Mol Metab:GLP-1 improves the ability of astrocytes to support neurons by promoting aerobic glycolysis in Alzheimer's disease

Alzheimer's dissease (AD) is characterized by the overproduction of β-amyloid (Aβ) peptides, Aβ toxicity has long been considered the main cause of AD, and in recent years researchers have found that the impact of brain energy deficiency and metabolic changes on AD should not be underestimated

The glycolytic regulatory effect of GLP-1 in AD was investigated and its neuroprotective mechanism

The study found that GLP-1 improved cognitive function levels and oxidative stress in the brain of 5× FAD mice by enhancing aerobic glycolysis and reducing oxidative phosphorylation, and could alleviate Aβ-induced decrease in astrocyte glycolysis, which led to decreased OXPHOS levels as well as reactive oxygen species (ROS) production

Experimental results of the experimental design

GLP-1 improved spatial cognition in 5×FAD mice

5× FAD mice are transgenic mice transfected with 5 FAD mutations that develop amyloid plaques and gliomas

The authors observed the number of neurons by Nicholas staining, and then the synaptic structure

Next, the authors conducted a proteomic analysis to compare the proteomics profiles of the three groups with a view to investigating the specific mechanisms

Figure 1 GLP-1 improves spatial cognitive performance in 5×FAD mice

MWM evaluation of mouse cognitive function, including escape latency (A) and quadrant (B) during the traversal target (A) probe test and representative swimming trajectories (C)

2.

In the proteomic analysis, the authors identified a total of 7013 proteins in the cortical regions of the 3 groups of mice, of which 300 proteins were expressed differently between the WT group and the AD group, 275 proteins were expressed differently between the AD group and the AD+Lira group (Figures 2C and 2D), and 49 proteins had significant differences

The metabolic pathways in the AD brain were significantly altered and also changed after the GLP-1 treatment, and after further detection, the expression of several bioenetic proteins in the AD group treated with GLP-1 changed, such as PDK2, NDUFB2, ACAA2 and GSTK1 expression decreased, while GSTK1 was upregulated (Figure 2E

In addition to the above proteins, the other 14 are involved in protein transport, proteolysis, and protein glycosylation, and partially reverse the effects of

Figure 2 GLP-1 alters the proteomics profile of the brains of 5× FAD mice

(A-B) The volcanic map of the changes in cortical protein expression changes in KEGG enriched by KEGG is different from the expression of proteins (DEPs), and the intensity of red indicates the significance of

GLP-1 improves aerobic glycolysis in the COrtex of 5× FAD mice and reduces oxidative stress

The authors evaluated glycolysis and OXPHOS levels in three groups and found that glycolysis was suppressed in the brains of the AD group, while OXPHOS was upregulated

An upregulation of OXPHOS levels, a decrease in ATP production, and an increase in ROS levels (Figures 3H and 3F), while a decrease in glutathione content (Figure 3G), indicates an excessive redox state

The PI3K/Akt pathway is deeply involved in energy processes, and a decrease in Akt phosphorylation has been found to be associated with impaired cognitive function in

Figure 3 GLP-1 improves aerobic glycolysis in 5×FAD mice and relieves oxidative stress in the cortex

(A-D) analysis of corticolytic levels in three groups, relative expression of glycolytic enzymes detected by Western blotting (A); Glycolysis of lactic acid (B) and NAD+ (C-D

4.
GLP-1 improves aerobic glycolysis and relieves oxidative stress in astrocytes

Astrocytes mainly use glycolysis to produce ATP, and numerous studies have shown that astrocyte dysfunction leads to AD
.
To assess the effect of GLP-1 on astrocytes, the authors constructed an AD cell model Aβ1-42
treating astrocytes with soluble oligomers.

Consistent with the results of animal experiments, GLP-1 relieved Aβ-induced glycolysis inhibition and increased the expression of glycolytic enzymes such as PKM2 and HK2 (Figure 4A) as well as glycolytic products (Figure 4B-D
).
GLP-1 increases Akt phosphorylation levels in Aβ-treated astrocytes (Figure 4E); However, when the PI3K/Akt pathway is inhibited by the PI3K inhibitor LY294002, the glycolytic effect produced by GLP-1 stimulation is reduced
.
LY294002 reduced the expression of PDK2 and HIF1α (Figures 4E and 4I), and transcription of Ldha, Pkm, and hexokinase II was inhibited (Figure 4J); Intracellular lactate and NAD+ levels were also reduced with inhibition of the PI3K/Akt pathway (Figure 4F-H
).

