH grants R01DK060591 to ARS and R24DK090962 to

ARS and RME, and a mentor-based postdoctoral fellowship from the American Diabetes

Association to BL. This work was also funded by grants from the National Institutes of

Health (DK057978), the Leona M. and Harry B. Helmsley Charitable Trust, the Glenn

Foundation for Medical Research, and the Ellison Medical Foundation to RME. RME is

an investigator of the Howard Hughes Medical Institute and March of Dimes Chair in

Molecular and Developmental Biology at the Salk Institute.

DUALITY OF INTEREST

No potential conflicts of interest relevant to this article were reported.

AUTHOR CONTRIBUTIONS

BL and ARS developed the overall research plan. BL generated the figures and wrote the

manuscript. BL, DB, AC, LC, ZM conducted experiments, researched data, performed

analysis and interpretation. YY and KF carried out experiments. DB and ARS contributed

to discussion, interpretation and reviewed/edited manuscript. MD, RTY, CL, RME

performed RNA-Seq and data analysis. ZM and JL performed the primary hepatocyte

experiment. ARS is the guarantor of this work and, as such, had full access to all the data

in the study and takes responsibility for the integrity of the data and the accuracy of the

data analysis.

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Figure Legends

Figure 1. High Fat Diet Increases Hepatic Glycogen in Fasted Mice

(A) Glycogen content in the liver of C57BL/6J mice on HFD for 12 weeks and sacrificed

after 16hrs fasting followed by 6hrs refeeding (n=8 each group).

(B) Quantitative real-time PCR showing relative mRNA level of PTG, SREBP1 and

GYS2 from the liver of mice in (A).

(C) Representative western blots from the liver of mice in (A). WCL, whole cell lysate;

N.E., nuclear extracts; AMPD, after an amylose pull-down assay.

(D) Hepatic GP activity and (E) GS activity from ND and HFD mice sacrificed after

16hrs fasting followed by 6hrs refeeding (n=6-8 mice each group).

Values are expressed as mean ± SE.*indicates statistical significance by student t-test

(*P<0.05).

Figure 2. The PTG Promoter is Regulated by SREBP1 and mTORC1

(A) HEK293 cells were cotransfected with nuclear active form of SREBP-1c (1-471) and

the indicated reporter constructs of the PTG promoter. Cells were harvested 24 hrs after

transfection and assayed for luciferase activity. Results were normalized (Renilla

luciferase) and expressed as fold increase over the basal value of each construct.

(B) TSC2 MEF cells were transfected with the indicated luciferase reporter constructs of

the PTG promoter and treated with Rapamycin (100nM) or DMSO before harvest.

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(C) A schematic representation of the PTG promoter region (top) and Chromatin

immunoprecipitation (CHIP) experiment showing increased SREBP1 activity in TSC2-/-

cells (bottom). Cross-linked chromatin was immunoprecipitated with SREBP1 antibody

and extensively washed before elution and precipitation of DNA. Quantitative PCR was

performed with the indicated primer pairs. Primer sequences were listed in the Research

Design and Methods.

(D) Effect of Rheb and rapamycin on the PTG promoter activity. HEK293 cells were

cotransfected with Rheb or vector and the indicated luciferase reporter construct of the

PTG promoter harboring the deletion of the putative SREBP1 binding site. Cells were

subsequently treated with DMSO or rapamycin (100nM) for 24hrs before harvest and

assayed for luciferase activity. Results were normalized (Renilla luciferase) and

expressed as fold increase over the basal value of each construct.

All the Values are expressed as mean ± SE (n=3). *indicates statistical significance by

student t-test (*P<0.05).

Figure 3. Transcriptional Regulation of PTG expression by SREBP1 is Required for

the Increased Glycogen Produced by mTORC1 Activation

(A) Western blots showing SREBP1 knockdown in TSC2 MEFs. Cells were transfected

with lentiviral mediated control shRNA or shRNAs targeting SREBP1 and selected for

cells stably expressing shRNAs. Stable cells were lysed and subjected to western blotting

against indicated antibodies.

(B) Quantitative real-time PCR showing relative mRNA level of SREBP1 and PTG from

cells in (A).

(C) Glycogen content of cells in (A).

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(D) 14C-Glucose conversion into glycogen of cells in (A).

