The citrus flavonoid nobiletin confers protection from metabolic dysregulation in high-fat-fed mice independent of AMPK

Abbreviations: ACC, acetyl-CoA carboxylase, AMPK, adenosine monophosphate activated kinase; AUC, area under the curve; BAT, brown adipose tissue; Cpt1, carnitine palmitoyltransferase 1; CE, cholesteryl ester; eWAT, epididymal white adipose tissue; FA, fatty acid, FC, free cholesterol; FPLC, fast-protein liquid chromatography; HFHC, high fat, cholesterol-containing; iWAT, inguinal white adipose tissue; Ldlr, low density lipoprotein receptor: Pcg1a, peroxisome proliferator receptor gamma co-activator 1a; RER, respiratory exchange ratio; SEM, standard error of the mean; Srebp-1c, sterol response element binding protein 1c; TC, total cholesterol; TG, triglyceride ABSTRACT Obesity, dyslipidemia, and insulin resistance-the increasingly common metabolic syndrome-are risk factors for cardiovascular disease and type 2 diabetes that warrant novel therapeutic interventions. The flavonoid nobiletin displays potent lipid-lowering and insulin-sensitizing properties in mice with metabolic dysfunction. However, the mechanisms by which nobiletin mediates metabolic protection are not clearly established. The central role of AMP-activated protein kinase (AMPK) as an energy sensor suggests that AMPK is a target of nobiletin. We tested the hypothesis that metabolic protection by nobiletin required phosphorylation of AMPK and acetyl-CoA carboxylase (ACC) in mouse hepatocytes, in mice deficient in hepatic AMPK ( (cid:36)(cid:80)(cid:83)(cid:78)(cid:533)(cid:20) -/- ) , in mice incapable of inhibitory phosphorylation of ACC ( AccDKI ) and in mice with adipocyte-specific AMPK deficiency ( i (cid:533) 1 (cid:533) 2AKO ). We fed mice a high-fat/high-cholesterol diet with or without nobiletin. Nobiletin increased phosphorylation of AMPK and ACC in primary mouse hepatocytes, which was associated with increased fatty acid (FA) oxidation and attenuated FA synthesis. Despite loss of ACC phosphorylation in (cid:36)(cid:80)(cid:83)(cid:78)(cid:533)(cid:20) -/- hepatocytes, nobiletin suppressed FA synthesis and enhanced FA oxidation. Acute injection of nobiletin into mice did not increase phosphorylation of either AMPK or ACC in liver. In mice fed a high-fat diet, nobiletin robustly prevented obesity, hepatic steatosis, dyslipidemia and insulin resistance, and it improved energy expenditure in (cid:36)(cid:80)(cid:83)(cid:78)(cid:533)(cid:20) -/- , AccDKI and i (cid:533) 1 (cid:533) 2AKO mice to the same extent as in wild-type controls. Thus, the beneficial metabolic effects of nobiletin in vivo are conferred independently of hepatic or adipocyte AMPK activation. These studies further underscore the therapeutic potential of nobiletin and begin to clarify possible mechanisms. appropriate secondary anti-Rabbit anti-Mouse IgG Donkey anti-Rabbit IRDye® anti-Rabbit blots using HRP-linked antibodies, detection was determined using enhanced chemiluminescence reagent and quantification performed using an Imaging Densitometer For blots using LI-COR secondary antibodies, imaging and quantitation immunoblots the LI-COR nobiletin-mediated of metabolic in HFHC fed-AccDKI mice and in WT clearly demonstrating that the protective effects of nobiletin were independent of inhibitory phosphorylation of ACC. These results differ from the effects of metformin. In contrast to nobiletin, HFD-fed AccDKI mice were refractory to the lipid-lowering and insulin-sensitizing effects of metformin and metformin had no effect on body weight or adiposity in either genotype (36). These findings imply that the mechanisms underlying nobiletin-induced metabolic protection differ from those of metformin.


