Melinjo (Gnetum gnemon L.) seed extract for treatment of sleep/wake fragmentation in diet-induced obese mice.

Dietary supplementation with melinjo (Gnetum gnemon L.) seed extract (MSE) has been an integral part of an anti-obesity therapeutic regimen. To examine the relationship between anti-obesity and sleep, we explored the effect of MSE on sleep structure in high-fat diet (HFD)-induced obese mice. Although HFD did not alter the total amount of daily sleep, it significantly reduced the average duration of non-rapid eye movement (NREM) sleep and wakefulness episodes and significantly increased the number of these episodes. These findings indicate fragmented NREM sleep due to repeated brief awakenings in the HFD-fed mice. When 1% (w/v) MSE was given to HFD-fed mice, their weight or sleep structure were comparable to those of ND-fed mice, proving that dietary MSE completely hindered HFD-induced weight gain and sleep/wake fragmentation. Our data provide compelling evidence that MSE is a novel and promising dietary supplement that restores obesity-induced sleep architecture changes in mice.

Behavioral characteristics of dopamine D5 receptor knockout mice.

Major psychiatric disorders such as attention-deficit/hyperactivity disorder and schizophrenia are often accompanied by elevated impulsivity. However, anti-impulsive drug treatments are still limited. To explore a novel molecular target, we examined the role of dopamine D5 receptors in impulse control using mice that completely lack D5 receptors (D5KO mice). We also measured spontaneous activity and learning/memory ability because these deficits could confound the assessment of impulsivity. We found small but significant effects of D5 receptor knockout on home cage activity only at specific times of the day. In addition, an analysis using the q-learning model revealed that D5KO mice displayed lower behavioral adjustment after impulsive actions. However, our results also showed that baseline impulsive actions and the effects of an anti-impulsive drug in D5KO mice were comparable to those in wild-type littermates. Moreover, unlike previous studies that used other D5 receptor-deficient mouse lines, we did not observe reductions in locomotor activity, working memory deficits, or severe learning deficits in our line of D5KO mice. These findings demonstrate that D5 receptors are dispensable for impulse control. Our results also indicate that time series analysis and detailed analysis of the learning process are necessary to clarify the behavioral functions of D5 receptors.

Melanin-concentrating hormone-producing neurons in the hypothalamus regulate brown adipose tissue and thus contribute to energy expenditure.

KEY POINTS: Melanin-concentrating hormone (MCH) neuron-ablated mice exhibit increased energy expenditure and reduced fat weight. Increased brown adipose tissue (BAT) activity and locomotor activity-independent energy expenditure contributed to body weight reduction in MCH neuron-ablated mice. MCH neurons send inhibitory input to the medullary raphe nucleus to modulate BAT activity. ABSTRACT: Hypothalamic melanin-concentrating hormone (MCH) peptide robustly affects energy homeostasis. However, it is unclear whether and how MCH-producing neurons, which contain and release a variety of neuropeptides/transmitters, regulate energy expenditure in the central nervous system and peripheral tissues. We thus examined the regulation of energy expenditure by MCH neurons, focusing on interscapular brown adipose tissue (BAT) activity. MCH neuron-ablated mice exhibited reduced body weight, increased oxygen consumption, and increased BAT activity, which improved locomotor activity-independent energy expenditure. Trans-neuronal retrograde tracing with the recombinant pseudorabies virus revealed that MCH neurons innervate BAT via the sympathetic premotor region in the medullary raphe nucleus (MRN). MRN neurons were activated by MCH neuron ablation. Therefore, endogenous MCH neuron activity negatively modulates energy expenditure via BAT inhibition. MRN neurons might receive inhibitory input from MCH neurons to suppress BAT activity.

Salmon acyl-ghrelin increases food intake and reduces doxorubicin-induced myocardial apoptosis in rats, likely by anti-oxidative activity.

