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. 2023 Feb 1;102(5):546–554. doi: 10.1177/00220345221148983

AMPK Activation Restores Salivary Function Following Radiation Treatment

RK Meyer 1, KE Gilman 1, BA Rheinheimer 1, L Meeks 1, KH Limesand 1,✉
PMCID: PMC10249004  PMID: 36726289

Abstract

Head and neck cancers represent a significant portion of cancer diagnoses, with head and neck cancer incidence increasing in some parts of the world. Typical treatment of early-stage head and neck cancers includes either surgery or radiotherapy; however, advanced cases often require surgery followed by radiation and chemotherapy. Salivary gland damage following radiotherapy leads to severe and chronic hypofunction with decreased salivary output, xerostomia, impaired ability to chew and swallow, increased risk of developing oral mucositis, and malnutrition. There is currently no standard of care for radiation-induced salivary gland dysfunction, and treatment is often limited to palliative treatment that provides only temporary relief. Adenosine monophosphate (AMP)–activated protein kinase (AMPK) is an enzyme that activates catabolic processes and has been shown to influence the cell cycle, proliferation, and autophagy. In the present study, we found that radiation (IR) treatment decreases tissue levels of phosphorylated AMPK following radiation and decreases intracellular NAD+ and AMP while increasing intracellular adenosine triphosphate. Furthermore, expression of sirtuin 1 (SIRT1) and nicotinamide phosphoribosyl transferase (NAMPT) was lower 5 d following IR. Treatment with AMPK activators, 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) and metformin, attenuated compensatory proliferation (days 6, 7, and 30) following IR and reversed chronic (day 30) salivary gland dysfunction post-IR. In addition, treatment with metformin or AICAR increased markers of apical/basolateral polarity (phosphorylated aPKCζT560-positive area) and differentiation (amylase-positive area) within irradiated parotid glands to levels similar to untreated controls. Taken together, these data suggest that AMPK may be a novel therapeutic target for treatment of radiation-induced salivary damage.

Keywords: salivary glands, metformin, xerostomia, radiotherapy, saliva, salivary dysfunction

Introduction

Approximately 55,000 new cases of head and neck cancer will be diagnosed next year in the United States (Siegel et al. 2022). Despite increasing incidence of head and neck cancers, treatment options remain limited. Standard treatment often includes surgery and radiotherapy (Cramer et al. 2019). Although treatment options and survival rates vary depending on location and severity of malignancy, thousands of cancer survivors retain impaired quality of life following treatment due to secondary damage to surrounding tissues, including salivary glands. Radiotherapy-induced salivary gland damage chronically affects function, typically resulting in decreased salivary output, difficulty eating, oral mucositis, and malnutrition (Grundmann et al. 2009). Treatment options are commonly reserved to palliative care, with few options available to directly treat or prevent damage to the gland (Jensen et al. 2019).

Compensatory proliferation in the wound-healing response is a process conserved across tissue types and has been implicated in the cellular response to radiation. While proliferation following tissue damage is typically considered beneficial to replace apoptotic and/or necrotic cells, the proliferative response in radiation-treated salivary glands occurs concomitantly with significant reductions in salivary output (Grundmann et al. 2010; Morgan-Bathke, Harris, et al. 2014). In addition to compensatory proliferation, cytoskeletal disruptions and disorganization of the Par3-aPKC polarity complex have been associated with deficient wound healing and correlate with a significant decrease in saliva production (Abreu-Blanco et al. 2012; Niessen et al. 2013; Chibly et al. 2018; Wong et al. 2018). Previous work indicated that administration of insulin-like growth factor 1 (IGF-1) to mice following exposure to 5-Gy radiation restores glandular function and salivary output (Grundmann et al. 2010; Chibly et al. 2018; Limesand et al. 2009). Functional restoration of the gland is associated with attenuation of proliferative responses in IGF-1–treated irradiated mice (Grundmann et al. 2010), possibly indicating that the newly dividing cells have limited functional capability and are therefore not contributing to saliva production. Taken together, these data suggest that reducing proliferative cells in the parotid salivary gland may play a role in improving functionality following radiation therapy.