INTRODUCTION
5 AMP-activated protein kinase (AMPK) is a gulation cellular energy homeostasis (12,13). Multiple levels of hormonal, nutritional and cytokine stimuli mediate the activation of AMPK in most tissues, leading to inhibition of anabolic processes and stimulation of ATP-generating catabolic processes (14,15). Specifically, phosphorylation of the alpha catalytic subunit of AMPK at Thr172, results in inhibitory phosphorylation of acetyl-CoA carboxylase (ACC)1 at Ser79 and ACC2 at Ser212, which decreases the conversion of acetyl-CoA to malonyl-CoA, the rate-limiting step in de novo FA synthesis (14). Malonyl-CoA also functions as an allosteric inhibitor of CPT1, a protein that facilitates the rate-limiting transport of FA into the mitochondria for FA oxidation (14). Thus, AMPK-mediated phosphorylation of ACC not only suppresses FA synthesis, but also relieves the repression of FA oxidation by malonyl-CoA. AMPK indirectly upregulates FA oxidation by increasing mitochondrial (15). AMPK also mediates suppression of fatty acid synthase (FAS) through phosphorylation and inactivation of SREBP-1c (16).
A variety of drugs, xenobiotics, polyphenols and flavonoids activate AMPK, including A-769662, salicylate, metformin, berberine, quercetin, resveratrol, and genistein (17)(18)(19). Enhanced phosphorylation of AMPK and ACC in HepG2 cells or primary mouse hepatocytes by metformin, A-769662 or resveratrol increased FA-oxidation, decreased FA-synthesis and reduced cellular TG (18,20), effects similar to HepG2 cells exposed to nobiletin (7). Recent studies in cultured HepG2 cells reported that nobiletin blunted palmitate-induced lipogenesis and inhibited the protein expressions of SREBP-1c and FAS via phosphorylation of AMPK and ACC (21,22). Also, the metabolic protection associated with nobiletin treatment in mouse models is similar to the effects of pharmacological activation of AMPK, as observed (23)(24)(25). Taken together, these observations suggest that AMPK activation may be a target of nobiletin. One of the objectives of the present study was to determine the requirement of nobiletin to activate AMPK and improve lipid metabolism in cultured hepatocytes, in mouse liver following acute nobiletin administration, and in 6 chronically treated mice with or without genetic inactivation of hepatic AMPK ( -/-) or phosphorylation-defective ACC1 and 2 (AccDKI).
Current understanding of the role of AMPK in adipose tissue is primarily based on studies conducted in cultured cells (26)(27)(28) and in mice lacking single subunits of AMPK, that retain significant residual AMPK activity (29)(30)(31). Recently, Motillo et al reported that inducible deletion of both in mouse adipocytes ( -adrenergic agonists to stimulate browning of white adipose tissue (WAT) and amplified diet-induced hepatic steatosis and insulin resistance (32). In cultured 3T3-L1 white adipocytes, nobiletin was shown to stimulate browning (33), reduce cellular TG content, and inhibit adipogenesis through phosphorylation of AMPK (33)(34)(35). Therefore, we hypothesized that activation of adipocyte AMPK was an integral part of nobiletin's mechanism of action, and that adipocyte-specific AMPK deficiency would compromise metabolic protection mediated by nobiletin.
In primary C57BL/6 (WT) hepatocytes, nobiletin increased the phosphorylation of AMPK and ACC, which was associated with suppressed FA synthesis and increased FA oxidation. Despite the loss of ACC phosphorylation in -/hepatocytes, nobiletin was still able to suppress FA synthesis and enhance FA oxidation. Acute injection of nobiletin into mice did not increase phosphorylation of either AMPK or ACC in liver. In mice fed a high-fat diet, nobiletin supplementation robustly prevented metabolic dysregulation in -/-, AccDKI and i 1 2AKO mice to the same extent as in wild-type controls.
Thus, metabolic protection by nobiletin in vivo is conferred independently of hepatic or adipocyte AMPK. 7

Animals and diets
Male Ldlr -/mice on a C57BL/6J background (Jackson Laboratory, Bar Harbor, ME) were bred in-house.
Male -/mice on a C57BL/6J background and littermate controls were generated as described previously (30).