All the Values are expressed as mean ± SE (n=3). *indicates statistical significance by


Figure 4. PTG Levels Control Glycogen Synthesis and Contribute to Feedback

Regulation of mTORC1 and SREBP1.

(A) Western blots showing PTG knockdown in TSC2 MEFs. Cells were transfected with

lentiviral mediated control shRNA or shRNAs targeting PTG and selected for cells stably

expressing shRNAs. Stable cells were lysed and subjected to western blotting against

indicated antibodies.

(B) Quantitative real-time PCR showing relative mRNA level of PTG and SREBP1 from

cells in (A).

(C) Glycogen content of cells in (A).

(D) 14C-Glucose conversion into glycogen of cells in (A).

(E) 2-DG uptake of cells in (A).

(F) Glycogen levels of HEK293A cells stably overexpressing Flag-tagged PTG.

(G) Representative western blots showing activation of mTORC1 activity by

overexpressing PTG. Numbers above blots indicate relative band intensity as quantified

by Image J software.

Values are expressed as mean ± SE (n=3 experiments). *indicates statistical significance

by student t-test (*P<0.05) n.s. indicates not significant by student t-test.

Figure 5. PTG Ablation Results in Decreased Glucose Incorporation into Glycogen

and Lipids.

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14C-glucose incorporation into glycogen (A), lipids (B) and CO2 (C) in primary

hepatocytes isolated from 6-8 week old PTG WT or KO mice on ND. Values are

expressed as mean ± SE (n=3 triplicate experiments). * indicates statistical significance

by student t-test (*P<0.05)

3H-glucose incorporation into glycogen (D) and lipids (E) during a hyperinsulinemic-

euglycemic clamp study using PTG WT or KO mice on HFD for 8-10 weeks.

Values are expressed as mean ± SE (n=10-12 mice each group). * indicates statistical

significance by student t-test (*P<0.05)

Figure 6. PTG Ablation Reduces Hepatic Steatosis in vivo after Long Term HFD.

(A) Glycogen content and (B) TG levels in the liver of WT and PTG KO mice on HFD

for 24 weeks and fasted overnight (n=8-10 mice each group).

(C) Representative H&E staining of liver sections of mice in (A).

(D) Quantitative real-time PCR showing relative mRNA level of SREBP1 and FAS from

mice in (A).

Quantitative real-time PCR showing relative mRNA level of glucose metabolism genes

(E) and inflammatory genes and macrophage markers (F) from mice in (A).

Figure 7. PTG Ablation Improves Glucose Tolerance and Insulin Sensitivity in vivo

after Long Term HFD.

(A) Fasting blood glucose and insulin levels of WT and PTG KO mice on HFD for 24

weeks and fasted overnight.

(B) Oral glucose tolerance test, (C) Insulin tolerance test and (D) Pyruvate tolerance test

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of WT and PTG KO mice on HFD for 24 weeks.

(E) Representative blot (Left) and quantification (right) from the liver of mice in (A).

All the Values are expressed as mean ± SE (n=8-10). *indicates statistical significance by


Figure 8. A PTG-SREBP1/mTORC1 Feedback Mechanism Regulates Lipogenesis

(A) Quantitative real-time PCR showing relative mRNA level of SREBP1 and its target

genes from the liver of mice on HFD for 12 weeks and fasted overnight (n=8-10 mice

each group).

(B) GO term enrichment analysis of RNA-seq data from the livers of mice in (A).

(C) Unbiased hierarchical clustering analysis of RNA-Seq data from the livers of mice in

(A) showing down-regulation of SREBP1 hepatic target genes in PTG KO mice on HFD .

Genes shown on the heatmap are listed in Supplementary Table 2.

(D) A schematic model showing the feedback regulation of PTG and glycogen on

mTORC1/SREBP and lipogenesis.

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A

pGSS6K

pS6K

PTG

S6

m-SREBP1

GS

pS6RalA

Fasted Refed Fasted RefedND HFD

B

WCL

AMPD

Figure1. High Fat Diet Increases Hepatic Glycogen in Fasted Mice.

