Nicotinamide N-methyltransferase upregulation contributes to palmitate-elicited peroxisome proliferator-activated receptor transactivation in hepatocytes

Abstract

Peroxisome proliferator-activated receptor γ (PPARγ) plays a pivotal role in regulating lipid metabolism and hepatic PPARγ transactivation contributes to fatty liver development. Fatty acids (FAs) are well-known endogenous ligands for PPARγ. Palmitate, a 16-C saturated FA (SFA) and the most abundant SFA in human circulation, is a strong inducer of hepatic lipotoxicity, a central pathogenic factor for various fatty liver diseases. In this study, using both alpha mouse liver 12 (AML12) and primary mouse hepatocytes, we investigated the effects of palmitate on hepatic PPARγ transactivation and underlying mechanisms, as well as the role of PPARγ transactivation in palmitate-induced hepatic lipotoxicity, all of which remain ambiguous currently. Our data revealed that palmitate exposure was concomitant with both PPARγ transactivation and upregulation of nicotinamide N-methyltransferase (NNMT), a methyltransferase catalyzing the degradation of nicotinamide, the predominant precursor for cellular NAD⁺ biosynthesis. Importantly, we discovered that PPARγ transactivation by palmitate was blunted by NNMT inhibition, suggesting that NNMT upregulation plays a mechanistic role in PPARγ transactivation. Further investigations uncovered that palmitate exposure is associated with intracellular NAD⁺ decline and NAD⁺ replenishment with NAD⁺-enhancing agents, nicotinamide and nicotinamide riboside, obstructed palmitate-induced PPARγ transactivation, implying that cellular NAD⁺ decline resulted from NNMT upregulation represents a potential mechanism behind palmitate-elicited PPARγ transactivation. At last, our data showed that the PPARγ transactivation marginally ameliorated palmitate-induced intracellular triacylglycerol accumulation and cell death. Collectively, our data provided the first-line evidence supporting that NNMT upregulation plays a mechanistic role in palmitate-elicited PPARγ transactivation, potentially through reducing cellular NAD⁺ contents.

NEW & NOTEWORTHY Hepatic PPARγ transactivation contributes to fatty liver development. Saturated fatty acids (SFAs) induce hepatic lipotoxicity. Here, we investigated whether and how palmitate, the most abundant SFA in the human blood, affects PPARγ transactivation in hepatocytes. We reported for the first time that upregulation of nicotinamide N-methyltransferase (NNMT), a methyltransferase catalyzing the degradation of nicotinamide, the predominant precursor for cellular NAD⁺ biosynthesis, plays a mechanistic role in regulating palmitate-elicited PPARγ transactivation through reducing intracellular NAD⁺ contents.

INTRODUCTION

Peroxisome proliferator-activated receptor γ (PPARγ) is a nuclear receptor of the nuclear hormone receptor superfamily, whose transactivation plays an important role in regulating both adipogenesis and lipogenesis (1–3). Many genes involved in lipid uptake, intracellular trafficking, esterification, formation, and storage, such as fatty acid-binding protein 4 (Fabp4), cell death activator CIDE-3 (Cidec), and perilipin 2 (Plin2), are the direct targets of PPARγ (4). In adipose tissue, PPARγ plays a central role in regulating adipocyte differentiation and proliferation (5). Although basal expression is relatively low in hepatocytes, accumulated evidence supports that the hepatic PPARγ expression level positively correlates with fatty liver development (6–8). PPARγ has two isoforms, PPARγ1 and PPARγ2, and the liver predominantly expresses PPARγ2 (9). Liver PPARγ overexpression is coupled with severe lipid accumulation and inflammation in mouse fatty liver disease models (10–12). On the contrary, liver-specific PPARγ gene knockout alleviates liver pathologies of both alcoholic liver disease (ALD) and nonalcoholic fatty liver disease (NAFLD) in both dietary and genetic mouse models, concomitant with lowered expression of genes involved in lipogenesis including Srebf1, cluster of differentiation 36 (CD36), Adrb, Fabp4, Cidec, Plin4, and Fasn (8, 13–17), suggesting that hepatic PPARγ transactivation plays an important role in the pathogenesis of metabolic fatty liver diseases.

Hepatic lipotoxicity plays a pivotal role in the pathogenesis of various fatty liver diseases, including both ALD and NAFLD (18–20). In comparison to most unsaturated fatty acids, saturated fatty acids (SFA), such as palmitate (the most abundant SFA in human circulation), are more cytotoxic to most cell types, including hepatocytes (21, 22). Accordingly, exogenous palmitate exposure has been widely used in various cell culture systems to investigate lipotoxicity (23–26). Although it has been well-documented that PPARγ is activated in response to a variety of long-chain fatty acids (27–30), few studies were conducted to examine whether and how palmitate affects PPARγ transactivation in hepatocytes (30). Similarly, the role of PPARγ (de)activation in palmitate-induced lipotoxic process remains unclear (31–33).

Nicotinamide N-methyltransferase (NNMT) is a methyltransferase that catalyzes S-adenosylmethionine (SAM)-dependent methylation and subsequent degradation of nicotinamide, a predominant precursor for cellular NAD⁺ regeneration via the salvage pathway (34). Abundantly expressed in the liver and adipose tissue, NNMT is emerging to play a critical role in regulating metabolism and energy homeostasis. Whole body NNMT overexpression worsened hepatic steatosis upon chronic high-fat diet feeding (35), whereas NNMT knockdown was protective against diet-induced obesity in mice (36), and genetic NNMT deficiency improved insulin sensitivity in diet-induced obesity in mice (37). We are the first to identify activating transcription factor 4 (ATF4) as a key transcription factor in regulating hepatic NNMT expression and to demonstrate that adenoviral NNMT knockdown protects against fatty liver development in mice upon chronic alcohol exposure (38).