One of the most frequently studied metabolic regulators, adenosine monophosphate (AMP)–activated protein kinase (AMPK), modulates flux through metabolic pathways in response to low cellular energy states (Hardie et al. 1998; Ke et al. 2018). AMPK is activated in a 2-step process involving increased binding of AMP and phosphorylation on threonine172 by LKB1 or CAMKK2 (Herzig and Shaw 2018). Active AMPK increases production of NAD+ via inducing expression of nicotinamide phosphoribosyltransferase (NAMPT), a key enzyme in NAD biosynthesis (Garten et al. 2015); the AMPK-mediated increase in NAD+ enhances the activity of the NAD-dependent histone deacetylase sirtuin 1 (SIRT1) (Cantó et al. 2009) that plays a regulatory role in autophagy induction (Lee 2019). Previous work has implicated autophagy in amelioration of radiation-induced salivary gland dysfunction (Morgan-Bathke, Hill, et al. 2014). Autophagy-deficient mice have a more severe response to targeted head and neck radiation, with decreased acute and chronic salivary flow rates following 5-Gy radiation. While autophagy is not acutely activated following radiation (IR), IGF-1 pretreatment induces autophagy and improves salivary function. Autophagy induction likely acts, in part, via inhibition of mammalian target of rapamycin (mTOR) signaling, which is increased 4 to 5 d following IR. Indeed, irradiated mice treated with the rapalogue CCI-779 have improved salivary function 30 d following radiation; parotid salivary glands from these mice also show fewer proliferative cells compared to irradiated controls (Morgan-Bathke, Harris, et al. 2014). AMPK activation is also known to activate autophagy, in part, due to inhibition of mTOR1 signaling (Imamura et al. 2001; Jones et al. 2005). While AMPK activation has not been studied in the salivary gland following radiation exposure, treatment with metformin, a drug known to activate AMPK, improves salivary flow rate in a mouse model of Sjögren’s syndrome (Kim et al. 2019), indicating a potential therapeutic use for metformin in the salivary gland response to radiation. Taken together, the role of AMPK in the inhibition of proliferation and activation of autophagy implicates this enzyme as a prime molecular target to improve radiation-induced salivary gland dysfunction.

Based on preliminary evidence suggesting a role for AMPK in salivary secretion, the present study aimed to 1) assess changes in NAD+ and AMP levels in parotid salivary glands following IR, 2) characterize the levels of phosphorylated AMPK and enzymes in downstream pathways following IR, 3) determine the effect of pharmacological activation of AMPK with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or metformin on radiation-induced compensatory proliferation in parotid cells, and 4) evaluate the effect of AICAR or metformin treatment on chronic salivary output following radiation exposure. We hypothesize that treatment with AICAR or metformin will attenuate the well-characterized proliferative response following IR, and pharmacological AMPK activation with AICAR or metformin will improve salivary output when administered following radiation.

Materials and Methods

Mice

FVB mice were purchased from Jackson Laboratories and maintained in accordance with protocols approved by the University of Arizona Institutional Animal Care and Use Committee. This study conforms to the ARRIVE (Animal Research: Reporting of In Vivo Experiments) guidelines. Administration of metformin (Sigma-Aldrich) or AICAR (Toronto Research Chemicals) was performed via oral gavage (metformin, 100 mg/kg body weight) or intraperitoneal injection (AICAR, 500 mg/kg body weight).

Radiation Treatment

The head and neck region of mice received single (5-Gy) or fractionated (2 Gy/d × 5 d) dose of radiation using a 60Cobalt Teletherapy instrument (Atomic Energy of Canada Limited Theratron-80) as previously described (Limesand et al. 2010; Limesand et al. 2009).

NAD+ and AMP Metabolite Analysis

Mice were unirradiated or received a 5-Gy dose of radiation. Parotid glands were removed at day 5 following IR, snap frozen, and sent to Metabolon for metabolomics analysis by liquid chromatography–mass spectrometry (LC-MS) as previously described (Meeks et al. 2021).