ND

HFD

G

lyco

gen

cont

ent

(μm

ol g

luco

se/g

tiss

ue)

120

100

80

60

40

20

0Fasted Refed

0.0

0.5

1.0

4.5

Fasted Refed

Rel

ativ

e m

RN

A

Ppp1r3c

0

2

4

6

8

10

12

14

16

Fasted Refed

Rel

ativ

e m

RN

A

Srebf1

ND HFD

ND HFD 4.0

3.5

3.0

2.5

2.0

1.5

*

*

*

*

N.E. CREB

C

0 2 4 6 8

10 12 14

Fasted Refed

pS6K

/S6K

0 2 4 6 8

10 12 14 16 ND

HFD

pS6/

S6

Fasted Refed

ND HFD

* *

0 1 2 3 4 5 6

PTG

Fasted Refed

ND HFD *

*

0

5

10

15

20

Fasted Refed

m-S

RE

BP

1 ND HFD

*

0

0.4

0.8

1.2

1.6

Rel

ativ

e m

RN

A

Gys2

Fasted Refed

0.2

0.6

1.0

1.4

0

0.2

0.4

0.6

0.8

1.0

GS

Act

ivity

ratio

(-/+

G6P

)

0

0.2

0.4

0.6

0.8

1.0

Fasted

Refe

d

*

ND HFD

0

GP

activ

ity (n

mol

/min

/mg

prot

ein) 25

20

15

10

5

0

-AMP+AMP

GP

activ

ity ra

tio(-

/+A

MP

)

ND-Fas

ted

HFD-Refe

d

HFD-Fas

ted

ND-Refe

d

ND HFD

GS

act

ivity

(nm

ol/m

in/m

g pr

otei

n) 14

12

10

8

6

0

4

2

-G6P+G6P

ND-Fas

ted

HFD-Refe

d

HFD-Fas

ted

ND-Refe

d

Fasted

Refe

d

1.2*

ND HFD

ED

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WT -70 agggaccgGTTCACGTGATC tggctttgat -41

M1 -70 agggaccg------------------------- tggctttgat -41M2 -70 agggaccgGTTGTCGTGATCtggctttgat -41M3 -70 agggaccgGTTGTCGACATCtggctttgat -41

0

0.5

1.0

1.5

2.0

2.5

3.0

WT M1 M2

Rel

ativ

e Lu

c A

ctiv

ity

(fold

incr

ease

)

VectorSREBP-1c

AB

+/++/+-/- -/-

TSC2 DMSOTSC2 RapaTSC2 DMSOTSC2 Rapa

WT M1M3

* * *

Rel

ativ

e Lu

c A

ctiv

ity(F

old

incr

ease

)

WT M1

Vector DMSOVector RapaRheb DMSORheb Rapa

Rel

ativ

e Lu

cife

rase

Act

ivity

(F

old

chan

ge)

* *

0

0.5

1.0

1.5

2.0

2.5

DC

TSS

-2015

0.00

0.01

0.02

0.03

0.04

0.05

0.06

0.07

Pre

cipi

tatio

n re

lativ

e to

inpu

t (%

)

TSC2+/+ TSC2-/-

SREBP binding sitePrimer pairs

1 2 4 3

1 2 3 4

*

*

0

0.5

1.0

1.5

2.0

2.5

Figure 2. The PTG promoter is regulated by SREBP1 and mTORC1.

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(µmol

/mg

prot

ein)

Contro

l

SREBP1-sh2

Contro

l

SREBP1-sh2

0.6

0

40

60

80

100

120

(fmol

/mg

prot

ein/

60m

in)

Gly

coge

n sy

nthe

sis

Gly

coge

n co

nten

t

TSC2

m-SREBP1

PTG

GS

RalA

GS

pGS

TSC2 TSC2

WCL

AMPD

+/+ -/-A

C

Figure 3. Transcriptional Regulation of PTG expression by SREBP1 is Required for the Increased Glycogen Produced by mTORC1 Activation

0.5

0.4

0.3

0.2

0.1

0

TSC2+/+TSC2-/-

CREBN.E.

Con

trol

SR

EB

P1-

sh1

SR

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P1-

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SR

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P1-

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SR

EB

P1-

sh2

B

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RN

A

2.0

1.6

1.2

0.8

0.4

0

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l

SREBP1-sh1

SREBP1-sh2

0

0.5

1.0

1.5

2.0

2.5

Srebf1 Ppp1r3c

* * *

* *

**

* *

Contro

l

SREBP1-sh1

SREBP1-sh2

* *

D

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TSC2

PTG

GS

RalA

m-SREBP1

GS

pGS

TSC2 TSC2

WCL

AMPD

+/+ -/-A

C

0

0.1

0.2

0.3

0.4

0.5

(µmol

/mg

prot

ein)

0.6

Gly

coge

n co

nten

t

0

0.5

1.0

1.5

2.0

2.5

(nm

ol/m

g pr

otei

n/60

min

)G

lyco

gen

synt

hesi

s

TSC2+/+TSC2-/-

Contro

l

PTG-sh

1

PTG-sh

2

Contro

l

PTG-sh

1

PTG-sh

2

* * *

CREBN.E.

