May Yun Wang

Lipoapoptosis in beta-cells of obese prediabetic fa/fa rats. Role of serine palmitoyltransferase overexpression.

We reported that the lipoapoptosis of beta-cells observed in fat-laden islets of obese fa/fa Zucker Diabetic Fatty (ZDF) rats results from overproduction of ceramide, an initiator of the apoptotic cascade and is induced by long-chain fatty acids (FA). Whereas the ceramide of cytokine-induced apoptosis may be derived from sphingomyelin hydrolysis, FA-induced ceramide overproduction seems to be derived from FA. We therefore semiquantified mRNA of serine palmitoyltransferase (SPT), which catalyzes the first step in ceramide synthesis. It was 2–3-fold higher in fa/fa islets than in+/+ controls. [3H]Ceramide formation from [3H]serine was 2.2–4.5-fold higher in fa/faislets. Triacsin-C, which blocks palmitoyl-CoA synthesis, andl-cycloserine, which blocks SPT activity, completely blocked [3H]ceramide formation from [3H]serine. Islets of fa/fa rats are unresponsive to the lipopenic action of leptin, which normally depletes fat and prevents FA up-regulation of SPT. To determine the role of leptin unresponsiveness in the SPT overexpression, we transferred wild type OB-Rb cDNA to their islets; now leptin completely blocked the exaggerated FA-induced increase of SPT mRNA while reducing the fat content. Beta-cell lipoapoptosis was partially prevented in vivo by treating prediabetic ZDF rats withl-cycloserine for 2 weeks. Ceramide content and DNA fragmentation both declined 40–50%. We conclude that lipoapoptosis of ZDF rats is mediated by enhanced ceramide synthesis from FA and that blockade by SPT inhibitors prevents lipoapoptosis.

Novel Form of Lipolysis Induced by Leptin

Hyperleptinemia causes disappearance of body fat without a rise in free fatty acids (FFA) or ketones, suggesting that leptin can deplete adipocytes of fat without releasing FFA. To test this, we measured FFA and glycerol released from adipocytes obtained from normal lean Zucker diabetic fatty rats (+/+) and incubated for 0, 3, 6, or 24 h in either 20 ng/ml recombinant leptin or 100 nm norepinephrine (NE). Whereas NE increased both FFA and glycerol release from adipocytes of +/+ rats, leptin increased glycerol release in +/+ adipocytes without a parallel increase in FFA release. In adipocytes of obese Zucker diabetic fatty rats (fa/fa) with defective leptin receptors, NE increased both FFA and glycerol release, but leptin had no effect on either. Leptin significantly lowered the mRNA of leptin and fatty acid synthase of adipocytes (FAS) (p< 0.05), and up-regulated the mRNA of peroxisome proliferator-activated receptor (PPAR)-α, carnitine palmitoyl transferase-1, (CPT-1), and acyl CoA oxidase (ACO) (p< 0.05). NE (100 nm) also lowered leptin mRNA (p < 0.05) but did not affect FAS, PPARα, ACO, or CPT-1 expression. We conclude that in normal adipocytes leptin directly decreases FAS expression, increases PPARα and the enzymes of FFA oxidation, and stimulates a novel form of lipolysis in which glycerol is released without a proportional release of FFA.

Leptin therapy in insulin-deficient type I diabetes

In nonobese diabetic mice with uncontrolled type 1 diabetes, leptin therapy alone or combined with low-dose insulin reverses the catabolic state through suppression of hyperglucagonemia. Additionally, it mimics the anabolic actions of insulin monotherapy and normalizes hemoglobin A1c with far less glucose variability. We show that leptin therapy, like insulin, normalizes the levels of a wide array of hepatic intermediary metabolites in multiple chemical classes, including acylcarnitines, organic acids (tricarboxylic acid cycle intermediates), amino acids, and acyl CoAs. In contrast to insulin monotherapy, however, leptin lowers both lipogenic and cholesterologenic transcription factors and enzymes and reduces plasma and tissue lipids. The results imply that leptin administration may have multiple short- and long-term advantages over insulin monotherapy for type 1 diabetes.

Glucagon receptor knockout prevents insulin-deficient type 1 diabetes in mice.

