Hideto Kojima

Identification of a gene, ABCG5, important in the regulation of dietary cholesterol absorption.

The molecular mechanisms regulating the amount of dietary cholesterol retained in the body, as well as the bodys ability to exclude selectively other dietary sterols, are poorly understood. An average western diet will contain about 250–500 mg of dietary cholesterol and about 200–400 mg of non-cholesterol sterols. About 50–60% of the dietary cholesterol is absorbed and retained by the normal human body, but less than 1% of the non-cholesterol sterols are retained. Thus, there exists a subtle mechanism that allows the body to distinguish between cholesterol and non-cholesterol sterols. In sitosterolemia, a rare autosomal recessive disorder, affected individuals hyperabsorb not only cholesterol but also all other sterols, including plant and shellfish sterols from the intestine. The major plant sterol species is sitosterol; hence the name of the disorder. Consequently, patients with this disease have very high levels of plant sterols in the plasma and develop tendon and tuberous xanthomas, accelerated atherosclerosis, and premature coronary artery disease. We previously mapped the STSL locus to human chromosome 2p21 (ref. 4) and further localized it to a region of less than 2 cM bounded by markers D2S2294 and D2S2291 (M.-H.L. et al., manuscript submitted). We now report that a new member of the ABC transporter family, ABCG5, is mutant in nine unrelated sitosterolemia patients.

NeuroD-betacellulin gene therapy induces islet neogenesis in the liver and reverses diabetes in mice.

To explore induced islet neogenesis in the liver as a strategy for the treatment of diabetes, we used helper-dependent adenovirus (HDAD) to deliver the pancreatic duodenal homeobox-1 gene (Ipf1; also known as Pdx-1) to streptozotocin (STZ)-treated diabetic mice. HDAD is relatively nontoxic as it is devoid of genes encoding viral protein. Mice treated with HDAD-Ipf1 developed fulminant hepatitis, however, because of the exocrine-differentiating activity of Ipf1. The diabetes of STZ mice was partially reversed by HDAD-mediated transfer of NeuroD (Neurod), a factor downstream of Ipf1, and completely reversed by a combination of Neurod and betacellulin (Btc), without producing hepatitis. Treated mice were healthy and normoglycemic for the duration of the experiment (>120 d). We detected in the liver insulin and other islet-specific transcripts, including proinsulin-processing enzymes, β-cell–specific glucokinase and sulfonylurea receptor. Immunocytochemistry detected the presence of insulin, glucagon, pancreatic polypeptide and somatostatin-producing cells organized into islet clusters; immuno-electron microscopy showed typical insulin-containing granules. Our data suggest that Neurod-Btc gene therapy is a promising regimen to induce islet neogenesis for the treatment of insulin-dependent diabetes.

Oral Administration of Tetrahydrobiopterin Prevents Endothelial Dysfunction and Vascular Oxidative Stress in the Aortas of Insulin-Resistant Rats

We have reported that a deficiency of tetrahydrobiopterin (BH4), an active cofactor of endothelial NO synthase (eNOS), contributes to the endothelial dysfunction through reduced eNOS activity and increased superoxide anion (O2−) generation in the insulin-resistant state. To further confirm this hypothesis, we investigated the effects of dietary treatment with BH4 on endothelium-dependent arterial relaxation and vascular oxidative stress in the aortas of insulin-resistant rats. Oral supplementation of BH4 (10 mg · kg−1 · d−1) for 8 weeks significantly increased the BH4 content in cardiovascular tissues of rats fed high levels of fructose (fructose-fed rats). Impairment of endothelium-dependent arterial relaxation in the aortic strips of the fructose-fed rats was reversed with BH4 treatment. The BH4 treatment was associated with a 2-fold increase in eNOS activity as well as a 70% reduction in endothelial O2− production compared with those in fructose-fed rats. The BH4 treatment also partially improved the insulin sensitivity and blood pressure, as well as the serum triglyceride concentration, in the fructose-fed rats. Moreover, BH4 treatment of the fructose-fed rats markedly reduced the lipid peroxide content of both aortic and cardiac tissues and inhibited the activation of 2 redox-sensitive transcription factors, nuclear factor-&kgr;B and activating protein-1, which were increased in fructose-fed rats. The BH4 treatment of control rats did not have any significant effects on these parameters. These results indicate that BH4 augmentation is essential for the restoration of eNOS function and the reduction of vascular oxidative stress in insulin-resistant rats.

Neurogenin3 is sufficient for transdetermination of hepatic progenitor cells into neo-islets in vivo but not transdifferentiation of hepatocytes.

