Diet-induced obesity impairs endometrial stromal cell decidualization: a potential role for impaired autophagy.

STUDY QUESTION: What effect does diet-induced obesity have on endometrial stromal cell (ESC) decidualization? SUMMARY ANSWER: Diet-induced obesity impairs ESC decidualization. WHAT IS KNOWN ALREADY: Decidualization is important for successful implantation and subsequent health of the pregnancy. Compared with normal-weight women, obese women have lower pregnancy rates (both spontaneous and by assisted reproductive technology), higher rates of early pregnancy loss and poorer oocyte quality. STUDY DESIGN, SIZE, DURATION: Beginning at 6 weeks of age, female C57Bl/6J mice were fed either a high-fat/high-sugar diet (HF/HS; 58% Fat Energy/Sucrose) or a diet of standard mouse chow (CON; 13% Fat) for 12 weeks. At this point, metabolic parameters were measured. Some of the mice (n = 9 HF/HS and 9 CON) were mated with reproductively competent males, and implantation sites were assessed. Other mice (n = 11 HF/HS and 10 CON) were mated with vasectomized males, and artificial decidualization was induced. For in vitro human studies of primary ESCs, endometrial tissue was obtained via biopsy from normo-ovulatory patients without history of infertility (obese = BMI > 30 kg/m(2), n = 11 and lean = BMI < 25 kg/m(2), n = 7) and from patients consented for hysterectomies for a benign indication (n = 4). In vitro studies were also performed with immortalized human ESCs. ESCs were decidualized in culture for nine 9 days in the presence or absence of palmitic acid (PA), and the degree of decidualization was assessed by measuring expression of decidualization markers. PARTICIPANTS/MATERIALS, SETTING, METHODS: The sizes of implantation sites and fetuses were analyzed in mice mated with reproductively competent males. In mice mated with vasectomized males, decidualization was induced, and uterine tissues were analyzed via hematoxylin and eosin staining, quantitative RT-PCR (RT-qPCR), and western blots. Human ESCs were cultured in vitro and induced to decidualize by treatment with cAMP and medroxyprogesterone. The level of expression of decidualization markers was assessed by RT-qPCR (mRNA) and western blotting (protein). ATP content of ESCs was measured, and levels of autophagy were assessed by western blotting of the autophagy regulators acetyl coa carboxylase (ACC) and ULK1 (Ser 317). Autophagic flux was measured by western blot of the marker LC3b-II. MAIN RESULTS AND THE ROLE OF CHANCE: Mice exposed to an HF/HS diet became obese and metabolically impaired. HF/HS-exposed mice mated to reproductively competent males had smaller implantation sites in early pregnancy (P <0.001) and larger fetuses at term (P <0.05) than CON-exposed mice. In the artificial decidualization experiments, mice exposed to the HF/HS diet developed 50% smaller deciduomas than mice exposed to CON diet (P< 0.001). Human ESCs cultured in the presence of PA had markedly decreased mRNA expression of the decidualization markers, decidual prolactin (PRL) (P< 0.0001) and insulin-like growth factor binding protein 1 (IGFBP1) (P< 0.0001). Expression of PRL and IGFBP1 by mRNA were also significantly lower in early follicular phase ESCs of obese women than in those of normal-weight women (P< 0.05). Protein expression of phosphorylated ACC and phosphorylated ULK1, both activated forms, were lower in deciduomas of HF/HS mice than in those of control mice (P < 0.01). In immortalized human ESCs, LC3b-II levels were higher in decidualized cells than in controls, indicating increased autophagy. PA treatment abrogated this increase. LIMITATIONS, REASONS FOR CAUTION: Many aspects of obesity and metabolic impairment could contribute to the decidualization defects observed in the HF/HS-exposed mice. Although our findings suggest that both autophagy and decidualization are impaired by exposure to PA, the underlying mechanisms should be elucidated. Finally, our human patient sample size was small. WIDER IMPLICATIONS OF THE FINDINGS: Although many factors contribute to poor reproductive outcome and early pregnancy loss in obese women, our study suggests the importance of decidualization defects. Such defects may contribute to compromised endometrial receptivity and poor implantation. If defects in autophagy contribute to impaired decidualization, therapeutics could be developed to improve this process and thus improve implantation and pregnancy outcomes in obese women. STUDY FUNDING/COMPETING INTERESTS: Grants include NIH 5T32HD040135-12 (J.S.R.), R01 HD065435 (K.H.M.), NIH T32 HD049305 (J.L.S.) and ACOG Research Grant (M.B.S.). The authors report no conflicts of interest.

SIRT1 selectively exerts the metabolic protective effects of hepatocyte nicotinamide phosphoribosyltransferase.