OBJECTIVE To determine the role of glucagon action in the metabolic phenotype of untreated insulin deficiency. RESEARCH DESIGN AND METHODS We compared pertinent clinical and metabolic parameters in glucagon receptor-null (Gcgr−/−) mice and wild-type (Gcgr+/+) controls after equivalent destruction of β-cells. We used a double dose of streptozotocin to maximize β-cell destruction. RESULTS Gcgr+/+ mice became hyperglycemic (>500 mg/dL), hyperketonemic, polyuric, and cachectic and had to be killed after 6 weeks. Despite comparable β-cell destruction in Gcgr−/− mice, none of the foregoing clinical or laboratory manifestations of diabetes appeared. There was marked α-cell hyperplasia and hyperglucagonemia (∼1,200 pg/mL), but hepatic phosphorylated cAMP response element binding protein and phosphoenolpyruvate carboxykinase mRNA were profoundly reduced compared with Gcgr+/+ mice with diabetes—evidence that glucagon action had been effectively blocked. Fasting glucose levels and oral and intraperitoneal glucose tolerance tests were normal. Both fasting and nonfasting free fatty acid levels and nonfasting β-hydroxy butyrate levels were lower. CONCLUSIONS We conclude that blocking glucagon action prevents the deadly metabolic and clinical derangements of type 1 diabetic mice.

Rapid transformation of white adipocytes into fat-oxidizing machines

Adenovirus-induced hyperleptinemia rapidly depletes body fat in normal rats without increasing free fatty acids and ketogenesis, implying that fat-storing adipocytes are oxidizing the fat. To analyze the ultrastructural changes of adipocytes accompanying this functional transformation, we examined the fat tissue by electron microscopy. After 14 days of hyperleptinemia, adipocytes had become shrunken, fatless, and encased in a thick basement-membrane-like matrix. They were crowded with mitochondria that were much smaller than those of brown adipocytes. Their gene expression profile revealed striking up-regulation of peroxisome proliferator-activated receptor γ coactivator 1α (an up-regulator of mitochondrial biogenesis not normally expressed in white fat), increased uncoupling proteins-1 and -2, and down-regulation of lipogenic enzymes. Phosphorylation of both acetyl CoA carboxylase and AMP-activated protein kinase was increased, thus explaining the increase in fatty acid oxidation. The ability to transform adipocytes into unique fat-burning cells may suggest novel therapeutic strategies for obesity.

Making insulin-deficient type 1 diabetic rodents thrive without insulin

Terminally ill insulin-deficient rodents with uncontrolled diabetes due to autoimmune or chemical destruction of β-cells were made hyperleptinemic by adenoviral transfer of the leptin gene. Within ≈10 days their severe hyperglycemia and ketosis were corrected. Despite the lack of insulin, moribund animals resumed linear growth and appeared normal. Normoglycemia persisted 10–80 days without other treatment; normal physiological conditions lasted for ≈175 days despite reappearance of moderate hyperglycemia. Inhibition of gluconeogenesis by suppression of hyperglucagonemia and reduction of hepatic cAMP response element-binding protein, phoshoenolpyruvate carboxykinase, and peroxisome proliferator-activated receptor-γ-coactivator-1α may explain the anticatabolic effect. Up-regulation of insulin-like growth factor 1 (IGF-1) expression and plasma levels and increasing IGF-1 receptor phosphorylation in muscle may explain the increased insulin receptor substrate 1, PI3K, and ERK phosphorylation in skeletal muscle. These findings suggest that leptin reverses the catabolic consequences of total lack of insulin, potentially by suppressing glucagon action on liver and enhancing the insulinomimetic actions of IGF-1 on skeletal muscle, and suggest strategies for making type 1 diabetes insulin-independent.

PPARα is necessary for the lipopenic action of hyperleptinemia on white adipose and liver tissue

Adenovirus-induced hyperleptinemia causes rapid disappearance of body fat in normal rats, presumably by up-regulating fatty acid oxidation within white adipocytes. To determine the role of peroxisomal proliferation-activated receptor (PPAR)α expression, which was increased during the rapid loss of fat, we infused adenovirus–leptin into PPARα−/− and PPARα+/+ mice. Despite similar degrees of hyperleptinemia and reduction in food intake, epididymal fat pad weight declined 55% in wild-type but only 6% in PPARα−/− mice; liver triacylglycerol fell 39% in the wild-type group but was unchanged in PPAR−/− mice. Carnitine palmitoyl transferase-1 mRNA rose 52% in the wild-type mice but did not increase in PPARα−/− mice. PPARγ coactivator-1α rose 3-fold in the fat and 46% in the liver of wild-type mice but was unchanged in PPARα−/− mice. Although AMP-activated protein kinase could not be implicated in the lipopenic actions of hyperleptinemia, acetyl CoA carboxylase protein was reduced in the liver of wild-type but not in PPARα−/− mice. Thus, in PPARα−/− mice, up-regulation of carnitine palmitoyl transferase-1 mRNA in fat, down-regulation of acetyl CoA carboxylase in liver, and up-regulation of PPARγ coactivator-1α mRNA in both tissues are abolished, as is the reduction in their triacylglycerol content.