Con

trol

PTG

-sh1

PTG

-sh2

Con

trol

PTG

-sh1

PTG

-sh2

Figure 4. PTG Levels Control Glycogen Synthesis and Contribute to Feedback Regulation of mTORC1 and SREBP1.

B

0

0.5

1.0

1.5

2.0

2.5

3.0

Ppp1r3c

0

0.5

1.0

1.5

2.0

2.5

Srebf1

Rel

ativ

e m

RN

A

Contro

l

PTG-sh

1

PTG-sh

2Con

trol

PTG-sh

1

PTG-sh

2

** *

*

* *

0

10

20

30

40

50

60

70

80

2-D

G u

ptak

e (µ

mol

/g p

rote

in/5

min

)

Contro

l

PTG-sh

1

PTG-sh

2

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S6K

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Flag-PTG

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S6K

pS6K

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IP:

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PTG stab

le29

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20

15

10

5

0

mm

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luco

se/m

g pr

otei

n

F

IgG S6K IgG S6K

IB

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1.3 2.0 2.5 1.8 1.0 1.1 1.3 1.4

1.5 2.0 2.3 1.1 1.3 1.0

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PP1

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1.5 1.8 1.6 1.4

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0.2

0.4

0.6

0.8

1.0

1.2

1.4

WT KO 14C

glu

cose

inco

rpor

atio

n in

to g

lyco

gen

0

0.2

0.4

0.6

0.8

1.0

1.2

WT KO 0

0.2

0.4

0.6

0.8

1.0

1.2

WT KO

14C

glu

cose

inco

rpor

atio

n in

to li

pids

14C

glu

cose

inco

rpor

atio

n in

to C

O2

0

0.2

0.4

0.6

0.8

1.0

1.2

WT KO

3H g

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se in

corp

orat

ion

into

gly

coge

n

0

0.2

0.4

0.6

0.8

1.0

1.2

WT KO

3H g

luco

se in

corp

orat

ion

into

lipi

ds

A B C

D E

Figure 5. PTG Ablation Results in Decreased Glucose Incorporation into Glycogen and Lipids.

* *

*

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5

10

15

20

25

30

35

ND HFD

AG

lyco

gen

cont

ent

(μm

ol g

luco

se/g

tiss

ue) WT

KO

0

40

80

120

160

200

ND HFDTG

(μ

mol

gly

cero

l/g t

issu

e) **** * WT

KO

WT KO

ND

HFD

CB* *

*

WTKO

WTKO

Srebf1 Fasn

2.5

2.0

1.5

1.0

0.5

0.0

2.5

2.0

1.5

1.0

0.5

0.0ND HFD ND HFD

Rel

ativ

e ge

ne e

xpre

ssio

n

Rel

ativ

e ge

ne e

xpre

ssio

n

#

*

D

0

1

2

3

4

5

6

G6pc Pck1 Pdk4 Pklr Gck

ND-WT

HFD-WT

HFD-KOR

elat

ive

gene

exp

ress

ion

glucose metabolism genes

#

#

#

*

E

Tnf Emr1 Itgax Ccl5 Ccl2

ND-WT

HFD-WT

HFD-KO

0

1

2

3

4

5

Rel

ativ

e ge

ne e

xpre

ssio

n

inflammatory genes and macrophage markers

*

#

##

#

#

*

*

*

F

Figure 6. PTG Ablation Reduces Hepatic Steatosis in vivo after Long Term HFD.

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Diabetes

PTG

RalA

pGSS6K

S6pS6

pS6K

GS

m-SREBP1

WT KOWT KOND HFD

GS

WCL

AMPD

CREBN.E.

A

Figure 7. PTG Ablation Improves Glucose Tolerance and Insulin Sensitivity in vivo afterLong Term HFD.