The transcription factor Neurogenin3 (Ngn3) is required for islet-cell type specification. Here, we show that hepatic gene transfer of Ngn3 transiently induces insulin in terminally differentiated hepatocytes but fails to transdifferentiate them, i.e., switch their lineage into islet cells. However, Ngn3 leads to long-term diabetes reversal in mice due to the emergence of periportal islet-like cell clusters. These neo-islets display glycemia-regulated insulin, beta-cell-specific transcripts, and an islet-specific transcription cascade, and they produce all four major islet hormones. They appear to arise from hepatic progenitor cells, most likely endoderm-derived oval cells. Thus, transfer of a single lineage-defining transcription factor, Ngn3, is sufficient to induce cell-lineage switching from a hepatic to an islet lineage in these progenitor cells, a process consistent with transdetermination, i.e, lineage switching in lineage-determined, but not terminally differentiated, cells. This paradigm of induced transdetermination of receptive progenitor cells in vivo may be generally applicable to therapeutic organogenesis for multiple diseases, including diabetes.

ArticleNeurogenin3 Is Sufficient for Transdetermination of Hepatic Progenitor Cells into Neo-Islets In Vivo but Not Transdifferentiation of Hepatocytes

The transcription factor Neurogenin3 (Ngn3) is required for islet-cell type specification. Here, we show that hepatic gene transfer of Ngn3 transiently induces insulin in terminally differentiated hepatocytes but fails to transdifferentiate them, i.e., switch their lineage into islet cells. However, Ngn3 leads to long-term diabetes reversal in mice due to the emergence of periportal islet-like cell clusters. These neo-islets display glycemia-regulated insulin, beta-cell-specific transcripts, and an islet-specific transcription cascade, and they produce all four major islet hormones. They appear to arise from hepatic progenitor cells, most likely endoderm-derived oval cells. Thus, transfer of a single lineage-defining transcription factor, Ngn3, is sufficient to induce cell-lineage switching from a hepatic to an islet lineage in these progenitor cells, a process consistent with transdetermination, i.e, lineage switching in lineage-determined, but not terminally differentiated, cells. This paradigm of induced transdetermination of receptive progenitor cells in vivo may be generally applicable to therapeutic organogenesis for multiple diseases, including diabetes.

Endothelium-specific activation of NAD(P)H oxidase in aortas of exogenously hyperinsulinemic rats.

To examine the effects of chronic hyperinsulinemia on vascular tissues, we examined the production of superoxide anion ([Formula: see text]) in the aortic tissues of control and exogenously hyperinsulinemic rats performed by the implantation of an insulin pellet for 4 wk. [Formula: see text]production by aortic segments from hyperinsulinemic rats was 2.4-fold (lucigenin chemiluminescence method) and 1.7-fold (cytochrome c method) of that of control rats without any differences in [Formula: see text]degrading activities in aortic tissues, respectively ( P < 0.025). The increment was completely abolished in the presence of either 100 μmol/l apocynin (an inhibitor of NADPH oxidase) or 10 μmol/l diphenyleneiodonium (an inhibitor of flavin-containing enzyme) and was exclusively endothelium dependent. Consistently, NAD(P)H oxidase activities in endothelial homogenate in hyperinsulinemic rats were dose dependently stimulated above the values of control rats, although these activities in nonendothelial homogenate were not significantly stimulated by insulin. Furthermore, an insulin effect was also demonstrated 1 h after exposing aortic tissues to insulin. These results indicate that[Formula: see text] production specifically increases in endothelium of aortic tissues in chronic hyperinsulinemic rats through the activation of NAD(P)H oxidase.To examine the effects of chronic hyperinsulinemia on vascular tissues, we examined the production of superoxide anion (O(-2)) in the aortic tissues of control and exogenously hyperinsulinemic rats performed by the implantation of an insulin pellet for 4 wk. O(-2) production by aortic segments from hyperinsulinemic rats was 2. 4-fold (lucigenin chemiluminescence method) and 1.7-fold (cytochrome c method) of that of control rats without any differences in O(-2) degrading activities in aortic tissues, respectively (P < 0.025). The increment was completely abolished in the presence of either 100 micromol/l apocynin (an inhibitor of NADPH oxidase) or 10 micromol/l diphenyleneiodonium (an inhibitor of flavin-containing enzyme) and was exclusively endothelium dependent. Consistently, NAD(P)H oxidase activities in endothelial homogenate in hyperinsulinemic rats were dose dependently stimulated above the values of control rats, although these activities in nonendothelial homogenate were not significantly stimulated by insulin. Furthermore, an insulin effect was also demonstrated 1 h after exposing aortic tissues to insulin. These results indicate that O(-2) production specifically increases in endothelium of aortic tissues in chronic hyperinsulinemic rats through the activation of NAD(P)H oxidase.