In this study, using palmitate exposure of cultured hepatocytes [alpha mouse liver 12 (AML12) cells and primary mouse hepatocytes] as our in vitro model, we investigated the effect and underlying mechanism(s) of palmitate on hepatic PPARγ activation. We demonstrate that the intracellular NAD⁺ decline resulting from NNMT upregulation contributes to palmitate-induced PPARγ activation in hepatocytes. Intriguingly, PPARγ transactivation seems not to play a principal role in palmitate-induced intracellular triacylglycerol (TAG) accumulation and cell death.

MATERIALS AND METHODS

Reagents

Palmitic acid and oleic acid-bovine serum albumin (BSA) solutions were purchased from Sigma-Aldrich (St. Louis, MO). JBSNF-000088 was purchased from MCE (Monmouth Junction, NJ), and II399 was kindly provided by Dr. Rong Huang from Purdue University. Triacsin C was obtained from ENZO life sciences (Farmingdale, NY). 2-Fluoropalmitic acid was obtained from Cayman Chemical (Ann Arbor, MI). Nicotinamide riboside chloride was purchased from MedKoo Biosciences (Morrisville, NC). The antibodies for PPARγ (No. 2443), NNMT (ab11978), and β-actin (AC026) were obtained from Cell Signaling Technology (Beverly), Abcam (Boston, MA), and ABclonal (Woburn, MA), respectively. Detailed information about reagents, antibodies, and assay kits used in this study is listed in Table 1.

Table 1.

Information on chemicals, antibodies, and assay kits used in this study

Chemical/Reagent	Brand	Cat. No.	Batch or Lot No.
BSA (Albumin)	Sigma	A6003	JSLCP4267
Palmitate	Sigma	P5595-100G	110k03013
Oleic Acid	Sigma	O3008-5ml	JSLCC7923
Triacsin C	ENZO	BML-E1218-0100	4021908
2-fluoro Palmitic Acid	Cayman Chemical	90380	0437938-35
II399	Provided by Prof. Rong Huang from Purdue University
JBSNF-000088	MCE	HY-112584/CS-W000376	55756
Nicotinamide Riboside Chloride	MedKoo Biosciences	329479	A9T037K28
Nicotinamide	Sigma	N0636-100G	079K1404

siRNA	Brand	Cat. No.	Batch or Lot No.
Control	Santa Cruz	sc37007	J0522
NNMT (m)	Santa Cruz	sc61214	JD2407
PPARG (m)	Santa Cruz	sc29456	E1920

Antibody	Brand	Cat. No.	Batch or Lot No.	Dilutions
Actin	ABClonal	AC026	9100026001	1:100,000
PPARG	Cell Signaling Technology	2430S	2	1:1,000
NNMT	Abcam	Ab119758	GR3233469-24	1:1,000
IRDye 800CW Goat anti-Rabbit	LI-COR	926-32211	D00804-07	1:20,000
IRDye 800CW Goat anti-Mouse	LI-COR	926-32210	C80710-15	1:20,000

Kit	Brand	Cat. No.	Batch or Lot No.
PPAR gamma Transcription Factor Assay Kit	Abcam	AB133101	1033310-1
CyQUANT LDH Cytotoxicity Assay	Thermo Fisher	C20301	2488970
NAD+/NADH Quantification Colorimetric Kit	Biovision	K337-100	6B27K03370

BSA, bovine serum albumin; NNMT, nicotinamide N-methyltransferase; PPARγ, peroxisome proliferator-activated receptor γ.

Palmitate-BSA Solution Preparation

Palmitate-BSA solution was made from palmitic acid powder. First, palmitic acid (0.1 g) was dissolved in 20 mL ethanol. Then NaOH solution (1.6 M, 244 μL) was added to generate sodium palmitate, a mixture with gelatinous precipitate. The whole mixture was air-dried overnight before completely dissolved in 20 mL PBS at 80°C. Then 20 mL of 20% BSA solution (in PBS) was added and stirred for 4 h. Finally, a sterile palmitate-BSA solution was obtained by filtering the solution through a 0.2-µm filter membrane.

Cell Culture

AML12 (alpha mouse liver 12) cells were purchased from American Type Culture Collection (ATCC; Manassas, VA). Cells were cultured in DMEM: F12 medium (Life Technologies, NY) supplemented with 10% fetal bovine serum (RMBIO, MT), 10 µg/mL insulin, 5.5 µg/mL transferrin, 5 ng/mL selenium, and 40 ng/mL dexamethasone at 37°C in a humidified atmosphere with 5% CO₂ in the air.

Primary Mouse Hepatocyte Isolation and Culture

All animal studies were approved by the Institutional Animal Care and Use Committee at the University of Illinois Chicago (Chicago, IL) and consistent with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory Animals. Male C57BL/6 mice (Charles RiverLab, Chicago, IL) at 10-wk-old were used for primary hepatocyte isolation. Primary mouse hepatocytes were isolated and cultured as previously described by Lee et al. (39).

siRNA Transfection

AML12 hepatocytes were plated in six-well or 24-well plates and allowed to adhere for 24 h before transfection with siRNA. Transfection was performed according to Invitrogen’s Lip 2000 protocol. Briefly, hepatocytes were cultured to 60%–70% confluence and transfected with 2 μL siRNA (20 mM) using 2 μL Lipo2000. As a control, cells were transfected with scrambled siRNA under the same conditions. The results of transfection were identified by RT-PCR following a 12-h siRNA transfection.

