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Background & Aims: Liver inflammation, fibrosis, and dyslipidemia are common features in patients with chronic hepatitis C virus (HCV) infection. Because peroxisome proliferator-activated receptor @a (PPAR@a) is highly expressed in the liver and is involved in the regulation of lipid metabolism and inflammation, we sought to determine whether HCV infection may locally impair PPAR@a expression and activity. Methods: PPAR@a expression was investigated in liver biopsy specimens of 86 untreated patients with HCV infection and controls, by using real-time polymerase chain reaction (PCR), Western blot analysis, and immunohistochemistry. PPAR@a activity was assessed by quantification of the key gene target carnitine palmitoyl acyl-CoA transferase 1 (CPT1A) messenger RNA (mRNA).The influence of HCV core protein on PPAR@a mRNA expression was analyzed in vitro by real-time PCR in HCV core-expressing HepG2 cells activated with the PPAR@a ligand fenofibric acid. Results: Hepatic concentrations of PPAR@a and CPT1A expressed by hepatocytes were impaired profoundly in the livers of untreated patients with HCV infection compared with controls. A mean decrease of 85% in PPAR@a mRNA expression paralleled with a lack of CPT1A mRNA induction also were observed in HCV core-expressing HepG2 cells compared with controls. Conclusions: HCV infection is related to altered expression and function of the anti-inflammatory nuclear receptor PPAR@a. These results identify hepatic PPAR@a as one mechanism underlying the pathogenesis of HCV infection, and as a new therapeutic target in traditional treatment of HCV-induced liver injury.

The intracellular fatty acid content of insulin-sensitive target tissues determines in part their insulin sensitivity. Uptake of fatty acids into cells is a controlled process determined in part by a regulated import/export system that is controlled at least by two key groups of proteins, i.e. the fatty acid transport protein (FATP) and acyl-CoA synthetase (ACS), which facilitate, respectively, the transport of fatty acids across the cell membrane and catalyze their esterification to prevent their efflux. Previously it was shown that the expression of the FATP-1 and ACS genes was controlled by insulin and by peroxisome proliferator-activated receptor (PPAR) agonists in liver or in adipose tissue. The aim of this investigation was to determine the effects of retinoic acid derivatives on the expression of FATP-1 and ACS. In several cultured cell lines, it was shown that the expression of both the FATP-1 and ACS mRNAs was specifically induced at the transcriptional level by selective retinoid X receptor (RXR) but not by retinoic acid receptor (RAR) ligands. This effect was most pronounced in hepatoma cell lines. A similar induction of FATP-1 and ACS mRNA levels was also observed in vivo in Zucker diabetic fatty rats treated with the RXR agonist, LGD1069 (4-[1-(3,5,5,8,8-pentamethyl-5,6,7, 8-tetrahydro-2-naphthyl)ethenyl]benzoic acid). Through the use of heterodimer-selective compounds, it was demonstrated that the modulatory effect of these rexinoids on FATP-1 and ACS gene expression was mediated through activation of RXR in the context of the PPAR-RXR heterodimer. The observation that both RXR and PPAR agonists can stimulate the transcription of genes implicated in lipid metabolism, suggest that rexinoids may also act as lipid-modifying agents and support a role of the permissive PPAR-RXR heterodimer in the control of insulin sensitivity.

