Atsushi Mitsumoto

Up-Regulation of Stearoyl-CoA Desaturase 1 Increases Liver MUFA Content in Obese Zucker but Not Goto-Kakizaki Rats

The Goto-Kakizaki (GK) rat is an animal model for spontaneous-onset, non-obese type 2 diabetes. Despite abundant evidence about disorders in metabolism, little information is available about fatty acid metabolism in the liver of GK rats. This study aimed to investigate the characteristics of the fatty acid profile, particularly MUFA, and the mechanism underlying the alterations in fatty acid profiles in the liver of GK rats. The activities of enzymes that participate in the biosynthesis of MUFA, expressions of genes encoding these enzymes, and the fatty acid profile in the liver were compared with those of obese Zucker (fa/fa) (ZF) rats, which are obese and non-diabetic. Stearoyl-CoA desaturase (SCD) activity and SCD1 gene expression were considerably up-regulated in GK rats, and these levels were largely comparable to those in ZF rats. However, the proportions and contents of oleic acid and palmitoleic acid were very low considering the highly elevated activity of SCD in the liver of GK rats, when compared with ZF rats. Palmitoyl-CoA chain elongation (PCE) activity and fatty acid elongase (Elovl6) gene expression were markedly up-regulated in ZF rats, whereas PCE activity was up-regulated much less and Elovl6 gene expression was unchanged in GK rats. These results suggest the possibility that up-regulation of gene expression of Elovl6 along with SCD1 is indispensable to elevate the proportions and contents of oleic acid in the liver.

Role of transient receptor potential melastatin 2 (TRPM2) channels in visceral nociception and hypersensitivity

Transient receptor potential melastatin 2 (TRPM2) is a thermosensitive, Ca2+-permeable cation channel. TRPM2 contributes to the pathogenesis of inflammatory bowel disease, and inflammatory and neuropathic pain. We hypothesized that TRPM2 is important for visceral nociception and the development of visceral hypersensitivity. Therefore, we investigated the expression of TRPM2 channels and their involvement in visceral nociception in normal physiology and under pathological conditions that cause visceral hypersensitivity in rats. TRPM2 immunoreactivities were detected in the mucosa and muscle layer of the rat gastrointestinal tract. TRPM2 immunopositive cell bodies were almost completely co-localized with calretinin- and NeuN-positive cells in the myenteric plexus. We found that the majority of the TRPM2-immunoreactive cells were double-labeled with the retrograde marker fluorogold in lumbar 6/sacral 1 dorsal root ganglia (DRG), indicating that TRPM2 is expressed in spinal primary afferents innervating the distal colon. Subtypes of TRPM2-immunopositive DRG neurons were labeled by the A-fiber marker NF200, the C-fiber marker IB4, substance P, calcitonin gene-related peptide, or P2X3 receptor. We found that oral administration of the TRPM2 inhibitor econazole (30mg/kg) reduced the visceromotor response (VMR) to noxious colorectal distention (CRD) at 80mmHg in control rats. Expression of TRPM2 in the mucosa of the distal colon was increased in a trinitrobenzene sulfonic acid-induced colitis model. The VMR to CRD significantly increased in colitis model rats compared with control rats at 40, 60, and 80mmHg. Econazole restored visceral hypersensitivity to the control level. Furthermore, TRPM2-deficient mice showed significantly attenuated trinitrobenzene sulfonic acid induced visceral hypersensitivity compared with wild-type mice. In conclusion, TRPM2 channels contribute to visceral nociception in response to noxious stimuli under normal conditions and visceral hypersensitivity in pathological conditions.

Increased Lipid Synthesis and Decreased β-Oxidation in the Liver of SHR/NDmcr-cp (cp/cp) Rats, an Animal Model of Metabolic Syndrome