Calorie restriction abates aging and cardiometabolic disease by activating metabolic signaling pathways, including nicotinamide adenine dinucleotide (NAD+) biosynthesis and salvage. Nicotinamide phosphoribosyltransferase (NAMPT) is rate-limiting in NAD+ salvage, yet hepatocyte NAMPT actions during fasting and metabolic duress remain unclear. We demonstrate that hepatocyte NAMPT is upregulated in fasting mice, and in isolated hepatocytes subjected to nutrient withdrawal. Mice lacking hepatocyte NAMPT exhibit defective FGF21 activation and thermal regulation during fasting, and are sensitized to diet-induced glucose intolerance. Hepatocyte NAMPT overexpression induced FGF21 and adipose browning, improved glucose homeostasis, and attenuated dyslipidemia in obese mice. Hepatocyte SIRT1 deletion reversed hepatocyte NAMPT effects on dark-cycle thermogenesis, and hepatic FGF21 expression, but SIRT1 was dispensable for NAMPT insulin-sensitizing, anti-dyslipidemic, and light-cycle thermogenic effects. Hepatocyte NAMPT thus conveys key aspects of the fasting response, which selectively dissociate through hepatocyte SIRT1. Modulating hepatocyte NAD+ is thus a potential mechanism through which to attenuate fasting-responsive disease.

Hepatic arginase 2 (Arg2) is sufficient to convey the therapeutic metabolic effects of fasting.

Caloric restriction and intermittent fasting are emerging therapeutic strategies against obesity, insulin resistance and their complications. However, the effectors that drive this response are not completely defined. Here we identify arginase 2 (Arg2) as a fasting-induced hepatocyte factor that protects against hepatic and peripheral fat accumulation, hepatic inflammatory responses, and insulin and glucose intolerance in obese murine models. Arg2 is upregulated in fasting conditions and upon treatment with the hepatocyte glucose transporter inhibitor trehalose. Hepatocyte-specific Arg2 overexpression enhances basal thermogenesis, and protects from weight gain, insulin resistance, glucose intolerance, hepatic steatosis and hepatic inflammation in diabetic mouse models. Arg2 suppresses expression of the regulator of G-protein signalling (RGS) 16, and genetic RGS16 reconstitution reverses the effects of Arg2 overexpression. We conclude that hepatocyte Arg2 is a critical effector of the hepatic glucose fasting response and define a therapeutic target to mitigate the complications of obesity and non-alcoholic fatty liver disease.

TFEB-dependent induction of thermogenesis by the hepatocyte SLC2A inhibitor trehalose.

The macroautophagy/autophagy-inducing disaccharide, trehalose, has been proposed to be a promising therapeutic agent against neurodegenerative and cardiometabolic diseases. We recently showed that trehalose attenuates hepatic steatosis in part by blocking hepatocyte glucose transport to induce hepatocyte autophagic flux. However, although every major demonstration of trehalose action invokes activating autophagic flux as its primary function, the mechanism of action of trehalose in whole-body energy metabolism remains poorly defined. Here, we demonstrate that trehalose induces hepatocyte TFEB (transcription factor EB)-dependent thermogenesis in vivo, concomitant with upregulation of hepatic and white adipose expression of UCP1 (uncoupling protein 1 [mitochondrial, protein carrier]). Mechanistically, we provide evidence that hepatocyte fasting transcriptional and metabolic responses depend upon PPARGC1A (peroxisome proliferative activated receptor, gamma, coactivator 1 alpha), TFEB, and FGF21 (fibroblast growth factor 21) signaling. Strikingly, hepatocyte-selective TFEB knockdown abrogated trehalose induction of thermogenesis and white adipose tissue UCP1 upregulation in vivo. In contrast, we found that trehalose action on thermogenesis was independent of LEP (leptin) and the autophagy pathway, as there was robust thermogenic induction in trehalose-treated ob/ob, Becn1, Atg16l1, and Epg5 mutant mice. We conclude that trehalose induces metabolically favorable effects on whole-body thermogenesis in part via hepatocyte-centered fasting-like mechanisms that appear to be independent of autophagic flux. Our findings elucidate a novel mechanism by which trehalose acts as a metabolic therapeutic agent by activating hepatic fasting responses. More broadly, the hepatic glucose fasting response may be of clinical utility against overnutrition-driven disease, such as obesity and type 2 diabetes mellitus.

Hepatocyte ALOXE3 is induced during adaptive fasting and enhances insulin sensitivity by activating hepatic PPARγ.