However, the role of OXPHOS has the opposite result, and hippocampal experiments have detected OXPHOS levels in different groups, all assessed
by measuring oxygen consumption rate (OCR) in real time.
Oligomycin is an ATP synthase inhibitor; Cyanide p-(trifluoromethoxyphenylhydrazone) (FCCP) is an H ion carrier; Rotenone/antimycin A is an electron transport chain inhibitor, the addition of which can assess basic or maximum OCR
.

As shown in Figure 4K, the maximum OCR in the Aβ group is elevated and increases over time, and the production of mitochondrial ATP increases, suggesting that the OXOS level and energy supply are low when the mitochondrial activation glycolytic inflow is insufficient
.
GLP-1 reduces OXPHOS and reduces mitochondrial stress, but when the PI3K/Akt pathway is inhibited, OXPHOS levels rebound, and these results reveal the role of
the PI3K/Akt pathway in GLP-1 modulating glycolysis and OXPHOS.

Astrocytes absorb glutamate in the synaptic cleft, where the synaptic cleatamine synthase (GS) is converted into glutamine, which is then delivered to neurons, or glutamate can be metabolized into a-ketoglutaric acid and inter-TCA
.
The study found that Aβ treatment led to a decrease in glutamate uptake in astrocytes, while decreased expression of EAAT2, a type of transporter of glutamate; However, GLP-1 did not improve the glutamate uptake capacity of astrocytes, although it upregulated GS expression (Figure 5
).

Fig.
4 GLP-1 improves astrocyte aerobic glycolysis and reduces oxidative stress through the PI3K/Akt pathway

5 mM Aβ1-42 treated with astrocytes for 24 h or incubated with 100 nM GLP-1 for 2 h
after treatment with Aβ1-42 treatment.
After 1 h incubation of GLP-1 and Aβ1-42, pretreat astrocytes with LY294002 at 10 mM in the Aβ+GLP-1+LY group
.
(A-D) evaluation of glycolysis levels in three groups, including Western blotting detection of relative expression of glycolytic enzymes (A); Lactic glycolysis (B) and NAD+ (C-D
).
(E) Western blotting detects the relative expression
of PI3K, Akt, PDK2 and HIF-1α in the cortex.
(F-H) to evaluate glycolysis levels, including lactate glycolysis (F) and NAD+ (G-H
).
(I) Immunofluorescence images
of HIF-1α and GFAP in three groups.
(J) Transcriptional levels of glycolyzymes, including Hk1, Hk2, Pkm, Pfkmb3 and Ldha
.
(F) Assess oxidative phosphorylation levels
.
The addition
of oligomycin, FCCP, and rotenone/antimycin A to hippocampal assays to calculate consumption rate (OCR), maximum OCR, and mitochondrial ATP yield.

Figure 5 GLP-1 did not enhance glutamate uptake in in vitro Aβ-treated astrocytes

Astrocytes are treated with 5 mM Aβ1-42 for 24 h or incubated with 100 nM GLP-1 for 24 h
.
(A-D) Western blotting evaluates expression levels
of EAAT1, EAAT2 and GS.
(E) Glutamate is absorbed by astrocytes uptake of different groups
.

5.
GLP-1 improves the supporting effect of astrocytes on neurons by improving the aerobic glycolysis of astrocytes

Astrocytes support the survival and function of neurons by providing lactate as an energy substrate for neurons, maintaining redox balance
.
We added 2-deoxyglucose (2-DG) to evaluate its effects on
neurons.
When the role of GLP-1 in astrocyte glycolysis is inhibited, such as lactic acid and NAD+ levels decrease (Figures 6A-C
).
2-DG is rapidly transmembrane transport phosphorylation to form 2-deoxyglucose-6-phosphate that cannot be phosphorylated, which is then further metabolized by glycolytic enzymes, thereby hindering the glycolysis process
.
Activation of GLP-1 to alleviate OXPHOS hinders the presence of 2-DG, manifested by decreased PDH phosphorylation (Figure 6D) and elevated OCR values (Figure 6J-M
).
Consistent with this, the production of total mitochondrial ROS is elevated (Figure 6F-H) and glutathione levels are decreased (Figure 6I), indicating disruption of the redox equilibrium state of astrocytes
.