The isoprenoids farnesol and juvenile hormone III (JH), metabolites of the cholesterol biosynthetic pathway, have been shown to stimulate fetal epidermal development in rodents. In this study we determined whether this effect might be attributed to a direct induction of keratinocytes differentiation and examined the mechanisms responsible for these effects. Rates of cornified envelope formation, a marker of keratinocyte terminal differentiation, as well as protein and mRNA levels of two proteins required for cornified envelope formation, involucrin (INV) and transglutaminase, increased 2- to 3-fold in normal human keratinocytes (NHK) treated with either farnesol or JH, even at low calcium concentrations (0.03 mM), which otherwise inhibit differentiation. In contrast, neither cholesterol nor mevalonate affected INV or transglutaminase mRNA levels. Effects of farnesol and JH on INV and transglutaminase mRNA levels were additive with high calcium concentrations (1.2 mM) that independently stimulate keratinocyte differentiation. In contrast, keratinocyte DNA synthesis was inhibited by these compounds. Both farnesol and JH stimulated INV and transglutaminase promoter activity, suggesting regulation at the transcriptional level. A series of truncation and deletion experiments revealed a farnesol-responsive region (-2452 to -1880 base pairs (bp)) in the INV gene. This region contained an AP-1 site. A single base pair mutation of the AP-1 site at -2116 to -2110 bp abolished farnesol responsiveness, identical to effects by peroxisome proliferator-activated receptor (PPARalpha) activators. Farnesoid X-activated receptor mRNA was not detected in NHK, but farnesol treatment increased activities of both a PPAR response element and PPARalpha mRNA levels in NHK. Furthermore, the increase in PPRE activity by farnesol was dependent upon PPARalpha in CV-1 cells. Finally, topical applications of farnesol increased mRNA and protein levels of the differentiation-specific genes, profilaggrin and loricrin, determined by immunohistochemistry and in situ hybridization, in wild-type but not in PPARalpha-/- murine epidermis. These findings suggest a novel role for selected isoprenoid cholesterol intermediates in the regulation of differentiation-specific gene transcription and a convergence of PPARalpha with the cholesterol synthetic pathway.

The peroxisome proliferator-activated receptor gamma (PPARγ) quickly evolved over the last decade from a new orphan receptor to one of the best characterized nuclear receptors. This fast pace in PPARγresearch was triggered by two main discoveries. Firstly, that PPARγwas shown to have a key role in adipogenesis and be a master controller of the “thrifty gene response” leading to efficient energy storage. Secondly, the discovery that its synthetic ligands, the thiazolidinediones, are promising insulin sensitizing drugs, which are currently being developed for the treatment of Type II (non-insulin-dependent) diabetes mellitus. More recently this nuclear receptor emerged from a role limited to metabolism (diabetes and obesity) to a power player in general transcriptional control of numerous cellular processes, with implications in cell cycle control, carcinogenesis, inflammation, atherosclerosis and immunomodulation. This widened role of PPARγwill certainly initiate a new flurry of research, which will not only refine our current often partial knowledge of PPARγbut more importantly also establish that this receptor has a definite role as a primary link adapting cellular, tissue and whole body homeostasis to energy stores. [Diabetologia (1999) 42: 1033–1049]The peroxisome proliferator-activated receptor gamma (PPARγ) quickly evolved over the last decade from a new orphan receptor to one of the best characterized nuclear receptors. This fast pace in PPARγresearch was triggered by two main discoveries. Firstly, that PPARγwas shown to have a key role in adipogenesis and be a master controller of the “thrifty gene response” leading to efficient energy storage. Secondly, the discovery that its synthetic ligands, the thiazolidinediones, are promising insulin sensitizing drugs, which are currently being developed for the treatment of Type II (non-insulin-dependent) diabetes mellitus. More recently this nuclear receptor emerged from a role limited to metabolism (diabetes and obesity) to a power player in general transcriptional control of numerous cellular processes, with implications in cell cycle control, carcinogenesis, inflammation, atherosclerosis and immunomodulation. This widened role of PPARγwill certainly initiate a new flurry of research, which will not only refine our current often partial knowledge of PPARγbut more importantly also establish that this receptor has a definite role as a primary link adapting cellular, tissue and whole body homeostasis to energy stores. [Diabetologia (1999) 42: 1033–1049]