We had reported that orally administered ghrelin-containing salmon stomach extract prevents doxorubicin (DOX)-induced cardiotoxicity. In this study, we investigated the binding affinity of salmon ghrelin to rat ghrelin receptor and the cardioprotective effects of subcutaneous (sc) injected synthetic salmon ghrelin in rats with DOX-induced acute heart failure in order to clarify the potential efficacy of salmon ghrelin. Intracellular calcium mobilization assay was performed on rat GHS-R1a-expressing CHO cells to reveal ghrelin activity. Rats were divided into five groups; the normal control (I), and toxic control (II) groups were given saline (sc, twice daily), and the salmon acyl-ghrelin (sAG) (III), salmon unacylated-ghrelin (sUAG) (IV), and rat acyl-ghrelin (rAG) (V) groups were given corresponding synthetic ghrelins (sc, twice daily), respectively. After seven days of treatment, DOX (20 mg/kg BW) or saline was administered to the corresponding groups by intraperitoneal injection. The toxic control group was the negative control group for the DOX-induced cardiotoxicity groups. While sAG displayed similar affinity to rAG upon application to GHS-R1a-expressing cells, and also decreased DOX-induced apoptosis and increased food intake, sUAG did not. Both sAG and rAG improved DOX-induced deterioration, showing anti-oxidative activity. The anti-oxidative activity of sAG might contribute to the protective effects on cardiomyocytes. The results also suggest that, similar to rAG, sAG is a potent protectant against DOX-induced cardiotoxicity and a potential functional component in orally administered ghrelin-containing salmon stomach extract, which prevented DOX-induced cardiotoxicity in our previous study.

REM sleep-active MCH neurons are involved in forgetting hippocampus-dependent memories.

The neural mechanisms underlying memory regulation during sleep are not yet fully understood. We found that melanin concentrating hormone-producing neurons (MCH neurons) in the hypothalamus actively contribute to forgetting in rapid eye movement (REM) sleep. Hypothalamic MCH neurons densely innervated the dorsal hippocampus. Activation or inhibition of MCH neurons impaired or improved hippocampus-dependent memory, respectively. Activation of MCH nerve terminals in vitro reduced firing of hippocampal pyramidal neurons by increasing inhibitory inputs. Wake- and REM sleep-active MCH neurons were distinct populations that were randomly distributed in the hypothalamus. REM sleep state-dependent inhibition of MCH neurons impaired hippocampus-dependent memory without affecting sleep architecture or quality. REM sleep-active MCH neurons in the hypothalamus are thus involved in active forgetting in the hippocampus.

Melinjo (Gnetum gnemon L.) seed extract induces uncoupling protein 1 expression in brown fat and protects mice against diet-induced obesity, inflammation, and insulin resistance.

Dietary supplementation with melinjo (Gnetum gnemon L.) seed extract (MSE) has been proposed as an anti-obesity strategy. However, it remains unclear how MSE modulates energy balance. We tested the hypothesis that dietary MSE reduces energy intake and/or increases physical activity and metabolic thermogenesis in brown and white adipose tissue (BAT and WAT) in mice. Twenty-four C57BL/6 J mice were provided with normal diet, high-fat diet (HFD), or HFD with 1% MSE added, for 17 weeks. Food intake, spontaneous locomotor activity, hepatic triglyceride (TG) content, and blood parameters were examined. Mitochondrial thermogenesis-associated molecule and inflammatory marker expression levels in BAT and WAT were examined by quantitative PCR and western blotting. Dietary MSE did not affect energy intake or spontaneous locomotor activity, but significantly suppressed HFD-induced fat accumulation, hyperglycemia, and hyperinsulinemia. Homeostasis model assessment of insulin resistance score and hepatic TG content were both lower in the MSE-supplemented HFD-fed group than in the HFD-fed group, indicating reduced insulin resistance and a less fatty liver. Dietary MSE upregulated thermogenic uncoupling protein 1 (UCP1) and mitochondrial marker cytochrome c oxidase subunit IV protein expression in BAT; this was closely associated with sirtuin 1 mRNA induction. mRNAs of adipose inflammatory markers, such as monocyte chemotactic 1 and interleukin-1, were induced by HFD but suppressed by MSE. Considering that UCP1 protein expression is the most physiologically relevant parameter to assess the thermogenic capacities of BAT, our results indicate that dietary MSE supplementation induces BAT thermogenesis and reduces obesity-associated adipose tissue inflammation, hepatic steatosis, and insulin resistance.

