Cell treatments - Materials and Methods - THE 5' AMP-ACTIVATED PROTEIN KINASE IN BOVINE MAMMARY

2. Manuscript

2.2 Materials and Methods

2.2.3 Cell treatments

A-769662 was dissolved in dimethyl sulfoxide (DMSO). For treatment, A-769662 or vehicle control (DMSO) was added to lactogenic differentiated BMEC to a final concentration of 100 μM for 16 h.

To test the effect of glucose and acetate on AMPK activation, cells were incubated in DMEM medium containing 4 mM glucose and 1 mM sodium acetate (control), or control medium lacking glucose or acetate, or both, for 4 h. To define the sensitivity of AMPK to glucose levels, BMEC were treated with DMEM without sodium acetate containing 1, 2 or 4 mM glucose, or without glucose, for 4 h. For time course experiments, cells were incubated in DMEM media without glucose for 0, 1, 2, or 4 h.

37 2.2.4 Lipid synthesis assay

Lipid synthesis in BMEC was determined by quantifying the incorporation of [³H]-labeled acetate into total lipids. Methods were adapted from McFadden (2009) and modified as described. After 12 h of treatment, cells were incubated with [³H]-labeled acetate (0.084 Ci/mmol) at a final concentration of 1 μCi/mL for 4 h. Cells were then lysed in 0.1% SDS in PBS. A portion of the cell lysate was retained for measurement of protein concentration.

Total lipids were extracted from cell lysates using hexane:isopropanol (3:2 vol/vol). The solvent layer was combined with scintillation cocktail (Universol, MP Biomedical; Solon, OH) for quantification of radioactive label incorporation into lipid using a Tri-Carb liquid scintillation counter (Perkin-Elmer). Lipid synthesis was expressed as cpm of acetate incorporated per μg of protein.

2.2.5 Lactose synthesis assay

Lactose synthesis in BMEC was determined by quantifying the incorporation of radioactive glucose into lactose. Methods were adapted from Kwon et al. (1981) and Mellenberger et al. (1973) as described. After 12 h of treatment, cells were incubated with [U-¹⁴C]-D-glucose (263 mCi/mmol) at a final concentration of 1 μCi/mL for 4 h. A portion of the cell lysate was retained for measurement of protein concentration. Total lactose was extracted from spent media using 10 M solution of MgCl2 and 10 N NaOH. The precipitate layer was lactose-Mg(OH)2 slurries for quantification of radioactive label incorporation into lactose using a Tri-Carb liquid scintillation counter (Perkin-Elmer).

Lactose synthesis was expressed as cpm of glucose incorporated per μg of protein.

2.2.6 Immunoblotting

Cells were rinsed once with ice-cold PBS prior to harvest. Cellular proteins were extracted in ice-cold lysis buffer containing 1% (vol/vol) Triton X-100, 50 mM Tris-HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM β-glycerophosphate, 50 mM NaF, 10 mM Na4P207, 2 mM Na3VO4 and 1× protease inhibitor cocktail on ice for 15 min. Cell lysates

were cleared by centrifugation at 15,000 × g at 4°C for 10 min. An aliquot of cleared cell lysate was used to measure protein concentration using a BCA protein assay kit with bovine serum albumin as standard according to manufacturer’s instructions. An aliquot of cleared cell lysate was mixed with 5× Laemli sample buffer and then heated at 95℃ for 5 min. Equal amounts of protein were resolved by SDS-PAGE and then transferred onto a PVDF membranes. The membranes were blocked in 5% (wt/vol) non-fat milk in Tris-buffered saline containing 0.1% (vol/vol) Tween 20 (TBS-T) at room temperature for 1 h and then incubated primary antibodies raised against phospho-specific proteins diluted in 5% non-fat milk in T at 4°C with constant rocking overnight. After washing 6× in TBS-T for 5 min, membranes were incubated with secondary antibodies diluted 1: 10,000 in 5% non-fat milk in TBS-T at room temperature for 1 h with constant shaking. After washing in 6× in TBS-T, the bound horse radish peroxidase-linked secondary antibodies were visualized by chemiluminescence (Clarity ECL; Bio-Rad Laboratories). The band signal intensity was quantified using the Image Lab Software (Bio-Rad Laboratories). After detection of the phospho-specific signal, the antibodies were stripped off the membranes by incubation in 62.5 mM Tris-HCl, pH 6.8, 2% (wt/vol) SDS, and 100 mM 2-mercaptoethanol at 50°C for 30 min with constant rocking. The membranes were washed, blocked, and re-probed with primary antibodies that recognized total proteins, irrespective of the phosphorylation state. For determination of protein phosphorylation, the intensity of phosphorylated signals was normalized to that of total protein. For determination of protein abundance, total protein levels were normalized to α-tubulin. For SREBP-1c processing, the mature (cleaved) SREBP-1c was normalized to total SREBP-1c (precursor + mature).