0

1

2

3

4

5

6

Fast

ing

insu

lin (n

g/m

l)

020406080

100120140160180200

ND HFD ND HFD

Fast

ing

gluc

ose

(mg/

dL)

% o

f bas

al b

lood

glu

cose

0

50

100

150

200

250

300

0 30 60 90 1200 30 60 90 120 0 30 60 90 120

HFD-WTHFD-KO

ND-WTND-KO

HFD-WTHFD-KO

% o

f bas

al b

lood

glu

cose 120

100

80

60

40

20

0

120

100

80

60

40

20

0

Time after injection (min) Time after injection (min) Time after injection (min)B

lood

glu

cose

(mg/

dL)

PTTITT ITT

B

C D

E *G

S in

AMPD

PTG

in A

MP

D **

WTKO

*

pS6K

/S6K

m-S

RE

BP

1

0

1

2

3

0

1.0

2.0 * *

0

1

2

3

4

ND HFD

* *

0

1

2

3 * *

ND HFD

0.5

1.5

050

100150200250300350400

0 30 60 90 1200

100

200

300

400

500

600

ND-WTND-KO

HFD-WTHFD-KOB

lood

glu

cose

(mg/

dL)

Time after injection (min)

Blo

od g

luco

se (m

g/dL

)

0 30 60 90 120Time after injection (min)

OGTT OGTT* * * *

** * *

**

* *

* * * * * *

WTKO

*

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Diabetes

C

WT KO

mTORC1

HFD

Glycogenesis

FASNSCD1

LDLRHMGCR

SREBP1

Lipogenesis

Glycogen

PTG

TG

GS

0

5

10

15

20

25

30

sterol

meta

bolic

proc

ess

steroi

d meta

bolic

proc

ess

chole

sterol

meta

bolic

proc

ess

sterol

bios

ynthe

tic pr

oces

s

chole

sterol

bios

ynthe

tic pr

oces

s

steroi

d bios

ynthe

tic pr

oces

s

oxida

tion r

educ

tion

lipid

biosy

ntheti

c proc

ess

acyl-

CoA m

etabo

lic pr

oces

s

isopre

noid

biosy

ntheti

c proc

ess

cofac

tor m

etabo

lic pr

oces

s

fatty

acid

metabo

lic pr

oces

s

Fold

enr

ichm

ent

GO Term

A

D

> 3 2 1 0

-1 -2

> -3

Fold change?

?

?

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

ND HFD

Rel

ativ

e m

RN

ASrebf1

0.00.20.40.60.81.01.21.41.61.82.0

ND HFDR

elat

ive

mR

NA

Fasn

0.00.20.40.60.81.01.21.41.61.8

ND HFD

Rel

ativ

e m

RN

A

Ldlr

0.00.20.40.60.81.01.21.41.6

ND HFD

Rel

ativ

e m

RN

A

Hmgcr

P=0.06

WTKO

B

Glucose Insulin

Figure 8. A PTG-SREBP1/mTORC1 Feedback Mechanism Regulates Lipogenesis

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SUPPLEMENTARY DATA

Supplementary Figure Legends

Supplementary Figure 1. Metabolic phenotypes of C57BL/6J mice fed normal chow

diet (ND) or high fat diet (HFD).

Male mice aged 8-10 weeks were put on HFD for 12 weeks (n=8 each group). (A)

Growth curve. (B) Fasting glucose level. (C) Fasting insulin level. (D) Glucose tolerance

test. (E) Insulin tolerance test. (F) Representative Western blots showing pAKT (S473)

levels from liver lysates of ND and HFD mice sacrificed after 16hrs fasting followed by

6hrs refeeding. (G) Representative Western blots showing pAKT (S473) levels from liver

lysates of ND and HFD mice fasted overnight and sacrificed 20 min after intraperitoneal

injections of insulin (2U/kg body weight). (H) Quantitative real-time PCR showing

hepatic gluconeogenic gene expression.

Values are expressed as mean ± SE.*indicates statistical significance by student t-

test (*P<0.05).

Supplementary Figure 2. Glycogen synthesis is similarly regulated in MEFs, 293

and liver cell lines.

Quantitative real-time PCR showing GL (A) and GM (B) gene expression in TSC: PTG

knockdown MEFs.

(C) Glycogen content in 293A or Hepa1c cells transiently overexpressing WT PTG or

PTG mutants incompetent to bind to GS or PP1.