Liver lipid analyses
Lipids from liver dissected free of fat and connective tissue and stored at -80 o C were extracted as described previously (10).

Glucose and insulin tolerance tests
Glucose tolerance tests were performed following a 6 h fast by intraperitoneal injection (i.p.) with 15% glucose in 0.9% NaCl (1 g/kg body weight) (6). Blood samples for glucose analyses (glucometer) were taken up to 120 min post-injection. An insulin tolerance test was conducted following a 5 h fast by i.p.

11
Blood samples for glucose analyses were obtained up to 60 min post-injection. Glucose tolerance was determined from the incremental change in blood glucose from baseline concentrations and insulin tolerance was determined from the percent change in blood glucose from baseline concentrations (10).

Metabolic cage studies
Energy expenditure, respiratory exchange ratio (RER) and ambulatory activity were assessed using the Comprehensive Laboratory Animal Monitoring System (CLAMS; Columbus Instruments, Columbus, OH) as described previously (10). Mice were housed in metabolic cages with free access to food and water and acclimatized for 48 h. For the subsequent 24 h, every 10 min, data on O 2 consumption (VO 2 ; milliliters per hour) and CO 2 production (VCO 2 ; milliliters per hour) were collected. The RER was derived from the ratio of VCO 2 to VO 2 , and energy expenditure was determined as (3.815 + 1.232 × RER) × VO 2 and expressed as ANCOVA-adjusted energy expenditure in kilocalories per hour. ANCOVA adjustments to energy expenditure were made based on body weight, which allowed for the determination of differences in energy expenditure independent of group differences in body weight (10). Ambulatory activity was measured as infrared beam breaks in the X, Y, and Z axes per hour.

Activation of AMPK in vivo
Activation of AMPK in vivo was assessed in chow-fed WT, -/and Ldlr mice following i.p.

Immunoblotting
Cell lysates from HepG2 cells or primary mouse hepatocytes were prepared using a minor modification of a previously described method (39,40). Briefly, total cell or tissue lysates from snap frozen, freeze- 13

Gene expression
RNA was extracted from iWAT tissue using TriZol reagent and was reverse-transcribed to cDNA using previously published methods (6). PCR primers and TaqMan probes for Ucp1, Cd137, Tbx1, Serca2b, Ampkb1, Ampkb2 and Ppia were obtained from Life Technologies (Burlington, ON). mRNA expression of each gene was determined by quantitative real-time PCR on an ABI ViiA 7 detection system (Applied Biosystems, Streetsville, CA) using the standard curve method, as previously published (6). mRNA expression levels were normalized to the expression of Ppia (32).

Statistical analysis
Data is presented as mean ± SEM. Statistical analyses were performed using GraphPad Prism 8. A oneway ANOVA with post hoc Tukey's test was used to test for differences between groups, except in the case of parameters measured over time where a two-way repeated measures ANOVA with post hoc Tukey's test was used. Significance thresholds were P < 0.05. Different letters indicate significant differences. A Student's t-test was used to assess differences in ANCOVA-adjusted energy expenditure; an asterisk indicates significant differences (P < 0.05).