Biochemical Assays

The CyQUANT LDH (lactate dehydrogenase) Cytotoxicity Assay Kit (Life Technologies, OR) was used to monitor cytotoxicity resulting from palmitate exposure. In brief, AML12 cells were seeded at 1 × 10⁵ in 24-well plates overnight. After the indicated treatments, LDH concentrations in the culture media were detected according to the manufacturer’s instructions. The intracellular total nicotinamide adenine dinucleotide (NAD) contents were measured by NAD⁺/NADH Quantitation Colorimetric Kit (BioVision, CA) according to the manufacturer’s instructions. For intracellular TAG measurement, total intracellular lipids were collected using 300 μL of 0.25 M NaOH solution. Then hexane-isopropanol (3:2, 1 mL) reagent was added to extract the total lipid, followed by centrifugation at 10,000 rpm for 10 min. The supernatants were aliquoted and dried in air before being dissolved in the Triglyceride Liquicolor reagent (Stanbio Laboratory, TX) at 37°C. The absorbance at OD500 nm was measured. The relative TAG contents were calculated after standardization by protein concentrations.

Cell Lysates and Western Blotting

Total proteins from AML12 hepatocytes were obtained using RIPA lysis buffer with 2 mM PMSF, 1 mM sodium orthovanadate, and protease inhibitor cocktail (all from Santa Cruz Biotechnology, TX). Equal amounts of protein (40 µg) were subjected to 10% SDS-PAGE and transferred to Odyssey Nitrocellulose Membranes (LI-COR, NE). β-Actin protein was used as the housekeeping loading control. The membranes were blocked with 5% (wt./vol.) nonfat dry milk-PBS solution and probed with specific primary antibodies. The primary antibody for NNMT (Table 1) has been validated by us in both cultured hepatocytes and mouse liver tissue under either knockout or overexpression manipulation (38). Similarly, the primary antibody for PPARγ (Table 1) has been validated by Dr. Jose Cordoba-Chacon (one of the coauthors) in their previous studies with PPARγ knockout mice (17). IRDye secondary antibodies (LI-COR, NE) were applied for signal detection. Immunoreactive bands of predicted molecular mass were visualized using a LI-COR Odyssey CLx system (Lincoln, NE).

Quantitative Real-Time RT-PCR

TRIzol reagent (Life Technologies, CA) was used to isolate total RNA from AML12 hepatocytes, and 0.8 µg total RNA was used for reverse-transcription through a high-capacity cDNA reverse transcription kit (Applied Biosystems, Vilnius, LT). The cDNA was amplified in MicroAmp Optical 384-well reaction plates with a SYBR Green PCR Master Mix (Applied Biosystems, Warrington, UK) on a Life Technologies ABI ViiA7 sequence detection system. Relative gene expression was calculated after normalization by a housekeeping gene (mouse 18 s rRNA) by implementing the 2^−ΔΔCT algorithm.

Primer Sequences

Rn18s: 5′- GTAACCCGTTGAACCCCATT-3′ and 5′- CCATCCAATCGGTAGTAGCG-3′; Nnmt: 5′- CTTTGGGTCCAGACACTGTGCA-3′ and 5′- CCAGAGCCAATGTCAATCAGGAG-3′; Pparg: 5′- GTACTGTCGGTTTCAGAAGTGCC-3′ and 5′- ATCTCCGCCAACAGCTTCTCCT-3′; Cd36: 5′- GGACATTGAGATTCTTTTCCTCTG-3′ and 5′- GCAAAGGCATTGGCTGGAAGAAC-3′; Plin2: 5′- GACAGGATGGAGGAAAGACTGC-3′ and 5′- GGTAGTCGTCACCACATCCTTC-3′; Cidec: 5′- TCGGAAGGTTCGCAAAGGCATC-3′ and 5′- CTCCACGATTGTGCCATCTTCC-3′; Fabp4: 5′- TGAAATCACCGCAGACGACAGG-3′ and 5′- GCTTGTCACCATCTCGTTTTCTC-3′.

PPARγ Transactivity Assay

PPARγ transactivity was measured using a commercially available PPARγ transcription factor assay kit from Abcam (ab133101, Waltham, MA) according to the manufacturer’s instructions.

Statistics

GraphPad Prism 9 was used for the statistical analysis of data. All data were expressed as means ± SD. Statistical analysis was performed using a two-tail unpaired student’s t test for the comparison of two groups, or one-way ANOVA with Tukey’s post hoc test for three or more groups. Values of P < 0.05 were considered statistically significant.

RESULTS

Palmitate Exposure Elicits PPARγ Transactivation in Hepatocytes

The effect of exogenous palmitate on hepatic PPARγ gene expression was first examined in AML12 hepatocytes exposed to various concentrations of palmitate (0, 0.2, 0.4, 0.6 mM) for different durations (4, 8, 16 h). The mRNA levels of PPARγ were measured by real-time q-PCR and shown in Fig. 1A. At the 4-h time point, only 0.6 mM palmitate led to a significant increase in PPARγ gene expression, but both 0.4 mM and 0.6 mM palmitate significantly increased PPARγ mRNA at the 8-h time point. After a 16-h exposure, palmitate at all three concentrations upregulated PPARγ gene expression significantly. Then, we examined the effect of palmitate exposure on PPARγ protein abundance by exposing hepatocytes to 0.4 mM palmitate for 16 h. As shown in Fig. 1, B and C, in line with many previous reports (8, 30), hepatocytes predominantly express PPARγ2, and exogenous palmitate exposure significantly increased PPARγ2 protein expression. The effects of palmitate exposure on PPARγ transactivity were subsequently measured using an ELISA assay kit and the results were shown in Fig. 1D. Palmitate exposure augmented PPARγ transactivity dramatically in AML12 cells. Correspondingly, several signature PPARγ target genes, including CD36, Plin2, and Cidec, were significantly upregulated upon a 16-h palmitate challenge (Fig. 1E). Similarly, exogenous palmitate-induced PPARγ transactivation in mouse primary hepatocytes was also observed (Fig. 1F).

