Regulation of hepatokine gene expression in response to fasting and feeding: Influence of PPAR-α and insulin-dependent signalling in hepatocytes.

AIM: In hepatocytes, the peroxisome proliferator-activated receptor (PPAR)-α and insulin receptor (IR) are critical for transcriptional responses to fasting and feeding, respectively. The present report analyzes the effects of nutritional status (fasting vs feeding) on the expression of a large panel of hepatokines in hepatocyte-specific PPAR-α (Pparαhep-/-) and IR (IRhep-/-) null mice. METHODS: Pparαhep-/- and IRhep-/- mice, and their wild-type littermates, were subjected to fasting or feeding metabolic challenges, then analyzed for hepatokine gene expression. Experiments were conducted in mice of both genders. RESULTS: Our data confirmed that PPAR-α is essential for regulating fasting-induced Fgf21 and Angptl4 expression. In mice lacking PPAR-α, fasting led to increased Igfbp1 and Gdf15 gene expression. In the absence of hepatic IR, feeding induced overexpression of Igfbp1, follistatin (Fst) and adropin (Enho), and reduced activin E (Inhbe) expression. Gender had only a modest influence on hepatokine gene expression in the liver. CONCLUSION: The present results highlight the potential roles of hepatokines as a class of hormones that substantially influence nutritional regulation in both female and male mice.

The role of the lipogenic pathway in the development of hepatic steatosis.

Non-alcoholic fatty liver disease (NAFLD) represents a wide spectrum of diseases, ranging from simple fatty liver (hepatic steatosis) through steatosis with inflammation and necrosis to cirrhosis. NAFLD, which is strongly associated with obesity, insulin resistance and type 2 diabetes, is now well recognized as being part of the metabolic syndrome. The metabolic pathways leading to the development of hepatic steatosis are multiple, including enhanced non-esterified fatty acid release from adipose tissue (lipolysis), increased de novo fatty acids (lipogenesis) and decreased beta-oxidation. Recently, several mouse models have helped to clarify the molecular mechanisms leading to the development of hepatic steatosis in the pathogenesis of NAFLD. This review describes the models that have provided evidence implicating lipogenesis in the development and/or prevention of hepatic steatosis.

Phosphoenolpyruvate carboxykinase is necessary for the integration of hepatic energy metabolism.

We used an allelogenic Cre/loxP gene targeting strategy in mice to determine the role of cytosolic phosphoenolpyruvate carboxykinase (PEPCK) in hepatic energy metabolism. Mice that lack this enzyme die within 3 days of birth, while mice with at least a 90% global reduction of PEPCK, or a liver-specific knockout of PEPCK, are viable. Surprisingly, in both cases these animals remain euglycemic after a 24-h fast. However, mice without hepatic PEPCK develop hepatic steatosis after fasting despite up-regulation of a variety of genes encoding free fatty acid-oxidizing enzymes. Also, marked alterations in the expression of hepatic genes involved in energy metabolism occur in the absence of any changes in plasma hormone concentrations. Given that a ninefold elevation of the hepatic malate concentration occurs in the liver-specific PEPCK knockout mice, we suggest that one or more intermediary metabolites may directly regulate expression of the affected genes. Thus, hepatic PEPCK may function more as an integrator of hepatic energy metabolism than as a determinant of gluconeogenesis.

Hepatocyte-specific mutation establishes retinoid X receptor alpha as a heterodimeric integrator of multiple physiological processes in the liver.

A large number of physiological processes in the adult liver are regulated by nuclear receptors that require heterodimerization with retinoid X receptors (RXRs). In this study, we have used cre-mediated recombination to disrupt the mouse RXRalpha gene specifically in hepatocytes. Although such mice are viable, molecular and biochemical parameters indicate that every one of the examined metabolic pathways in the liver (mediated by RXR heterodimerization with PPARalpha, CARbeta, PXR, LXR, and FXR) is compromised in the absence of RXRalpha. These data demonstrate the presence of a complex circuitry in which RXRalpha is integrated into a number of diverse physiological pathways as a common regulatory component of cholesterol, fatty acid, bile acid, steroid, and xenobiotic metabolism and homeostasis.

