Shigeru Nishida

Long‐term melatonin administration reduces hyperinsulinemia and improves the altered fatty‐acid compositions in type 2 diabetic rats via the restoration of Δ‐5 desaturase activity

The objective of this study was to investigate the effect of long‐term melatonin administration on plasma levels of triglycerides, insulin and leptin, and on the fatty‐acid metabolism of plasma and hepatic lipids in type 2 diabetic rats. Otsuka Long‐Evans Tokushima Fatty (OLETF) rats, an animal model of type 2 diabetes mellitus, were divided into two groups: one untreated (n=6), and one implanted with time‐releasing melatonin pellets (1.1 mg/day for 30 wk) under the abdominal skin (n=6). Age‐matched Long‐Evans Tokushima Otsuka (LETO) rats (n=6) were used as healthy controls. The untreated diabetic rats had the increased plasma levels of triglycerides, cholesterol, insulin and leptin at 35 wk, as compared with the healthy control rats (n=6). The diabetic rats also had augmented ratios of 20:3n‐6/20:4n‐6 fatty acids, owing to diminished activity of Δ‐5 desaturase, an insulin‐permissive enzyme, in the liver. Melatonin administration to OLETF rats reduced the hypertriglyceridemia (−39%, P < 0.05), hyperinsulinemia (−33%, P < 0.01) and hyperleptinemia (–43%, P < 0.01), and restored hepatic Δ‐5 desaturase activity (148%, P < 0.005). This resulted in a return to normal ratios of 20:3n‐6/20:4n‐6 fatty acids in plasma and hepatic lipids. There was a significant correlation (r=0.64, P < 0.005) between plasma levels of insulin and the ratios of 20:3n‐6/20:4n‐6 in plasma phospholipids of all rats in the three groups. Thus, subcutaneous implantation of a melatonin‐releasing pellet thus resulted in improved lipid metabolism in diabetic rats, probably through restored insulin resistance.ALP, alkaline phosphataseGOT, glutamic oxalacetic transaminaseGPT, glutamic pyruvic transaminaseHPLC, high performance liquid chromatographyLETO rats, Long‐Evans Tokushima Otsuka ratsMUFA, monounsaturated fatty acidOLETF rats, Otsuka Long‐Evans Tokushima Fatty ratsPUFA, polyunsaturated fatty acidVLDL, very low density lipoprotein

Effect of pinealectomy on plasma levels of insulin and leptin and on hepatic lipids in type 2 diabetic rats

Abstract: We previously reported that pharmacological melatonin administration to type 2 diabetic rats reduces hyperinsulinemia and improves the altered fatty‐acid metabolism. To determine whether melatonin deficiency exacerbates diabetes‐associated conditions, we investigated the effect of pinealectomy (i.e. melatonin‐deficiency) on plasma hormone levels and lipid metabolism in type 2 diabetic Otsuka Long‐Evans Tokushima Fatty (OLETF) rats. We compared levels of insulin and leptin, and hepatic lipids in pinealectomized OLETF (PO) rats, sham‐operated OLETF (SO) rats and sham‐operated healthy Long‐Evans Tokushima Otsuka (LETO) (SL) rats 16 and 30 wk after the operation. Plasma glucose and triglycerides were increased in SO and PO rats 30 wk after operation compared with age‐matched SL rats. Pinealectomy caused an increase in free cholesterol among the plasma lipids, as compared with SO rats. Sixteen weeks after pinealectomy, typical hyperinsulinemia was observed in PO rats (3.47‐fold increase, P < 0.01) as compared with SL rats, whereas at 30 wk, the plasma levels of insulin in PO and SO rats had decreased and there was no significant difference among the three groups. Hepatic triglycerides were increased (1.54‐fold, P < 0.005) in PO rats, compared with SO rats. Hepatic acyl‐CoA synthetase (ACS) activity was significantly augmented in PO rats at 30 wk (10%, P < 0.01 versus SO group), while microsomal triglyceride transfer protein (MTP) decreased (−27% versus SO, P < 0.05); thus, the increased ACS activity and decreased MTP might have a role in the accumulation of hepatic triglycerides in PO rats. In summary, pinealectomy causes severe hyperinsulinemia and accumulation of triglycerides in the liver, probably owing to the loss of the nocturnal melatonin surge.