Adenosine Triphosphate Cell Assay

Adenosine triphosphate (ATP) concentration was determined by ATP bioluminescence assay (Roche) following the manufacturer’s instructions. See Appendix for more information.

AMP and ATP Tissue Assay

Parotid glands were removed; 1 gland was homogenized in 400 µL AMP assay buffer while the second gland was homogenized in 100 μL ATP assay buffer (Abcam). AMP and ATP concentrations were determined following the manufacturers’ instructions.

Western Blot

Parotid glands were removed, protein was extracted, and sodium dodecyl–sulfate polyacrylamide gel electrophoresis was conducted as previously described (Gilman et al. 2021). Refer to the Appendix for antibody information.

RNA Isolation and Quantitative Reverse Transcription Polymerase Chain Reaction

Parotid glands were removed from mice at days 3, 5, and 30 after IR. RNA was isolated as previously described (Gilman et al. 2019). Refer to the Appendix for primer information.

Blood Glucose

Following a 4-h fast, the distal 1 mm of tail was cut, and glucose was measured from 10 to 20 µL whole blood by the CVS Health Advanced Blood Glucose Meter (CVS Health).

Histology and Immunofluorescent Staining

Salivary glands were removed, fixed in 10% (v/v) formalin for 24 h, and sent to IDEXX Bioresearch for embedding. Immunofluorescent staining and quantification were performed as described in the Appendix.

Saliva Collection

Stimulated whole saliva collection was performed on days 3 and 30 following IR as previously described (Gilman et al. 2021).

Statistical Analysis

Statistical analysis was performed using GraphPad Prism 6 software (GraphPad Software). Manual cell counts from immunofluorescent stained slides were analyzed by analysis of variance (ANOVA) with Tukey’s multiple comparisons test. Percent positive staining area was analyzed by ANOVA followed by Bonferroni’s post hoc comparisons. Quantitative reverse transcription polymerase chain reaction (qRT-PCR) data were normalized to loading controls (15S) and analyzed with a t test comparing irradiated values to untreated. Densitometry data from Western blot were analyzed using a t test. Saliva collection data were normalized to untreated values for each collection day and compared with a 1-way ANOVA with a Newman–Keuls multiple comparisons test. Treatment groups denoted with different letters above the graph are significantly different from each other while data from groups without statistical differences are indicated with the same letter.

Results

Radiation Treatment Leads to Suppression of the AMPK Pathway

AMPK is primarily activated in response to changes in intracellular metabolism, specifically an increase in AMP/ATP ratio and decreased NAD+ availability. Radiation exposure has been shown to affect metabolism within a whole organism or specific tissue (Manna et al. 2013; Golla et al. 2017). To determine the effect of radiation on metabolites involved in AMPK activation, untargeted metabolomics was performed on parotid glands from irradiated and untreated (UT) mice when compensatory proliferation begins (day 5 following IR) (Meeks et al. 2021). We observed a ~50% decrease in NAD+ levels and a ~30% decrease in AMP levels in irradiated parotid glands compared to untreated controls (Fig. 1A, B). Further, radiation significantly increased ATP levels in parotid glands (Fig. 1C). To determine the effect of radiation on AMPK activation, we measured phosphorylated (activated) AMPKT172 at days 3 and 5 following IR. Phosphorylated-AMPK protein levels decreased 3 and 5 d following IR in parotid glands compared to untreated (Fig. 1D, E). AMPK activity has also been shown to regulate NAMPT expression and SIRT1 activity (Cantó et al. 2009; Brandauer et al. 2013). SIRT1 and NAMPT expression was significantly lower in mouse parotid salivary glands at day 5 following radiation (Fig. 1F). These data suggest radiation affects cellular energy homeostasis at early time points, leading to a reduction in AMPK signaling.

Figure 1.

Figure 1.