Dysregulated CRTC1 activity is a novel component of PGE2 signaling that contributes to colon cancer growth.

First identified as a dedicated CREB (cAMP response element-binding protein) co-activator, CRTC1 (CREB-regulated transcription co-activator 1) has been widely implicated in various neuronal functions because of its predominant expression in the brain. However, recent evidences converge to indicate that CRTC1 is aberrantly activated in an expanding number of adult malignancies. In this study, we provide strong evidences of enhanced CRTC1 protein content and transcriptional activity in mouse models of sporadic (APC(min/+) mice) or colitis-associated colon cancer azoxymethane/dextran sulfate sodium (AOM/DSS-treated mice), and in human colorectal tumors specimens compared with adjacent normal mucosa. Among signals that could trigger CRTC1 activation during colonic carcinogenesis, we demonstrate that treatment with cyclooxygenase 2 (COX2) inhibitors reduced nuclear CRTC1 active form levels in colonic tumors of APC(min/+) or AOM/DSS mice. In accordance, prostaglandins E2 (PGE2) exposure to human colon cancer cell lines promoted CRTC1 dephosphorylation and parallel nuclear translocation, resulting in enhanced CRTC1 transcriptional activity, through EP1 and EP2 receptors signaling and consecutive calcineurin and protein kinase A activation. In vitro CRTC1 loss of function in colon cancer cell lines was associated with reduced viability and cell division rate as well as enhanced chemotherapy-induced apoptosis on PGE2 treatment. Conversely, CRTC1 stable overexpression significantly increased colonic xenografts tumor growth, therefore demonstrating the role of CRTC1 signaling in colon cancer progression. Identification of the transcriptional program triggered by enhanced CRTC1 expression during colonic carcinogenesis, revealed some notable pro-tumorigenic CRTC1 target genes including NR4A2, COX2, amphiregulin (AREG) and IL-6. Finally, we demonstrate that COX2, AREG and IL-6 promoter activities triggered by CRTC1 are dependent on functional AP1 and CREB transcriptional partners. Overall, our study establishes CRTC1 as new mediator of PGE2 signaling, unravels the importance of its dysregulation in colon cancer and strengthens its use as a bona fide cancer marker.

The effects of hyperinsulinemia and hyperglycemia on GLUT4 and hexokinase II mRNA and protein in rat skeletal muscle and adipose tissue.

The GLUT4 glucose transporter and type II hexokinase are predominantly expressed in skeletal muscle and adipose tissue. The effects of insulin and glucose on the expression of GLUT4 and HKII were studied in vivo by using the euglycemic-hyperinsulinemic and hyperglycemic-hyperinsulinemic clamp methods. The clamps were maintained in conscious rats for 6 or 24 h after a 1-day starvation period. Adipose tissue GLUT4 mRNA was increased 4-fold after 6 h and 23-fold after 24 h of hyperinsulinemia; HKII mRNA was increased by four- and eightfold after 6 and 24 h, respectively. In contrast, GLUT4 mRNA was not significantly changed in skeletal muscle by either the euglycemic- or hyperglycemic-hyperinsulinemic clamps. Each of these treatments resulted in a fourfold induction of HKII mRNA. No changes of GLUT4 protein and hexokinase activity were detected after 6 h of hyperinsulinemia in either skeletal muscle or adipose tissue. After 24 h of hyperinsulinemia, adipose tissue GLUT4 protein had doubled, whereas skeletal muscle GLUT4 was unchanged. In contrast, hexokinase activity increased by two- to eightfold in skeletal muscle and adipose tissue. Hyperinsulinemia alone was sufficient to mediate the effects observed, because no additional effects were seen when hyperglycemia accompanied hyperinsulinemia. These results reveal the lack of coordinate regulation of GLUT4 and HKII in adipose tissue and skeletal muscle. Whereas hyperinsulinemia increases both GLUT4 and HKII mRNA and protein levels in adipose tissue, this treatment increases HKII mRNA and protein in skeletal muscle, but has no effect on GLUT4 in this tissue.

Glucokinase gene locus transgenic mice are resistant to the development of obesity-induced type 2 diabetes.