Figure 1.

Palmitate exposure triggers PPARγ transactivation in hepatocytes. A: time-course and dose-dependent effects of palmitate on PPARγ gene expression in AML12 cells. AML12 hepatocytes were treated with palmitate at indicated doses for 4, 8, and 12 h, respectively. PPARγ gene expression was quantified by real-time qPCR. Data are expressed as means ± SD, n = 3 separate experiments. Differences between groups were determined by one-way ANOVA with Tukey’s post hoc test. Bars with different letters differ significantly (P < 0.05). B and C: the effect of palmitate on PPARγ protein abundance. AML12 hepatocytes were treated with 0.4 mM palmitate for 16 h. Protein abundance of PPARγ and β-actin was determined by Western blotting and densitometric analysis of the ratio of PPARγ to β-actin conducted. Data are expressed as means ± SD, n = 3 separate experiments. Two-tail unpaired Student’s t test was used for statistical evaluation (**P<0.01 vs. control). D: palmitate exposure triggers PPARγ transactivation in AML12 hepatocytes. AML12 cells were treated with 0.4 mM palmitate for 8 h. Nuclear proteins were extracted and subjected to ELISA assay for PPARγ transactivity. Data were expressed as means ± SD, n = 3 separate experiments. Differences between groups were determined by two-tail unpaired Student’s t test (***P<0.001 vs. control). E: palmitate exposure upregulates PPARγ target genes. AML12 cells were treated with 0.4 mM palmitate for 16 h. The mRNA levels of three signature PPARγ target genes, Cd36, Plin2, and Cidec, were measured by real-time qPCR. Data were expressed as means ± SD, n = 3 separate experiments. Differences between groups were determined by two-tail unpaired Student’s t test (***P<0.001, ****P<0.0001 vs. control). F: palmitate exposure triggers PPARγ transactivation in primary mouse hepatocytes. Primary mouse hepatocytes were isolated, cultured, and treated with 0.4 mM for 16 h. The mRNA levels of signature PPARγ target genes, Cd36, Plin2, Cidec, and Fabp4, were measured by real-time qPCR. Data were expressed as means ± SD, n = 3 separate experiments. Differences between groups were determined by two-tail unpaired Student’s t test (*P < 0.05, **P < 0.01, ****P< 0.0001 vs. control). AML12, alpha mouse liver 12; PA, palmitate; PPARγ, peroxisome proliferator-activated receptor γ; UT, untreated.

Palmitate Upregulates NNMT Expression in Hepatocytes

Nicotinamide N-methyltransferase (NNMT) catalyzes S-adenosylmethionine (SAM)-dependent methylation (degradation) of nicotinamide, a predominant precursor for cellular NAD⁺ regeneration via the salvage pathway (Fig. 2A). Abundantly expressed in the liver, NNMT is an emerging regulator for metabolism and energy homeostasis. Next, we examined the effect of palmitate exposure on NNMT expression in AML12 cells. As shown in Fig. 2, B and C, palmitate exposure increased NNMT expression at both mRNA (Fig. 2B) and protein levels (Fig. 2C).

Figure 2.

Palmitate exposure upregulates NNMT expression in hepatocytes. A: schematic illustration of nicotinamide metabolism. B: time-course and dose-dependent effects of palmitate on NNMT gene expression in AML12 cells. AML12 cells were treated with palmitate at indicated doses for 4, 8, and 12 h, respectively. NNMT gene expression was quantified by real-time qPCR. Data are expressed as means ± SD, n = 3 separate experiments. Differences between groups were determined by one-way ANOVA with Tukey’s post hoc test. Bars with different letters differ significantly (P < 0.05). C: the effect of palmitate on NNMT protein abundance. AML12 hepatocytes were treated with 0.4 mM palmitate for 16 h. Protein abundance was determined by Western blotting. AML12, alpha mouse liver 12; NAMPT, nicotinamide phosphoribosyltransferase; NMNAT, nicotinamide mononucleotide adenylyl transferase; NNMT, nicotinamide N-methyltransferase; NR, nicotinamide riboside; PA, palmitate; SAH, S-adenosylhomocysteine; SAM, S-adenosylmethionine; UT, untreated.

Oleate Affects Neither PPARγ Transactivation nor NNMT Expression in Hepatocytes

Palmitate (a 16-C saturated fatty acid) and oleate (an 18-C monounsaturated fatty acid) are the most abundant free fatty acids in the human circulation. To determine if unsaturated fatty acid oleate has a similar effect on NNMT upregulation and PPARγ transactivation in hepatocytes, we treated AML12 with oleate (0.4 mM) for 16 h and examined the mRNA levels of NNMT, PPARγ, as well as three signature PPARγ-target genes by real-time qPCR. As shown in Fig. 3A, no changes in NNMT gene expression were observed in response to oleate exposure. In contrast to what observed from palmitate exposure experiments, oleate treatment indeed downregulated PPARγ expression (Fig. 3B); however, it failed to affect the expression of three signature PPARγ-target genes measured (Fig. 3C).

Figure 3.