2.2.7 Subcellular fractionation

Nuclear and cytoplasmic fraction were isolated from cellular extracts using the NE-PER Nuclear and Cytoplasmic Extraction Kit (ThermoFisher Scientific) according to manufacturer’s instructions. Protein concentration in each fraction was measured using the BCA method. Nuclear and cytoplasmic fractions were subjected to immunoblotting and probed for SREBP-1c, GAPDH, or LAMIN-A/C, as described previously.

2.2.8 RNA extraction and quantitative real-time PCR

Total RNA was extracted from BMEC using the TRI-Reagent (Sigma), according to manufacturer’s instructions. RNA yield was quantified by measuring absorbance at 260 nm in a spectrophotometer (BioTek; Winooski, VT). RNA integrity was determined by bleach agarose gel electrophoresis as described in (Aranda, 2012). Total RNA (1 µg) was reverse-transcribed using the iScript cDNA synthesis kit (Bio-Rad Laboratories) in a T100 thermal cycler (Bio-Rad Laboratories), according to manufacturer’s instructions.

Quantitative real-time PCR (qPCR) was performed using SsoAdvanced Universal SYBR green (Bio-Rad Laboratories) and gene-specific primers in a CFX96 Touch Real Time PCR System (Bio-Rad Laboratories). Samples for each experimental condition were run in duplicate. Relative gene expression was determined using the geometric mean of at least two reference genes (GAPDH, ATCB or PPIA) and calculated using the ΔΔCq method. Experiment were conducted using the CFX Maestro Software (Bio-Rad Laboratories).

2.2.9 Statistical analysis

Data are presented as mean ± SEM. Results are from at least 6 replicates per treatment group from BMEC derived from 3 independent cows (2 replicates/cow) in a randomized complete block design. Data were analyzed by ANOVA using PROC MIXED in SAS (Version 9.4; SAS Institute, 2011). The statistical model included the fixed effect of treatment, random effect of cow (block) and the residual random error. For glucose and acetate deprivation experiments, treatment means were separated by least square differences. For time course and dose response experiments, treatments we compared to 0 h and 4 mM glucose, respectively, by least square differences. Treatment differences were considered significant at P < 0.05 and a statistical trend at 0.05 ≤ P ≤ 0.10.

2.3. Results

2.3.1 Effect of A-769662 on phosphorylation of AMPK, TSC2 and ACC in BMEC To investigate the role of AMPK in lipid and lactose synthesis by BMEC, we treated cells with 100 µM of A-769662, a new allosteric activator of AMPK. Unlike other pharmacological agents, A-769662 directly activates AMPK by mimicking both effects of AMP (Göransson et al., 2007). We assessed AMPK activation by measuring phosphorylation of AMPKα (Thr172) and two downstream targets: ACC1 (Ser79) and TSC2 (Ser1387) (Gwinn et al., 2008). We found that AMPKα Thr172 phosphorylation was unaffected by incubation of BMEC in medium containing 100 µM A-769662 (Figure 2.1).

In contrast, the phosphorylation of ACC1 at Ser79 and TSC2 at Ser1387 were 144% (P

= 0.015) and 26% (P = 0.048) higher, respectively, in cells treated with A-769662 for 16 h compared to vehicle (Figure 2.1). This was not unexpected, as ACC1 Ser79 phosphorylation is a more sensitive marker of AMPK activity than AMPKα Thr172 phosphorylation by this compound in intact cells (Göransson et al., 2007). Thus, treatment of BMEC with A-769662 enhanced AMPK activation in BMEC.

2.3.2 Effect of A-769662 on de novo lipid synthesis and lipogenic gene expression in BMEC

To determine the effect of AMPK activation on de novo lipid synthesis, we measured the incorporation radioactive acetate into total cellular lipids in BMEC treated with A-769662 or vehicle for 16 h. AMPK activation by A-769662 decreased lipid synthesis in BMEC (P

= 0.04) (Figure 2.2).