Aims/hypothesis. The objectives of the present investigation were to examine: 1) whether a Pro115Glnvariant in the peroxisome proliferator-activated receptor-γ2 (PPAR-γ2) is associated with juvenile-onset obesity among Danish Caucasianmen and 2) whether the relation of a Pro12Alapolymorphism in PPAR-γ2with BMI and long-term weight regulation differ between lean and obese subjects within the same cohort. Methods. The Pro115Glnand Pro112Alavariants were examined using PCR and RFLP in a group of 752 subjects with a Body Mass Index (BMI) of 31.0 kg/m2or more and in 869 non-obese control subjects. Results. We did not find Pro115Glnin any of the 1621 male subjects we examined. Among the males with juvenile-onset obesity, the allelic frequency of the Pro12Alapolymorphism was 14 % (95 % confidence interval: 12–16 %) compared with 16 % (14–17 %) among the non-obese control subjects (NS). Heterozygosity of the codon 12variant was not associated with differences in BMI or changes in body weight regulation during follow up in lean or obese subjects. In the group of obese subjects, 21 homozygous Ala12Alacarriers had, however, a higher BMI (38.9 ± 5.4 kg/m2(means ± SD) vs 35.5 ± 5.5 kg/m2, p= 0.008) and a higher weight gain (0.27 ± 0.24 kg · m–2· year–1vs 0.10 ± 0.24 kg · m–2· year–1, p= 0.004), compared with wild-type carriers. Moreover, within the control group of 869 men the 14 homozygous carriers of the variant had a lower BMI (24.4 ± 2.7 kg/m2vs 26.2 ± 3.7 kg/m2, p= 0.005) and a slower increase in BMI (0.11 ± 0.11 kg · m–2· year–1vs 0.17 ± 0.11 kg · m–2· year–1, p= 0.002) compared with wild-type carriers. Conclusion/interpretation. The codon 12variant of PPAR-γ2is not intrinsically associated with juvenile obesity. The variant may in its homozygous form interact, however, with various combinations of genetic and environmental factors in lean and obese subjects to cause divergent modulating effects on BMI and long-term body weight control. [Diabetologia (1999) 42: 892–895]Aims/hypothesis. The objectives of the present investigation were to examine: 1) whether a Pro115Glnvariant in the peroxisome proliferator-activated receptor-γ2 (PPAR-γ2) is associated with juvenile-onset obesity among Danish Caucasianmen and 2) whether the relation of a Pro12Alapolymorphism in PPAR-γ2with BMI and long-term weight regulation differ between lean and obese subjects within the same cohort. Methods. The Pro115Glnand Pro112Alavariants were examined using PCR and RFLP in a group of 752 subjects with a Body Mass Index (BMI) of 31.0 kg/m2or more and in 869 non-obese control subjects. Results. We did not find Pro115Glnin any of the 1621 male subjects we examined. Among the males with juvenile-onset obesity, the allelic frequency of the Pro12Alapolymorphism was 14 % (95 % confidence interval: 12–16 %) compared with 16 % (14–17 %) among the non-obese control subjects (NS). Heterozygosity of the codon 12variant was not associated with differences in BMI or changes in body weight regulation during follow up in lean or obese subjects. In the group of obese subjects, 21 homozygous Ala12Alacarriers had, however, a higher BMI (38.9 ± 5.4 kg/m2(means ± SD) vs 35.5 ± 5.5 kg/m2, p= 0.008) and a higher weight gain (0.27 ± 0.24 kg · m–2· year–1vs 0.10 ± 0.24 kg · m–2· year–1, p= 0.004), compared with wild-type carriers. Moreover, within the control group of 869 men the 14 homozygous carriers of the variant had a lower BMI (24.4 ± 2.7 kg/m2vs 26.2 ± 3.7 kg/m2, p= 0.005) and a slower increase in BMI (0.11 ± 0.11 kg · m–2· year–1vs 0.17 ± 0.11 kg · m–2· year–1, p= 0.002) compared with wild-type carriers. Conclusion/interpretation. The codon 12variant of PPAR-γ2is not intrinsically associated with juvenile obesity. The variant may in its homozygous form interact, however, with various combinations of genetic and environmental factors in lean and obese subjects to cause divergent modulating effects on BMI and long-term body weight control. [Diabetologia (1999) 42: 892–895]