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Supplementary Figure 3. PTG Ablation Results in Decreased Glycogen and Lipid

levels in vivo after Short-term HFD without Changing Glucose and Insulin

Sensitivity.

(A) Genotyping of transgenic mouse showing homozygous deletion of the PTG gene.

(B) PTG homozygous knockout mouse has improved survival rate at birth with more

backcross. Heterozygous PTG knockout mice maintained on a mixed background were

sequentially backcrossed with C57BL/6J mice and birth ratio of homozygous female,

male, and total PTG knockout mice were calculated from genotyping of over 200 pups

generated from heterozygous breeding pairs at each backcross generation. *indicates

statistical significance by CHITEST relative to expected Mendelian ratio of 25% each

group (*P<0.05).

(C) Growth curves and (D) food intake for wild type (WT) or PTG knockout (KO) mice.

Mice were fed a normal chow diet (ND) for 8 weeks before a high fat diet (HFD) feeding

of 24 weeks. Body weights were measured every other week. Food intake was measured

for consecutively three weeks (n=8-10 mice each group).

(E) Glycogen content and (F) TG levels in the liver of WT and PTG KO mice on HFD

for 12 weeks and fasted overnight (n=7-10 mice each group).

(G) Quantitative real time PCR showing relative mRNA expression level of PTG

(Ppp1r3c), SREBP1 (Srebf1) and GL (Ppp1r3c) in the same mice as in (E) and (F).

(H) Oral glucose tolerance test and (I) Insulin tolerance test of WT and PTG KO mice on

HFD for 12 weeks.

Supplementary Figure 4. GS and GP activity is not changed in PTG KO mice.

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(A) Hepatic GS activity (-/+ G6P) and (B) GP activity (-/+AMP) from ND and HFD

PTG mice sacrificed after 16hrs fasting (n=8-10 mice each group).

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0

50

100

150

200

250

HFD

Fasting glucose (mg/dL)

Fasting insulin (ng/ml)

0

100

200

300

400

500

600

0 15 30 45 60 75 90 105 120

Glucose (mg/dL)

HFD

ND

ND HFDND

Glucose (% of basal)

0

20

40

60

80

100

120

0 15 30 45 60 75 90 105 120

HFD

ND

Fasted Refed Fasted Refed

ND HFD

- - + + + - - + + +

ND HFD

insulin

20

22

24

26

28

30

32

34

36

38

40

0 2 4 6 8 10 12

Body Weight (g)

Weeks on HFD

ND

HFD

A B C

0

0.5

1.0

1.5

2.0

2.5

3.0

D E

F G

AKT

pAKT

(S473)

AKT

pAKT

(S473)

* ***

* *

*

*

*

* **

*

*

*

**

Time after injection (min) Time after injection (min)

0.2

0.4

0.6

0.8

1.0

1.2

Fasted Refed

Relative mRNA

PEPCK ND HFD

0

0.2

0.4

0.6

0.8

1.0

1.2

1.4

1.6

1.8

Fasted Refed

Relative mRNA

G6PC

0

0.5

1.0

1.5

2.0

2.5

Fasted Refed

Relative mRNA

PDK4

0

H

Suppementary Figure 1. Metabolic phenotypes of C57BL/6J mice fed normal chow diet (ND) or high fat diet (HFD).

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A

B

PTG: WT KO Het

PTG KO mouse ratio (%)

0

25

50

BC1 BC2 BC3

FemaleMaleTotal

300 bp D

* * *

200 bp

C

0

1

2

3

4

ND HFD

Food intake

WTKO

(g/mouse/day)

Body weight (g)

0 2 4 6 8 10 12 1416 18 20 22 24

60

50

40

30

20

10

0

HFD-WT

HFD-KO

ND-WT

ND-KO

Weeks on HFD

0

5

10

15

20

25

30

35

ND HFD

E

**

***

glycogen content

(µmol glucose/g tissue)

0

50

100

150

200

250

ND HFD

TG (µmol glycerol/g tissue)

**** *

050100150200250300350400450

Glucose (mg/dL)

WTKO

HFD

0

50

100

150

200

250

300

0 15 30 45 60 75 90 105 120

Glucose (mg/dL)


WTKO

ND

0 15 30 45 60 75 90 105 120Time after injection (min)

0

20

40

60

80

100

120

0 15 30 45 60 75 90 105 120

WTKO

ND



0

20

40

60

80

100

120


WT

KO

HFD

0 15 30 45 60 75 90 105 120Time after injection (min)

F G

H

Supplementary Figure 3. PTG Ablation Results in Decreased Glycogen and Lipid levels in vivo after

Short-term HFD without Changing Glucose and Insulin Sensitivity .