Oleate exposure fails to induce PPARγ transactivation and NNMT expression in hepatocytes. AML12 hepatocytes were exposed to 0.4 mM oleate for 16 h. Both NNMT (A) and PPARγ (B) gene expression was determined by qPCR. C: the mRNA levels of signature PPARγ target genes, Plin2, Cidec, and Fabp4 were measured by real-time qPCR. Data were expressed as means ± SD, n = 4 separate experiments. Differences between groups were determined by two-tail unpaired Student’s t test (*P<0.05 vs. control). AML12, alpha mouse liver 12; NNMT, nicotinamide N-methyltransferase; OA, oleate; PPARγ, peroxisome proliferator-activated receptor γ; UT, untreated.

NNMT Upregulation Contributes to Palmitate-Induced PPARγ Activation

Hepatic PPARγ activation plays a pivotal role in the regulation of lipid metabolism and the development of various fatty liver diseases (4). We previously reported that the genetic knockdown of NNMT was protective against fatty liver development in response to chronic alcohol exposure in mice (38). Our observations that palmitate exposure of hepatocytes causes both PPARγ transactivation and NNMT upregulation prompted us to explore the potential causal relationship between these two events. We first pretreated AML12 cells with either JBSNF000088 or II399 (chemical inhibitors for NNMT) for 4 h before palmitate exposure for 16 h (40, 41). PPARγ transactivation status was determined by ELISA assay and the detection of PPARγ signature target genes via real-time qPCR, respectively. As shown in Fig. 4A, both NNMT inhibitors blunted palmitate-elicited PPARγ activation. Accordingly, NNMT inhibitors compromised palmitate-elicited upregulation of Pparg, Cd36, Plin2, and Cidec (Fig. 4B). To consolidate this observation, we then transfected AML12 cells with either scrambled siRNA (siCtrl) or siRNA for NNMT (siNnmt) for overnight, followed by palmitate exposure for 16 h. In line with the observations from chemical inhibitor experiments, genetic NNMT knockdown attenuated palmitate-elicited PPARγ transactivation as well (Fig. 4B). Finally, we also examined the effect of PPARγ activation on NNMT expression via transfecting AML12 cells with either control siRNA (siCtrl) or siRNA for PPARγ (siPparg) for overnight, followed by palmitate exposure for 16 h. As shown in Fig. 4C, PPARγ siRNA knockdown alleviated palmitate-induced NNMT upregulation, suggesting the existence of a feed-forward regulatory loop between PPARγ activity and NNMT expression, which amplifies the palmitate-triggered PPARγ transactivation in hepatocytes.

Figure 4.

NNMT upregulation contributes to palmitate-induced PPARγ transactivation in AML12 hepatocytes. A: pharmacological inhibition of NNMT blunted palmitate-induced PPARγ transactivation in AML12 hepatocytes. AML12 cells were pretreated with or without NNMT inhibitor JBSNF000088 (10 μM) or II399 (10 μM) for 4 h before palmitate (0.4 mM) exposure for 8 h. Nuclear proteins were extracted and subjected to ELISA assay for PPARγ transactivity. Data are expressed as means ± SD, n = 3 separate experiments. Differences between groups were determined by one-way ANOVA with Tukey’s post hoc test. Bars with different letters differ significantly (P < 0.05). B: pharmacological inhibition of NNMT blunted palmitate-induced PPARγ target gene upregulation in AML12 hepatocytes. AML12 cells were pretreated with or without NNMT inhibitor JBSNF000088 (10 μM) or II399 (10 μM) for 4 h before palmitate (0.4 mM) exposure for 16 h. The mRNA levels of PPARγ and PPARγ target genes, Cd36, Plin2, Cidec, and Fabp4, were measured by real-time qPCR. Data are expressed as means ± SD, n = 3 separate experiments. Differences between groups were determined by one-way ANOVA with Tukey’s post hoc test. Bars with different letters differ significantly (P < 0.05). C: genetic knockdown of NNMT gene attenuated palmitate-induced PPARγ transactivation in AML12 hepatocytes. AML12 hepatocytes were transfected with either scrambled siRNA or NNMT siRNA for 24 h before palmitate (0.4 mM) exposure for 16 h. The mRNA levels of NNMT, PPARγ, as well as four PPARγ target genes (Cd36, Plin2, Cidec, and Fabp4) were determined by real-time qPCR. Data are expressed as means ± SD, n = 5 separate experiments. Differences between groups were determined by one-way ANOVA with Tukey’s post hoc test. Bars with different letters differ significantly (P < 0.05). D: PPARγ gene knockdown alleviated palmitate-induced NNMT upregulation in AML12 hepatocytes. AML12 cells were transfected with either scrambled siRNA or PPARγ siRNA for 24 h before being treated with palmitate (0.4 mM) for 16 h. Both NNMT and PPARγ mRNA levels were quantified by real-time qPCR. All data are expressed as means ± SD, n = 4 separate experiments. Differences between groups were determined by one-way ANOVA with Tukey’s post hoc test. Bars with different letters differ significantly for an individual gene (P < 0.05). AML12, alpha mouse liver 12; NNMT, nicotinamide N-methyltransferase; PA, palmitate; PPARγ, peroxisome proliferator-activated receptor γ; UT, untreated.