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Radiation treatment leads to suppression of the adenosine monophosphate (AMP)–activated protein kinase (AMPK) pathway. FVB mice were untreated (UT) or exposed to 5-Gy radiation (IR) and sacrificed at days 3 or 5 following radiation. (A) NAD+ and (B) AMP levels were determined via liquid chromatography–mass spectrometry in untreated (n = 4) or irradiated (n = 4) parotid salivary glands. (C) Primary cultures of parotid gland cells were prepared and left untreated (n = 6) or received 5-Gy IR (n = 5). Cell lysates were collected at day 3 post-IR, and intracellular ATP levels were determined with a luciferin–luciferase assay. (D) Protein was extracted from untreated and day 3 or 5 irradiated parotid glands, and levels of phosphorylated AMPK (p-AMPK), total AMPK (t-AMPK), and ERK 1/2 (loading control) were determined by immunoblot. (E) Densitometry data from untreated (n = 4), day 3 (n = 3), or day 5 (n = 3) irradiated parotid glands. p-AMPK levels were normalized to t-AMPK levels and are presented as mean ± SEM. Statistical differences were determined with a 1-way analysis of variance followed by Tukey’s post hoc test. Groups with different letter designations are significantly different from each other, P < 0.05. (F) Complementary DNA was prepared from untreated (n = 4) or 5-Gy irradiated parotid glands at day 5 following radiation (n = 4) and quantitative reverse transcription polymerase chain reaction was performed with primers specific to sirtuin 1 (SIRT1) and nicotinamide phosphoribosyltransferase (NAMPT). Data were normalized to 15S ribosomal RNA as an internal control and calculated as fold change relative to the average of untreated mice. (A–C, F) Data are presented as mean ± SEM, and statistical differences between groups were determined with an unpaired t test, *P < 0.05, **P < 0.01, ***P < 0.001.

Treatment with Metformin or AICAR Following Radiation Activates AMPK in Parotid Glands

To confirm the AMPK-activating effect of AICAR and metformin in our model of radiation, we treated irradiated mice with AICAR or metformin at days 4, 5, and 6 following radiation. We measured intracellular AMP and ATP levels at days 7 and 30 following radiation and found no significant differences in AMP levels between groups (Fig. 2A, B; Appendix Fig. 1A, B). Mice treated with AICAR or metformin had lower fasting blood glucose at day 7 following IR compared to UT (Fig. 2C), with no differences between groups at day 30 (Appendix Fig. 1C). To confirm that AICAR and metformin treatment activates AMPK in parotid glands, we measured phosphorylated AMPK at days 7 and 30 following IR. Radiation decreased phospho-AMPK in parotid glands at day 7 compared to UT, while AICAR or metformin treatment increased phospho-AMPK levels compared to IR (Fig. 2D, E). At day 30, phospho-AMPK levels were similar between UT and IR; however, metformin or AICAR treatment significantly increased phospho-AMPK levels compared to IR (Appendix Fig. 1D, E). There were no differences between groups in the expression of SIRT1 and NAMPT at day 30 following radiation (Appendix Fig. 1F, G). These data indicate that administration of AICAR or metformin following radiation induces AMPK activation independent of changes to intracellular AMP and ATP levels.

Figure 2.

Figure 2.

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Treatment with metformin or 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) following radiation activates adenosine monophosphate (AMP)–activated protein kinase (AMPK) in parotid glands. FVB mice were untreated (UT, n = 4) or exposed to 5-Gy radiation (n = 4) with additional mice receiving AICAR (n = 3) or metformin (n = 4) injections on days 4, 5, and 6 after radiation. Parotid salivary glands were collected at day 7 following radiation. Levels of (A) AMP and (B) adenosine triphosphate (ATP) were evaluated from tissue lysates via colorimetric assays as described in the Materials and Methods. (C) Blood was collected from mice in indicated treatment groups on day 7 after radiation (IR), and glucose levels were measured via handheld glucometer following a 4-h fast. (D–E) Levels of phosphorylated AMPK (p-AMPK), total AMPK (t-AMPK), and ERK 1/2 (loading control) were evaluated via immunoblot. p-AMPK levels were normalized to t-AMPK levels. (A–E) Data are presented as mean ± SEM. Statistical differences were determined with a 1-way analysis of variance followed by Tukey’s post hoc test. Groups with different letter designations are significantly different from each other, P < 0.05.