0.0

0.5

1.0

1.5

2.0

2.5

3.0

ND HFD

Relative mRNA

Ppp1r3c

0.0

0.5

1.0

1.5

2.0

2.5

3.0

3.5

4.0

ND HFD

Relative mRNA

Srebf1* *

*

WT

KO

0.0

0.5

1.0

1.5

2.0

2.5

ND HFD

Relative mRNA

Ppp1r3b**

WTKO

WTKO

I

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Supplementary Table 1. List of Real Time PCR Primers

*Triplicates of each sample were prepared and normalized to RPLP0 (primer1) to

determine relative expression levels. In the case of TSC2 knockout MEFs, we observed

significant changes in the levels using this primer so we used RPLP0 (primer2) for

normalization.

Name Gene Symbol Primer Sequence

FAS Fasn F: 5'-GGAGGTGGTGATAGCCGGTAT-3'

R: 5'-TGGGTAATCCATAGAGCCCAG-3'

HMGCR Hmgcr F: 5’- AGCTTGCCCGAATTGTATGTG-3’

R: 5’-TCTGTTGTGAACCATGTGACTTC -3’

LDLR Ldlr F: 5’- AGTGGCCCCGAATCATTGAC-3’

R: 5’-CTAACTAAACACCAGACAGAGGC -3’

PTG Ppp1r3c F: 5’-TGATCCATGTGCTAGATCCACG-3’

R: 5’-ACTCTGCGATTTGGCTTCCTG-3’

GYS1 Gys1 F: 5’-CGCTGGAAGGGTGAGCTTT-3’

R: 5’-GAAGTGGGCAACCACATACG-3’

GYS2 Gys2 F: 5’- ACCAAGGCCAAAACGACAG-3’

R: 5’-GGGCTCACATTGTTCTACTTGA-3’

GM Ppp1r3a F: 5’-GCTTCCCGGAGAGTTTCCTTT-3’

R: 5’-CACGGCTTTCTGGACTTGGA-3’

GL Ppp1r3b F: 5’-GTGGACATCCAATACAGCTACAG-3’

R: 5’- CCGAGAACACTTTCACCATTGT-3’

PEPCK Pck1 F: 5'-CTGCATAACGGTCTGGACTTC-3'

R: 5’-CAGCAACTGCCCGTACTCC-3’

PDK4 Pdk4 F: 5'-GGAGTGTTCACTAAGCGGTCA-3'

R: 5'-AGGGAGGTCGAGCTGTTCTC-3'

PKLR Pklr 5'-TCAAGGCAGGGATGAACATTG-3'

5'-CACGGGTCTGTAGCTGAGTG-3'

GCK Gck F: 5'-ATGGCTGTGGATACTACAAGGA-3'

R: 5'-TTCAGGCCACGGTCCATCT-3'

TNFa Tnf F: 5'-ACGGCATGGATCTCAAAGAC-3'

R: 5'-AGATAGCAAATCGGCTGACG-3'

F4/80 Emr1 F: 5'-CTGGGATCCTACAGCTGCTC-3'

R: 5'-AGGAGCCTGGTACATTGGTG-3'

CD11C Itgax F: 5'-CTGGATAGCCTTTCTTCTGCTG-3'

R: 5'-GCACACTGTGTCCGAACTCA-3'

Rantes Ccl5 F: 5'-GCTGCTTTGCCTACCTCTCC-3'

R: 5'-TCGAGTGACAAACACGACTGC-3'

MCP-1 Ccl2 F: 5'-TTAAAAACCTGGATCGGAACCAA-3' R: 5'-GCATTAGCTTCAGATTTACGGGT-3'

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Supplementary Table 2. List of Genes Shown on the Heatmap from Figure 8C.