Adipogenic capacity and the susceptibility to type 2 diabetes and metabolic syndrome

To determine whether adipocyte storage capacity influences the onset and severity of type 2 diabetes and other components of the metabolic syndrome, we made normal and db/db mice resistant to obesity by overexpressing leptin receptor-b on the aP2-Lepr-b promoter. On a 4% diet, these mice have no phenotype, but on a 60% fat diet, they resist diet-induced obesity because constitutive adipocyte-specific overexpression of Lepr-b prevents obesity via the antilipogenic autocrine/paracrine action of leptin on adipocytes. After 8 months on the same 60% fat diet, body fat of transgenic mice was 70% below WT controls. Cardiac and liver fat was elevated in the transgenics, and their hyperinsulinemia was more marked, suggesting greater insulin resistance. The aP2-Lepr-b transgene also prevented obesity in db/db mice; at 10 weeks of age their body fat was half that of the db/db mice. This lack of obesity was attributable to reduced expression of sterol regulatory element binding protein-1c and its target lipogenic enzymes in adipose tissue and a 6-fold increase in Pref-1 mRNA. Severe diabetes was present in transgenics at 4 weeks of age, 10 weeks before db/db controls. Echocardiographic evidence of cardiomyopathy appeared at 10 weeks, weeks before the db/db mice. Histologically, loss of β cells and myocardial fibrosis was present in the transgenic group at least 6 weeks before the db/db mice. These results suggest that the expression level of genes that regulate the adipogenic response to overnutrition profoundly influences the age of onset and severity of diet-induced type 2 diabetes and co-morbidities.

Metabolic manifestations of insulin deficiency do not occur without glucagon action

To determine unambiguously if suppression of glucagon action will eliminate manifestations of diabetes, we expressed glucagon receptors in livers of glucagon receptor-null (GcgR−/−) mice before and after β-cell destruction by high-dose streptozotocin. Wild type (WT) mice developed fatal diabetic ketoacidosis after streptozotocin, whereas GcgR−/− mice with similar β-cell destruction remained clinically normal without hyperglycemia, impaired glucose tolerance, or hepatic glycogen depletion. Restoration of receptor expression using adenovirus containing the GcgR cDNA restored hepatic GcgR, phospho-cAMP response element binding protein (P-CREB), and phosphoenol pyruvate carboxykinase, markers of glucagon action, rose dramatically and severe hyperglycemia appeared. When GcgR mRNA spontaneously disappeared 7 d later, P-CREB declined and hyperglycemia disappeared. In conclusion, the metabolic manifestations of diabetes cannot occur without glucagon action and, once present, disappear promptly when glucagon action is abolished. Glucagon suppression should be a major therapeutic goal in diabetes.

Regeneration of pancreatic islets in vivo by ultrasound-targeted gene therapy

This study uses a novel approach to gene therapy in which plasmid DNA is targeted to the pancreas in vivo using ultrasound-targeted microbubble destruction (UTMD) to achieve islet regeneration. Intravenous microbubbles carrying plasmids are destroyed within the pancreatic microcirculation by ultrasound, achieving local gene expression that is further targeted to β-cells by a modified rat insulin promoter (RIP3.1). A series of genes implicated in endocrine development were delivered to rats 2 days after streptozotocin-induced diabetes. The genes, PAX4, Nkx2.2, Nkx6.1, Ngn3 and Mafa, produced α-cell hyperplasia, but no significant improvement in β-cell mass or blood glucose level 30 days after UTMD. In contrast, RIP3.1-NeuroD1 promoted islet regeneration from surviving β-cells, with normalization of glucose, insulin and C-peptide levels at 30 days. In a longer-term experiment, four of six rats had a return of diabetes at 90 days, accompanied by β-cell apoptosis on Tunel staining. Pretreatment with the JNK inhibitor SP600125 successfully blocked β-cell apoptosis and resulted in restoration of β-cell mass and normalization of blood glucose level for up to 90 days. This technique allows in vivo islet regeneration, restoration of β-cell mass and normalization of blood sugar, insulin and C-peptide in rats without viruses.