Metabolic effects of melatonin on odative stress and dbetes mellitus

Melatonin, which is synthesized in the pineal gland and other tissues, has a variety of physiological, immunological, and biochemical functions. It is a direct scavenger of free radicals and has indirect antioxidant effects due to its stimulation of the expression and activity of antioxidative enzymes such as glutathione peroxidase, superoxide dismutase and catalase, and NO synthase, in mammalian cells. Melatonin also reduces serum lipid levels in mammalian species, and helps to prevent oxidative stress in diabetic subjects. long-term melatonin administration to diabetic rats reduced their hyperlipidemia and hyperinsulinemia, and restored their altered ratios of polyunsaturated fatty acid in serum and tissues. It was recently reported that melatonin enhanced insulin-receptor kinase and IRS-1 phosphorylation, suggesting the potential existence of signaling pathway cross-talk between melatonin and insulin. Because TNF-α has been shown to impair insulin action by suppressing insulin receptortyrosine kinase activity and its IRS-lyrosine phosphorylation in peripheral tissues such as skeletal muscle cells, it was speculated that melatonin might counteract TNF-α-associated insulin resistance in type 2 diabetes. This review will focus on the physiological and metabolic effects of melatonin and highlight its potential use for the treatment of cholesterol/lipid and carbohydrate disorders.

Modulation of Bile Acid Metabolism by 1α-Hydroxyvitamin D3 Administration in Mice

The vitamin D receptor (VDR) is a nuclear receptor for the active form of vitamin D3 and mediates regulation of calcium homeostasis. Bile acids, such as lithocholic acid, have been identified as additional endogenous VDR ligands. The in vivo role of VDR in bile acid metabolism has not been elucidated. We investigated potential effects of in vivo VDR activation on bile acid metabolism by feeding mice bile acid-supplemented chow and then treating them with 1α-hydroxyvitamin D3 [1α(OH)D3]. We administered 1α(OH)D3 via gavage to mice fed chow supplemented with 0.4% cholic acid (CA), chenodeoxycholic acid (CDCA), deoxycholic acid (DCA), or lithocholic acid (LCA) and examined liver and plasma bile acid composition with gas chromatography-mass spectrometry analysis. 1α(OH)D3 treatment reduced hepatic bile acids in mice fed CDCA- and DCA-supplemented chow but was less effective in mice fed chow supplemented with LCA or CA. 1α(OH)D3 administration also decreased plasma bile acids in mice fed bile acids, such as DCA. The effect of 1α(OH)D3 administration in decreasing liver bile acid composition was observed in mice under fasting conditions and was associated with increased urinary excretion and increased expression of bile acid transporters, such as renal multidrug resistance-associated protein 4. These findings indicate that pharmacological activation of VDR enhances metabolism of bile acids, especially urinary excretion. The results confirm that VDR acts a regulator of bile acid metabolism in vivo.

PXR Prevents Cholesterol Gallstone Disease by Regulating Biosynthesis and Transport of Bile Salts

BACKGROUND & AIMS Cholesterol gallstone disease (CGD) results from a biochemical imbalance of lipids and bile salts in the gallbladder bile. We investigated whether the xenobiotic receptor pregnane X receptor (PXR) has a role in pathogenesis of CGD. METHODS Wild-type, PXR-null (PXR-/-), and CGD-sensitive C57L mice were placed on a lithogenic diet and then analyzed for CGD at the biochemical, histological, and gene-regulation levels. RESULTS Loss of PXR sensitized mice to lithogenic diet-induced CGD, characterized by decreases in biliary concentrations of bile salts and phospholipids and an increases in the cholesterol saturation index and formation of cholesterol crystals. The decreased bile acid pool size in PXR-/- mice that received lithogenic diets was associated with reduced expression of cholesterol 7α-hydroxylase, the rate-limiting enzyme of cholesterol catabolism and bile acid formation. The reduced expression of cholesterol 7α-hydroxylase most likely resulted from activation of farnesoid X receptor and induction of fibroblast growth factor 15 in the intestine. In C57L mice given the PXR agonist, pregnenolone-16α-carbonitrile, or the herbal medicine, St Johns wort, cholesterol precipitation was prevented by increases in concentrations of biliary bile salt and a reduced cholesterol saturation index. PXR prevented CGD via its coordinate regulation of the biosynthesis and transport of bile salts in the liver and intestine. CONCLUSIONS PXR maintains biliary bile acid homeostasis and may be developed as a therapeutic target for CGD.