AMPK is known to exert metabolic control on lipid synthesis by modulating the expression of lipogenic genes. To test this possibility, we measured the expression of key lipogenic genes in response to treatment with A-769662 for 16 h by qPCR. The mRNA expression of ACC1, encoded by ACACA, was not affected by A-769662 (Figure 2.3.A). The transcript abundance of fatty acid synthase (gene name FASN) was 17% higher in cells

treated with A-769662 compared to control, but the difference was not significant (Figure 2.3). In addition to de novo fatty acid synthesis, BMEC take up preformed fatty acids for incorporation into milk lipids. Fatty acid binding protein 3 (FABP3) is one of the most abundant isoforms of a family of intracellular lipid-binding proteins that bind reversibly to FAs to facilitate their intracellular trafficking and metabolism in the lactating bovine mammary gland (Specht et al., 1996, Bionaz and Loor, 2008b). FABP3 mRNA expression did not change in response to A-769662 (Figure 2.3). Both de novo synthesized and preformed can be desaturated by stearoyl-CoA desaturase-1 (SCD1), which introduces a double bond between carbon 9 and 10 of FA-CoAs (Jacobs et al., 2013). The expression of SCD1 was 64% higher than vehicle (P = 0.04) (Figure 2.3). Activation of AMPK by A-769662 significantly increased SREBF1 mRNA expression by 42% (P = 0.04) (Figure 2.3), consistent with a previous study in MAC-T cells using AICAR (McFadden and Corl, 2009). These results suggest that SREBP-1c may be involved in mediating the effect of AMPK on lipogenic gene expression in BMEC.

2.3.3 Effect of A-769662 on SREBP-1c protein abundance, proteolytic processing and subcellular localization in BMEC

To examine the potential role of SREBP-1c on AMPK, we investigated the effect of A769662 on SREBP-1c abundance, proteolytic processing and subcellular localization in BMEC. As shown in Figure 2.4, treatment of A-769662 tended to increase total (precursor plus mature) SREBP-1c protein abundance by 30% (P = 0.077). the proportion of mature SREBP-1c in total SREBP-1c, including precursor and mature forms, from A-769662 treated cells exhibited a 19% less expression compared to vehicle (P = 0.0005). In addition, there was a nominal decline in the abundance of mature SREBP-1c in both nuclear and cytoplasmic fractions in cells treated with A-769662 compared to vehicle control, but the differences were not significant (P = 0.12 and P = 0.56, respectively) (Figure 2.4).

2.3.4 Effect of A-769662 on lactose synthesis and glycolytic gene expression in BMEC

To investigate the potential role for AMPK in regulation of lactose synthesis, we measured radioactive glucose incorporation into lactose. Treatment of BMEC with A-769662 significantly decreased the incorporation of radiolabelled glucose into lactose (P = 0.01) (Figure 2.5). To investigate changes in the expression of genes involved in glucose metabolism in BMEC, we measured the expression of key glycolytic genes in response to treatment with A-769662 for 16 h by qPCR. The mRNA expression of VEGF-A, marker of HIF-1α activity, increased by 46% in response to A-769662 treatment (P = 0.02) (Figure 9.A). The transcript abundance of GLUT1, encoded by SLC2A1, was not affected by A-769662 (Figure 2.6). This results contrasts with a previous study in goat mammary epithelial cells treated with AICAR (Zhang et al., 2011). Glucose-6-phosphate dehydrogenase (G6PD), 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 1 (PFKFB1) and phosphoglycerate kinase 1 (PGK1) are rate-limiting enzymes in the glycolysis pathway, however, the mRNA expression of them were insignificantly decreased in A-769662-treated BMEC compared to vehicle (Figure 2.6). Thus, AMPK inhibits lactose synthesis, but does not affect the expression of glycolytic genes.

2.3.5 Effect of glucose and acetate deprivation on AMPK activity in BMEC

Both acetate and glucose are major energy sources used by BMEC for maintenance functions and synthesis of milk components. To examine the effect of energy-yielding substrate depletion on AMPK activation, we measured phosphorylation of AMPK and its substrates in BMEC deprived of glucose or acetate alone, or in combination, for 4 h.

Deprivation of glucose or acetate promoted phosphorylation of AMPKα at Thr172 by 84%

(P = 0.01) or 58% (P = 0.05), respectively, compared to vehicle (Figure 2.7). Although deprivation of energy substrates, to a certain extent, nominally increased phosphorylation of ACC at Ser79 and TSC2 at Ser1387, only deprivation of both glucose and acetate caused a significant change on TSC2 (P = 0.03) (Figure 2.7). Thus, the shortage of acetate and glucose in BMEC cause the activation of AMPK.