SHR/NDmcr-cp (cp/cp) rats (SHR/NDcp) are an animal model of metabolic syndrome. A previous study of ours revealed drastic increases in the mass of palmitic (16:0), oleic (18:1n-9), palmitoleic (16:1n-7), cis-vaccenic (18:1n-7) and 5,8,11-eicosatrienoic acids in the liver of SHR/NDcp. However, detailed information on the class of lipid accumulated and the mechanism responsible for the overproduction of the accumulated lipid in the liver was not obtained. This study aimed to characterize the class of lipid accumulated and to explore the mechanism underlying the lipid accumulation in the liver of SHR/NDcp, in comparison with SHR/NDmcr-cp (+/+) (lean hypertensive littermates of SHR/NDcp) and Wistar Kyoto rats. In the liver of SHR/NDcp, de novo synthesis of fatty acids (16:0, 18:1n-9 and 16:1n-7) and triacylglycerol (TAG) synthesis were up-regulated and fatty acid β-oxidation was down-regulated. These perturbations of lipid metabolism caused fat accumulation in hepatocytes and accumulation of TAG, which were enriched with 16:0, 18:1n-9 and 16:1n-7, in the liver of SHR/NDcp. On the other hand, no changes were found in hepatic contents of diacylglycerol and unesterified fatty acid (FFA); among FFA, there were no differences in the hepatic concentrations of unesterified 16:0 and stearic acid between SHR/NDcp and two other groups of rats. Moreover, little change was brought about in the expression of genes responsive to endoplasmic reticulum stress in the liver of SHR/NDcp. These results may reinforce the pathophysiological role of stearoyl-CoA desaturase 1 and fatty acid elongase 6 in the liver of SHR/NDcp.

Impairment of Heme Biosynthesis Induces Short Circadian Period in Body Temperature Rhythms in Mice

It has been demonstrated that the function of mammalian clock gene transcripts is controlled by the binding of heme in vitro. To examine the effects of heme on biological rhythms in vivo, we measured locomotor activity (LA) and core body temperature (T(b)) in a mouse model of porphyria with impaired heme biosynthesis by feeding mice a griseofulvin (GF)-containing diet. Mice fed with a 2.0% GF-containing diet (GF2.0) transiently exhibited phase advance or phase advance-like phenomenon by 1-3 h in terms of the biological rhythms of T(b) or LA, respectively (both, P < 0.05) while mice were kept under conditions of a light/dark cycle (12 h:12 h). We also observed a transient, ~0.3 h shortening of the period of circadian T(b) rhythms in mice kept under conditions of constant darkness (P < 0.01). Interestingly, the observed duration of abnormal circadian rhythms in GF2.0 mice lasted between 1 and 3 wk after the onset of GF ingestion; this finding correlated well with the extent of impairment of heme biosynthesis. When we examined the effects of therapeutic agents for acute porphyria, heme, and hypertonic glucose on the pathological status of GF2.0 mice, it was found that the intraperitoneal administration of heme (10 mg·kg(-1)·day(-1)) or glucose (9 g·kg(-1)·day(-1)) for 7 days partially reversed (50%) increases in urinary δ-aminolevulinic acids levels associated with acute porphyria. Treatment with heme, but not with glucose, suppressed the phase advance (-like phenomenon) in the diurnal rhythms (P < 0.05) and restored the decrease of heme (P < 0.01) in GF2.0 mice. These results suggest that impairments of heme biosynthesis, in particular a decrease in heme, may affect phase and period of circadian rhythms in animals.

Regulation of palmitoyl-CoA chain elongation by clofibric acid in the liver of zucker fa/fa rats

The regulation of palmitoyl-CoA chain elongation (PCE) by clofibric acid [2-(4-chlorophenoxy)-2-methylpropionic acid] was investigated in comparison with stearoyl-CoA desaturase (SCD) in the liver of obese Zucker fa/fa rats. The proportion of oleic acid in the hepatic lipids of Zucker obese rats is 2.7 times higher than that of lean littermates. The activities of PCE and SCD in the liver of Zucker obese rats were markedly higher than in lean rats, and the hepatic uptake of 2-deoxyglucose (2-DG) was also higher in Zucker obese rats compared with lean rats. The increased activities of SCD and PCE in Zucker obese rats were due to the enhanced expression of mRNA of rELO1. The proportion of oleic acid in the liver was significantly increased by the administration of clofibric acid to Zucker obese rats, and the hepatic PCE activity and rELO2 mRNA expression, but not the SCD activity or SCD1 mRNA expression, were increased in response to clofibric acid treatment. By contrast, the activities of both PCE and SCD and the mRNA expression of SCD1 and rELO2 in the liver were increased by the treatment of Zucker lean rats with clofibric acid. Multiple regression analysis, which was performed to determine the relationships involving PCE activity, SCD activity, and the proportion of oleic acid, revealed that the three parameters were significantly correlated and that the standardized partial regression coefficient of PCE was higher than that of SCD. These results indicate that oleic acid is synthesized by the concerted action of PCE and SCD and that PCE plays a crucial role in the formation of oleic acid when Zucker fa/fa rats are given clofibric acid.