The hepatic glucose fasting response is gaining traction as a therapeutic pathway to enhance hepatic and whole-host metabolism. However, the mechanisms underlying these metabolic effects remain unclear. Here, we demonstrate the epidermal-type lipoxygenase, eLOX3 (encoded by its gene, Aloxe3), is a potentially novel effector of the therapeutic fasting response. We show that Aloxe3 is activated during fasting, glucose withdrawal, or trehalose/trehalose analogue treatment. Hepatocyte-specific Aloxe3 expression reduced weight gain and hepatic steatosis in diet-induced and genetically obese (db/db) mouse models. Aloxe3 expression, moreover, enhanced basal thermogenesis and abrogated insulin resistance in db/db diabetic mice. Targeted metabolomics demonstrated accumulation of the PPARγ ligand 12-KETE in hepatocytes overexpressing Aloxe3. Strikingly, PPARγ inhibition reversed hepatic Aloxe3-mediated insulin sensitization, suppression of hepatocellular ATP production and oxygen consumption, and gene induction of PPARγ coactivator-1α (PGC1α) expression. Moreover, hepatocyte-specific PPARγ deletion reversed the therapeutic effect of hepatic Aloxe3 expression on diet-induced insulin intolerance. Aloxe3 is, therefore, a potentially novel effector of the hepatocellular fasting response that leverages both PPARγ-mediated and pleiotropic effects to augment hepatic and whole-host metabolism, and it is, thus, a promising target to ameliorate metabolic disease.

Enhanced Hepatic PPARα Activity Links GLUT8 Deficiency to Augmented Peripheral Fasting Responses in Male Mice.

The adaptive fasting response is invoked as a promising cardiometabolic and neurodegenerative therapeutic pathway. We and others have defined the carbohydrate transporter glucose transporter 8 (GLUT8) as a critical regulator of hepatic and whole-organism metabolic homeostasis in the overfed and diabetic states. However, the functions of this critical transporter in the physiological fasting response remain poorly understood. Here, we tested the hypothesis that GLUT8 modulates the adaptive hepatic fasting response. We demonstrate that mice with targeted Slc2a8 disruption exhibit enhanced thermogenesis, ketogenesis, and peripheral lipid mobilization during fasting. These metabolic enhancements were observed in the context of mildly impaired hepatic mitochondrial oxidative metabolism in vivo and in vitro. Mechanistically, we show that hepatic peroxisome proliferator-activated receptor α (PPARα) and its transcriptional fasting response target hepatokine, fibroblast growth factor (FGF)21, are cell-autonomously hyperactivated in GLUT8-deficient liver and in isolated primary murine hepatocytes during nutrient depletion. Hepatic PPARα knockdown in GLUT8-deficient mice normalized the enhanced ketogenic and FGF21 secretory responses and decreased mitochondrial respiratory function without altering the hyperthermic response to fasting. Our data demonstrate that hepatocyte GLUT8 regulates adaptive fasting in part through regulation of the PPARα signaling cascade. Moreover, the ketotic and thermic responses to fasting are differentially encoded within the GLUT8-PPARα communication axis. GLUT8 therefore represents a therapeutic target that can be leveraged against cardiometabolic disease.

SLC2A8 (GLUT8) is a mammalian trehalose transporter required for trehalose-induced autophagy.

Trehalose is a disaccharide demonstrated to mitigate disease burden in multiple murine neurodegenerative models. We recently revealed that trehalose rapidly induces hepatic autophagy and abrogates hepatic steatosis by inhibiting hexose transport via the SLC2A family of facilitative transporters. Prior studies, however, postulate that intracellular trehalose is sufficient to induce cellular autophagy. The objective of the current study was to identify the means by which trehalose accesses the hepatocyte cytoplasm, and define the distal signaling mechanisms by which trehalose induces autophagy. We provide gas chromatographic/mass spectrometric, fluorescence microscopic and radiolabeled uptake evidence that trehalose traverses the plasma membrane via SLC2A8 (GLUT8), a homolog of the trehalose transporter-1 (Tret1). Moreover, GLUT8-deficient hepatocytes and GLUT8-deficient mice exposed to trehalose resisted trehalose-induced AMP-activated protein kinase (AMPK) phosphorylation and autophagic induction in vitro and in vivo. Although trehalose profoundly attenuated mTORC1 signaling, trehalose-induced mTORC1 suppression was insufficient to activate autophagy in the absence of AMPK or GLUT8. Strikingly, transient, heterologous Tret1 overexpression reconstituted autophagic flux and AMPK signaling defects in GLUT8-deficient hepatocyte cultures. Together, these data suggest that cytoplasmic trehalose access is carrier-mediated, and that GLUT8 is a mammalian trehalose transporter required for hepatocyte trehalose-induced autophagy and signal transduction.

Trehalose inhibits solute carrier 2A (SLC2A) proteins to induce autophagy and prevent hepatic steatosis.

Trehalose is a naturally occurring disaccharide that has gained attention for its ability to induce cellular autophagy and mitigate diseases related to pathological protein aggregation. Despite decades of ubiquitous use as a nutraceutical, preservative, and humectant, its mechanism of action remains elusive. We showed that trehalose inhibited members of the SLC2A (also known as GLUT) family of glucose transporters. Trehalose-mediated inhibition of glucose transport induced AMPK (adenosine 5'-monophosphate-activated protein kinase)-dependent autophagy and regression of hepatic steatosis in vivo and a reduction in the accumulation of lipid droplets in primary murine hepatocyte cultures. Our data indicated that trehalose triggers beneficial cellular autophagy by inhibiting glucose transport.