Transgenic mice that overexpress the entire glucokinase (GK) gene locus have been previously shown to be mildly hypoglycemic and to have improved tolerance to glucose. To determine whether increased GK might also prevent or diminish diabetes in diet-induced obese animals, we examined the effect of feeding these mice a high-fat high-simple carbohydrate low-fiber diet (HF diet) for 30 weeks. In response to this diet, both normal and transgenic mice became obese and had similar BMIs (5.3 +/- 0.1 and 5.0 +/- 0.1 kg/m2 in transgenic and non-transgenic mice, respectively). The blood glucose concentration of the control mice increased linearly with time and reached 17.0 +/- 1.3 mmol/l at the 30th week. In contrast, the blood glucose of GK transgenic mice rose to only 9.7 +/- 1.2 mmol/l at the 15th week, after which it returned to 7.6 +/- 1.0 mmol/l by the 30th week. The plasma insulin concentration was also lower in the GK transgenic animals (232 +/- 79 pmol/l) than in the controls (595 +/- 77 pmol/l), but there was no difference in plasma glucagon concentrations. Together, these data indicate that increased GK levels dramatically lessen the development of both hyperglycemia and hyperinsulinemia associated with the feeding of an HF diet.

Isolation and characterization of the mouse cytosolic phosphoenolpyruvate carboxykinase (GTP) gene: evidence for tissue-specific hypersensitive sites.

A 72 kilobase pair DNA fragment that contains the mouse phosphoenolpyruvate carboxykinase (PEPCK) gene locus, pck1, was isolated from a genomic bacterial artificial chromosome library. The region from approximately -5.5 to +6.6 kilobase pairs relative to the pck1 transcription start site was sequenced and exhibits a high degree of homology to the rat and human genes. Additionally, the chromatin structure of the PEPCK gene in mouse liver resembles that seen in rat. Backcross panel analysis of a microsatellite sequence confirms that the gene is located on chromosome 2. Hypersensitive site analysis was performed on nuclei isolated from the adipocyte cell line 3T3-F442A in the preadipose and adipose states. Several hypersensitive sites are present in the undifferentiated 3T3-F442A cells, before PEPCK mRNA is detected. The same sites are present after differentiation, however, the sensitivity of mHS 3 increases relative to the others. We conclude that the chromatin is open in 3T3-F442A cells and that factors are able to bind in the undifferentiated state but that something else is required for transcription.

Role of the liver in the control of carbohydrate and lipid homeostasis.

The liver plays a unique role in controlling carbohydrate metabolism by maintaining glucose concentrations in a normal range over both short and long periods of times. In type 2 diabetes, alterations in hepatic glucose metabolism are observed, i.e. increased post-absorptive glucose production and impaired suppression of glucose production together with diminished glucose uptake following carbohydrate ingestion. The simultaneous overproduction of glucose and fatty acids in liver further stimulates the secretion of insulin by the pancreatic B cells, and elicits further peripheral insulin resistance thereby establishing a vicious circle. The present review will focus on some of the genetically-altered mouse models that have helped identify enzymes or transcription factors that are essential for maintaining either glucose or lipid homeostasis in liver. Among these mouse models, we will discuss transgenic mice overexpressing key gluconeogenic enzymes (PEPCK, G6Pase) or transcription factors (Foxo1, Pgc1-alpha) that control de novo glucose synthesis. In addition, since the possibility of controlling hepatic glucose utilization as a treatment of type 2 diabetes has been explored we will review some of the strategies proved to be valuable for improving the hyperglycemic phenotype.

[Glucose transporters. Physiology and physiopathology]. / Transporteurs de glucose. Physiologie et physiopathologie.