Gene Symbols Cyp7a1

Hmox1

Aco2

Apoa2

Mttp

Apoa5

Elovl3

Pal2g6

Irs2

Tm7sf2

Nr0b2

Pck1

Fdps

Insig1

Aldoc

Idi1

Sqle

Acss2

Acat2

Ebp

Fads1

Idh1

Dbi

Pmvk

Pgd

Ggps1

Pcyt1a

Ucp2

Acly

Ldlr

Fasn

Nss

Nsdhl

Mid1ip1

Cyb5b

Hsd17b7

Dhcr24

Fdft1

Tmem97

Rdh11

Srebf2

Dhcr7

Mvk

Thrsp

Mmab

Hmgcr

Pfkfb1

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Supplementary Method

Enzyme activity assay. GS activity was determined by measuring incorporation of 14C-

glucose from UDP- 14C-glucose into glycogen in the absence or presence of G6P as

described previously (5). GP activity was assayed by measuring incorporation of 14C-

glucose from 14C-glucose-1-phosphate into glycogen in the absence or presence of AMP

(6). Activity ratios represent the activity measured in the absence of the allosteric effector

(G6P for GS or AMP for GP) divided by that in the presence of the allosteric effector.

Primary hepatocyte isolation. Primary hepatocyte isolation and cultures were carried

out as previously described (7). Cells were starved in serum-free media for 3 hr prior to

the incubation with 14C-glucose for 1hr. Glucose conversion into glycogen, lipid and

CO2 assay was performed as described in (8,9). Values are normalized to the protein

content of hepatocytes and averaged from three independent experiments using primary

hepatocytes isolated from 3 WT and 4 PTG KO mice on ND individually.

Euglycemic hyperinsulinemic clamp experiments. Euglycemic hyperinsulinemic

studies were performed by the Animal Phenotyping Core of the Nutrition Obesity

Research Center from University of Michigan, as previously described (10). Liver

glycogen and lipids were extracted and 3H counts were calculated for in vivo glucose

incorporation experiment.

Supplementary References

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1. Crosson SM, Khan A, Printen J, Pessin JE, Saltiel AR. PTG gene deletion causes

impaired glycogen synthesis and. Journal of Clinical Investigation.

2003;111(9):1423–32.

2. Cheng A, Zhang M, Crosson SM, Bao ZQ, Saltiel AR. Regulation of the mouse

protein targeting to glycogen (PTG) promoter by the FoxA2 forkhead protein and

by 3’,5'-cyclic adenosine 5'-monophosphate in H4IIE hepatoma cells.

Endocrinology. 2006 Jul 1;147(7):3606–12.

3. Zhang H, Cicchetti G, Onda H, Koon HB, Asrican K, Bajraszewski N, et al. Loss

of Tsc1/Tsc2 activates mTOR and disrupts PI3K-Akt signaling through

downregulation of PDGFR. The Journal of clinical investigation. 2003

Oct;112(8):1223–33.

4. Lois C, Hong EJ, Pease S, Brown EJ, Baltimore D. Germline transmission and

tissue-specific expression of transgenes delivered by lentiviral vectors. Science

(New York, N.Y.). 2002 Feb 1;295(5556):868–72.

5. Thomas JA, Schlender KK, Larner J. A rapid filter paper assay for UDPglucose-

glycogen glucosyltransferase, including an improved biosynthesis of UDP-14C-

glucose. Analytical biochemistry. 1968 Oct 24;25(1):486–99.

6. Gilboe DP, Larson KL, Nuttall FQ. Radioactive method for the assay of glycogen

phosphorylases. Analytical biochemistry. 1972 May;47(1):20–7.

7. Liu C, Li S, Liu T, Borjigin J, Lin JD. Transcriptional coactivator PGC-1alpha

integrates the mammalian clock and energy metabolism. Nature. 2007 May

24;447(7143):477–81.

8. Greenberg CC, Meredith KN, Yan L, Brady MJ. Protein targeting to glycogen

overexpression results in the specific enhancement of glycogen storage in 3T3-L1

adipocytes. The Journal of biological chemistry. 2003 Aug 15;278(33):30835–42.

9. Meng ZX, Yin Y, Lv JH, Sha M, Lin Y, Gao L, et al. Aberrant activation of liver

X receptors impairs pancreatic beta cell function through upregulation of sterol

regulatory element-binding protein 1c in mouse islets and rodent cell lines.

Diabetologia. 2012 Jun;55(6):1733–44.

10. Bracy DP, Ayala JE, Malabanan C, James FD, Ansari T, Fueger PT, et al.

Hyperinsulinemic-euglycemic clamps in conscious, unrestrained mice. Journal of

visualized experiments. 2011 Jan;(57).

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