Treatment with AICAR or Metformin Decreases Compensatory Proliferation Following Radiation

Previous research restoring salivary gland function demonstrated a decrease in compensatory proliferation following IR that occurs concomitantly with improved salivary output (Grundmann et al. 2010; Morgan-Bathke, Hill, et al. 2014). Further, AMPK activation inhibits proliferation by decreasing flux through anabolic pathways that require excessive ATP, inhibition of mTOR signaling, and activation of p53 (Jones et al. 2005; Agarwal et al. 2015; Hao et al. 2018). To determine if AMPK activation has a similar effect on proliferation in the parotid salivary gland, we treated mice with 1 dose of AICAR or metformin 5 d following IR and collected glands 24 h later (day 6 after IR) for Ki67 staining. Metformin and AICAR treatment attenuated the proliferative response to radiation, exhibiting a 65% and 63% decrease in Ki67+ cells compared to IR alone, respectively (Fig. 3A–E). Given that previous models of functional restoration have administered multiple doses of therapeutic compound to reverse salivary gland dysfunction, mice were treated with 3 doses of AICAR or metformin at days 4, 5, and 6 following radiation, and glands were collected 24 h after the last dose (day 7 after IR; Fig. 3F–J). Treatment with metformin or AICAR over 3 d decreased the proliferative response to radiation at day 7 following IR by 43% and 40%, respectively (Fig. 3F–J). These data indicate that AMPK activation following IR attenuates the proliferative response to radiation in parotid salivary glands.

Figure 3.

Figure 3.

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5-Aminoimidazole-4-carboxamide ribonucleotide (AICAR) or metformin treatment decreases compensatory proliferation of parotid glands following radiation. FVB mice were untreated or exposed to 5-Gy radiation, with a subset of mice receiving AICAR (500 mg/kg) or metformin (100 mg/kg) on day 5 (A–E) or days 4, 5, and 6 (F–J). Parotid salivary glands were collected at indicated time points, and immunohistochemistry for Ki67 was performed. (A–D) Representative images of Ki67-positive cells (green) over total nuclei stained with DAPI (blue) in (A) untreated (n = 4), (B) day 6 following 5-Gy radiation (n = 4), (C) day 6 following 5-Gy radiation and metformin on day 5 (100 mg/kg, 1 dose), or (D) day 6 following 5-Gy radiation and AICAR on day 5 (500 mg/kg, 1 dose) (n = 5). (E) Quantification of Ki67+ nuclei as a percentage of the total number of nuclei from 5 fields of view per mouse. (F–I) Representative images of Ki67-positive cells (green) over total nuclei stained with DAPI (blue) in (F) untreated (n = 4); (G) day 7 following 5-Gy radiation (n = 4); (H) day 7 following 5-Gy radiation and metformin on days 4, 5, and 6 (100 mg/kg, 3 doses) (n = 4); or (I) day 7 following 5-Gy radiation and AICAR on days 4, 5, and 6 (500 mg/kg, 3 doses) (n = 4). (J) Data are presented as mean ± SEM, and statistical differences were determined with a 1-way analysis of variance followed by Tukey’s post hoc test, P < 0.05.

AICAR or Metformin Treatment Increases Salivary Output, Increases Amylase and Phosphorylated aPKCζ Area, and Decreases Proliferation 30 d Posttreatment

Reduced proliferation in irradiated salivary glands correlates with improved salivary output in previous models of restoration (Grundmann et al. 2010; Morgan-Bathke, Hill, et al. 2014; Chibly et al. 2018). To determine the restorative potential of pharmacological AMPK activation, we irradiated mice and treated with AICAR or metformin (Fig. 4A). Radiation treatment decreases salivary output by 29% or 26% at days 3 or 30, respectively, following IR compared to UT (Fig. 4B, C). Treatment with metformin or AICAR improves salivary output compared to IR, with no differences from UT control salivary output (Fig. 4C). To determine the effect of metformin or AICAR treatment on salivary output with fractionated doses of radiation, we treated mice with 2Gy/d for 5 d and administered metformin or AICAR at days 4, 5, and 6 following the final radiation dose (Fig. 4D). Fractionated radiation decreased salivary output at day 30 following radiation by 33% compared to UT, while metformin or AICAR treatment improved salivary output at day 30, with no significant differences from UT (Fig. 4E).