To assess the sensitivity of AMPK to by glucose levels, we measured phosphorylation of AMPK and its downstream targets in the BMEC treated without or with 1, 2, or 4 mM for 4 h. The results showed that BMEC exposed to low glucose concentration did not show a significant change in phosphorylation of AMPK and its downstream targets compared to 4 mM glucose level, the significance only occurred in the glucose depletion treatment (P = 0.05) (Figure 2.8). When reducing the glucose levels from 4 to 2 or 1 mM, there was no change in the phosphorylation of ACC, mTOR and TSC2. However, glucose deprivation increased the phosphorylation of AMPK targets TSC2 and ACC by 50% (P = 0.003) and 249% (P = 0.01), respectively, but decreased the phosphorylation of mTOR by 70% (P = 0.01) (Figure 2.8). Thus, complete glucose deprivation was required to trigger AMPK activation in BMEC, suggesting other metabolic fuels maintain ATP/AMP levels at low glucose levels.

Finally, we examined the time-dependent activation of AMPK by glucose deprivation. The phosphorylation of AMPK and its downstream targets were measured in BMEC deprived of glucose for 0, 1, 2, or 4 h. Phosphorylation of AMPK increased gradually with time up to 62% (P = 0.01) by 4 h of glucose deprivation compared to 0 h (Figure 2.9).

Phosphorylation of mTOR at Ser2448, ACC at Ser79 and TSC2 at Ser1387 followed a similar temporal patter to that of AMPK at Thr172 upon glucose withdrawal. TSC2 phosphorylation was only significantly different after 4 h of glucose deprivation (P = 0.001), whereas required at least 2 h in AMPK-mediated phosphorylation of ACC (P = 0.02) (Figure 2.9).

2.4. Discussion

AMPK is a cellular energy gauge that sense energy stress. Previous studies have established a role for AMPK on the control of nutrient metabolism and mammary component synthesis in BMEC. Burgos et al. (2013) used 2-deoxy-D-glucose, a non-metabolizable glucose analog that inhibits glycolysis, to demonstrate that AMPK activation inhibits mammary protein synthesis through mTOR complex 1 (mTORC1) in MAC-T cells. McFadden and Corl (2009) showed that AMPK activation blunted de novo

lipid synthesis in MAC-T cells treated with the AMP-analog AICAR, but the mechanisms were not established. In this study, we used a novel allosteric activator of AMPK to confirm this observation and extending it by demonstrating that SREBP-1c may mediate the effect of AMPK on lipid synthesis by modulating SREBP-1c proteolytic processing and nuclear translocation. Collectively, these studies support an important role for AMPK on the regulation of lipid synthesis in BMEC.

The mechanism by which AMPK activation reduces de novo lipid synthesis. In MAC-T cells, the inhibition of de novo lipid synthesis by AICAR was accompanied by increased mRNA expression of several lipogenic genes including SREBF1, FASN and FABP3 by 66%, 38% and 56%, respectively (McFadden and Corl, 2009). In partial agreement, we found that the mRNA expression of SREBF1 and SCD1 was increased by 36% and 41%, respectively, in response to A-769662, whereas the expression of other lipogenic genes (FASN, DGAT1, ACACA, and FABP3) did not differ significantly from controls in our study.

In the present study, treatment of BMEC with A-769662 increased the phosphorylation of ACCα by 144%, while the mRNA expression of ACACA was unaffected, which was consistent with a previous study in MAC-T cells by McFadden and Corl (2009). They also observed a significant decrease in ACC activity in response to increased ACCα phosphorylation. The reduction in ACC activity most likely contributed to the significant decrease in total lipid synthesis in response to treatment of A-769662 in our study. The difference in some of the mRNA targets may relate to the expression level of other co-activators or differing sensitivities between primary BMEC and Mac-T cell line.

AMPK activation modulates SREBP-1c expression by inhibiting the trafficking of SREBP to the nucleus, thereby controlling its lipogenic activity. Although the result showed activation of AMPK by A-769662 increased SREBF1 gene expression and protein abundance, the proportion of mature SREBP-1c decreased. Quan et al. (2013) found that activation of AMPK suppressed 1c nuclear translocation and repressed SREBP-1c target gene expression, resulting in decreased lipogenesis and lipid accumulation in HepG2 cells and primary hepatocytes. When activated by metformin, AMPK suppressed SREBP-1c cleavage and nuclear translocation in hepatocytes (Li et al., 2011). In the

present study, the abundance of mature SREBP-1c in both nuclear and cytoplasmic factions were synchronously decreased by AMPK activation, suggesting a similar mechanism may operate in BMEC. The SREBP-1c targets that mediate AMPK inhibition of lipid synthesis have yet to be identified. In addition, whether other lipogenic transcription factors such as LXRα cooperate with SREBP-1c to regulate lipogenesis in BMEC downstream of AMPK remains to be determined.