Abstract.: Peroxisome proliferator-activated receptors (PPARs) are nuclear receptors and have been initially described as molecular targets for compounds which induce peroxisome proliferation. The interest of researchers for PPARs increased dramatically when these receptors were shown to be directly activated by a number of medically relevant compounds. These compounds include: the fibrate class of hypolidemic drugs, the thiazolidinediones, which are insulin sensitizers used as orally active antidiabetic agents, certain non-steroidal anti-inflammatory drugs (NSAIDs), and naturally occurring fatty acid-derived molecules. Rapidly, it was demonstrated that PPARs are key regulators of lipid homeostasis and provide a molecular link between nutrition and gene regulation. Recently, detailed studies of PPAR expression profiles in different tissues pointed to the roles these receptors play in inflammation control and cell proliferation. In this review we will focus on the new insights gained into these two areas and we will also discuss our current knowledge of the regulation of PPAR transcriptional activity by cofactors.

Background & Aims: Abnormalities of fat in the mesentery including adipose tissue hypertrophy and fat wrapping have been long recognized on surgical specimens as characteristic features of Crohn's disease. However, the importance, origin, and significance of the mesenteric fat hypertrophy in this chronic inflammatory disease are unknown. Peroxisome proliferator-activated receptor @c (PPAR@c) is a crucial factor involved in the homeostasis of adipose tissue, a major source of biologically active mediators. Methods: Intra-abdominal fat accumulation was quantified using a magnetic resonance imaging method in patients with Crohn's disease and controls. PPAR@c and inflammatory cytokines synthesized by mesenteric adipose tissues were assessed by quantitative polymerase chain reaction, in situ hybridization, and immunohistochemistry. Results: In vivo, patients with Crohn's disease have an important accumulation of intra-abdominal fat. This mesenteric obesity, present from the onset of the disease, is associated with overexpression of PPAR@c and tumor necrosis factor (TNF)-@a, synthesized, at least in part, by adipocytes. Conclusions: These results suggest that confined increased PPAR@c mesenteric concentrations could lead to the mesenteric fat hypertrophy, which could actively participate through the synthesis of TNF-@a in the inflammatory response. GASTROENTEROLOGY 1999;117:73-81

The long-chain acyl-coenzyme A synthetase (ACS) gene gives rise to three transcripts containing different first exons preceded by specific regulatory regions A, B, and C. Exon-specific oligonucleotide hybridization indicated that only A-ACS mRNA is expressed in rat liver. Fibrate administration induced liver C-ACS strongly and A-ACS mRNA to a lesser extent. B-ACS mRNA remained undetectable. In primary rat hepatocytes and Fa-32 hepatoma cells C-ACS mRNA increased after treatment with fenofibric acid, alpha-bromopalmitate, tetradecylthioacetic acid, or alpha-linolenic acid. Nuclear run-on experiments indicated that fenofibric acid and alpha-bromopalmitate act at the transcriptional level. Transient transfections showed a 3.4-, 2.3-, and 2.2-fold induction of C-ACS promoter activity after fenofibric acid, alpha-bromopalmitate, and tetradecylthioacetic acid, respectively. Unilateral deletion and site-directed mutagenesis identified a peroxisome proliferator activator receptor (PPAR)-responsive element (PPRE) mediating the responsiveness to fibrates and fatty acids. This ACS PPRE contains three imperfect half sites spaced by 1 and 3 oligonucleotides and binds PPAR.retinoid X receptor heterodimers in gel retardation assays. In conclusion, the regulation of C-ACS mRNA expression by fibrates and fatty acids is mediated by PPAR.retinoid X receptor heterodimers interacting through a PPRE in the C-ACS promoters. PPAR therefore occupies a key position in the transcriptional control of a pivotal enzyme controlling the channeling of fatty acids into various metabolic pathways.

