Michiko Yoshii

Endoplasmic reticulum stress induces leptin resistance

Leptin is an important circulating signal for inhibiting food intake and body weight gain. In recent years, “leptin resistance” has been considered to be one of the main causes of obesity. However, the detailed mechanisms of leptin resistance are poorly understood. Increasing evidence has suggested that stress signals, which impair endoplasmic reticulum (ER) function, lead to an accumulation of unfolded proteins, which results in ER stress. In the present study, we hypothesized that ER stress is involved in leptin resistance. Tunicamycin, thapsigargin, or brefeldin A was used to induce ER stress. The activation status of leptin signals was measured by Western blotting analysis using a phospho-(Tyr705) signal transducer and activator of transcription 3 (STAT3) antibody. We observed that ER stress markedly inhibited leptin-induced STAT3 phosphorylation. In contrast, ER stress did not affect leptin-induced c-Jun NH2-terminal kinase activation. These results suggest that ER stress induces leptin resistance. ER stress-induced leptin resistance was mediated through protein tyrosine phosphatase 1B but not through suppressors of cytokine signaling 3. It is noteworthy that a chemical chaperone, which could improve the protein-folding capacity, reversed ER stress-induced leptin resistance. Moreover, homocysteine, which induces ER stress, caused leptin resistance both in vitro and in vivo. Together, these findings suggest that the pathological mechanism of leptin resistance is derived from ER stress.

Leptin induced GRP78 expression through the PI3K-mTOR pathway in neuronal cells

Leptin is a circulating hormone that plays a critical role in regulating energy expenditure and food intake. Evidence to suggest the involvement of endoplasmic reticulum (ER) stress in the development of obesity is increasing. To adapt against ER stress, cells trigger the unfolded protein response (UPR). The 78 kDa glucose-regulated protein (GRP78) is an ER chaperone that protects cells against ER stress by inducing protein folding. In the present study, we hypothesized that leptin may activate UPR and protect against ER stress associated with obesity. SH-SY5Y, a human neuroblastoma cell line stably transfected with the Ob-Rb leptin receptor (SH-SY5Y-ObRb), was treated with leptin. We demonstrated that leptin induced GRP78 expression. We then validated the mechanism responsible for the leptin-induced expression of GRP78. Interestingly, leptin-induced GRP78 expression was not dependent on IRE1-XBP1 pathway. On the other hand, the PI3K inhibitor, LY294002, and mTOR inhibitor, rapamycin, inhibited the leptin-induced expression of GRP78. These results suggested that the leptin-induced expression of GRP78 may be dependent on the PI3K-mTOR pathway. Leptin specifically induced GRP78 because the induction of the ER-apoptotic marker, CHOP, was not detected in leptin-treated cells. Therefore, leptin may upregulate the expression of GRP78, thereby protecting against ER stress associated with obesity.

Bile acid profiles in bile, urine, and feces of a patient with cerebrotendinous xanthomatosis

Bile acid profiles of bile, urine, and feces obtained from a patient with cerebrotendinous xanthomatosis on the same day have been analyzed by gas-liquid chromatography-mass spectrometry after fractionation into groups by mode of conjugation by an ion-exchange chromatography. The predominant biliary bile acid was cholic acid conjugated with glycine and taurine. Lesser amounts of the amino acid conjugates of chenodeoxycholic acid, ursodeoxycholic acid, 7-ketodeoxycholic acid, allocholic acid, and deoxycholic acid, and of unconjugated norcholic acid and allonorcholic acid were also present in the bile. The major fecal bile acid was 7-epicholic acid. Relatively large amounts of bile acids were excreted in the urine. Unconjugated 7-epicholic acid, norcholic acid, allonorcholic acid, and cholic acid predominated. The bile acid profiles of the patient were different from those of normal subjects and should be useful for the diagnosis.