Figure 4.

Figure 4.

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Adenosine monophosphate (AMP)–activated protein kinase (AMPK) activation with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or metformin increases salivary output following radiation. (A, D) Timeline of radiation and AICAR or metformin administration for salivary output experiments following (A) single-day radiation (5-Gy × 1 d) exposure or (D) fractionated radiation (2 Gy × 5 d) exposure. (A, B) FVB mice were randomly assigned to receive no treatment (UT; n = 22) or 5-Gy radiation (IR; n = 30). Carbachol-stimulated saliva (0.25 mg/kg) was collected 3 d following radiation. (C) Irradiated mice were then randomized to receive 3 doses of either AICAR (500 mg/kg; n = 10) or metformin (100 mg/kg; n = 8) on days 4, 5, and 6 following radiation or no further treatment (n = 12), and saliva was collected 30 d following radiation. (D, E) FVB mice were randomly assigned to receive no treatment (UT; n = 10) or 2 Gy fractionated radiation (IR; n = 30). Irradiated mice were then randomized to receive 3 doses of either AICAR (500 mg/kg; n = 10) or metformin (100 mg/kg; n = 10) on days 4, 5, and 6 following the last dose of radiation or no further treatment (n = 10), and saliva was collected 30 d following the last dose of radiation. Data are combined from multiple independent experiments and presented as mean ± SEM. Statistical differences were determined with a 1-way analysis of variance with Newman–Keuls multiple comparisons test,P < 0.05.

To evaluate the role of AMPK activation in the redifferentiation response of salivary glands to radiation, we evaluated the proportion of amylase-producing cells 30 d following radiation treatment. Radiation treatment decreases amylase-positive area by ~33% compared to UT (Fig. 5A–E). Treatment with AICAR or metformin at days 4, 5, and 6 following radiation restored amylase area to levels observed in unirradiated mice (Fig. 5A–E). In addition, radiation treatment decreased a marker of apical/basolateral polarity (phosphorylated aPKCζT560-positive area) by 41% compared to UT (Fig. 5F–J). Treatment with AICAR or metformin increased phosphorylated aPKCζT560 area, albeit levels in metformin mice were lower than unirradiated mice (Fig. 5F–J). To further assess the effect of pharmacological AMPK activation on parotid gland proliferation, we counted Ki67+ cells 30 d post-IR with and without metformin or ACIAR treatment. Radiation increased Ki67+ parotid cells by 65% at day 30 following IR compared to UT (Fig. 5K–O), and treatment with AICAR or metformin reduced Ki67+ parotid cells to UT levels (Fig. 5K–O). Taken together, these data implicate a role for AMPK activation in restoring salivary gland function following radiation.

Figure 5.

Figure 5.