We have isolated a cDNA corresponding to the hamster peroxisome proliferator-activated receptor haPPAR@c, a member of the steroid nuclear hormone receptor superfamily of transcription factors. haPPAR@c mRNA is highly expressed in adipose tissue, and is expressed in lung, heart, kidney, liver and spleen to a lower extent. Thus, haPPAR@c may function in activating the transcription of target genes in a variety of tissues, including those not particularly subjected to peroxisomal @b-oxidation. haPPAR@c binds efficiently in the presence of retinoid X receptor @a (RXR@a) to a peroxisome proliferator response element ( PPRE) first identified in the acyl-CoA oxidase (ACO) promoter, the ratelimiting enzyme of peroxisomal @b-oxidation. The gene (ACO) encoding this enzyme has been previously shown to be under the transcriptional control of mouse PPAR (mPPAR). Although binding of haPPAR@c/RXR@a on the PRE of the ACO promoter in vitro is similar to that observed for mPPAR/RXR@a, we show that the transcriptional activities of mPPAR and haPPAR@c are regulated differently in vivo in response to peroxisome proliferators and heterodimerization with RXR.

Summary THP-1 is a human monocytic leukemia cell line. After treatment with phorbol esters, THP-1 cells differentiate into macrophage-like cells which mimic native monocyte-derived macrophages in several respects. Compared to other human myeloid cell lines, such as HL-60, U937, KG-1, or HEL cell lines, differentiated THP-1 cells behave more like native monocyte-derived macrophages. Because of these characteristics, the THP-1 cell line provides a valuable model for studying the mechanisms involved in macrophage differentiation, and for exploring the regulation of macrophage-specific genes as they relate to physiological functions displayed by these cells.

Transcription of the low-density lipoprotein receptor (LDL-R) gene in the human monocytic leukemic cell line THP-1 and in the human hepatocarcinoma cell line Hep-G2 is regulated by second messengers of the diacylglycerol-protein kinase C (DAG-PKC), inositol 1,4,5-triphosphate-Ca2+, and cyclic AMP pathways. Exogenous phospholipase C (which releases DAG and inositol 1,4,5-triphosphate), PKC activators (phorbol esters and DAG), Ca2+ ionophores, and a cyclic AMP analog all transiently induced accumulation of LDL-R mRNA. The effects of these three signal-transducing pathways were to a large extent additive. Furthermore, PKC stimulation effected an increase in LDL binding, which suggested that the increase in LDL-R mRNA resulted in an increase in functional cell surface receptor activity. These results suggest that uptake of cholesterol by these cells is under control of both intracellular cholesterol levels and external signals.

The peroxisome proliferator-activated receptor-alpha (PPARalpha) controls gene expression in response to a diverse class of compounds collectively referred to as peroxisome proliferators. Whereas most known peroxisome proliferators are of exogenous origin and include hypolipidemic drugs and other industrial chemicals, several endogenous PPARalpha activators have been identified such as fatty acids and steroids. The latter finding and the fact that PPARalpha modulates target genes encoding enzymes involved in lipid metabolism suggest a role for PPARalpha in lipid metabolism. This was investigated in the PPARalpha-deficient mouse model. Basal levels of total serum cholesterol, high density lipoprotein cholesterol, hepatic apolipoprotein A-I mRNA, and serum apolipoprotein A-I in PPARalpha-deficient mice are significantly higher compared with wild-type controls. Treatment with the fibrate Wy 14,643 decreased apoA-I serum levels and hepatic mRNA levels in wild-type mice, whereas no effect was detected in the PPARalpha-deficient mice. Administration of the fibrate Wy 14,643 to wild-type mice results in marked depression of hepatic apolipoprotein C-III mRNA and serum triglycerides compared with untreated controls. In contrast, PPARalpha-deficient mice were unaffected by Wy 14,643 treatment. These studies demonstrate that PPARalpha modulates basal levels of serum cholesterol, in particular high density lipoprotein cholesterol, and establish that fibrate-induced modulation in hepatic apolipoprotein A-I, C-III mRNA, and serum triglycerides observed in wild-type mice is mediated by PPARalpha.