Nobiletin reduces lipogenesis and increases fatty oxidation independent of AMPK in primary mouse hepatocytes
As the metabolic protection by nobiletin has been demonstrated in mouse models, we next examined the ability of nobiletin to increase the phosphorylation of AMPK and ACC in primary mouse hepatocytes from C57BL/6J mice. Initial dose-response studies in isolated hepatocytes cultured in normal glucose media showed that nobiletin increased pAMPK (2-fold) and increased pACC ( The functional significance of AMPK activation by nobiletin was assessed in hepatocytes isolated from -/at WT mice. The ability of nobiletin, A769662 and salicylate to increase the phosphorylation of ACC was lost in -/hepatocytes, indicating that ACC phosphorylation was dependent on AMPK ( Fig. 1F). However, nobiletin suppressed lipogenesis to the same extent in both WT (-31%) and -/-(-29%) hepatocytes (Fig. 1G). In contrast, lipogenesis was inhibited by metformin (-69%) and A-769662 (-77%) in WT, but not in -/hepatocytes. Nobiletin increased palmitate oxidation similarly (24%) in hepatocytes from both WT and -/mice, whereas salicylate and A-769662 increased palmitate oxidation in WT hepatocytes (32% and 18%, respectively) but had no effect in -/hepatocytes ( Fig. 1H). This suggests that nobiletin's ability to decrease lipogenesis and stimulate FA-oxidation in primary hepatocytes is independent of AMPK or ACC phosphorylation.

Nobiletin does not acutely activate AMPK in vivo
Acute administration of salicylate and A-769662 to mice has been shown to activate hepatic AMPK and ACC (38). The ability of nobiletin to acutely activate AMPK in vivo was evaluated using a fasting, feeding, i.p. injection and re-fasting protocol (23) in chow-fed C57BL/6 WT, chow-fed Ampk -/and HFHC-fed Ldlr -/mice. In livers isolated 90 min after the injection of nobiletin, the phosphorylation of AMPK or ACC were not affected in chow-fed WT mice, chow-fed -/mice or HFHC-diet-fed Fig. S1A-E). Under the same conditions, A-769662 had no effect on hepatic ACC phosphorylation in chow-fed -/mice, but increased phosphorylation of AMPK and ACC in the other two groups of mice (Supplemental Fig. S1A-E). This suggests that nobiletin's ability to confer metabolic protection is not through acute activation of AMPK.

Nobiletin prevents metabolic dysregulation in HFHC diet-fed -/mice
Treatment of mice with nobiletin, increases energy expenditure, induces weight loss, lowers insulin resistance and decreases liver and plasma lipids (7); effects that are analogous to pharmacological activation of AMPK (13,18,(23)(24)(25). To investigate whether metabolic protection by nobiletin was mediated through AMPK , WT and -/littermates starting at 10 weeks of age were fed a HFHC diet with or without nobiletin for 12 weeks.
-/mice have a ~90% reduction in liver AMPK activity (30) and as anticipated levels of AMPK, pAMPK and pACC in the livers of -/mice were markedly reduced compared to WT mice ( Fig. 2A). Mice lacking hepatic AMPK gained a similar amount of weight in response to the HFHC diet as the WT mice (Fig. 2B). Nobiletin supplementation prevented HFHC diet-induced weight gain in -/to the same extent as in WT mice. Caloric intake was unaffected by nobiletin in both genotypes (Fig. 2C). The HFHC diet increased eWAT depots to similar levels in -/and WT mice (Fig. 2D). The striking reduction in eWAT in nobiletin supplemented mice (~56%) was similar in each genotype (Fig. 2D).
Energy expenditure and RER did not differ between HFHC-fed -/and WT mice (Fig. 2E, F).
Nobiletin induced a ~20% increase in ANCOVA-adjusted energy expenditure in both WT and  (Fig. 2J, K). Nobiletin had no effect on HDL-C in controls, but decreased HDL-C by 46% in -/mice (Fig. 2L).
There was no difference in fasting blood glucose or glucose tolerance between -/and WT mice ( Fig. 3A,B). Addition of nobiletin to the HFHC diet decreased fasting blood glucose (-17%) and improved glucose tolerance (46%) in WT mice. In -/mice, nobiletin also decreased fasting blood glucose (-21%) and improved glucose tolerance (33%), although the decrease in iGTT AUC in -/mice did not reach statistical significance (Fig. 3C). Insulin resistance was similar between -/and WT mice (Fig. 3E, F) and nobiletin decreased plasma insulin (~80%) and improved insulin tolerance AUC (~29%) to a similar degree in both -/and WT mice. Nobiletin induced striking reductions in hepatic TG and CE in both genotypes, although the extent of reduction was greater in control mice (Fig.   3G,H). Nobiletin supplementation increased hepatic FA oxidation in both genotypes (Fig. 3I).