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Adenosine monophosphate (AMP)–activated protein kinase activation with 5-aminoimidazole-4-carboxamide ribonucleotide (AICAR) or metformin increases amylase and phosphorylated aPKCζ area and decreases proliferation following radiation. FVB mice were untreated or exposed to 5-Gy radiation with a subset of mice receiving metformin (100 mg/kg) or AICAR (500 mg/kg) on days 4, 5, and 6 following radiation. Parotid salivary glands were collected 30 d following radiation, and immunofluorescence for amylase, phosphorylated aPKCζT560, or Ki67 was performed. (A–D) Representative images of amylase-positive acinar cells (red) in (A) untreated (n = 4), (B) 5-Gy radiation (n = 4), (C) 5-Gy radiation plus metformin (n = 4), and (D) 5-Gy radiation plus AICAR (n = 4). (E) The percentage of amylase-positive area was determined using ImageJ software from at least 20 fields of view per mouse. The graph represents the percentage of positive amylase area as a percentage of the total area. (F–I) Representative images of phosphorylated aPKCζ-positive acinar cells (red) in (F) untreated (n = 3), (G) 5-Gy radiation (n = 3), (H) 5-Gy radiation plus metformin (n = 3), and (I) 5-Gy radiation plus AICAR (n = 3). (J) The percentage of positive phosphorylated aPKCζ was determined using ImageJ software from at least 20 fields of view per mouse. The graph represents the percentage of positive phosphorylated aPKCζ as a percentage of the total area. Graphs represent the mean ± SEM. Significant differences were determined via a 1-way analysis of variance (ANOVA) followed by Bonferroni’s post hoc comparisons. (K–N) Representative images of Ki67-positive acinar cells (green) over total nuclei stained with DAPI (blue) in (K) untreated (n = 5), (L) 5-Gy radiation (n = 5), (M) 5-Gy radiation plus metformin (n = 5), and (N) 5-Gy radiation plus AICAR (n = 5). (O) Quantification of Ki67+ nuclei as a percentage of the total number of nuclei from 5 fields of view per mouse. Data are presented as mean ± SEM, and statistical differences were determined with a 1-way ANOVA followed by Tukey’s post hoc test, P < 0.05.

Discussion

Chronic hyposalivation remains an incurable side effect of radiation therapy; therefore, research into novel therapeutic targets is necessary. In the present study, we found that AMPK activation was decreased at days 3, 5, and 7 following IR, under conditions of altered cellular energy homeostasis evidenced by decreased intracellular NAD+ levels and the AMP/ATP ratio (Fig. 1). Postradiation treatment with pharmacological AMPK activators, AICAR or metformin, improved salivary output 30 d following radiation (Fig. 4C). This work provides a potential new model using AMPK activation for functional salivary gland restoration that may prove safer and more cost-effective than some proposed therapies, including gene therapy and surgical strategies (Sasportas et al. 2013; Sood et al. 2014). Although research using metformin in a salivary gland damage model has not been described, metformin has been widely studied in other models of epithelial wound healing. Han et al. (2017) found significantly improved cutaneous wound healing in diabetic mice treated with metformin. Further, in an aged-mouse model of cutaneous would healing, metformin, as well as AMPK activator resveratrol, improved the speed of cutaneous wound healing (Zhao et al. 2017). Activation of AMPK by metformin and resveratrol likely has multiple consequences, including mTOR inhibition, autophagy induction, and subsequent inhibition of protein synthesis and proliferation. Persistent compensatory proliferation in irradiated salivary glands is associated with negative functional outcomes (Grundmann et al. 2010; Morgan-Bathke, Hill, et al. 2014; Chibly et al. 2018); inhibition of proliferation and associated anabolic processes may improve cell homeostasis and restore acinar cell function in irradiated salivary glands.

AMPK activation leads to inhibition of mTOR activity through phosphorylation of TSC2 and Raptor and activation of ULK1, which collectively stimulates autophagy. Decreases in AMPK activation (Fig. 1) align with previous work demonstrating increased mTOR activity and lack of autophagy induction following IR (Morgan-Bathke, Hill, et al. 2014). Autophagy-deficient mice have exacerbated radiation-induced salivary gland dysfunction, and treatment with a rapalogue (CCI-779) that inhibits mTOR activity restores salivary gland function (Morgan-Bathke, Harris, et al. 2014; Morgan-Bathke, Hill, et al. 2014). Correspondingly, activation of AMPK and inhibition of mTOR lead to reductions in compensatory proliferation and restoration of salivary gland function (Figs. 3, 4; Morgan-Bathke, Hill, et al. 2014). In addition, AMPK activation has been shown to regulate mitophagy and the removal of defective mitochondria (Herzig and Shaw 2018). We have recently demonstrated that radiation leads to a reduction of genes involved in lipid β-oxidation and oxidative phosphorylation suggestive of a mitochondrial dysfunction phenotype (Meeks et al. 2021). Therefore, restoration of salivary gland function by AICAR or metformin may involve the stimulation of mitophagy and subsequent mitochondrial biogenesis in order to restore mitochondrial function.