Glucose transport is an important step in the regulation of glucose homeostasis. Two types of transport systems are described: active transport accumulates glucose in specific cells, whereas facilitative transport equilibrates blood glucose and intracellular glucose inside all mammalian cells. At the present time, different levels of facilitative transport regulation are known. Facilitative transport is achieved by 5 different isoforms; each isoform has its own characteristics and is subjected to tissue-specific regulation. Alteration of glucose transporters expression may be involved in a physiopathological situation such as diabetes which is characterized by insulin resistance of peripheral tissues and impaired insulin secretion by beta pancreatic cells. Thus, Glut 2 expression is reduced in the beta cells of diabetic rats. The reduction of Glut 2 expression correlates with, and may contribute to the loss of glucose-stimulated insulin secretion. However, Glut 2 expression in liver remains unaltered. The insulin resistance of peripheral tissues may be explained in adipose tissue by a decrease in Glut 4 expression. In skeletal muscle, Glut 4 expression remains constant whatever the physiological or physiopathological situation.

Probabilistic assessment of exposure to nail cosmetics in French consumers.

The aim of this study was to assess probabilistic exposure to nail cosmetics in French consumers. The exposure assessment was performed with base coat, polish, top coat and remover. This work was done for adult and child consumers. Dermal, inhalation and oral routes were taken into account for varnishes. Exposure evaluation was performed for the inhalation route with polish remover. The main route of exposure to varnishes was the ungual route. Inhalation was the secondary route of exposure, followed by dermal and oral routes. Polish contributed most to exposure, regardless of the route of exposure. For this nail product, P50 and P95 values by ungual route were respectively equal to 1.74 mg(kg bw week)(-1) and 8.55 mg(kg bw week)(-1) for women aged 18-34 years. Exposure to polish by inhalation route was equal to 0.70 mg(kg bw week)(-1) (P50) and 5.27 mg(kg bw week)(-1) (P95). P50 and P95 values by inhalation route were respectively equal to 0.08 mg(kg bw week)(-1) and 1.14 mg(kg bw week)(-1) for consumers aged 18-34 years exposed to polish remover. This work provided current exposure data for nail cosmetics, and a basis for future toxicological studies of the uptake of substances contained in nail cosmetics in order to assess systemic exposure.

Cell-specific roles of glucokinase in glucose homeostasis.

Mutations in the glucokinase (GK) gene cause two different diseases of blood glucose regulation: maturity onset diabetes of the young, type 2 (MODY-2) and persistent hyperinsulinemic hypoglycemia of infancy (PHHI). To gain further understanding of the pathophysiology of these disorders, we have used both transgenic and gene-targeting strategies to explore the relationship between GK gene expression in specific tissues and the blood glucose concentration. These studies, which have included the use of aCre/loxP gene-targeting strategy to perform both pancreatic beta-cell- and hepatocyte-specific knockouts of GK, clearly demonstrate multiple, cell-specific roles for this hexokinase that, together, contribute to the maintainance of euglycemia. In the pancreatic beta cell, GK functions as the glucose sensor, determining the threshold for insulin secretion. Mice lacking GK in the pancreatic beta cell die within 3 days of birth of profound hyperglycemia. In the liver, GK facilitates hepatic glucose uptake during hyperglycemia and is essential for the appropriate regulation of a network of glucose-responsive genes. While mice lacking hepatic GK are viable, and are only mildly hyperglycemic when fasted, they also have impaired insulin secretion in response to hyperglycemia. The mechanisms that enable hepatic GK to affect beta-cell function are not yet understood. Thus, the hyperglycemia that occurs in MODY-2 is due to impaired GK function in both the liver and pancreatic beta cell, although the defect in beta-cell function is clearly more dominant. Whether defects in GK gene expression also impair glucose sensing by neurons in the brain or enteroendocrine cells in gut, two other sites known to express GK, remains to be determined. Moreover, whether the pathophysiology of PHHI also involves multitissue dysfunction remains to be explored.

Nutritional regulation of glucose transporter in muscle and adipose tissue of weaned rats.

