Citrulline counteracts overweight- and aging-related effects on adiponectin and leptin gene expression in rat white adipose tissue.

We recently demonstrated that citrulline (CIT) reduced the expression of inflammatory genes in cultured explants from retroperitoneal (RET) white adipose tissue (WAT) from young (2-4 months) but not old (25 months) rats. Here we show that in RET WAT from old rats and high-fat-diet-fed (HFD) young rats, the basal expression of the leptin gene was increased (275-345%) whereas that of the adiponectin gene was decreased (48-60%), when compared to those from control-diet-fed (CD) young rats. We show also that in RET WAT from old rats, a diet supplemented with CIT for 3 months reduced macrophage (F4/80, CD68) and inflammation (interleukin-6, tumor necrosis factor-α) marker genes 23-97%. CIT supplementation lowered leptin mRNA 62% and increased adiponectin mRNA 232%. In cultured explants of RET WAT from 4 month-old CD, 4 month-old HFD and 25-month-old CD rats, the exposure to 2.5 mmol/L CIT for 24 h up-regulated adiponectin gene expression 151%, 362% and 216% respectively. In contrast, leptin gene expression was down-regulated 66% selectively in CIT-treated explants from 25-month-old CD rats. These results further support the proposed beneficial effect of CIT to counteract the deleterious effects of aging and overweight on the metabolic, inflammatory and endocrine functions of WAT.

Citrulline reduces glyceroneogenesis and induces fatty acid release in visceral adipose tissue from overweight rats.

SCOPE: High-fat diet (HFD) increases visceral adipose tissue (AT). Our aim was to evaluate whether citrulline (CIT) affected nonesterified fatty acid (NEFA) metabolism in AT from HFD-fed rats. METHODS AND RESULTS: Rats were fed for 8 weeks with either a control diet (CD) or HFD. Retroperitoneal AT explants were exposed to 2.5 mmol/L CIT for 24 h. We analyzed lipolysis, beta-oxidation, glyceroneogenesis, and the expression of the key associated enzymes. CIT doubled NEFA release selectively in HFD AT. Phosphorylation of hormone-sensitive lipase was upregulated 50 and 100% by CIT in CD and HFD AT, respectively. Under CIT, beta-oxidation increased similarly whatever the diet, whereas glyceroneogenesis, which permits NEFA re-esterification, was downregulated 50 and 80% in CD and HFD AT, respectively. In the latter, the important decrease in re-esterification probably explains the rise of NEFA release. A pretreatment with the nitric oxide synthase inhibitor N ω-nitro-l-arginine methyl ester abolished CIT effects. CONCLUSION: These results demonstrate direct lipolytic and antiglyceroneogenic effects of CIT on CD and HFD AT. The selective CIT-mediated NEFA release from HFD AT was probably the consequence of the drastic decrease in glyceroneogenesis and nitric oxide was a mediator of CIT effects. These results provide evidence for a direct action of CIT on AT to reduce overweight.

Citrulline induces fatty acid release selectively in visceral adipose tissue from old rats.

SCOPE: During aging, increased visceral adipose tissue (AT) mass may result in impaired metabolic status. A citrulline (CIT)-supplemented diet reduces AT mass in old rats. We hypothesized that CIT could directly affect fatty acid (FA) metabolism in retroperitoneal AT. METHODS AND RESULTS: A 24-h exposure of AT explants from old (25 months) rats to 2.5 mM CIT induced a 50% rise in glycerol and FA release, which was not observed in explants from young (2 months) animals. The phosphorylated form of hormone-sensitive lipase, a key lipolytic enzyme, was 1.5-fold higher in CIT-treated explants from old and young rats, whereas glyceroneogenesis, that provides glycerol-3P requested for FA re-esterification, and its key enzyme phosphoenolpyruvate carboxykinase, were down-regulated 40-70%. Specifically in young rats, beta-oxidation capacity and gene expressions of carnitine palmitoyl transferase 1-b and very long chain acyl-CoA dehydrogenase were strongly up-regulated by CIT. In contrast, in old rats, while glyceroneogenesis was lower, beta-oxidation was not affected, enabling increased FA release. CONCLUSION: Hence, in visceral AT, CIT exerts a specific induction of the beta-oxidation capacity in young rats and a selective stimulation of FA release in old rats, therefore providing a direct mechanism of CIT action to reduce AT mass.

Rapid nitration of adipocyte phosphoenolpyruvate carboxykinase by leptin reduces glyceroneogenesis and induces fatty acid release.