Royal jelly ameliorates diet-induced obesity and glucose intolerance by promoting brown adipose tissue thermogenesis in mice.

INTRODUCTION: Identification of thermogenic food ingredients is potentially a useful strategy for the prevention of obesity and related metabolic disorders. It has been reported that royal jelly (RJ) supplementation improves insulin sensitivity; however, its impacts on energy expenditure and adiposity remain elusive. We investigated anti-obesity effects of RJ supplementation and their relation to physical activity levels and thermogenic capacities of brown (BAT) and white adipose tissue (WAT). METHODS: C57BL/6J mice were fed under four different experimental conditions for 17 weeks: normal diet (ND), high fat diet (HFD), HFD with 5% RJ, and HFD with 5% honey bee larva powder (BL). Spontaneous locomotor activity, hepatic triglyceride (TG) content, and blood parameters were examined. Gene and protein expressions of thermogenic uncoupling protein 1 (UCP1) and mitochondrial cytochrome c oxidase subunit IV (COX-IV) in BAT and WAT were investigated by qPCR and Western blotting analysis, respectively. RESULTS: Dietary RJ, but not BL, suppressed HFD-induced accumulations of WAT and hepatic TG without modifying food intake. Consistently, RJ improved hyperglycemia and the homeostasis model assessment-insulin resistance (HOMA-IR). Although dietary RJ and BL unchanged locomotor activity, gene and protein expressions of UCP1 and COX-IV in BAT were increased in the RJ group compared to the other experimental groups. Neither the RJ nor BL treatment induced browning of WAT. CONCLUSION: Our results indicate that dietary RJ ameliorates diet-induced obesity, hyperglycemia, and hepatic steatosis by promoting metabolic thermogenesis in BAT in mice. RJ may be a novel promising food ingredient to combat obesity and metabolic disorders.

Progesterone dose-dependently modulates hepatocyte growth factor production in 3T3-L1 mouse preadipocytes.

It is well documented that estrogen is predominant inducer of hepatocyte growth factor (HGF) in a variety of cell types. However, the effect of progesterone (P) remains to be elusive. Thus, in the present study, we examined the effect of P and combined effect of P and 17ß-estradiol (E2) on HGF expression and production in 3T3-L1 fibroblastic preadipocytes and mature adipocytes, as a model of stromal cells. Northern blot analysis showed that hgf mRNA expressed in preadipocytes was notably higher than that of mature adipocytes, and increased by treatment of preadipocytes with E2 or 10 nM P, but not with 1,000 nM P. The E2-induced hgf mRNA expression was enhanced by 10 nM P, but suppressed by 1,000 nM P. Western blot analysis revealed that biological active forms of HGF protein was found in the preadipocyte culture medium, while the lesser amount of HGF precursor protein was detected in the mature adipocyte culture medium. The amounts of HGF were changed dependently on the hgf mRNA expression levels. These results indicate that HGF production is intricately regulated by E2 and P at the transcriptional levels in 3T3-L1 cells, and may explain the changes in the HGF production during the mammary gland development, especially decrease in HGF expression during pregnancy when P concentration is high.

Study on the Increased Probability of Detecting Adverse Drug Reactions Based on Bayes' Theorem: Evaluation of the Usefulness of Information on the Onset Timing of Adverse Drug Reactions.

In order to avoid adverse drug reactions (ADRs), pharmacists are reconstructing ADR-related information based on various types of data gathered from patients, and then providing this information to patients. Among the data provided to patients is the time-to-onset of ADRs after starting the medication (i.e., ADR onset timing information). However, a quantitative evaluation of the effect of onset timing information offered by pharmacists on the probability of ADRs occurring in patients receiving this information has not been reported to date. In this study, we extracted 40 ADR-drug combinations from the data in the Japanese Adverse Drug Event Report database. By applying Bayes' theorem to these combinations, we quantitatively evaluated the usefulness of onset timing information as an ADR detection predictor. As a result, when information on days after taking medication was added, 54 ADR-drug combinations showed a likelihood ratio (LR) in excess of 2. In particular, when considering the ADR-drug combination of anaphylactic shock with levofloxacin or loxoprofen, the number of days elapsed between start of medication and the onset of the ADR was 0, which corresponded to increased likelihood ratios (LRs) of 138.7301 or 58.4516, respectively. When information from 1-7 d after starting medication was added to the combination of liver disorder and acetaminophen, the LR was 11.1775. The results of this study indicate the clinical usefulness of offering information on ADR onset timing.