Comparative studies on ω-hydroxylation of 5β-cholestane-3α, 7α,12α-triol in the mitochondrial and microsomal fraction of the liver from several vertebrates

Abstract The activity and the stereospecificity of ω-hydroxylation, a hydroxylation at one of the two terminal methyl groups of 5β-cholestane-3α,7α, 12α-triol, which is thought to be the first step in side-chain degradation resulting in the formation of cholic acid, was elucidated in mitochondria and microsomes of the liver from several evolutionarily primitive vertebrates, fish, frogs, turtles, and chickens in addition to such mammals as rats, hamsters, and rabbits. The detection of ω-hydroxylation products (25R)- and (25S)-5β-cholestane-3α, 7α, 12α, 26-tetrols as well as the separation of their two isomers was facilitated using high-performance liquid chromatography after conversion to 9-anthroyl derivatives. All the mammals examined, except for the rat, exhibited predominant activity in the mitochondrial fraction. Although the hydroxylation activity was somewhat lower in the primitive vertebrates, it was present in the mitochondria more than in the microsomes. Furthermore, the stereospecific formation of a 25R-isomer was detected in the mitochondrial fraction of most animals estimated. However, activity in the carp liver was seven times higher in the microsomes than in the mitochondria, and the hydroxylation product was almost always a 25R-isomer. ω-Hydroxylation activity could not be detected in rainbow trout, suggesting the existence of another biosynthetic pathway, not via 26-hydroxylation, as in the 25-hydroxylation pathway, for the production of bile acid.

Bile acid sulfonate and 7-alkylated bile acid analogs : effect on intestinal absorption of taurocholate and cholesterol 7α-hydroxylase activity in cultured rat hepatocytes

The effects of sulfonate analogs of cholic (C), chenodeoxycholic (CDC), and ursodeoxycholic acid (UDC) and three 7-alkylated CDCs--7-methyl-, 7-ethyl-, and 7-propyl-CDCs--on taurocholate absorption from rat terminal ileum in situ and on cholesterol 7alpha-hydroxylase activity in primary culture of the rat liver were investigated. The sulfonate analogs of two dihydroxy bile acids CDC and UDC, but not C, significantly decreased the absorption of taurocholate. Taurine conjugates of 7-alkylated CDC slightly decreased the taurocholate absorption, and tauro-7-propyl-CDC significantly suppressed the absorption. Although the sulfonate analogs of C and CDC reduced cholesterol 7alpha-hydroxylase activity by 40% and 60% compared to control, UDC-sulfonate analog did not affect enzymatic activity. These results were consistent with those of the lead compounds, C, CDC, and UDC. The introduction of methyl group at C-7 position of CDC attenuated the reduction in cholesterol 7alpha-hydroxylase activity by CDC. However, elongation of the alkyl group resulted in an inhibitory effect. The present study revealed the following: 1) bile acid sulfonates act on cholesterol and bile acid metabolism in a similar manner as taurine conjugated bile acids; and 2) the biologic properties of CDC could be altered by the introduction of alkyl group at C-7 position.

Comparative studies of metabolism of simultaneously administered chenodeoxycholic acid and ursodeoxycholic acid in hamsters.

We present the comparative studies of metabolism of chenodeoxycholic acid and ursodeoxycholic acid and their taurine conjugates in the liver and fecal culture from hamsters. When [24-14C]chenodeoxycholic acid and [11,12-3H]ursodeoxycholic acid were simultaneously instilled into the jujunal loop of bile fistula hamsters, both bile acids administered were recovered mainly as their conjugates with taurine and glycine in the fistula bile. The recovery of chenodeoxycholic acid was slightly but significantly higher than that of ursodeoxycholic acid. Chenodeoxycholic acid was more efficiently conjugated with glycine than ursodeoxycholic acid. The glycine/taurine ratio in the biliary chenodeoxycholic acid was 1.9, and that in ursodeoxycholic acid was 1.6. In addition, as much as 6.2% of ursodeoxycholic acid was excreted as the unconjugated form; on the other hand only 2.4% of unconjugated chenodeoxycholic acid was excreted. When [24-14C]chenodeoxycholyltaurine and [11,12-3H]ursodeoxycholyltaurine were simultaneously administered into the ileum loop of bile fistula hamsters, both bile salts were absorbed and secreted efficiently into the bile at the same rate. These results indicate that slightly lower recovery of ursodeoxycholic acid in the bile could be due to the less effective conjugation of ursodeoxycholic acid than chenodeoxycholic acid in the liver. Deconjugation by fecal culture from a hamster proceeded more rapidly in chenodeoxycholyltaurine than ursodeoxycholyltaurine. 7-Dehyroxylation to form lithocholic acid by fecal culture was also faster in chenodeoxycholic acid than ursodeoxycholic acid. The formation of 7-oxolithocholic acid from ursodeoxycholic acid was lesser than from chenodeoxycholic acid. In summary, bacterial deconjugation followed by 7-dehydroxylation to form lithocholic acid seems to be achieved more efficiently with chenodeoxycholic acid than ursodeoxycholic acid.