The role of glucose transporters GLUT-1 and GLUT-4 in the development of insulin sensitivity at weaning in rat skeletal muscles and adipose tissue was studied in relation to the nutritional changes when suckling rats shift from a high-fat (HF) to a high-carbohydrate (HCHO) diet. Insulin stimulated the translocation of GLUT-4 protein from an intracellular pool to the plasma membrane in adipocytes from suckling and HCHO- or HF-weaned rats. The GLUT-4 protein and the insulin stimulation were threefold higher in adipocytes from HCHO-weaned rats than in suckling or HF-weaned rats. GLUT-4 mRNA and protein were low in adipose tissue and skeletal muscles of suckling rats and increased two- to threefold in HCHO-weaned rats. This increase was prevented in HF-weaned rats. GLUT-1 mRNA was not affected in both tissues by the developmental stage or the nutritional environment. After feeding HCHO to a suckling rat, GLUT-4 mRNA was threefold increased in 6 days and reached a peak after 4 days in both tissues. The insulin sensitivity of glucose transport in rats at weaning might be conferred by an enhanced expression of GLUT-4, which can be induced within a few hours after feeding a HCHO diet.

Glucose transporter expression in rat mammary gland.

The expression of different glucose transporter isoforms was measured during the development and differentiation of the rat mammary gland. Before conception, when the mammary gland is mainly composed of adipocytes, Glut 4 and Glut 1 mRNAs and proteins were present. During pregnancy, the expression of Glut 4 decreased progressively, whereas that of Glut 1 increased. In the lactating mammary gland only Glut 1 was present, and was expressed at a high level. The absence of Glut 4 suggests that glucose transport is not regulated by insulin in the lactating rat mammary gland.

Effects of increased glucokinase gene copy number on glucose homeostasis and hepatic glucose metabolism.

The relationship between glucokinase (GK) gene copy number and glucose homeostasis was studied in transgenic mice with additional copies of the entire GK gene locus (Niswender, K. D., Postic, C., Jetton, T. L., Bennett, B. D., Piston, D. W., Efrat, S., and Magnuson, M. A. (1997) J. Biol. Chem. 272, 22564-22569). The plasma glucose concentration was reduced by 25 +/- 3% and 37 +/- 4% in mice with one or two extra copies of the gene locus, respectively. The basis for the hypoglycemic phenotype was determined using metabolic tracer techniques in chronically cannulated, conscious mice with one extra GK gene copy. Under basal conditions (6-h fasted) transgenic mice had a lower blood glucose concentration (-12 +/- 1%) and a slightly higher glucose turnover rate (+8 +/- 3%), resulting in a significantly higher glucose clearance rate (+21 +/- 2%). Plasma insulin levels were not different, suggesting that increased glucose clearance was due to augmented hepatic, not islet, GK gene expression. Under hyperglycemic clamp conditions the transgenic mice had glucose turnover and clearance rates similar to the controls, but showed a lower plasma insulin response (-48 +/- 5%). Net hepatic glycogen synthesis was markedly elevated (+360%), whereas skeletal muscle glycogen synthesis was decreased (-40%). These results indicate that increased GK gene dosage leads to increased hepatic glucose metabolism and, consequently, a lower plasma glucose concentration. Increased insulin secretion was not observed, even though the transgene is expressed in islets, because hypoglycemia causes a down-regulation in islet GK content (Niswender, K. D., Postic, C., Jetton, T. L., Bennett, B. D., Piston, D. W., Efrat, S., and Magnuson, M. A. (1997) J. Biol. Chem. 272, in press).

Variable expression of hepatic glucokinase in mice is due to a regulational locus that cosegregates with the glucokinase gene.

The Gk activity locus affects expression of hepatic glucokinase (GK) in mice. Analysis of microsatellites within the mouse GK gene locus revealed two major haplotypes in 19 of 22 inbred strains predictive of either high or low hepatic GK gene expression. C3H/HeJ mice, a high-activity strain, and two other wild-derived strains contain less common haplotypes. No coding sequence differences were found in hepatic GK-coding sequences from representative high and low Gk activity strains, thereby excluding kinetic abnormalities as the basis for hepatic GK activity differences. Screening of approximately 10 kb of potential regulatory DNA, including all eight known and three of four newly identified DNase I-hypersensitive sites, by restriction enzyme fingerprinting-single-strand conformation analysis revealed a tetranucleotide microsatellite, the length of which was also predictive of the Gk activity phenotype. This tetranucleotide repeat is located in the first intron of the hepatic transcription unit and lies close to a newly identified liver-specific DNase I-hypersensitive site. These results indicate that the Gk activity alleles are a regulational locus associated with the GK gene locus.