Lipoprotein lipase (LPL), an enzyme which hydrolyzes triglycerides and participates in the catabolism of remnant lipoproteins, plays a crucial role in energy and lipid metabolism. The goal of this study was to analyze the expression and regulation of the LPL gene in human adrenals. Reverse transcriptase-polymerase chain reaction amplification and sequence analysis demonstrated the presence of LPL mRNA in fetal and adult human adrenal cortex. Furthermore, the human adrenocortical carcinoma cell line, NCI-H295, expresses LPL mRNA and protein, which is localized to the outer cellular membrane as demonstrated by immunofluorescence confocal microscopy and can be released in the medium by heparin addition. To asses whether the LPL gene is regulated by agents regulating adrenal steroidogenesis, NCI-H295 cells were treated with activators of second messenger systems. Whereas the calcium-ionophore A23187 did not affect LPL gene expression, treatment with phorbol 12-myristate 13-acetate decreased LPL mRNA levels in a time- and dose-dependent manner. This decrease after phorbol 12-myristate 13-acetate was associated with diminished heparin-releasable LPL mass and activity in the culture medium. Addition of the cAMP analog 8-Br-cAMP to NCI-H295 cells resulted in a rapid, but transient dose-dependent induction of LPL mRNA. Treatment with the protein synthesis inhibitor cycloheximide gradually induced, whereas simultaneous addition of cAMP and cycloheximide superinduced LPL mRNA levels. Nuclear run-on analysis indicated that the effects of cAMP and cycloheximide occurred at the transcriptional and post-transcriptional level, respectively. Transient co-transfection assays demonstrated that the first 230 base pairs of the proximal LPL promoter contain a cAMP-responsive element activated by protein kinase A and transcription factors belonging to the CREB/CREM family. These data indicate that LPL is expressed in human adrenal cortex and regulated in NCI-H295 adrenocortical carcinoma cells by activators of the protein kinase A and protein kinase C second messenger pathways in a manner comparable to P450scc, which catalyzes the first step in adrenal steroidogenesis. These observations suggest a role for LPL in adrenal energy and/or lipid metabolism and possibly in steroidogenesis.

Certain missense substitutions on the human lipase (hLPL) gene produce mutated proteins that are retained in different compartments along the secretory pathway. The purpose of the present study was to elucidate whether the C-terminal domain of the hLPL molecule could be important for secretion. We constructed by site-directed mutagenesis three carboxy-terminal mutated (F388-->Stop, K428-->Stop and K441-->Stop) hLPL cDNAs that were expressed in COS1 cells. Immunoblotting of cell extracts showed that all three constructs led to similar levels of protein. Both wild type (WT) hLPL and the truncated K441-->Stop hLPL were secreted to the extracellular medium, and presented a similar intracellular distribution pattern as shown by immunofluorescence. Neither F388-->Stop nor K428-->Stop hLPL protein was detected in cell medium. Immunofluorescence experiments showed that both truncated hLPL were retained within an intracellular compartment, which became larger. Double immunofluorescence analysis using antibodies against LPL and antiprotein disulfide isomerase as a marker showed that the truncated K428-->Stop hLPL was retained within the rough endoplasmic reticulum. This truncated protein was not found in other compartments in the secretory pathway, such as Golgi complex and lysosomes, indicating that it did not exit the endoplasmic reticulum. Further analysis of the C-terminal region of the LPL molecular model showed both that F388-->Stop and K428-->Stop hLPL truncated proteins are highly hydrophobic. As retention of secretory proteins in the rough endoplasmic reticulum is a quality control mechanism of the secretory pathway, we conclude that the C-terminal domain of hLPL is critical for correct intracellular processing of the newly synthesized protein.