Intracellular Metabolism of Palmitate is Required for Both NNMT Upregulation and PPARγ Transactivation

The role of palmitate intracellular metabolism in NNMT expression and PPARγ transactivation was subsequently investigated. Acyl-CoA synthetase catalyzes the initial step of intracellular palmitate metabolism, converting palmitate to palmitoyl-CoA. To test whether the intracellular metabolism is required for palmitate-elicited NNMT upregulation and PPARγ transactivation, we pretreated AML12 cells with Triacsin C (Tri C, 10 µM), a chemical inhibitor of long fatty acid acyl-CoA synthetase, for 2 h before palmitate exposure for 16 more hours. As shown in Fig. 5A, acyl-CoA synthetase inhibition by Tri C blunted palmitate-elicited NNMT upregulation. In accordance, Tri C pretreatment attenuated palmitate-elicited PPARγ expression at both mRNA (Fig. 5B) and protein levels (Fig. 5, C and D). Correspondingly, Tri C attenuated palmitate-elicited PPARγ transactivation, evidenced by the compromised increase of all tested signature PPARγ-target genes (Fig. 5B). To further corroborate this notion, another chemical inhibitor of long fatty acid acyl-CoA synthetase, 2-fluoropalmitic acid (FP, 10 and 20 µM, respectively), was used. As shown in Fig. 5E, FP pretreatments attenuated palmitate-elicited upregulation of PPARγ target genes, confirming that the conversion to palmitoyl-CoA is required for palmitate-elicited PPARγ transactivation.

Figure 5.

Intracellular palmitate metabolism is required for both NNMT upregulation and PPARγ transactivation. AML12 hepatocytes were pretreated with or without Triacsin C (Tri C, 10 µM), a chemical inhibitor of long chain fatty acyl-CoA synthetase, for 2 h before palmitate (0.4 mM) exposure for 16 h. A: NNMT gene expression. B: mRNA levels of PPARγ and its target genes, Cd36, Plin2, Cidec, and Fabp4. C and D: protein abundance of PPARγ and β-actin were determined by Western blotting and densitometric analysis of the ratio of PPARγ to actin conducted. Data are expressed as means ± SD, n = 3–4 separate experiments. Differences between groups were determined by one-way ANOVA with Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001 vs. control). E: AML12 hepatocytes were pretreated with or without 2-fluoropalmitic acid (FP, 10 and 20 mM, respectively), another chemical inhibitor of long chain fatty acyl-CoA synthetase, for 2 h before palmitate (0.4 mM) exposure for 16 h. mRNA levels of PPARγ and its target genes. Data are expressed as means ± SD, n = 4 separate experiments. Differences between groups were determined by one-way ANOVA with Tukey’s post hoc test. Bars with different letters differ significantly for an individual gene (P < 0.05). AML12, alpha mouse liver 12; NNMT, nicotinamide N-methyltransferase; PA, palmitate; PPARγ, peroxisome proliferator-activated receptor γ; UT, untreated.

Intracellular NAD Decline Due to NNMT Overexpression Contributes to Palmitate-Elicited PPARγ Deactivation in Hepatocytes

Nicotinamide is the predominant precursor for cellular NAD⁺ regeneration via the salvage pathway of NAD⁺ biosynthesis (Fig. 2A). NNMT competes with nicotinamide phosphoribosyltransferase (NAMPT), the rate-limiting enzyme in the salvage pathway, for substrate nicotinamide. Therefore, NNMT overexpression/overactivation has the potential to reduce intracellular NAD⁺ concentrations. We previously reported that NNMT overexpression led to intracellular total NAD reduction in AML12 cells, whereas NNMT knockdown in both hepatocytes and mice was associated with incremental hepatic total NAD contents (38). The observation that palmitate upregulates NNMT expression prompted us to posit that palmitate exposure can decrease cellular total NAD contents. To test our hypothesis, we measured intracellular total NAD contents in AML12 cells after a 16-h palmitate exposure. As expected, palmitate exposure significantly decreased cellular total NAD concentration (Fig. 6A). To determine whether reduced intracellular NAD levels contribute to palmitate-triggered PPARγ transactivation, we pretreated AML12 cells for 2 h with two well-established NAD⁺-enhancing agents, nicotinamide riboside (NR) and nicotinamide (NAM; Fig. 2A), respectively, followed by palmitate exposure for 16 h. As shown in Fig. 6B, both NR and NAM pretreatment blunted palmitate-induced PPARγ transactivation. Importantly, both NR and NAM pretreatment protected hepatocytes against palmitate-induced cell death in AML12 cells (Fig. 6C).

Figure 6.

Intracellular NAD decline contributes to palmitate-induced PPARγ transactivation and cell death. A: palmitate exposure decreased cellular total NAD contents in AML12 hepatocytes. AML12 cells were treated with 0.4 mM palmitate for 16 h. Intracellular total NAD contents were measured. Data are shown as means ± SD, n = 4 separate experiments. Two-tail unpaired Student’s t test was used for statistical evaluation (*P < 0.05 vs. control). B: NAD replenishment prevented palmitate-induced PPARγ transactivation. AML12 hepatocytes were pretreated with or without nicotinamide riboside (NR, 5 mM) or nicotinamide (NAM, 5 mM) for 2 h before palmitate exposure (0.4 mM) for 16 h. The mRNA levels of PPARγ and its target genes, Plin2 and Cidec, were determined by real-time qPCR. C: NAD replenishment prevented palmitate-induced cell death. AML12 hepatocytes were pretreated with or without nicotinamide riboside (NR, 5 mM) or nicotinamide (NAM, 5 mM) for 2 h before palmitate exposure (0.4 mM) for 16 h. Cell death was determined by LDH release measurement. Data are expressed as means ± SD, n = 4 separate experiments. Differences between groups were determined by one-way ANOVA with Tukey’s post hoc test (*P < 0.05, **P < 0.01, ***P < 0.001 vs. control). AML12, alpha mouse liver 12; PA, palmitate; PPARγ, peroxisome proliferator-activated receptor γ; UT, untreated.