Activation of AMPK can regulate genes that control glucose metabolism (Magnoni et al., 2012). VEGF-A, a known marker of HIF-1α activity, significantly increase by 168% in BMEC treated with A-769662. HIF-1α stimulates glucose uptake and the expression of glycolytic enzymes, which is consistent with the function of AMPK. However, treatment of BMEC with A-769662 did not alter the mRNA expression of the genes encoding for GLUT1 or enzymes involved in glycolysis. This may relate to the relative low importance of glycolysis as a flux of glucose in the mammary gland of dairy cows. Zhang et al. (2011) reported that intracellular glucose levels, which was interpreted to indicate heightened glucose uptake, as well as mRNA abundance of GLUT1 and glycogen synthase, increased after incubation of goat mammary epithelial cells with AICAR. The change in intracellular glucose level is detected in response to AICAR, in that upon entry to cells, glucose is rapidly phosphorylated by hexokinase in BMEC (Xiao and Cant, 2005). This effect could also be explained by a decrease in glucose utilization. In this regard, we found that lactose synthesis was markedly reduced by activation of AMPK, which is in agreement with physiological role of AMPK that inhibits energy consuming processes to modulate BMEC energy utilization. Further research is needed to identify the mechanisms by which AMPK inhibits lactose synthesis in BMEC.

AMPK is sensitive to cellular energy status in various cell types, and the decrease in energy charge activates AMPK. In dairy cows, mammary cells utilizes acetate and glucose a major sources of energy. In earlier work, Appuhamy et al. (2014) tested the effect of essential amino acid in the absence or presence of acetate (5 mM) or glucose (17.5 mM) on mTORC1 signaling and protein synthesis in Mac-T cells and bovine mammary tissue slices. They found that provision of essential amino acids with or without

glucose reduced AMPKα Thr172 phosphorylation relative to nutrient-deprived Mac-T cells.

In the present study, deprivation BMEC of glucose and acetate deprivation differentially affected the phosphorylation of AMPK and its downstream targets, with glucose deprivation having the greatest on AMPKα Thr172 phosphorylation. Dose response experiments revealed that AMPK was activated when glucose is absent from medium.

Furthermore, time course experiments revealed that AMPK only responded to long-term depletion of glucose supply. These results suggest that oxidation of other metabolic fuels, possibly amino acids, can allow BMEC to cope with low-level or short-term decrease in glucose supply. There is evidence to suggest that AMPK senses intracellular glucose levels directly, in addition to cellular energy stress. The physiological relevance of AMPK as a sensor of glucose and cellular energy status and its role on regulation of milk composition will require feeding trials or infusion studies in dairy cows.

2.5 Conclusions

We concluded that the allosteric activation of AMPK by A-769662 decreased the synthesis of both lipids and lactose in BMEC. The AMPK-induced inhibition of lipid synthesis in BMEC was accompanied by increased phosphorylation of ACCα and transcriptional changes in lipogenic genes. The effect of gene transcription was likely mediated by SREBP1-c, as AMPK activation increased the mRNA and protein abundance, but reduced proteolytic processing and nuclear transport, and presumably transcriptional activity. Deprivation of glucose and acetate, to a lesser extent, led to activation of AMPK, as judged by phosphorylation at Thr172 and its downstream targets, but changes occurred only with medium completely devoid of glucose and after 2-4 hours, suggesting that other metabolic fuels could temporarily make up for the inadequate glucose supply.

The physiological relevance of AMPK as a sensor of glucose and cellular energy status and its role in the control of milk components synthesis in vivo will require control feeding trials in dairy cows.

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Figure 2.1. Effect of A-769662 on phosphorylation of AMPK, TSC2 and ACC in BMEC.

Cells were treated with 100 μM A-769662 (A76) or vehicle control (Ctrl) for 16 h.

Phosphorylation of AMPKα at Thr172 (A), TSC2 at Ser1387 (B) and ACC at Ser79 (C) were measured by immunoblot analysis. The densities of phosphorylated AMPK, TSC2 and ACC were normalized to total AMPK, TSC2 and ACC protein levels, respectively.

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