Identification of 3,6,7,12-tetrahydroxy-5β-cholan-24-oic acids in human biologic fluids

Unusual bile acids, 3 alpha, 6 alpha, 7 alpha, 12 alpha-, and 3 alpha, 6 beta, 7 beta, 12 alpha-tetrahyroxy-5 beta-cholan-24-oic acids, were identified in all amniotic fluid (four samples) and urine (six samples) from adult patients with cholestatic liver disease by gas-liquid chromatography/mass spectrometry. For the certain identification of these bile acids in the biologic samples, the chemical syntheses of 3 alpha, 6 beta, 7 alpha, 12 alpha- and 3 alpha, 6 beta, 7 beta, 12 alpha-tetrahydroxy-5 beta-cholan-24-oic acids were conducted.

Synthesis and metabolism of sodium 3α,7α-dihydroxy-25,26-bishomo-5β-cholane-26-sulfonate in the hamster

This paper reports the chemical synthesis of a new bile acid analogue, namely sodium 3α,7α-dihydroxy-25,26-bishomo-5β-cholane-26-sulfonate (bishomoCDC-sul) from chenodeoxycholic acid and describes its metabolism in the hamster. The structure of the new compound was confirmed by proton and carbon-13 nuclear magnetic resonance spectroscopy. After intravenous infusion of [3H]-labeled sulfonate into bile fistula hamsters, it was extracted by the liver and secreted into the bile; more than 65% of the radioactivity was recovered in the bile within 1 h. Following intraduodenal administration of the [3H]sulfonate and [14C]chenodeoxycholyltaurine, both compounds were excreted into the bile more slowly; only 41 and 43% of the radioactivity, respectively, were recovered in the bile during the four-hour experimental period. In contrast, when the labeled compounds were injected into the terminal ileum, both the sulfonate and chenodeoxycholyltaurine were repidly absorbed and secreted into the bile; 84 and 97%, respectively, of the radioactivity were recovered during a four-hour period. Chromatographic analysis demonstrated that in these short-term experiments most (>95%) of the sulfonate was secreted into the bile without biotrasformation regardless of the route of administration. When infused intravenously at increasing doses, bishomoCDC-sul induced cholestasis at an infusion rate of 1 μmol/min/kg. These results suggest that sodium 3α,7α-dihydroxy-25,26-bishomo-5β-cholane-26-sulfonate was absorbed from the terminal ileum by active transport, extracted by the liver, and secreted into the bile in a manner similar to that of the natural bile acids.

Enhancing effect of 5 α-cyprinol sulfate on mucosal membrane permeability to sodium ampicillin in rats

Effect of 5 alpha-cyprinol sulfate, a bile alcohol sulfate specific to carp bile, on rectal membrane permeability to sodium ampicillin (AMP Na) was examined in rats. AMP Na is not easily absorbed through rat rectal membrane without aid. 5 alpha-Cyprinol sulfate significantly enhanced the rectal membrane permeability to AMP Na even at a low concentration (6.25 mM), though sodium taurocholate needed a higher concentration (25 mM). Co-administration of phosphatidylcholine significantly suppressed the enhancing action of both sodium taurocholate and 5 alpha-cyprinol sulfate. On the other hand, calcium ion did not suppress the action of 5 alpha-cyprinol sulfate, although it did clearly suppress the action of sodium taurocholate. In conclusion, 5 alpha-cyprinol sulfate was found to have a potent enhancing effect on mucosal membrane permeability to water-soluble compounds. The enhancing mechanism of 5 alpha-cyprinol sulfate appeared to be different from that of sodium taurocholate.

Mechanisms of the action of adenine on anti‐allergic effects in mast cells

Mast cells play an important role in allergic responses.