Abnormalities in the Metabolism of Fatty Acids and Triacylglycerols in the Liver of the Goto-Kakizaki Rat: A Model for Non-Obese Type 2 Diabetes.

The Goto-Kakizaki (GK) rat is widely used as an animal model for spontaneous-onset type 2 diabetes without obesity; nevertheless, little information is available on the metabolism of fatty acids and triacylglycerols (TAG) in their livers. We investigated the mechanisms underlying the alterations in the metabolism of fatty acids and TAG in their livers, in comparison with Zucker (fa/fa) rats, which are obese and insulin resistant. Lipid profiles, the expression of genes for enzymes and proteins related to the metabolism of fatty acid and TAG, de novo synthesis of fatty acids and TAG in vivo, fatty acid synthase activity in vitro, fatty acid oxidation in liver slices, and very-low-density-lipoprotein (VLDL)-TAG secretion in vivo were estimated. Our results revealed that (1) the TAG accumulation was moderate, (2) the de novo fatty acid synthesis was increased by upregulation of fatty acid synthase in a post-transcriptional manner, (3) fatty acid oxidation was also augmented through the induction of carnitine palmitoyltransferase 1a, and (4) the secretion rate of VLDL-TAG remained unchanged in the livers of GK rats. These results suggest that, despite the fact that GK rats exhibit non-obese type 2 diabetes, the upregulation of de novo lipogenesis is largely compensated by the upregulation of fatty acid oxidation, resulting in only moderate increase in TAG accumulation in the liver.

Time of Administration of Acute or Chronic Doses of Imipramine Affects its Antidepressant Action in Rats

The pathogenesis and therapeutics of depression are linked to the operation of the circadian system. Here, we studied the chronopharmacological action of a tricyclic antidepressant, imipramine. Male adult Wistar–Hannover rats were administered imipramine acutely or chronically in the morning or in the evening. The antidepressant action of imipramine was analyzed using the forced swim test (FST). A single dose of imipramine (30 mg/kg) in the morning, but not in the evening, reduced immobility and increased climbing in the FST. The plasma concentrations of imipramine and its metabolite, desipramine, were slightly higher in the morning than in the evening, which might explain the dosing time-dependent action of imipramine. Next, we analyzed the effect of chronic imipramine treatment. Rats received imipramine in the morning or in the evening for 2 weeks. The morning treatment resulted in larger effects in the FST than the evening treatment, and was effective at a dose that was ineffective when administered acutely. The levels of brain α-adrenergic receptors tended to decrease after chronic imipramine treatment. Imipramine might interact with noradrenergic neurons, and this interaction might chronically alter receptor expression. This alteration seemed greater in the morning than in the evening, which might explain the dosing time-dependent action of imipramine.

Short and long photoperiods differentially exacerbate corticosterone-induced physical and psychological symptoms in mice

Circadian disruption affects the pathogenesis and development of various diseases. Depression is one of the most common diseases that relate to circadian rhythm. In this study, we analyzed the effects of daily light/dark (LD) conditions on depression and other symptoms, and also analyzed the mixed effects of LD conditions and corticosterone treatment. Male adult C57BL/6 mice were treated with corticosterone in a normal LD cycle of 12 hours light and 12 hours dark (LD12 : 12), short day conditions of 6 hours light and 18 hours dark (LD6 : 18), or long day conditions of 21 hours light and 3 hours dark (LD21 : 3). The activity rhythms of mice in aberrant LD conditions were entrained within 2 weeks. After 6 weeks of exposure, several behavioral tests were conducted. Corticosterone induced body weight gain and depression-like symptoms. The short or long LD conditions had little effect on vehicle-treated mice behavior. However, the aberrant LD conditions exacerbated the corticosterone-induced symptoms. Mice treated with corticosterone in LD6 : 18 showed exacerbated depression-like symptoms in a novelty suppressed feeding test. On the other hand, LD21 : 3 did not show any effects on mood, but enhanced corticosterone-induced body weight gain. These results indicated that aberrant LD conditions could act as an exacerbating factor for corticosterone-induced symptoms, and that short and long photoperiods induce different psychological and physiological changes. This corticosterone + aberrant LD model could be a useful animal model for investigating the effect of LD conditions on depression, obesity, and other symptoms in stressful circumstances.