Altered Activities of Transcription Factors and Their Related Gene Expression in Cardiac Tissues of Diabetic Rats

Gene regulation in the cardiovascular tissues of diabetic subjects has been reported to be altered. To examine abnormal activities in transcription factors as a possible cause of this altered gene regulation, we studied the activity of two redox-sensitive transcription factors— nuclear factor-KB (NF-KB) and activating protein-1 (AP-1)—and the change in the mRNA content of heme oxygenase-1, which is regulated by these transcription factors in the cardiac tissues of rats with streptozotocin-induced diabetes. Increased activity of NF-KB and AP-1 but not nuclear transcription-activating factor, as determined by an electrophoretic mobility shift assay, was found in the hearts of 4-week diabetic rats. Glycemic control by a subcutaneous injection of insulin prevented these diabetes-induced changes in transcription factor activity. In accordance with these changes, the mRNA content of heme oxygenase-1 was increased fourfold in 4-week diabetic rats and threefold in 24-week diabetic rats as compared with control rats (P < 0.01 and P < 0.05, respectively). Insulin treatment also consistently prevented changes in the mRNA content of heme oxygenase-1. The oral administration of an antioxidant, probucol, to these diabetic rats partially prevented the elevation of the activity of both NFKB and AP-1, and normalized the mRNA content of heme oxygenase-1 without producing any change in the plasma glucose concentration. These results suggest that elevated oxidative stress is involved in the activation of the transcription factors NF-KB and AP-1 in the cardiac tissues of diabetic rats, and that these abnormal activities of transcription factors could be associated with the altered gene regulation observed in the cardiovascular tissues of diabetic rats.

Phosphorylation of carnitine palmitoyltransferase and activation by glucagon in isolated rat hepatocytes

Effects of glucagon and forskolin on the phosphorylation and changes of activity of carnitine palmitoyl‐transferase (CPT) have been studied in isolated rat hepatocytes using anti‐CPT immunoglobulin. When the activity was determined in lysed hepatocytes after glucagon or forskolin treatment, it was found to be stimulated 30–80% mainly through increased affinity for palmitoyl‐CoA. By SDS electrophoresis of the immuno‐precipitates, CPT subunit (M r 69000) was noted to be phosphorylated 4–5‐fold with glucagon (1.2 × 10−7 M) and forskolin (0.1 mM) over control. These results indicate that hepatic ketogenesis is regulated with glucagon by phosphorylation of CPT through cAMP‐dependent protein kinase.

Expression of a dominant negative SHP-2 in transgenic mice induces insulin resistance.

To elucidate the roles ofSHP-2, we generated transgenic (Tg) mice expressing a dominant negative mutant lacking protein tyrosine phosphatase domain (ΔPTP). On examining two lines of Tg mice identified by Southern blot, the transgene product was expressed in skeletal muscle, liver, and adipose tissues, and insulin-induced association of insulin receptor substrate 1 with endogenous SHP-2 was inhibited, confirming that ΔPTP has a dominant negative property. The intraperitoneal glucose loading test demonstrated an increase in blood glucose levels in Tg mice. Plasma insulin levels in Tg mice after 4 h fasting were 3 times greater with comparable blood glucose levels. To estimate insulin sensitivity by a constant glucose, insulin, and somatostatin infusion, steady state blood glucose levels were higher, suggesting the presence of insulin resistance. Furthermore, we observed the impairment of insulin-stimulated glucose uptake in muscle and adipocytes in the presence of physiological concentrations of insulin. Moreover, tyrosine phosphorylation of insulin receptor substrate-1 and stimulation of phosphatidylinositol 3-kinase and Akt kinase activities by insulin were attenuated in muscle and liver. These results indicate that the inhibition of endogenous SHP-2function by the overexpression of a dominant negative mutant may lead to impaired insulin sensitivity of glucose metabolism, and thusSHP-2 may function to modulate insulin signaling in target tissues.

Increased intestinal glucose absorption and postprandial hyperglycaemia at the early step of glucose intolerance in Otsuka Long-Evans Tokushima Fatty Rats

Summary Otsuka Long-Evans Tokushima Fatty (OLETF) rats are reported to be obese Type II (non-insulin-dependent) diabetic rats with insulin resistance and impaired insulin secretion. To investigate the contribution of intestinal glucose absorption to postprandial hyperglycaemia, we determined the plasma xylose concentrations after an 0.8 g/kg oral xylose load which was used as a test of small intestinal glucose absorption in 6-week-old OLETF rats and weight-matched Long-Evans Tokushima Otsuka (LETO) rats. An oral glucose tolerance test showed that OLETF rats developed hyperglycaemia at 60 and 90 min after the glucose load, though the fasting plasma glucose concentration, insulin concentration and insulin-induced in vivo glucose utilization rate were similar. Consistently, in an oral D-xylose loading test, the peak concentration of plasma xylose in OLETF rats was increased by 58.7 % compared with that of LETO rats (p < 0.005). The disappearance rate of plasma xylose concentrations after intravenous xylose loading did not differ between the two strains. Co-treatment with 0.4 g/kg phlorizin, a specific inhibitor of sodium-dependent glucose transporter 1 (SGLT1), abolished both plasma glucose and xylose concentrations after the loads. Morphological studies showed that both the small intestinal wet weight and surface area were 30 % larger in the OLETF rats than in the LETO rats. Furthermore, the SGLT1 mRNA content of OLETF rats also increased compared with LETO rats. These results suggest that an increased SGLT1 expression concomitant with intestinal hypertrophy in OLETF rats is partly associated with postprandial hyperglycaemia before the onset of insulin resistance and hyperinsulinaemia. [Diabetologia (1998) 41: 1459–1466]