Nobiletin attenuates hepatic steatosis and metabolic dysregulation in HFHC diet-fed AccDKI mice
AMPK, phosphorylates and inhibits ACC thereby reducing malonyl-CoA, a critical substrate for de novo lipogenesis and inhibitor of FA oxidation (12,13) AMPK activity in hepatocytes (30) and macrophages (41), residual AMPK in other tissues may be important for mediating the beneficial effects of nobiletin in vivo. Therefore, to assess the importance of AMPK phosphorylation of ACC in the metabolic protection mediated by nobiletin, we utilized AccDKI mice harboring alanine knock-in mutations at the AMPK phosphorylation sites in both Acc1 and Acc2 (36), which effectively prevent the inhibitory phosphorylation of ACC by activated AMPK in all tissues.
WT and AccDKI littermate mice were fed a HFHC diet with or without nobiletin supplementation for 18 weeks. Consistent with previous studies (36), phosphorylated ACC was completely absent in the livers of AccDKI mice fed either HFHC or HFHC supplemented with nobiletin (Fig. 4A). pAMPK and AMPK were not consistently affected by genotype or diet. AccDKI mice gained the same amount of body weight in response to the HFHC diet as in WT mice (Fig. 4B). Nobiletin prevented diet-induced weight gain in both AccDKI and WT mice. Caloric intake was unaffected by nobiletin treatment (Fig. 4C). eWAT depot weights were similar between AccDKI and WT mice and were markedly reduced with nobiletinsupplementation (~50%) in both genotypes (Fig. 4D).
Energy expenditure, RER and activity levels were not different between HFHC-fed AccDKI and WT mice ( Fig. 4E-G). Nobiletin induced a ~20% increase in total ANCOVA-adjusted energy expenditure in WT and AccDKI mice (Fig. 4E). RER and activity levels were unaffected by genotype or nobiletin treatment 18 (Fig. 4F,G). Plasma total cholesterol and TG were not different between HFHC-fed AccDKI and WT mice ( Fig. 4H,I). Nobiletin supplementation significantly decreased plasma cholesterol (~30%) and plasma TG levels (~25%) in both genotypes. FPLC profiles of plasma lipoproteins indicated that the nobiletininduced reductions in LDL-C (~33%) were similar in AccDKI and WT mice (Fig 4J, K). Nobiletin did not affect HDL-C in either genotype.