Retinoic acid modulates lipid accumulation glucose concentration dependently through inverse regulation of SREBP-1 expression in 3T3L1 adipocytes.

It is well known that retinoic acid (RA) suppresses adipogenesis, although there are some contradicting reports. In this study, we examined the effect of extracellular glucose on RA-induced suppression of adipogenesis in 3T3L1 cell culture. When the cells were cultured in normal glucose medium (NG), the addition of RA suppressed lipid accumulation. However, when cultured in high glucose medium (HG), addition of RA to the cells enhanced lipid accumulation. These changes were accompanied by parallel alterations in fatty acid synthase (FAS) and sterol regulatory element-binding protein (SREBP)-1 gene expression. Transfection of SREBP-1 siRNA suppressed RA-induced enhancement of lipid accumulation and FAS expression in the cells cultured with HG. Transfection of the nuclear form of SREBP-1a cDNA into the cells cultured with NG inhibited RA-induced suppression of lipid accumulation and FAS expression. Moreover, RA- and HG-induced SREBP-1a expression occurred at the early phase of adipogenesis and was dependent on glucocorticoid to induce liver X receptor (LXR) ß, peroxisomal proliferator-activated receptor (PPAR) γ and retinoid X receptor (RXR), the key nuclear factors influencing the SREBP-1a gene expression. These results suggest that RA suppresses and enhances lipid accumulation through extracellular glucose concentration-dependent modulation of SREBP-1 expression.

Efficacy and safety of sucroferric oxyhydroxide compared with sevelamer hydrochloride in Japanese haemodialysis patients with hyperphosphataemia: A randomized, open-label, multicentre, 12-week phase III study.

AIM: We aimed to investigate the non-inferiority of PA21 (sucroferric oxyhydroxide) to sevelamer hydrochloride (sevelamer) in terms of efficacy and safety in Japanese haemodialysis patients with hyperphosphataemia. METHODS: In this Phase III, open-label, multicentre study, 213 haemodialysis patients with hyperphosphataemia were randomized to PA21 or sevelamer treatment for 12 weeks. The primary outcome was adjusted serum phosphorus concentration at the end of treatment; the non-inferiority of PA21 was confirmed if the upper limit of the two-sided 95% confidence interval (CI) is ≤0.32 mmol/L. Secondary outcomes were corrected serum calcium and intact-parathyroid hormone concentrations. Adverse events (AEs) and adverse drug reactions (ADRs) were evaluated. RESULTS: The adjusted mean serum phosphorus concentration at the end of treatment confirmed the non-inferiority of PA21 for lowering serum phosphorus compared with sevelamer (1.62 vs 1.72 mmol/L; difference, -0.11 mmol/L; 95% CI, -0.20 to -0.02 mmol/L). The mean daily tablet intake was 5.6 ± 2.6 and 18.7 ± 7.1 tablets in the PA21 and sevelamer groups, respectively. The incidences of AEs and ADRs were not significantly different between the two groups. CONCLUSION: The non-inferiority of PA21 to sevelamer was confirmed for the treatment of Japanese haemodialysis patients with hyperphosphataemia. PA21 was effective, safe, and well tolerated, while having a considerably lower pill burden than sevelamer.

Dose-response efficacy and safety of PA21 in Japanese hemodialysis patients with hyperphosphatemia: a randomized, placebo-controlled, double-blind, Phase II study.

