Yoshihiko Izumida

Polyunsaturated fatty acids selectively suppress sterol regulatory element-binding protein-1 through proteolytic processing and autoloop regulatory circuit.

Sterol regulatory element-binding protein (SREBP)-1 is a key transcription factor for the regulation of lipogenic enzyme genes in the liver. Polyunsaturated fatty acids (PUFA) selectively suppress hepatic SREBP-1, but molecular mechanisms remain largely unknown. To gain insight into this regulation, we established in vivo reporter assays to assess the activities of Srebf1c transcription and proteolytic processing. Using these in vivo reporter assays, we showed that the primary mechanism for PUFA suppression of SREBP-1 is at the proteolytic processing level and that this suppression in turn decreases the mRNA transcription through lowering SREBP-1 binding to the SREBP-binding element on the promoter (“autoloop regulatory circuit”), although liver X receptor, an activator for Srebf1c transcription, is not involved in this regulation by PUFA. The mechanisms for PUFA suppression of SREBP-1 confirm that the autoloop regulation for transcription is crucial for the nutritional regulation of triglyceride synthesis.

Glycogen shortage during fasting triggers liver–brain–adipose neurocircuitry to facilitate fat utilization

During fasting, animals maintain their energy balance by shifting their energy source from carbohydrates to triglycerides. However, the trigger for this switch has not yet been entirely elucidated. Here we show that a selective hepatic vagotomy slows the speed of fat consumption by attenuating sympathetic nerve-mediated lipolysis in adipose tissue. Hepatic glycogen pre-loading by the adenoviral overexpression of glycogen synthase or the transcription factor TFE3 abolished this liver–brain–adipose axis activation. Moreover, the blockade of glycolysis through the knockdown of the glycogen phosphorylase gene and the resulting elevation in the glycogen content abolished the lipolytic signal from the liver, indicating that glycogen is the key to triggering this neurocircuitry. These results demonstrate that liver glycogen shortage activates a liver–brain–adipose neural axis that has an important role in switching the fuel source from glycogen to triglycerides under prolonged fasting conditions.

A Novel Method of Cryopreservation of Rat and Human Hepatocytes by Using Encapsulation Technique and Possible Use for Cell Transplantation

Encapsulated hepatocyte transplantation is a promising approach to cell transplantation without immunosuppression as an alternative to whole organ liver transplantation. However, the shortage of donor cells for hepatocyte transplantation has not been resolved, and at this critical point, it seems necessary to establish a method of hepatocyte cryopreservation to allow clinical application of hepatocyte transplantation and the development of a bioartificial liver system in the near future. In this study we demonstrated that cryopreserved microencapsulated rat and human hepatocytes can retain their hepatic function and that cryopreserved microencapsulated human hepatocytes transplanted into rat spleen remain viable without immunosuppression. Rat and human hepatocytes were isolated by a collagenase digestion method, and they were microencapsulated with poly-L-lysine. The microencapsulated rat hepatocytes were transferred to culture medium (DMEM containing 10% FBS and 10% DMSO) and immediately frozen in liquid nitrogen. A warm water bath (37°C) was used to thaw the microencapsulated hepatocytes. Hepatic function, drug metabolism, and cell morphology were assessed after 90 days of cryopreservation. After 1 week of cryopreservation, microencapsulated hepatocytes were cultured for up to 2 weeks to assess their hepatic function and morphology. The morphology of human hepatocytes was assessed after 30 days of cryopreservation. Cryopreserved human hepatocytes were transplanted into rat spleen to assess their morphology. Cryopreserved microencapsulated hepatocytes retained their viability and were strongly positive for expression of albumin, OAT2, CYP3A2, and CYP3A9. Two weeks after cultivation, the cryopreserved microencapsulated rat hepatocytes had retained their hepatic function (urea synthesis). Cryopreserved microencapsulated human hepatocytes also mainly survived and retained their hepatic function for at least 30 days after cryopreservation. Moreover, entrapped cryopreserved human hepatocytes also survived and expressed albumin in rat spleen after transplantation. We demonstrated a novel method of long-term cryopreservation of rat and human hepatocytes by using an encapsulation technique, with retention of biological activity and excellent survival of the cryopreserved microencapsulated human hepatocytes transplanted into rat spleen. We believe that this novel approach to hepatocytes cryopreservation provides a new direction in encapsulated cell therapy with the goal of clinical application in the near future.

Long-term maintenance of the drug transport activity in cryopreservation of microencapsulated rat hepatocytes.

Transplantation of isolated hepatocytes has been proposed to compensate for essential functions lacking in liver failure or for genetic defects that alter a specific liver metabolic pathway. Hepatocyte utilization for these purposes would be facilitated with a reliable, reproducible, and effective method of long-term hepatocyte storage. We have recently developed a simple new system for cryopreservation of hepatocytes that encapsulates alginate microspheres and maintains liver-specific function. The aim of this study was to elucidate the transport and drug-metabolizing enzyme activities of cryopreserved microencapsulated hepatocytes stored for a long time. Morphological examinations showed there is no apparent injury of the hepatocytes during cryopreservation processes. A drug-metabolizing enzyme (testosterone 6β-hydroxylase, a specific probe for CYP3A2) and drug transport activities [salicylate, allopurinol, and prostaglandin E2 (PGE2), typical substrates of rOat2] in cryopreserved microencapsulated hepatocytes were maintained up to 120 days. Our results thus demonstrate for the first time that cryopreservation of primary rat hepatocytes by the encapsulation technique allows long-term retention of drug metabolism and drug transport activities.