Since elevated concentrations of plasma high density lipoprotein (HDL) and its major apolipoprotein (apo), apoA-I, confer protection against atherosclerosis, considerable research efforts have focussed on the identification of factors regulating apoA-I gene expression in an attempt to increase its production. Nuclear receptors are interesting candidates because they are transcription factors whose activity is ligand-dependent. In the present study we identified the orphan receptor RORalpha1 as an activator of apoA-I gene transcription. In apoA-I-expressing intestinal Caco-2 cells, overexpression of the RORalpha1, but not the RORalpha2 or RORalpha3 isoforms, increased rat apoA-I gene transcription. Deletion and site-directed mutagenesis experiments identified a functional ROR-responsive element (RORE) in the rat and mouse apoA-I gene promoters, which overlaps with the TATA box. Gel shift experiments indicated that this RORE binds the RORalpha1 isoform, but not the RORalpha2 or RORalpha3 isoforms. Furthermore, compared with wild type mice, apoA-I mRNA levels were significantly lower in small intestines of staggerer mice homozygous for a deletion in the RORalpha gene. In addition, reverse transcriptase-polymerase chain reaction analysis revealed the expression of RORalpha in small intestinal epithelium and in Caco-2 cells. These data indicate a novel, physiological role for RORalpha1 in the regulation of genes involved in lipid and lipoprotein metabolism and possibly in the development of metabolic diseases, such as atherosclerosis.

Transcription of the low density lipoprotein receptor (LDL-R) and 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase genes was rapidly and transiently induced (8.5- and 2.3-fold, respectively) early during phorbol 12-myristate 13-acetate (PMA)-induced macrophage differentiation of the human monocytic leukemia cell line THP-1. The levels of mRNA coding for LDL-R and HMG-CoA reductase increased soon after induction, reached a maximum (12- and 7-fold increase, respectively) in 2-3 hr, and then rapidly returned to the low constitutive levels observed before induction. The stability of LDL-R mRNA did not change significantly during differentiation, whereas that of HMG-CoA reductase mRNA decreased by about 5-fold 6 hr after the addition of PMA. Transcriptional induction of both LDL-R and HMG-CoA reductase genes (5.6- and 2-fold, respectively) was also observed when undifferentiated cells were treated with cycloheximide (CHX), resulting in a transient increase in steady-state mRNA (7- and 3-fold, respectively). These results suggest that expression of the two genes is maintained at low constitutive levels in uninduced THP-1 cells by a protein with a short half-life. Superinduction of both genes occurred when PMA and CHX were added simultaneously. The induction of LDL-R and HMG-CoA reductase mRNAs during early macrophage differentiation is mediated by protein kinase C. It is hypothesized that protein kinase C acts directly or indirectly to inactivate the labile negative regulatory protein. Induction of LDL-R mRNA was also observed when the human hepatocarcinoma cell line Hep G2 was treated with PMA and CHX, suggesting that this mechanism of regulation may exist in several cell types.

While the molecular characterization of lipoprotein lipase (LPL) activation is progressing, the intracellular processing, transport, and secretion signals of LPL are still poorly known. The aim of this paper is to study are involvement of glycine 142 in LPL secretion and to elucidate the intracellular destination of the altered protein that remains inside the cell. We mutated the human LPL cDNA by site-directed mutagenesis in order to produce the G142e hLPL in which the glycine 142 was replaced by a glutamic acid. The wild type human LPL (WT hLPL) and the mutant G142E hLPL were expressed by transient transfection in COS1 cells. Using Western blot assays we identified a single band that had the same molecular weight for both proteins. However, Western blots of culture media did not reveal any specific band for the mutant protein, and ELISA experiments showed that the extracellular mass of the mutant LPL was only 25% of the WT protein, indicating defective secretion of the altered enzyme. Heparin increased LPL secretion in the case of the WT hLPL but did not have any stimulatory effect when acting on G142E hLPL-transfected cells. However, heparin-Sepharose chromatography revealed that both proteins presented the same heparin affinity. Metabolic labeling and radioimmunoprecipitation studies showed that both the WT and the mutant hLPL intracellular levels decreased upon chase time. Furthermore, leupeptin had a greater effect on the intracellular level of the mutant enzyme, thus indicating its higher intracellular degradation. Immunofluorescent studies using confocal microscopy indicated high colocalization of the LPL labeling and the Lamp1 lysosomal labeling in G142E hLPL-expressing cells. This result was confirmed using immunoelectron microscopy, which in addition showed gold labeling in Golgi stacks. This finding together with experiments performed with endoglycosidase H digestion of immunoprecipitated radiolabeled LPL, indicated that the mutant enzyme entered the Golgi compartment. The results reported in this paper show that the G142E hLPL is not efficiently secreted to the extracellular medium, but it is missorted to lysosomes for intracellular degradation. This finding suggests that lysosomal missorting might be a mechanism of cell quality control of secreted LPL.