BACKGROUND: Hyperphosphatemia is common in chronic kidney disease (CKD) and associated with mortality and morbidity. We aimed to evaluate the dose-dependent efficacy and safety of PA21 (sucroferric oxyhydroxide), an iron-based phosphate binder, in Japanese hemodialysis patients with hyperphosphatemia. METHODS: In this double-blind, multicenter, Phase II study, 183 patients were randomized to placebo or PA21 at doses of 250, 500, 750, or 1000 mg (based on iron content) three times/day for 6 weeks. The primary efficacy endpoint was the mean change in serum phosphorus levels from baseline to end of treatment in each group. Adverse reactions were evaluated. RESULTS: The change in serum phosphorus level was significantly greater in each PA21 group than in the placebo group (analysis of covariance: P < 0.001 for all groups). A dose-dependent change in serum phosphorus levels was observed in the PA21 groups. A notable decrease in mean serum phosphorus levels to the target level of ≤6 mg/dL was shown starting at Week 1 in all PA21 groups. The cumulative achievement rates for target serum phosphorus level at the end of treatment were generally >80 % in all PA21 groups. The major adverse reaction reported was diarrhea; however, most cases were mild. CONCLUSIONS: PA21 was an effective and safe treatment that decreased serum phosphorus levels starting at 1 week of treatment when administered as one 250-mg tablet three times/day. PA21 demonstrated a dose-dependent phosphorus lowering effect up to 3000 mg/day. PA21 may be a new treatment alternative with relatively low pill burden for Japanese hemodialysis patients with hyperphosphatemia.

Progressive Loss of the Orexin Neurons Reveals Dual Effects on Wakefulness.

STUDY OBJECTIVES: Narcolepsy is caused by loss of the orexin (also known as hypocretin) neurons. In addition to the orexin peptides, these neurons release additional neurotransmitters, which may produce complex effects on sleep/wake behavior. Currently, it remains unknown whether the orexin neurons promote the initiation as well as the maintenance of wakefulness, and whether the orexin neurons influence initiation or maintenance of sleep. To determine the effects of the orexin neurons on the dynamics of sleep/wake behavior, we analyzed sleep/wake architecture in a novel mouse model of acute orexin neuron loss. METHODS: We used survival analysis and other statistical methods to analyze sleep/wake architecture in orexin-tTA ; TetO diphtheria toxin A mice at different stages of orexin neuron degeneration. RESULTS: Progressive loss of the orexin neurons dramatically reduced survival of long wake bouts, but it also improved survival of brief wake bouts. In addition, with loss of the orexin neurons, mice were more likely to wake during the first 30 sec of nonrapid eye movement sleep and then less likely to return to sleep during the first 60 sec of wakefulness. CONCLUSIONS: These findings help explain the sleepiness and fragmented sleep that are characteristic of narcolepsy. Orexin neuron loss impairs survival of long wake bouts resulting in poor maintenance of wakefulness, but this neuronal loss also fragments sleep by increasing the risk of awakening at the beginning of sleep and then reducing the likelihood of quickly returning to sleep.

Organ-specific changes in norepinephrine turnover against various stress conditions in thermoneutral mice.

The effects of three stressors of different categories, namely cold exposure, immobilization, and lipopolysaccharide (LPS) treatment, on sympathetic nerve activity were examined by assessing its biochemical index norepinephrine (NE) turnover in peripheral organs of C57BL/6 mice. NE turnover was assessed by measuring the decrease in the organ NE concentration 3 h after inhibition of catecholamine biosynthesis with alpha-methyl-p-tyrosine. NE turnover in brown adipose tissue (BAT) in the room temperature (23 degrees C) control group was as high as that in the cold exposure (4 degrees C) group. Similarly, the mRNA level of the thermogenic marker uncoupling protein 1 (UCP1) in the room temperature control group was as high as that in the cold exposure group. As sympathetic stimulation upregulates the UCP1 mRNA level, we thought that sympathetic nerve tonus in BAT was already accelerated at room temperature. To exclude factors affecting basal sympathetic nerve activity, mice housed at thermoneutral temperature (30 degrees C) were used as controls for the subsequent experiments. In this condition, cold exposure accelerated NE turnover in the BAT, as well as heart and pancreas. The corticosterone level showed a higher trend in the cold exposure group in comparison to the control group. Immobilization accelerated NE turnover in the spleen, pancreas, and white adipose tissue and elevated the corticosterone level. LPS (3 mg/kg, i.p.) did not affect NE turnover in all peripheral organs but elevated the corticosterone level. In summary, the sympathetic nervous and adrenocortical responses to three stressors differed greatly. In particular, sympathetic responses showed clear organ-specific acceleration patterns. This important feature may improve our understanding of the multiplicity of biological responses.