Extracellular superoxide released from mitochondria mediates mast cell death by advanced glycation end products

Advanced glycation end products (AGEs) accumulate during aging and to higher extents under pathological conditions such as diabetes. Since we previously showed that mast cells expressed the AGE-binding protein, receptor for AGEs (RAGE) on their cell surface, we examined whether AGE affected mast cell survival. Herein, we demonstrate that mast cells undergo apoptosis in response to AGE. Glycated albumin (GA), an AGE, but not stimulation with the high-affinity IgE receptor (FcepsilonRI), can induce mast cell death, as measured by annexin V/propidium iodide double-staining. GA (> or =0.1 mg/ml) exhibited this pro-apoptotic activity in a concentration-dependent manner. GA and FcepsilonRI stimulation increased the cytosolic Ca(2+) levels to a similar extent, whereas GA, but not FcepsilonRI stimulation, caused mitochondrial Ca(2+) overload and membrane potential collapse, resulting in mitochondrial integrity disruption, cytochrome c release and caspase-3/7 activation. In addition, GA, but not FcepsilonRI stimulation, induced extracellular release of superoxide from mitochondria, and this release played a key role in the disruption of Ca(2+) homeostasis. Knockdown of RAGE expression using small interfering RNA abolished GA-induced apoptosis, mitochondrial Ca(2+) overload, and superoxide release, demonstrating that RAGE mediates the GA-induced mitochondrial death pathway. AGE-induced mast cell apoptosis may contribute to the immunocompromised and inflammatory conditions.

Induction of SREBP-1c mRNA by Differentiation and LXR Ligand in Human Keratinocytes

The epidermis is an active site of lipid metabolism, and the synthesis of fatty acids and cholesterol is required for cutaneous homeostasis. Liver X receptor-alpha (LXRalpha) and LXRbeta are nuclear receptors that are activated by oxysterols and regulate cholesterol and fatty acid metabolism. LXRs, predominantly LXRbeta, have been shown to be involved in keratinocyte differentiation and epidermal permeability barrier function. Although LXR regulates hepatic lipogenesis by inducing sterol-regulatory element-binding protein-1c (SREBP-1c), SREBP-1c induction by LXR in the epidermis has not been studied. In this study, we report that SREBP-1c mRNA increased during differentiation of human keratinocyte HaCaT cells and that LXR agonist effectively induced expression of LXR target genes, including SREBP-1c and ATP-binding cassette transporter A1, in differentiated HaCaT cells. Differentiation-associated and LXR-enhanced expression of SREBP-1c was also observed in malignant human keratinocyte A431 cells and primary human keratinocytes. A synthetic LXR antagonist inhibited confluency-dependent expression of SREBP-1c. Thus, SREBP-1c expression increases during keratinocyte differentiation, and LXR activation enhances its expression.

Diabetes-induced and age-related changes in fatty acid proportions of plasma lipids in rats

Diabetes-induced and age-related proportional changes in plasma fatty acids of triglycerides (TG), phospholipids (PL), and cholesteryl esters (CE) were investigated using streptozotocin-induced diabetic and control rats. Among n-6 fatty acids from diabetic rat plasma, increased proportions of 18∶2n-6 and 20∶3n-6 in all three lipid classes and of 18∶3n-6 in PL at 1–3 months old and in TG at 3–5 months old were observed. The proportions of 20∶4n-6 decreased in both PL and CE, but were unchanged in diabetic TG. Among the n-3 fatty acids, in the early stage, diabetes caused increases in the proportions of 18∶3n-3 in PL and CE and of 20∶5n-3 and 22∶6n-3 in TG, while 22∶5n-3 was decreased later in the disease course. These results suggest reduced Δ5-desaturase activities on 20∶3n-6 but not on 20∶4n-3, while Δ6-desaturase activity on 18∶2n-6 was essentially unaffected. Furthermore, the reduction in Δ9-desaturase activity in diabetic rats may well explain the decreases in the proportions of 16∶1n-7 and 18∶1n-7. However, the proportion of 18∶1n-9, another product of Δ9-desaturase, was significantly increased in CE and PL as compared to the controls. Thus, there was a discrepancy between our results and those of earlier studies with respect to the n-9, n-6, and n-3 fatty acid proportions of plasma lipids in diabetic rats. We also investigated age-related changes in the proportions of plasma fatty acids. Although rather small, age-related changes were evident in both diabetic and control rats.

Mast cell death induced by 24(S),25-epoxycholesterol

Mast cell is one of the central effectors in inflammatory responses. Recent studies suggest that a promising therapeutic approach for various inflammatory immune diseases, including rheumatoid arthritis, multiple sclerosis, and type I allergies, is to inhibit mast cell growth and/or survival. Studies also indicate that a balanced lipid metabolism is crucial for regulating the life span of cells. Oxysterol is a well-known regulator of lipid metabolism and has diverse functions, such as inhibition of the mevalonate isoprenoid pathway, efflux of free cholesterols, and synthesis of cholesterol esters. Here, we show that 24(S),25-epoxycholesterol, a representative endogenous oxysterol, induces apoptosis in bone marrow-derived murine mast cells. Furthermore, we have revealed, for the first time, that the accumulation of neutral lipids catalyzed by acyl-CoA:cholesterol acyltransferase in the cells was involved in induction of mast cell apoptosis. Our present findings confer new insights into the roles of lipid metabolism during oxysterol-mediated mast cell apoptosis.