Nobiletin prevents obesity and metabolic dysregulation in HFHC diet-fed mice
Nobiletin increases energy expenditure, protecting mice from obesity and insulin resistance (7). Brown and beige adipose tissue are important regulators of energy expenditure (32). A -/mice have modest reductions in AMPK activity in adipose tissue (30). However, studies in mice lacking both AMPK 1 and 2 subunits specifically in adipocytes have shown that AMPK is required for maintaining brown and beige adipose tissue thermogenesis; effects which are independent of ACC phosphorylation but instead involve the regulation of mitochondrial function (32). Therefore, to specifically investigate the role of adipocyte AMPK in the ability of nobiletin to correct diet-induced obesity and metabolic dysfunction, ( ) were fed a HFHC diet reduced in WAT of mice, but most striking was the marked reduction of phosphorylated ACC in mice fed either HFHC or HFHC plus nobiletin (Fig. 6A). The mRNA levels of Ampkb1 and 19 Ampkb2 in eWAT were decreased ~50% in mice (Fig. 6B, C), consistent with previous findings demonstrating that residual AMPK activity in adipose tissue is from other cell types (e.g. resident stromal vascular cells) (32). Also consistent with previous studies (32), in response to the HFHC diet, mice tended to gain slightly more weight compared to controls (Fig. 6D). Nobiletin prevented HFHC diet-induced weight gain to the same extent in both genotypes (Fig. 6D). Caloric intake was unaffected by nobiletin in both genotypes (Fig. 6E). eWAT and iWAT depot weights were similar in HFHC-fed and control mice and nobiletin supplementation reduced the weight of both depots to a similar degree (~56%) in both genotypes (Fig.6F, G). The histology of eWAT, iWAT and brown adipose tissue (BAT) did not show any gross genotype differences in HFHC fed-mice (Fig. 6H).
Nobiletin treatment resulted in smaller adipocytes in eWAT and iWAT and smaller lipid droplets in BAT of both and control mice (Fig. 6H). Quantitation of adipocyte area distribution, mean adipocyte area and adipocyte number in eWAT revealed no difference between HFHC-fed mice and controls (Fig. 6I-K). Nobiletin improved adipocyte area distribution and decreased mean adipocyte area and number to similar levels in both genotypes. Similar patterns were observed for nobiletin-induced reductions in iWAT adipocyte size and number in both mice and controls (Supplemental Fig. S2A-C). As adipose tissue browning of iWAT has been shown to be important for weight loss in some studies and may be modulated by nobiletin (42), we measured the expression of Ucp1, and other known regulators of adipose tissue browning. Despite the same nobiletin-induced decreases in iWAT mass (Fig. 6F), nobiletin treatment increased the mRNA of Ucp1 and Tbx1 in control iWAT, but not in iWAT. Furthermore, nobiletin increased Cd137 mRNA in iWAT but not control iWAT (Fig. 6L-O). This inconsistency in expression of browning markers between genotypes together with the marked reduction by nobiletin of adipose tissue depots in both genotypes suggests that the effect of nobiletin did not involve browning of WAT.
Parameters for total ANCOVA-adjusted energy expenditure, RER and activity levels were not different between HFHC-fed and control mice (Fig. 7A-C). Nobiletin induced a similar increase in 20 energy expenditure (~30%) in both and control mice (Fig. 7A). RER and activity levels were unaffected by genotype or nobiletin treatment (Fig. 7B,C). Plasma concentrations of total TG and cholesterol in HFHC-fed mice were not different from controls (Fig 7D, E). Nobiletin supplementation significantly decreased plasma TG and cholesterol to similar extents in each genotype (Fig. 7D, E). FPLC profiles of plasma lipoproteins indicated that the nobiletin-induced reductions in LDL-C (~65%) and HDL-C (~50%) were similar for and control mice (Fig. 7F-H).
Consistent with previous studies (32), fasting blood glucose, glucose intolerance, plasma insulin and insulin intolerance were elevated in HFHC diet-fed mice compared to controls, although in the present study, these genotype differences were not statistically significant ( Fig. 7I-N). Addition of nobiletin to the HFHC diet decreased fasting blood glucose (~36%) and improved glucose tolerance (~46%) similarly in both genotypes (Fig. 7I-K). Nobiletin decreased plasma insulin (~90%) and improved insulin tolerance (~27%) in mice to the same extent as in control mice (Fig. 7L-N). Dietinduced hepatic steatosis was similar in mice and control mice (Fig. 7O, P) Nobiletin significantly decreased TG (~86%), FC (~25%) and CE (~80%) such that the marked reduction of each lipid was similar in both genotypes. In Ampk -/mice, approximately 10% of hepatic AMPK activity is retained (30), which may be sufficient to mediate some downstream phosphorylation. Furthermore, it is possible that aspects of 23 nobiletin-mediated regulation of lipid metabolism involve ACC phosphorylation independent of AMPK.