Optogenetic manipulation of activity and temporally controlled cell-specific ablation reveal a role for MCH neurons in sleep/wake regulation.

Melanin-concentrating hormone (MCH) is a neuropeptide produced in neurons sparsely distributed in the lateral hypothalamic area. Recent studies have reported that MCH neurons are active during rapid eye movement (REM) sleep, but their physiological role in the regulation of sleep/wakefulness is not fully understood. To determine the physiological role of MCH neurons, newly developed transgenic mouse strains that enable manipulation of the activity and fate of MCH neurons in vivo were generated using the recently developed knockin-mediated enhanced gene expression by improved tetracycline-controlled gene induction system. The activity of these cells was controlled by optogenetics by expressing channelrhodopsin2 (E123T/T159C) or archaerhodopsin-T in MCH neurons. Acute optogenetic activation of MCH neurons at 10 Hz induced transitions from non-REM (NREM) to REM sleep and increased REM sleep time in conjunction with decreased NREM sleep. Activation of MCH neurons while mice were in NREM sleep induced REM sleep, but activation during wakefulness was ineffective. Acute optogenetic silencing of MCH neurons using archaerhodopsin-T had no effect on any vigilance states. Temporally controlled ablation of MCH neurons by cell-specific expression of diphtheria toxin A increased wakefulness and decreased NREM sleep duration without affecting REM sleep. Together, these results indicate that acute activation of MCH neurons is sufficient, but not necessary, to trigger the transition from NREM to REM sleep and that MCH neurons also play a role in the initiation and maintenance of NREM sleep.

Characteristics of sleep and wakefulness in wild-derived inbred mice.

Genetic variations in the wild-derived inbred mouse strains are more diverse than that of classical laboratory inbred mouse strains, including C57BL/6J (B6). The sleep/wake and monoamine properties of six wild-derived inbred mouse strains (PGN2, NJL, BLG2, KJR, MSM, HMI) were characterized and compared with those of B6 mice. All examined mice were nocturnal and had a polyphasic sleep pattern with a "main sleep period" identified during the light period. However, there were three sleep/wake phenotypic differences between the wild-derived mouse strains and B6 strain. First, the amount of sleep during the dark phase was comparable with that of B6 mice. However, the amount of sleep during the light phase was more varied among strains, in particular, NJL and HMI had significantly less sleep compared with that of B6 mice. Second, PGN2, NJL, BLG2, and KJR mice showed a "highly awake period" (in which the hourly total sleep time was <10%) immediately after the onset of the dark period, which was not seen in B6 mice. Third, relative to that of B6 mice, PGN2 and KJR mice showed longer duration of wakefulness episodes during the 12-h dark phase. Differences in whole brain noradrenaline, dopamine, and 5-hydroxy-tryptamine contents between the wild-derived mouse strains and B6 strain were also found. These identified phenotypes might be potentially under strong genetic control. Hence, wild-derived inbred mice could be useful for identifying the genetic factors underlying the regulation of sleep and wakefulness.

Hypothalamic prepro-orexin mRNA level is inversely correlated to the non-rapid eye movement sleep level in high-fat diet-induced obese mice.