Fatty acid (FA) release from white adipose tissue (WAT) is the result of the balance between triglyceride breakdown and FA re-esterification. The latter relies on the induction of cytosolic phosphoenolpyruvate carboxykinase (PEPCK-C), the key enzyme for glyceroneogenesis. We previously demonstrated that long-term (18 h) leptin treatment of rat epididymal WAT explants reduced glyceroneogenesis through nitric oxide (NO)-induced decrease in PEPCK-C expression. We investigated the effect of a short-term leptin treatment (2 h) on PEPCK-C expression and glyceroneogenesis in relation to NO production. We demonstrate that in WAT explants, leptin-induced NO synthase III (NOS III) phosphorylation was associated with reduced PEPCK-C level and glyceroneogenesis, leading to FA release, while PEPCK-C gene expression remained unaffected. These effects were absent in WAT explants from leptin receptor-deficient Zucker rat. Immunoprecipitation and western blot experiments showed that the leptin-induced decrease in PEPCK-C level was correlated with an increase in PEPCK-C nitration. All these effects were abolished by the NOS inhibitor Nω-nitro-L-arginine methyl ester and mimicked by the NO donor S-nitroso-N-acetyl-DL penicillamine. We propose a mechanism in which leptin activates NOS III and induces NO that nitrates PEPCK-C to reduce its level and glyceroneogenesis, therefore limiting FA re-esterification in WAT.

Early induction of a brown-like phenotype by rosiglitazone in the epicardial adipose tissue of fatty Zucker rats.

The epicardial adipose tissue (EAT) is "hypertrophied" in the obese. Thiazolidinediones are anti-diabetic, hypolipidemic drugs and are selective agonists for the gamma isoform of peroxisome proliferator-activated receptor (PPARγ). We evaluated the short-term effects of the prototype rosiglitazone (RSG, 5 mg kg(-1) day(-1) for 4 days) on the expression of the genes and proteins (by real-time PCR and Western blot) involved in fatty acid (FA) metabolism in EAT of the obese fatty Zucker rat and compared the levels of expression with those in retroperitoneal adipose tissue (RAT). The glyceroneogenic flux leading to fatty acid re-esterification was assessed by the incorporation of 14C from [1-14C]-pyruvate into neutral lipids. RSG upregulated the mRNA for phosphoenolpyruvate carboxykinase, pyruvate dehydrogenase kinase 4, glycerol kinase, adipocyte lipid binding protein, adipose tissue triglyceride lipase and lipoprotein lipase in both RAT and EAT with a resulting increase in glyceroneogenesis that, however, was more pronounced in EAT than in RAT. Under RSG, fatty acid output was decreased in both tissues but unexpectedly less so in EAT than in RAT. RSG also induced the expression of the key genes for fatty acid oxidation [carnitinepalmitoyl transferase-1, medium chain acyl dehydrogenase and very long chain acyl dehydrogenase (VLCAD)]in EAT and RAT with a resulting significant rise of  the expression of VLCAD protein. In addition, the expression of the genes encoding proteins involved in mitochondrial processing and density PPARγ coactivator 1 alpha (PGC-1α), NADH dehydrogenase 1 and cytochrome oxidase (COX4) were increased by RSG treatment only in EAT, with a resulting significant up-regulation of PGC1-α and COX4 protein. This was accompanied by a rise in the expression of PR domain containing 16 and uncoupling protein 1, two brown adipose tissue-specific proteins. In conclusion, this study reveals that PPAR-γ agonist could induce a rapid browning of the EAT that probably contributes to the increase in lipid turnover.

Glucose homeostasis in pre-diabetic NOD and lymphocyte-deficient NOD/SCID mice during gestation.