Recently, the LKB1–AMPK signaling axis has been shown to be intertwined with regulation of adherens and tight junctions, apical/basolateral polarity, and actin cytoskeleton assembly (Zhu et al. 2018; Tsukita et al. 2019). In cell culture models, LKB1 localization at adherens junctions has been shown to be critical in the activation of AMPK localized at tight junctions (Zhang et al. 2006; Sebbagh et al. 2009). In addition, pharmacological activation of AMPK via AICAR or metformin aids in the reestablishment of intestinal epithelial barrier function in experimental models of colitis or intestinal dysfunction caused by heat stress (Chen et al. 2018; Xia et al. 2019). The mechanisms of AMPK regulation of cellular junctions and polarity are currently unclear, especially in the context of salivary glands. For example, AMPK activation of the transcription factor caudal type homeobox 2 (CDX2) increased expression of junctional proteins (e.g., occludin, E-cadherin) (Sun et al. 2017, 2018); however, this family of transcription factors is expressed at a very low level in salivary glands (Salivary Gland Atlas). Following radiation damage to salivary glands, there is decreased apical/basolateral polarity, decreased E-cadherin/β-catenin association, and cytoskeletal disruptions 5 d after exposure (Chibly et al. 2018; Wong et al. 2018). Interestingly, decreases in AMPK activation (Fig. 1) precede this phenotype and may suggest a role in homeostatic maintenance of these structures. Treatment with AICAR or metformin following radiation increases levels of apical/basolateral polarity (Fig. 5), suggesting a reestablishment of epithelial differentiation that is critical for the restoration of saliva secretion. This mechanistic role of AMPK activation in salivary output provides additional support for clinical studies using metformin or other AMPK activators and broadens treatment options available for head and neck cancer patients undergoing radiotherapy.

In summary, radiation treatment leads to reductions in key components of the AMPK signaling pathways at acute time points (days 3–5) within salivary glands, notably phosphorylated AMPK, NAD+, AMP, Sirt1, and NAMPT levels compared to untreated glands. Activation of AMPK following IR reduces compensatory proliferation, increases differentiation markers, and restores both apical/basolateral polarity and salivary flow rates to levels comparable to unirradiated controls. This work provides a novel therapeutic target for functional restoration following radiotherapy that could eventually provide relief for those affected by chronic hyposalivation.

Author Contributions

R. Meyer, contributed to conception and design, data acquisition, analysis, and interpretation, drafted and critically revised the manuscript; K. Gilman, B.A. Rheinheimer, L. Meeks, contributed to data acquisition, analysis, and interpretation, critically revised the manuscript; K.H. Limesand, contributed to conception and design, data interpretation, drafted and critically revised the manuscript. All authors gave their final approval and agree to be accountable for all aspects of the work.

Supplemental Material

sj-docx-1-jdr-10.1177_00220345221148983 – Supplemental material for AMPK Activation Restores Salivary Function Following Radiation Treatment
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Supplemental material, sj-docx-1-jdr-10.1177_00220345221148983 for AMPK Activation Restores Salivary Function Following Radiation Treatment by R.K. Meyer, K.E. Gilman, B.A. Rheinheimer, L. Meeks and K.H. Limesand in Journal of Dental Research

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Supplemental material, sj-pdf-2-jdr-10.1177_00220345221148983 for AMPK Activation Restores Salivary Function Following Radiation Treatment by R.K. Meyer, K.E. Gilman, B.A. Rheinheimer, L. Meeks and K.H. Limesand in Journal of Dental Research

Footnotes

A supplemental appendix to this article is available online.

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding: The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This work was supported by the National Institute of Dental and Craniofacial Research (DE023534 and DE029506).

ORCID iD: B.A. Rheinheimer Inline graphichttps://orcid.org/0000-0001-9461-4967

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