Nevertheless, nobiletin was equally effective in preventing metabolic dysregulation in HFHC diet fed-AccDKI mice and in WT mice, clearly demonstrating that the protective effects of nobiletin were independent of inhibitory phosphorylation of ACC. These results differ from the effects of metformin. In contrast to nobiletin, HFD-fed AccDKI mice were refractory to the lipid-lowering and insulin-sensitizing effects of metformin and metformin had no effect on body weight or adiposity in either genotype (36).
These findings imply that the mechanisms underlying nobiletin-induced metabolic protection differ from those of metformin.
Obesity is associated with reduced AMPK activity in adipose tissue, and dysfunctional brown adipose tissue contributes to diet-induced obesity and insulin resistance (47)(48)(49). Recent studies reported that adult mice had impairments in cold tolerance -adrenergic activation of BAT and browning of WAT, due to impaired mitochondrial structure and function (32). In addition, hepatic steatosis, and glucose and insulin intolerance were amplified in high fat-fed mice (32). The mechanism(s) through which nobiletin regulates lipid metabolism, adiposity and insulin sensitivity are not fully understood. In mice, nobiletin suppressed hepatic FA synthesis and upregulated hepatic FAoxidation, independent of peroxisomal proliferation (7), however, the upstream effectors governing these effects have remained elusive. Experiments in cultured hepatocytes and murine liver indicated that  (7). In HepG2 cells, Qi et al (21) reported that nobiletin amplified glucose uptake and suppressed palmitate-induced lipogenesis, which required activation of AMPK in a clock gene-(Bmal1) dependent manner. The present study clearly demonstrates that any ROR-dependent metabolic effects of nobiletin in vivo do not require AMPK activation in the liver or adipose tissue.
Metabolites of nobiletin derived from gut microbes have been identified and shown to accumulate in mouse colonic mucosa at concentrations exceeding that of nobiletin by ~20-fold (43). These demethylated nobiletin metabolites have been shown to have stronger anti-cancer effects than the parent compound in human colon cancer cells (43), raising the possibility that metabolic protection by nobiletin in vivo is primarily conferred by these metabolites. Furthermore, these metabolites have been shown to inhibit the 25 induction of iNOS, the inflammatory response and scavenger receptor expression in LPS-treated macrophage cell lines (53)(54)(55), properties that may be related to cardiovascular protection. However, the molecular targets and cardiometabolic effects of these metabolites in vivo have not been reported. Thus, the extent to which microbiota-derived nobiletin metabolites contribute to the prevention of metabolic dysregulation and atherosclerosis in mice warrants further investigation.
In summary, the mechanism underlying the ability of nobiletin to achieve metabolic protection in mice is independent of AMPK activation, thereby bypassing the central regulator of cellular energy homeostasis.
In     www.jlr.org Figure 6. Nobiletin attenuates body weight and adiposity and normalizes adipocyte morphology in both HFHC-fed i and wild type mice. Wild type (WT) and i mice were fed a HFHC diet (HF) alone or HFHC plus nobiletin (+Nob) for 12 weeks, n=8-9 per group. A: Immunoblot of pAMPK, AMPK, pACC and ACC in liver lysates from WT and i mice fed HFHC (HF) or HFHC + nobiletin (N). Lysates were run on the same immunoblot. B: Adipose tissue (iWAT) Ampkb1 mRNA. C: Adipose tissue (iWAT) Ampkb2 mRNA. D: Body weight measured weekly. * or # indicate a statistical difference from nobiletin-treated mice within the same genotype, determined by two-way ANOVA with repeated measures analyses, P<0.05. E: Mean daily caloric intake measured weekly. F: Adiposity assessed as inguinal fat pad weight/total body weight. G: Adiposity assessed as epididymal fat pad weight/total body weight. H: Representative images of iWAT, eWAT and BAT stained with H&E. Scale bar is 100 . I: Frequency distribution of adipocyte area in eWAT. J: Mean adipocyte area in eWAT. K: Total adipocyte number in eWAT calculated as the mean number of cells per field of view X weight of eWAT. L-O: Adipose tissue (iWAT) Ucp1, Tbx1, Serca2b and Cd137 mRNA. Data represent the mean ± SEM. Different letters indicate statistical differences by ANOVA with post-hoc Tukey's test (P<0.05). N.S. indicates no significant differences.