BACKGROUND: Unlike other strains, spontaneously type 1 non-obese diabetic (NOD) experience transient hyperinsulinemia after weaning. The same applies for NOD/SCID mice, which lack functional lymphocytes, and unlike NOD mice, do not develop insulitis and diabetes like NOD mice. AIMS: Given that beta-cell stimulation is a natural feature of gestation, we hypothesized that glucose homeostasis is disturbed in gestate pre-diabetic NOD and non-diabetic NOD/SCID mice, which may accelerate the onset of diabetes and increase diabetes prevalence. METHODS: During gestation and postpartum, mice were analyzed under basal feed conditions followed by glucose injection (1 g/kg, i.p.) after overnight fast, using glucose tolerance test (GTT). Glycemia, corticosteronemia, blood and pancreatic insulin, glucagon levels, islet size, and islet morphology were evaluated. Glycemia and mortality were assessed after successive gestations in NOD mice mated for the first time at 2 different ages. RESULTS: 1. Basal glucagonemia rose markedly in first-gestation fed NOD mice. 2. beta-cell hyperactivity was present earlier in first-gestation non-diabetic fasted NOD and NOD/SCID mice than in age-matched C57BL/6 mice, assessed by increased insulin/glucose ratio after GTT. 3. Overnight fasting increased corticosteronemia rapidly and sharply in pre-diabetic gestate NOD and NOD/SCID mice. 4. Islet size increased in non-diabetic gestate NOD mice compared with C57BL/6 mice. 5. Successive gestations accelerated diabetes onset, and contributed to increased mortality in NOD mice. CONCLUSIONS: First-gestation pre-diabetic NOD and non-diabetic NOD/SCID mice exhibited beta-cell hyperactivity and deregulation of glucagon and/or corticosterone secretion. This amplified normally occurring insulin resistance, further exhausted maternal beta-cells, and accelerated diabetes in NOD mice.

The infrapatellar fat pad in knee osteoarthritis: an important source of interleukin-6 and its soluble receptor.

OBJECTIVE: Obesity is a potent risk factor in knee osteoarthritis (OA). It has been suggested that adipokines, secreted by adipose tissue (AT) and largely found in the synovial fluid of OA patients, derive in part from the infrapatellar fat pad (IFP), also known as Hoffa's fat pad. The goal of this study was to characterize IFP tissue in obese OA patients and to compare its features with thigh subcutaneous AT to determine whether the IFP contributes to local inflammation in knee OA via production of specific cytokines. METHODS: IFP and subcutaneous AT samples were obtained from 11 obese women (body mass index > or =30 kg/m2) with knee femorotibial OA. Gene expression was measured by real-time quantitative polymerase chain reaction. Cytokine concentrations in plasma and in conditioned media of cultured AT explants were determined by enzyme-linked immunosorbent assay or by Luminex xMAP technology. RESULTS: In IFP tissue versus subcutaneous AT, there was a decrease in the expression of genes for key enzymes implicated in adipocyte lipid metabolism, whereas the expression levels of genes for AT markers remained similar. A 2-fold increase in the expression of the gene for interleukin-6 (IL-6), a 2-fold increase in the release of IL-6, and a 3.6-fold increase in the release of soluble IL-6 receptor (sIL-6R) were observed in IFP samples, compared with subcutaneous AT, but the rates of secretion of other cytokines in IFP samples were similar to the rates in subcutaneous AT. In addition, leptin secretion was decreased by 40%, whereas adiponectin secretion was increased by 70%, in IFP samples versus subcutaneous AT. CONCLUSION: Our results indicate that the IFP cytokine profile typically found in OA patients could play a role in paracrine inflammation via the local production of IL-6/sIL-6R and that such a profile might contribute to damage in adjacent cartilage.

Pyruvate dehydrogenase kinase 4: regulation by thiazolidinediones and implication in glyceroneogenesis in adipose tissue.

OBJECTIVE: Pyruvate dehydrogenase complex (PDC) serves as the metabolic switch between glucose and fatty acid utilization. PDC activity is inhibited by PDC kinase (PDK). PDC shares the same substrate, i.e., pyruvate, as glyceroneogenesis, a pathway controlling fatty acid release from white adipose tissue (WAT). Thiazolidinediones activate glyceroneogenesis. We studied the regulation by rosiglitazone of PDK2 and PDK4 isoforms and tested the hypothesis that glyceroneogenesis could be controlled by PDK. RESEARCH DESIGN AND METHODS: Rosiglitazone was administered to Zucker fa/fa rats, and then PDK4 and PDK2 mRNAs were examined in subcutaneous, periepididymal, and retroperitoneal WAT, liver, and muscle by real-time RT-PCR. Cultured WAT explants from humans and rats and 3T3-F442A adipocytes were rosiglitazone-treated before analyses of PDK2 and PDK4 mRNA and protein. Small interfering RNA (siRNA) was transfected by electroporation. Glyceroneogenesis was determined using [1-(14)C]pyruvate incorporation into lipids. RESULTS: Rosiglitazone increased PDK4 mRNA in all WAT depots but not in liver and muscle. PDK2 transcript was not affected. This isoform selectivity was also found in ex vivo-treated explants. In 3T3-F442A adipocytes, Pdk4 expression was strongly and selectively induced by rosiglitazone in a direct and transcriptional manner, with a concentration required for half-maximal effect at 1 nmol/l. The use of dichloroacetic acid or leelamine, two PDK inhibitors, or a specific PDK4 siRNA demonstrated that PDK4 participated in glyceroneogenesis, therefore altering nonesterified fatty acid release in both basal and rosiglitazone-activated conditions. CONCLUSIONS: These data show that PDK4 upregulation in adipocytes participates in the hypolipidemic effect of thiazolidinediones through modulation of glyceroneogenesis.