Orexins are hypothalamic neuropeptides, which play important roles in the regulation and maintenance of sleep/wakefulness states and energy homeostasis. To evaluate whether alterations in orexin system is associated with the sleep/wakefulness abnormalities observed in obesity, we examined the mRNA expression of prepro-orexin, orexin receptor type 1 (orexin 1r), and orexin receptor type 2 (oxexin 2r) in the hypothalamus in mice fed with a normal diet (ND) and high-fat diet (HFD)-induced obese mice. We also compared their relationships with sleep/wakefulness. Twenty-four, 4-week-old, male C57BL/6J mice were divided randomly into three groups, which received the following: (1) ND for 17 weeks; (2) HFD for 17 weeks; and (3) ND for 7 weeks and HFD for a further 10 weeks. The body weights of mice fed the HFD for 10-17 weeks were 112-150% of the average body weight of the ND group. The daily amount of non-rapid eye movement (NREM) sleep increased significantly in HFD-fed mice. These changes were accompanied by increases in the number but decreases in the duration of each NREM sleep episode. In addition, brief awakenings (<20 s epoch) during NREM sleep was nearly 2-fold more frequent. The mRNA level of prepro-orexin in the hypothalamus was significantly reduced in HFD-induced obese mice, whereas the levels of orexin 1r and orexin 2r were unaffected. The daily amount of NREM sleep was negatively correlated with the hypothalamic prepro-orexin mRNA level, so these results suggest that the increased NREM sleep levels in HFD-induced obese mice are attributable to impaired orexin activity.

Thermogenic ability of uncoupling protein 1 in beige adipocytes in mice.

Chronic adrenergic activation leads to the emergence of beige adipocytes in some depots of white adipose tissue in mice. Despite their morphological similarities to brown adipocytes and their expression of uncoupling protein 1 (UCP1), a thermogenic protein exclusively expressed in brown adipocytes, the beige adipocytes have a gene expression pattern distinct from that of brown adipocytes. However, it is unclear whether the thermogenic function of beige adipocytes is different from that of classical brown adipocytes existing in brown adipose tissue. To examine the thermogenic ability of UCP1 expressed in beige and brown adipocytes, the adipocytes were isolated from the fat depots of C57BL/6J mice housed at 24°C (control group) or 10°C (cold-acclimated group) for 3 weeks. Morphological and gene expression analyses revealed that the adipocytes isolated from brown adipose tissue of both the control and cold-acclimated groups consisted mainly of brown adipocytes. These brown adipocytes contained large amounts of UCP1 and increased their oxygen consumption when stimulated with norepinephirine. Adipocytes isolated from the perigonadal white adipose tissues of both groups and the inguinal white adipose tissue of the control group were white adipocytes that showed no increase in oxygen consumption after norepinephrine stimulation. Adipocytes isolated from the inguinal white adipose tissue of the cold-acclimated group were a mixture of white and beige adipocytes, which expressed UCP1 and increased their oxygen consumption in response to norepinephrine. The UCP1 content and thermogenic ability of beige adipocytes estimated on the basis of their abundance in the cell mixture were similar to those of brown adipocytes. These results revealed that the inducible beige adipocytes have potent thermogenic ability comparable to classical brown adipocytes.

Proinsulin C-peptide activates α-enolase: implications for C-peptide--cell membrane interaction.

Proinsulin C-peptide shows beneficial effects on microvascular complications of Type 1 diabetes. However, the possible occurrence of membrane C-peptide receptor(s) has not been elucidated. The aim of this study was to identify and characterize membrane proteins to which C-peptide binds. The enzyme α-enolase was co-immunoprecipitated with C-peptide after chemical cross-linking to HL-60 cell surface proteins and identified by mass spectrometry. Recombinant α-enolase activity was modulated by C-peptide, with a significant decrease in K(m) for 2-phosphoglycerate without affecting V(max). The enzyme modulation by C-peptide was abolished when C-terminal basic lysine residue (K434) of the enzyme was replaced by neutral alanine or acidic glutamate, but not with basic arginine. The enzyme modulation by C-peptide was reproduced with the C-peptide fragments containing glutamate corresponding to position 27 (E27) of the full-length C-peptide. Addition of a lysine analogue to the assay and A31 cell culture abrogated the enzyme modulation and MAP kinase activation by C-peptide, respectively. The results indicate that C-peptide has the capacity to activate α-enolase through a specific interaction between E27 of the peptide and K434 of the enzyme. Since α-enolase plays a role as a cell surface receptor for plasminogen, it may conceivably also serve as a receptor for C-peptide in vivo.