The molecular mechanisms regulating the amount of dietary cholesterol retained in the body, as well as the body's 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 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.

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, beta-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.

Sitosterolemia is a rare autosomal recessive disorder characterized by (a) intestinal hyperabsorption of all sterols, including cholesterol and plant and shellfish sterols, and (b) impaired ability to excrete sterols into bile. Patients with this disease have expanded body pools of cholesterol and very elevated plasma plant-sterol species and frequently develop tendon and tuberous xanthomas, accelerated atherosclerosis, and premature coronary artery disease. In previous studies, we have mapped the STSL locus to human chromosome 2p21. Recently, we reported that a novel member of the ABC-transporter family, named "sterolin-1" and encoded by ABCG5, is mutated in 9 unrelated families with sitosterolemia; in the remaining 25 families, no mutations in sterolin-1 could be identified. We identified another ABC transporter, located <400 bp upstream of sterolin-1, in the opposite orientation. Mutational analyses revealed that this highly homologous protein, termed "sterolin-2" and encoded by ABCG8, is mutated in the remaining pedigrees. Thus, two highly homologous genes, located in a head-to-head configuration on chromosome 2p21, are involved as causes of sitosterolemia. These studies indicate that both sterolin-1 and sterolin-2 are indispensable for the regulation of sterol absorption and excretion. Identification of sterolin-1 and sterolin-2 as critical players in the regulation of dietary-sterol absorption and excretion identifies a new pathway of sterol transport.

Previous reports showed that recombinant fragments of adiponectin (adipo) displayed pharmacological effects when injected into rodents, but the relevance of these observations to the physiological function of adipo is unclear. We generated Adipo ؊/؊ mice by gene targeting. Adipo ؊/؊ mice are fertile with normal body and fat pad weights. Plasma glucose and insulin levels of Adipo ؊/؊ and Adipo ؉/؉ mice are similar under fasting conditions and during an intraperitoneal glucose tolerance test (GTT). Insulin tolerance test (ITT) also produces similar plasma glucose and insulin levels in the two groups of mice. Hyperinsulinemic-euglycemic clamp analysis showed that Adipo ؊/؊ and Adipo ؉/؉ mice have similar glucose infusion rates to maintain a similar serum glucose. High-fat diet feeding for 7 months led to similar weight gain and similar GTT and ITT responses. We next measured ␤-oxidation and found it to be significantly increased in muscle and liver of Adipo ؊/؊ mice. In conclusion, our study indicates that absence of adipo causes increased ␤-oxidation but does not cause glucose intolerance or insulin resistance in mice.

Insulin-producing cells normally occur only in the pancreas and thymus. Surprisingly, we found widespread insulin mRNA and protein expression in different diabetic mouse and rat models, including streptozotocin-treated mice and rats, ob͞ob mice, and mice fed high-fat diets. We detected in diabetic mice proinsulinand insulin-positive cells in the liver, adipose tissue, spleen, bone marrow, and thymus; many cells also produced glucagon, somatostatin, and pancreatic polypeptide. By in situ nucleic acid hybridization, diabetic, but not nondiabetic, mouse liver exhibited insulin transcript-positive cells, indicating that insulin was synthesized by these cells. In transgenic mice that express GFP driven by the mouse insulin promoter, streptozotocin-induced diabetes led to the appearance of GFP-positive cells in liver, adipose tissue, and bone marrow; the fluorescent signals showed complete concordance with the presence of immunoreactive proinsulin. Hyperglycemia produced by glucose injections in nondiabetic mice led to the appearance of proinsulin-and insulin-positive cells within 3 days. Bone marrow transplantation experiments showed that most of the extrapancreatic proinsulin-producing cells originated from the bone marrow. Immunoreactive proinsulin-and insulin-positive cells were also detected in the liver, adipose tissue, and bone marrow of diabetic rats, indicating that extrapancreatic, extrathymic insulin production occurs in more than one species. These observations have implications for the regulation of insulin gene expression, modulation of selftolerance by insulin gene expression, and strategies for the generation of insulin-producing cells for the treatment of diabetes.pancreatic islets ͉ hyperglycemia ͉ ob͞ob ͉ obesity ͉ bone marrow transplantation I nsulin is normally produced in highly specialized cells of the endocrine pancreas. The tissue-specific expression of insulin is tightly regulated at the transcriptional level, and the major regulatory elements are located in the 5Ј flanking region of the insulin gene (1). Insulin expression is specific to ␤ cells in the pancreatic islets, although proinsulin has also been detected in the fetal and postnatal thymus and spleen͞lymphoid tissues (2, 3), a pattern of promiscuous expression of normally tissuerestricted ''peripheral'' proteins in these tissues (4, 5). Immunoreactivity to insulin plays a key role in autoimmune diabetes in NOD mice (6, 7) and in type 1 diabetes in humans (7); expression of minute amounts of insulin in the thymus is believed to be important in the induction of tolerance to insulin and other ''self-antigens '' (8-11). In this study, we found insulin gene transcription and protein expression in multiple tissues outside the pancreas and thymus in mice and rats with diabetes. The widespread occurrence of extrapancreatic, extrathymic insulin gene products in diabetic animals is surprising; it suggests that we should revise our thinking on the regulation of insulin gene expression, the mechanism of immune tolerance to insulin, the pathophysi...

We have reported that a deficiency of tetrahydrobiopterin (BH(4)), an active cofactor of endothelial NO synthase (eNOS), contributes to the endothelial dysfunction through reduced eNOS activity and increased superoxide anion (O(2)(-)) generation in the insulin-resistant state. To further confirm this hypothesis, we investigated the effects of dietary treatment with BH(4) on endothelium-dependent arterial relaxation and vascular oxidative stress in the aortas of insulin-resistant rats. Oral supplementation of BH(4) (10 mg. kg(-1). d(-1)) for 8 weeks significantly increased the BH(4) 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 BH(4) treatment. The BH(4) treatment was associated with a 2-fold increase in eNOS activity as well as a 70% reduction in endothelial O(2)(-) production compared with those in fructose-fed rats. The BH(4) treatment also partially improved the insulin sensitivity and blood pressure, as well as the serum triglyceride concentration, in the fructose-fed rats. Moreover, BH(4) 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-kappaB and activating protein-1, which were increased in fructose-fed rats. The BH(4) treatment of control rats did not have any significant effects on these parameters. These results indicate that BH(4) augmentation is essential for the restoration of eNOS function and the reduction of vascular oxidative stress in insulin-resistant rats.

Summary 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, β-cell-specific transcripts, and an islet-specific transcription cascade, and 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 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.

Diabetic neuropathy is the most common microvascular complication of diabetes. Here we show that, in streptozotocin-induced diabetic rodents with neuropathy, a subpopulation of bonemarrow-derived cells marked by proinsulin expression migrates to and fuses with neurons in the sciatic nerve and dorsal root ganglion (DRG), resulting in neuronal dysfunction and accelerated apoptosis. The absence or presence of proinsulin expression, which identifies the fusion cells, and not the disease state (nondiabetic vs. diabetic) of the rats from which the DRG neurons are isolated determines whether the DRG neurons show normal or abnormal calcium homeostasis and apoptosis. These results suggest that bone-marrow-derived cells may play an important role in the pathogenesis of diabetic complications.diabetes ͉ diabetic complications ͉ dorsal root ganglion ͉ amputations ͉ bone marrow transplantation