Bromocriptine-induced hyperglycemia in nonobese diabetic mice: kinetics and mechanisms of action.

The effects of bromocriptine (10 mg/kg), known to inhibit prolactin secretion and lower autoimmune processes, were studied on glucose homeostasis in non-fasted non-obese diabetic mice, a spontaneous model of type 1 diabetes. Hyperglycemia was observed 120 and 240 min after i.p. but not s.c. injection. Bromocriptine administration i.p. led to rapid and marked hyperglycemia characterized by sexual dimorphism with males having higher glycemia than females. Bromocriptine induced a rapid but transient decrease in insulinemia in males only and biphasic increases in glucagon levels and a sustained stimulatory effect on circulating corticosterone in both sexes. Bromocriptine-induced hyperglycemia involved D2-dopaminergic receptors, as demonstrated by the inhibitory effect of the D2-dopamine antagonist, metoclopramide (10 mg/kg). Simultaneous injection of bromocriptine and metoclopramide also blocked the rise in blood corticosterone. In conclusion, by inducing hyperglycemia, i.p. bromocriptine administration to prediabetic autoimmune mice may counteract its beneficial anti-immunostimulatory effects.

Proposed involvement of adipocyte glyceroneogenesis and phosphoenolpyruvate carboxykinase in the metabolic syndrome.

Elevated concentration of plasma non-esterified fatty acids (NEFA) is now recognized as a key factor in the onset of insulin-resistance and type 2 diabetes mellitus. During fasting, circulating NEFAs arise from white adipose tissue (WAT) as a consequence of lipolysis from stored triacylglycerols. However, a significant part of these FAs (30-70%) is re-esterified within the adipocyte, so that a recycling occurs and net FA output is much less than << true >> lipolysis. Indeed, a balance between two antagonistic processes, lipolysis and FA re-esterification, controls the rate of net FA release from WAT. During fasting, re-esterification requires glyceroneogenesis defined as the de novo synthesis of glycerol-3-P from pyruvate, lactate or certain amino acids. The key enzyme in this process is the cytosolic isoform of phosphoenolpyruvate carboxykinase (PEPCK-C; EC 4.1.1.32). Recent advance has stressed the role of glyceroneogenesis and of PEPCK-C in FA release from WAT. Results indicate that glyceroneogenesis is indeed important to lipid homeostasis and that a disregulation in this pathway may have profound pathophysiological effects. The present review focuses on the regulation of glyceroneogenesis and of PEPCK-C gene expression and activity by FAs, retinoic acids, glucocorticoids and the hypolipidemic class of drugs, thiazolidinediones.

Increased transcriptional preproinsulin II beta-cell activity in neonatal nonobese diabetic mice: in situ hybridization analysis.

In the prediabetic nonobese diabetic (NOD) mouse, a spontaneous model of type 1 diabetes, we previously reported transient postweaning hyperinsulinemia followed by progressive islet hyperplasia. A modified in situ hybridization technique was used to determine whether these effects were accompanied by changes in insulin transcriptional activity as a function of age. We found that NOD neonates express higher levels of preproinsulin II primary transcripts than age-matched C57BL/6 mice, but this difference disappeared within the first wk of age. To manipulate insulin transcriptional activity in NOD neonates, NOD mothers were treated with insulin during the last two wk of gestation. A down-regulation of beta-cell hyperactivity was observed in female NOD neonates but not in male neonates. By contrast, the same insulin treatment applied to NODscid (severe combined immunodeficiency) mothers, devoid of functional lymphocytes but showing like NOD mice postweaning hyperinsulinemia, increased transcriptional beta-cell activity in both sexes of neonates. In conclusion, NOD mice exhibit successive and transient signs of beta-cell hyperactivity, reflected as early as birth by high transcriptional preproinsulin II activity and later, from weaning to around 10 wk of age, by hyperinsulinemia. Of note, when thinking in terms of in utero disease programming, the NOD neonatal transcriptional beta-cell hyperactivity could be modulated by environmental (maternal and/or fetal) factors.

Extracellular matrix distribution and islet morphology in the early postnatal pancreas: anomalies in the non-obese diabetic mouse.