The PPARγ Transactivation Partially Contributes to Palmitate-Induced Lipotoxicity in Hepatocytes

We previously reported that NNMT inhibition protected hepatocytes against palmitate-induced cell death in hepatocytes (42). To determine the role of PPARγ transactivation in palmitate-induced lipotoxicity, we transfected AML12 hepatocytes with either scrambled siRNA (siCtrl) or siRNA for PPARγ (siPparg) for overnight, followed by palmitate exposure for 16 h. The genetic knockdown of PPARγ resulted in a significant, albeit subtle, reduction of intracellular fat contents in hepatocytes at both basal (Fig. 7A) and palmitate-exposed conditions (Fig. 7B). Similarly, Pparg gene knockdown conferred partial protection against palmitate-induced cell death (Fig. 7C).

Figure 7.

The PPARγ transactivation minimally contributes to palmitate-induced lipotoxicity in hepatocytes. A: AML12 hepatocytes were transfected with either scramble or PPARγ siRNA for 24 h. Intracellular triacylglycerol (TG) contents were determined. B and C: AML12 hepatocytes were transfected with either scrambled siRNA or siRNA for PPARγ for overnight before palmitate (0.4 mM) exposure for 16 h. Intracellular TG contents (B) and media LDH levels (C) were determined. All data were expressed as means ± SD, n = 3 separate experiments. Differences between groups were determined by two-tail unpaired Student’s t test (*P < 0.05, **P < 0.01 vs. control). D: schematic illustration of the role and mechanism of NNMT upregulation in palmitate-induced hepatic PPARγ transactivation. Cellular NAD⁺ decline resulting from NNMT upregulation contributes to palmitate-elicited PPARγ transactivation in hepatocytes. ACSLs, long-chain acyl-CoA synthetases; AML12, alpha mouse liver 12; MNA, methylnicotinamide; NAM, nicotinamide; NAMPT, nicotinamide phosphoribosyltransferase; NNMT, nicotinamide N-methyltransferase; PPARγ, peroxisome proliferator-activated receptor γ.

DISCUSSION

Palmitate (a 16-carbon saturated fatty acid) and oleate (an 18-carbon monounsaturated fatty acid) are the most abundant free fatty acids in human circulation. We previously documented that exogenous palmitate exposure upregulated hepatic NNMT expression, which contributes to palmitate-induced lipotoxicity in hepatocytes (42). In the present study, we provided initial evidence that NNMT upregulation contributes to palmitate-elicited PPARγ transactivation in hepatocytes. Our data demonstrated that palmitate-induced PPARγ transactivation was blunted by both pharmacological and genetic NNMT inhibition, suggestive of mechanistic involvement of NNMT upregulation in palmitate-induced PPARγ transactivation. Noteworthily, our data also unraveled that a feedforward regulatory mechanism exists between NNMT expression and PPARγ activation in that PPARγ inhibition also compromised palmitate-induced NNMT upregulation. Our further investigations identified intracellular NAD⁺ decline resulting from NNMT upregulation as a potential mechanism accounting for palmitate-elicited PPARγ transactivation since NAD⁺ replenishment obstructed palmitate-induced PPARγ transactivation (Fig. 7D). At last, we obtained data suggesting that the PPARγ transactivation only minimally contributes to palmitate-induced intracellular triacylglycerol accumulation and cell death.

PPARγ has many natural modulators/ligands, including fatty acids. In cancer cell lines, polyunsaturated fatty acids (PUFAs; mainly arachidonic acid, eicosatetraenoic, and docosahexaenoic acid) increased PPARγ expression, resulting in inhibited proliferation and enhanced apoptosis of cancer cells (27, 43). Long-chain monounsaturated fatty acids-supplemented diet elevated PPARγ mRNA expression in mice adipose tissue (28). In hepatocytes, phytanic acid seems to be a strong ligand of PPARγ (29). Therefore, it is unequivocal that fatty acids affect PPARγ expression/transactivation in both fatty acid- and cell type-specific manner. Consistent with the previous study that palmitate, but not oleate, upregulated PPARγ in Huh7 cells and NAFLD mice model (30), our current study, wherein both nontransformed AML12 and primary mouse hepatocytes were used, provided direct evidence supporting that palmitate is a PPARγ activator in hepatocytes.

The observation that exogenous palmitate exposure upregulated NNMT expression at both mRNA and protein levels is consistent with our very recent report that NNMT upregulation contributed to palmitate-induced lipotoxicity in cultured hepatocytes (42). The observation that palmitate exposure instigated both NNMT upregulation and PPARγ transactivation led us to explore the potential causal link between these two events. The time-course study clearly demonstrated that in response to palmitate stimulation, NNMT upregulation appeared at an earlier time point (4-h; Fig. 2) than that for PPARγ gene expression (Fig. 1). Importantly, NNMT inhibition via both pharmacological and genetic approach blunted palmitate-elicited PPARγ transactivation, indicating that NNMT upregulation is an upstream event attributing to palmitate-induced PPARγ transactivation. Intriguingly, we also found that PPARγ gene knockdown attenuated palmitate-induced NNMT upregulation. Despite being somewhat unexpected, this observation points to the existence of a feedforward regulatory loop between NNMT expression and PPARγ transactivation when facing palmitate challenge, which amplifies the promotive effect of palmitate on PPARγ transactivity and consequential lipid metabolism.