Successful desensitization by glargine administration in a patient with insulin allergy: A case report

The patient was a 56-year-old man with type 2 diabetes and insulin allergy. He was administered glargine, which did not produce any allergic reactions, except for a small non-pruritic wheal. Thereafter, other insulin preparation could be administered. We consider this the first case of successful insulin desensitization by glargine administration.

Microencapsule technique protects hepatocytes from cryoinjury

Aim:  Hepatocyte transplantation is a potential alternative to whole organ liver transplantation. To realize this procedure, a hepatocyte bank system capable of supplying large numbers of hepatocytes must be established. We previously reported an easy method for cryopreserving hepatocytes using a microencapsulation technique. Here, we investigated how cryoinjury to microencapsulated hepatocytes could be avoided during cryopreservation.

Elevation of Serum Albumin Levels in Nagase Analbuminemic Rats by Allogeneic Bone Marrow Cell Transplantation

We investigated the feasibility of correcting the congenital absence of albumin in Nagase analbuminemic rats (NARs) by allogeneic bone marrow cell transplantation (BMT). Seven-week-old male NARs were used as recipients, and 6- to 8-week-old male Sprague-Dawley (SD) rats were used as allograft donors. NARs were divided into three groups: a BMT group (n = 10) in which bone marrow cells were infused into the liver; a hepatocyte transplantation (HCT) group (n = 8) in which hepatocytes were transplanted into the liver, and a control group (n = 8) in which PBS was injected into the portal vein. Serum albumin levels were measured as an indicator of the function of the grafted cells, and the phenotypic characteristics of the engrafted cells in the recipient’s liver were assessed with immunohistochemical and immunofluorescence techniques. At 8 weeks after cell transplantation, the serum albumin levels of the BMT group and HCT group were significantly higher than in the control group. The hepatocyte-like cells derived from bone marrow cells expressed albumin in liver of the NARs. According to this result, bone marrow cells can differentiate into hepatocyte-like cells in vivo. The results show that BMT is an effective treatment for congenital analbuminemia in a rat model and suggest that allogeneic BMT can be used as an efficient therapy for hereditary metabolic diseases.

Identification of human ELOVL5 enhancer regions controlled by SREBP

Fatty acid elongase 5 (ELOVL5) is an enzyme involved in the synthesis of polyunsaturated fatty acids. Sterol Regulatory Element-binding Protein (SREBP)-1 activates ELOVL5 and increases polyunsaturated fatty acid synthesis, which in turn negatively affects SREBP-1 expression. Thus, ELOVL5 has been established as an SREBP-1 target gene and an important component of the negative feedback loop of de novo lipogenesis. However, the human ELOVL5 promoter/enhancer has not been fully analyzed and the location of SREBP biding sites around the ELOVL5 gene has yet to be defined. Here we performed a detailed promoter/enhancer analysis of human ELOVL5 gene, and identified two new SREBP binding sites, one in the 10 kb upstream region and one in the exon 1. These two SRE motifs are conserved among mammals and the mechanism found in the present study by which SREBP activates ELOVL5 is considered to be common in mammals. Through these findings, we clarified the molecular mechanism how SREBP activates ELOVL5, an important regulator of de novo lipogenesis.

Autonomic nervous system-mediated effects of galanin-like peptide on lipid metabolism in liver and adipose tissue.

Galanin-like peptide (GALP) is a neuropeptide involved in the regulation of feeding behavior and energy metabolism in mammals. While a weight loss effect of GALP has been reported, its effects on lipid metabolism have not been investigated. The aim of this study was to determine if GALP regulates lipid metabolism in liver and adipose tissue via an action on the sympathetic nervous system. The respiratory exchange ratio of mice administered GALP intracerebroventricularly was lower than that of saline-treated animals, and fatty acid oxidation-related gene mRNA levels were increased in the liver. Even though the respiratory exchange ratio was reduced by GALP, this change was not significant when mice were treated with the sympatholytic drug, guanethidine. Lipolysis-related gene mRNA levels were increased in the adipose tissue of GALP-treated mice compared with saline-treated animals. These results show that GALP stimulates fatty acid β-oxidation in liver and lipolysis in adipose tissue, and suggest that the anti-obesity effect of GALP may be due to anorexigenic actions and improvement of lipid metabolism in peripheral tissues via the sympathetic nervous system.

Abrogation of neutral cholesterol ester hydrolytic activity causes adrenal enlargement.

We have previously demonstrated that neutral cholesterol ester hydrolase 1 (Nceh1) regulates foam cell formation and atherogenesis through the catalytic activity of cholesterol ester hydrolysis, and that Nceh1 and hormone-sensitive lipase (Lipe) are responsible for the majority of neutral cholesterol ester hydrolase activity in macrophages. There are several cholesterol ester-metabolizing tissues and cells other than macrophages, among which adrenocortical cells are also known to utilize the intracellular cholesterol for steroidogenesis. It has been believed that the mobilization of intracellular cholesterol ester in adrenal glands was facilitated solely by Lipe. We herein demonstrate that Nceh1 is also involved in cholesterol ester hydrolysis in adrenal glands. While Lipe deficiency remarkably reduced the neutral cholesterol ester hydrolase activity in adrenal glands as previously reported, additional inactivation of Nceh1 gene completely abrogated the activity. Adrenal glands were enlarged in proportion to the degree of reduced neutral cholesterol ester hydrolase activity, and the enlargement of adrenal glands and the accumulation of cholesterol esters were most pronounced in the Nceh1/Lipe double-deficient mice. Thus Nceh1 is involved in the adrenal cholesterol metabolism, and the cholesterol ester hydrolytic activity in adrenal glands is associated with the organ enlargement.