Peroxisome proliferator-activated receptors (PPARs) are nuclear hormone receptors that can be activated by fatty acids and peroxisome proliferators. The PPAR alpha subtype mediates the pleiotropic effects of these activators in liver and regulates several target genes involved in fatty acid catabolism. In primary hepatocytes cultured in vitro, the PPAR alpha gene is regulated at the transcriptional level by glucocorticoids. We investigated if this hormonal regulation also occurs in the whole animal in physiological situations leading to increased plasma corticosterone levels in rats. We show here that an immobilization stress is a potent and rapid stimulator of PPAR alpha expression in liver but not in hippocampus. The injection of the synthetic glucocorticoid dexamethasone into adult rats produces a similar increase in PPAR alpha expression in liver, whereas the administration of the antiglucocorticoid RU 486 inhibits the stress-dependent stimulation. We conclude that glucocorticoids are major mediators of the stress response. Consistent with this hormonal regulation, hepatic PPAR alpha mRNA and protein levels follow a diurnal rhythm, which parallels that of circulating corticosterone. To test the effects of variations in PPAR alpha expression on PPAR alpha target gene activity, high glucocorticoid-dependent PPAR alpha expression was mimicked in cultured primary hepatocytes. Under these conditions, hormonal stimulation of receptor expression synergizes with receptor activation by WY-14,643 to induce the expression of the PPAR alpha target gene acyl-CoA oxidase. Together, these results show that regulation of the PPAR alpha expression levels efficiently modulates PPAR activator signaling and thus may affect downstream metabolic pathways involved in lipid homeostasis.

Genetic studies in mice have identified the ob gene product as a potential signaling factor regulating body weight homeostasis and energy balance. It is suggested that modulation of ob gene expression results in changes in body weight and food intake. Glucocorticoids are shown to have important metabolic effects and to modulate food intake and body weight. In order to test the hypothesis that these metabolic effects of glucocorticoids are linked to changes in the expression of the ob gene, ob mRNA levels were evaluated in rats treated with different glucocorticosteroids at catabolic doses and correlated to the kinetics of changes in body weight gain and food intake. Results from time course experiments demonstrate that adipose tissue ob gene expression is rapidly induced by glucocorticosteroids. This induction is followed by a concordant decrease in body weight gain and food consumption. These data suggest that the catabolic effects of corticosteroids on body weight mass and food intake might be mediated by changes in ob expression. Modulation of ob expression may therefore constitute a mechanism through which hormonal, pharmacological, or other factors control body weight homeostasis.

Abstract. Changes in calcium concentration were induced by an infusion of disodium-EDTA or calcium in 2 members of a family suffering from hypocalciuric hypercalcaemia (FHH) associated with interstitial lung disease. These changes in calcium demonstrated an inverse linear relationship with the changes in serum parathyroid hormone (PTH). Infusion of EDTA in control subjects and in patients with an adenoma or hyperplasia of the parathyroid glands also showed inverse relationships between calcium and PTH. The correlation between serum calcium and serum PTH was significant over the range observed during the induced hypo- and/or hypercalcaemia in controls and in patients with FHH or adenoma. The regressions were, however, shifted relative to each other: in comparison with controls, the FHH was displaced upwards and to the right, although not as far as the adenomas. These findings suggest the existence of an elevated set point for extracellular calcium (or calciostat) in FHH.