Previously, we reported elevated numbers of macrophages in the pancreas of NOD mice, a spontaneous animal model for T1D, during the early postnatal period. Extracellular matrix plays an important role in the tissue trafficking and retention of macrophages as well as in postnatal pancreas development. Therefore, we have examined the expression and distribution of laminin and fibronectin, two major extracellular matrix proteins and their corresponding integrin receptors, in the pre-weaning pancreases of NOD mice and control mouse strains. In addition, we have characterized the pancreas morphology during this period, since the morphology of the pre-weaning pancreas before the onset of lymphocytic peri-insulitis, when the pancreas is still subject to developmental changes, has been poorly documented. We show that laminin labeling is mainly associated with exocrine tissue, whereas fibronectin labeling was mostly localized at the islet-ductal pole, islet periphery and in intralobular septa. Moreover, the protein expression level of fibronectin was increased in NOD pancreases at the early stage of postnatal development, as compared to pancreases of C57BL/6 and BALB/c mouse strains. Interestingly, pancreatic macrophages were essentially found at sites of intense fibronectin labeling. The increased fibronectin content in NOD neonatal pancreas coincided with altered islet morphology, histologically reflected by enlarged and irregular shaped islets and increased percentages of total endocrine area as compared to that of control strains. In conclusion, increased levels of the extracellular matrix protein fibronectin were found in the early postnatal NOD pancreas, and this is associated with an enhanced accumulation of macrophages and altered islet morphology.

Proapoptosis and antiapoptosis-related molecules during postnatal pancreas development in control and nonobese diabetic mice: relationship with innervation.

The mouse pancreas, an immature organ at birth, reaches its adult size and morphology after weaning (3 weeks of age). Around this time, apoptotic phenomena and various types of macrophages are normally present. During development, Fas-Fas ligand (FasL) interactions are known to play a role in apoptotic events involved in tissue remodeling and elimination of damaged cells, and macrophages are routinely observed near apoptotic cells. Apoptosis and Fas-FasL interactions are also thought to be involved in the pathogenesis of autoimmune diseases, particularly type 1 diabetes (T1D). Therefore, we used early postnatal mouse pancreata from three control strains (C57BL/6, DBA/2, BALB/c) and from two strains with the nonobese diabetic (NOD)-related genetic background (the spontaneous T1D NOD model and the lymphocyte-deficient NODscid strain) to study apoptotic phenomena together with the molecular and immunohistochemical expression of proapoptosis (Fas, FasL) and antiapoptosis (Bcl-2) proteins. First, although no major difference in the numbers of total pancreatic apoptotic cells was noted among strains, significantly more FasL(+) expression was detected immunohistochemically in mice with the NOD genetic background than in control pancreata from birth to 1 month of age. Second, FasL(+), Fas(+), and Bcl-2(+) structures seemed to be associated with innervation, regardless of the strain and age. Third, in control and NOD strains, nerves (identified by immunohistochemical labeling of peripherin or neurofilament 200), were often observed in periductular and peri-insular areas. Finally, some peripherin-positive nerves expressed the interferon-inducible protein-10 chemokine, and various types of macrophages were found to be in close proximity. These data highlight an overlooked, innervation-related aspect of normal mouse postnatal pancreas development with possible implications in T1D pathogenesis.

Nonobese diabetic (NOD) mouse dendritic cells stimulate insulin secretion by prediabetic islets.

In the Nonobese diabetic (NOD) mouse, a spontaneous model of type 1 diabetes, the pathogenic process is classically thought to start at 3-4 weeks of age with an accumulation of antigen-presentingcells (APC), especially CD11c+ dendritic cells (DC), around the pancreatic islets of Langerhans. Concomitantly, hyperinsulinemia and slight hyperglucagonemia are observed, which may be either the cause or consequence of the initial APC infiltration. To determine whether infiltrating DC can affect islet activity in control (C57BL/6) and NOD mice, we performed experiments in which islets and DC were isolated and co-cultured. We first showed that, immediately after isolation, islets from 8-week-old prediabetic NOD mice had significantly higher insulin and glucagon contents than those from C57BL/6 controls. Moreover, as is the case in vivo, prediabetic NOD mouse islets secrete more insulin in vitro at 11.1 mM glucose than C57BL/6 ones. In DC-islet co-cultures, insulin secretion was significantly increased for NOD mice only, while that of glucagon was not significantly affected. These findings indicate that NOD DC are good candidates for stimulating the NOD mouse beta-cell hyperactivity that is observed both in vivo and in vitro, and might, consequently, sensitize NOD islets to an autoimmune attack.