To assess the effect of GLP-1 on astrocyte neuron support, we conducted the astrocyte-neuronal co-culture Transwell experiment
.
Aβ-treated astrocytes reduce cell survival, while GLP-1 increases the ability of astrocytes to support neurons under the same experimental conditions (Figure 7A-B
).
GLP-1 treatment promotes axonal length and dendritic growth of neurons, while 2-DG eliminates inhibition of glycolysis produced by GLP-1 (Figure 7C-H
).
The expression of PSD95 and SYN is increased by expression after GLP-1 treatment and down-regulated by expression after 2-DG treatment (Figure 7I-K
).
These results underscore the importance of astrocyte glycolysis in neuronal growth and the increased aerobic glycolysis of GLP-1 by enhancing the supporting capacity of astrocytes
.

Fig.
6 Increased OXPHOS levels and oxidative stress in astrocytes after glycolysis inhibition

Treat cells with 5 mM Aβ1-42 for 24 h or with 100 nM GLP-1 for 2 h before treatment with Aβ1-42
.
In the Aβ+GLP-1+2-DG group, treat astrocytes with 10 mM 2-DG before incubating GLP-1 and Aβ1-42
.
(A-C) lactic acid glycolysis (A) and NAD+ (B and C).

(D) Western blotting detects the relative expression
of PDK2 and PDH.
(E) ASTR cells in different groups of ATP yield
.
(F-H) total ROS and mitochondrial ROS fluorescence images
of each group.
Mitochondria are labeled with MitoTracker Green; Total ROS and mitochondrial ROS are labeled
with DCFH-DA and MitoSox, respectively.
DCFH-DA scale: 100mm; For MitoTracker Green and MitoSox, ruler: 10mm
.
(J-M) Basal Oxygen Consumption (OCR), Maximum OCR, and Mitochondrial ATP Generation
for each group.

Figure 7 GLP-1 improves the ability of astrocytes to support neurons by increasing the aerobic glycolysis of astrocytes

Neurons are co-cultured with astrocytes and treated
with Aβ1-42, GLP-1, or 2-DG.
(A-B) Stain neurons with Hoechst3342 and PI to assess cell survival
.
Ruler: 75 μm
.
(C-H) fluorescent images of neurons in the cerebral cortex (C
).
Sholl neurotic intersection analysis (D); Neurite length (E), secondary branches (F), somatic cell protrusions (G), and axon length (H) were evaluated
by ImageJ software and The Seek analysis plug-in.
Analyze no less than 100 cells
per group.
Scale bars: 25 μm
.
(I-K) Western blotting detects the relative expression
of cortical neurons PSD95 with SYN.

discuss

In this study, we found that GLP-1 mediated alterations
in glucose metabolism in animal models of AD and astrocytes.
Enhanced glycolytic flow is accompanied by an increase in the production of glycolytic products (lactic acid and NAD+) and a decrease in mitochondrial ROS levels
in cases where OXPHOS is inhibited.
The effect of GLP-1 on metabolic phenotypic regulation is closely related to the activation of PI3K/Akt, and the transition from OXPHOS to aerobic glycolysis increases the ability of astrocytes to support neurons, manifested by increased cell viability and vigorous dendritic and axonal growth
.

This study confirms for the first time the role of GLP-1 in altering cellular glucose metabolism in astrocytes, revealing a new mechanism
for improving cognitive function in patients with AD.
In our study, GLP-1 mediated animal and cellular models of transitions from OXPHOS to glycolysis, an effect that may be one
of the mechanisms of action of GLP-1 neuroprotective.
GLP-1 has been found to regulate cellular energy expenditure through a variety of bioenergy pathways and to alter glycolysis
under the conditions of energy metabolism phenotype ischemia and hypoxia.
This mechanism helps cells cope with energy crises in aerobic situations, such as when oxygen supply is insufficient, such as myocardial ischemic injury, improving the cell's ability to adapt to adverse conditions and enhancing cell survival
.