Cellular catabolism of palmitate begins with a long-chain acyl-CoA synthetase-catalyzed reaction, which converts palmitate to palmitoyl-CoA before the mitochondrial entrance for β-oxidation. We previously documented that the metabolic conversation of palmitate to palmitoyl-CoA was indispensable for palmitate-elicited mechanistic target of rapamycin complex 1 (mTORC1)-ATF4 pathway activation and resultant NNMT upregulation (42). Here, a similar scenario was exhibited for palmitate-triggered PPARγ transactivation as the inhibition of long-chain acyl-CoA synthetase with its specific inhibitor, Triacsin C, attenuated not only NNMT upregulation but also PPARγ transactivation in response to palmitate exposure, suggesting that palmitate metabolism is required for inducing PPARγ transactivation.

NNMT is a methyltransferase, catalyzing nicotinamide methylation and subsequent degradative process (Fig. 2A). It has been well-established that nicotinamide is the predominant precursor for intracellular NAD⁺ biosynthesis via the salvage pathway, although NAD⁺ can also be synthesized from tryptophan via the de novo pathway. Nicotinamide phosphoribosyltransferase (NAMPT) is the first and the rate-limiting enzyme in the salvage pathway, converting nicotinamide to nicotinamide mononucleotide (NMN). As NNMT-catalyzed methylation reaction is the principal catabolic pathway for nicotinamide degradation, NNMT competes with NAMPT for nicotinamide availability. As a result, altered NNMT expression/activity may exert a profound impact on intracellular NAD⁺ homeostasis. In fact, we have demonstrated in our previous studies that NNMT overexpression in hepatocytes resulted in a significant reduction of intracellular NAD⁺ consents, whereas NNMT knockdown was associated with incremental hepatic NAD⁺ levels in both cultured hepatocytes and mice (38). In attempting to quest the mechanism(s) whereby NNMT upregulation mediates palmitate-triggered PPARγ transactivation, we examined the effect of altered intracellular NAD⁺ levels on palmitate-induced PPARγ transactivation and uncovered that intracellular NAD⁺ decline links NNMT upregulation to PPARγ transactivation in response to palmitate exposure. Our observations that palmitate exposure decreased intracellular total NAD⁺ levels and NAD⁺ replenishment, by either nicotinamide (NAM) or nicotinamide riboside (NR) supplementation, efficiently forestalled palmitate-induced PPARγ transactivation suggest that intracellular NAD⁺ decline due to NNMT overexpression contributes, at least partially, to palmitate-triggered PPARγ transactivation in hepatocytes. Given that intracellular NAD⁺ is a physiological activation of Sirt1, the founding member of the family of class III histone deacetylases (44, 45), and Sirt1 negatively mediates PPARγ activity (46), further studies are warranted to further elucidate whether Sirt1 inhibition is mechanistically involved in NAD⁺ reduction-mediated PPARγ transactivation in response to palmitate exposure.

Although NNMT inhibition has been reported by us to be protective against palmitate-induced cell death in hepatocytes (42), the role of PPARγ transactivation in palmitate-induced lipotoxicity remains ambiguous. Considering its critical role in cellular triacylglycerol synthesis and lipid droplet formation, it is reasonable to speculate that PPARγ transactivation would contribute to intracellular fat accumulation upon palmitate exposure. As anticipated, we showed here that PPARγ inhibition significantly, despite slightly, decreased cellular triacylglycerol contents at both basal and palmitate-supplemented conditions. It is generally accepted that the promotion of cellular triacylglycerol synthesis is a protective mechanism against cell death induced by saturated fatty acids, such as palmitate (47, 48). As a matter of fact, several reports have documented that PPARγ agonists exert a protective role against palmitate-induced cell death (31, 49, 50). Here, we found that PPARγ inhibition by siRNA transfection indeed provided a subtle protection against palmitate-induced cell death in AML12 hepatocytes (Fig. 7). Intriguingly, we also found that the pretreatment with rosiglitazone, a PPARγ agonist, also protected AML12 cells against palmitate-induced death (unpublished data). Although we are currently unable to provide a clear explanation for these contradictory observations, our data suggest that although NNMT upregulation is required for palmitate-elicited PPARγ transactivation, the PPARγ deactivation seems not to play a principal role in the antilipotoxic effect of NNMT inhibition against palmitate hepatotoxicity.

In conclusion, we here provide the first-line evidence supporting that palmitate is a PPARγ activator in hepatocytes and NNMT upregulation contributes to palmitate-elicited hepatic PPARγ transactivation. Our data unraveled that the intracellular NAD⁺ decline resulting from NNMT overexpression plays a mechanistic role in palmitate-elicited PPARγ transactivation. Given the well-established pathological role of hepatic PPARγ transactivation in fatty liver development, our study suggests the potential clinical benefits of NNMT inhibitors for the treatment of metabolic liver diseases, including both ALD and NAFLD.

DATA AVAILABILITY

Data will be made available upon reasonable request.

GRANTS

This work was in part funded by US NIH Grants NIAAA R01AA026603 (to Z.S.), NIAAA R01AA030255 (to Z.S.), and NIDDK R01DK131038 (to J.C-.C.).

DISCLOSURES

No conflicts of interest, financial or otherwise, are declared by the authors.

AUTHOR CONTRIBUTIONS

Q.S., R.H., and Z.S. conceived and designed research; Q.S., J.W., A.G., S.M.L., and I.D.I. performed experiments; Q.S., J.W., and A.G. analyzed data; Q.S., J.W., R.H., J.C-.C., and Z.S. interpreted results of experiments; Q.S., J.W., A.G., and S.M.L. prepared figures; Q.S. and J.W. drafted manuscript; R.H., J.C-.C., and Z.S. edited and revised manuscript; Z.S. approved final version of manuscript.