Geriatric neurodegenerative diseases (NDAs), particularly AD and Parkinson's disease, deficient glucose uptake and disorders of ATP production are drivers of disease progression
.
Lactic acid is produced by astrocytes, and glycolysis is an important alternative pathway when neurons face energy barriers, with glycolysis enhanced
after NDAs treatment 。 In the early stages of AD the authors found that HIF-1α expression was upregulated as well as an increase in the expression enzymes of several glycolysis, which is one of the mechanisms of toxicity in astrocytes and Aβ; However, the accumulation of Aβ and tau inhibits glycolysis, and this compensatory effect disappears in the late AD period, a situation that was also confirmed in this study, the accumulation of Aβ and tau leads to a state of insufficient energy and exacerbates oxidative stress, GLP-1 increases lactic acid production and alleviates the energy crisis
by improving the glycolysis process of astrocytes.

The effect of GLP-1 in enhancing aerobic glycolysis in AD is closely related to the PI3K/Akt pathway, which is deeply involved in energy metabolism and cell viability
.
GLP-1 has been reported to inhibit the PI3K/Akt pathway in β cells and nerve cells, which inhibits the PDK inhibitor FOXO and reduces OXPHOS levels
.
Consistent with previous studies, we found that GLP-1 increases Akt phosphorylation levels, leading to a higher degree of PDH phosphorylation, which downregulates OXPHOS and reduces oxidative stress; In addition, phosphorylation of Akt leads to activation of the mTOR-HIF-1α pathway, inducing increased expression of glycolytic enzymes and increased
glycolysis.
In a recent study, Sung et al.
found that the mTOR-HIF-1α pathway-mediated metabolic reprogramming is necessary for microglia activation, highlighting the function of
aerobic glycolysis in glials.
In this study, we reported on the GLP-1-mediated metabolic regulation mechanisms
in which the PI3K/Akt pathway is involved.

Enhancement of aerobic glycolysis enhances the ability
of astrocytes to support neurons by promoting synaptic growth.
It has been hypothesized that aerobic glycolysis protects energy-deficient neurons by providing lactate, and there is a wealth of evidence that lactate has beneficial effects on
neuronal cells.
Studies have shown that lactic acid supplementation can reduce brain damage in hypoxic-ischemic encephalopathy, increase hippocampal lactate concentration, improve neurogenesis, and alleviate depressive syndrome
.
It has also been reported that exercise can improve cognitive abilities by producing lactic acid, which crosses the blood-brain barrier and promotes the production
of BDNF.

Although some reports point out that the increased glycolytic capacity of neurons caused by the production of lactic acid through electron activation and glycolysis processes is not necessary for neuronal activity
.
In fact, chronic glycolysis enhancement can be harmful to neurons, as Herrero-Mendez's study shows, for neurons glycolysis leads to oxidative stress and apoptosis by upregulating the expression of Pfkfb3
.
The authors believe that in AD, when glucose metabolism is blocked and mitochondrial function is impaired, the shuttle of lactate during glial glycolysis becomes increasingly important for the survival of neurons, which plays an energy-replacement role
.
GLP-1 partially restores glycolytic function in astrocytes and increases lactic acid flux, which helps alleviate the energy crisis of neurons
.

In our study, lactate levels in the brains of AD mice did not differ significantly in the WT and AD+Lira groups, while Aβ treatment of astrocytes resulted in a significant reduction
in lactate levels.
This can be caused
by astrocyte-neuron interactions.
Lactic acid and NAD+ can be absorbed by neurons and enter the TCA cycle, so that the level of these substances in the brain is affected by the level of metabolism, richard et al.
also reported that although brain glycolysis varies in different courses of AD, the lactate content remains largely unchanged
.

Improving the balance of cellular redox states is the neuroprotective mechanism
of GLP-1.
The increase in glycolysis increases the antioxidant capacity of astrocytes and reduces the production
of reactive oxygen species.
Astrocytes are considered the main line of defense against oxidative damage, and glutathione is a key component of the astrocyte detoxification process
.
Neurons are essentially dependent on OXPHOS but have low antioxidant capacity, and astrocytes producing glutathione is an important protective mechanism
for neurons against oxidative stress.

conclusion

In summary, this study reveals that the neuroprotective mechanism of GLP-1 is closely related to its promotion of aerobic glycolysis and remission of OXPHOS activation and is closely related
to the activation of the PI3K/Akt pathway.
This study shows that energy regulation can be effective in slowing the progression of AD and also elucidates the possibility
of treating AD through energy regulation or in combination with other anti-Aβ drugs.