Correction of obesity and diabetes in genetically obese mice by leptin gene therapy

The ob/ob mouse is genetically deficient in leptin and exhibits both an obese and a mild non-insulin-dependent diabetic phenotype. To test the hypothesis that correction of the obese phenotype by leptin gene therapy will lead to the spontaneous correction of the diabetic phenotype, the ob/ob mouse was treated with a recombinant adenovirus expressing the mouse leptin cDNA. Treatment resulted in dramatic reductions in both food intake and body weight, as well as the normalization of serum insulin levels and glucose tolerance. The subsequent diminishment in serum leptin levels resulted in the rapid resumption of food intake and a gradual gain of body weight, which correlated with the gradual return of hyperinsulinemia and insulin resistance. These results not only demonstrated that the obese and diabetic phenotypes in the adult ob/ob mice are corrected by leptin gene treatment but also provide confirming evidence that body weight control may be critical in the long-term management of non-insulin-dependent diabetes mellitus in obese patients.

The Export of Major Histocompatibility Complex Class I Molecules from the Endoplasmic Reticulum of Rat Brown Adipose Cells Is Acutely Stimulated by Insulin

Major histocompatibility complex class I (MHC-I) molecules have been implicated in several nonimmunological functions including the regulation and intracellular trafficking of the insulin-responsive glucose transporter GLUT4. We have used confocal microscopy to compare the effects of insulin on the intracellular trafficking of MHC-I and GLUT4 in freshly isolated rat brown adipose cells. We also used a recombinant vaccinia virus (rVV) to express influenza virus hemagglutinin (HA) as a generic integral membrane glycoprotein to distinguish global versus specific enhancement of protein export from the endoplasmic reticulum (ER) in response to insulin. In the absence of insulin, MHC-I molecules largely colocalize with the ER-resident protein calnexin and remain distinct from intracellular pools of GLUT4. Surprisingly, insulin induces the rapid export of MHC-I molecules from the ER with a concomitant approximately three-fold increase in their level on the cell surface. This ER export is blocked by brefeldin A and wortmannin but is unaffected by cytochalasin D, indicating that insulin stimulates the rapid transport of MHC-I molecules from the ER to the plasma membrane via the Golgi complex in a phosphatidyl-inositol 3-kinase–dependent and actin-independent manner. We further show that the effect of insulin on MHC-I molecules is selective, because insulin does not affect the intracellular distribution or cell-surface localization of rVV-expressed HA. These results demonstrate that in rat brown adipose cells MHC-I molecule export from the ER is stimulated by insulin and provide the first evidence that the trafficking of MHC-I molecules is acutely regulated by a hormone.

Insulin Action on GLUT4 Traffic Visualized in Single 3T3-L1 Adipocytes by Using Ultra-fast MicroscopyV⃞

A novel imaging technology, high-speed microscopy, has been used to visualize the process of GLUT4 translocation in response to insulin in single 3T3-L1 adipocytes. A key advantage of this technology is that it requires extremely low light exposure times, allowing the quasi-continuous capture of information over 20–30 min without photobleaching or photodamage. The half-time for the accumulation of GLUT4-eGFP (enhanced green fluorescent protein) at the plasma membrane in a single cell was found to be of 5–7 min at 37°C. This half-time is substantially longer than that of exocytic vesicle fusion in neuroendocrine cells, suggesting that additional regulatory mechanisms are involved in the stimulation of GLUT4 translocation by insulin. Analysis of four-dimensional images (3-D over time) revealed that, in response to insulin, GLUT4-eGFP-enriched vesicles rapidly travel from the juxtanuclear region to the plasma membrane. In nontransfected adipocytes, impairment of microtubule and actin filament function inhibited insulin-stimulated glucose transport by 70 and 50%, respectively. When both filament systems were impaired insulin-stimulated glucose transport was completely inhibited. Taken together, the data suggest that the regulation of long-range motility of GLUT4-containing vesicles through the interaction with microtubule- and actin-based cytoskeletal networks plays an important role in the overall effect of insulin on GLUT4 translocation.

Hyposecretion of the adrenal androgen dehydroepiandrosterone sulfate and its relation to clinical variables in inflammatory arthritis

Hypothalamic–pituitary–adrenal underactivity has been reported in rheumatoid arthritis (RA). This phenomenon has implications with regard to the pathogenesis and treatment of the disease. The present study was designed to evaluate the secretion of the adrenal androgen dehydroepiandrosterone sulfate (DHEAS) and its relation to clinical variables in RA, spondyloarthropathy (Spa), and undifferentiated inflammatory arthritis (UIA). Eighty-seven patients (38 with RA, 29 with Spa, and 20 with UIA) were studied, of whom 54 were women. Only 12 patients (14%) had taken glucocorticoids previously. Age-matched, healthy women (134) and men (149) served as controls. Fasting blood samples were taken for determination of the erythrocyte sedimentation rate (ESR), serum DHEAS and insulin, and plasma glucose. Insulin resistance was estimated by the homeostasis-model assessment (HOMAIR). DHEAS concentrations were significantly decreased in both women and men with inflammatory arthritis (IA) (P < 0.001). In 24 patients (28%), DHEAS levels were below the lower extreme ranges found for controls. Multiple intergroup comparisons revealed similarly decreased concentrations in each disease subset in both women and men. After the ESR, previous glucocorticoid usage, current treatment with nonsteroidal anti-inflammatory drugs, duration of disease and HOMAIR were controlled for, the differences in DHEAS levels between patients and controls were markedly attenuated in women (P = 0.050) and were no longer present in men (P = 0.133). We concluded that low DHEAS concentrations are commonly encountered in IA and, in women, this may not be fully explainable by disease-related parameters. The role of hypoadrenalism in the pathophysiology of IA deserves further elucidation. DHEA replacement may be indicated in many patients with IA, even in those not taking glucocorticoids.

Overexpression of the LAR (leukocyte antigen-related) protein-tyrosine phosphatase in muscle causes insulin resistance

Previous reports indicate that the expression and/or activity of the protein-tyrosine phosphatase (PTP) LAR are increased in insulin-responsive tissues of obese, insulin-resistant humans and rodents, but it is not known whether these alterations contribute to the pathogenesis of insulin resistance. To address this question, we generated transgenic mice that overexpress human LAR, specifically in muscle, to levels comparable to those reported in insulin-resistant humans. In LAR-transgenic mice, fasting plasma insulin was increased 2.5-fold compared with wild-type controls, whereas fasting glucose was normal. Whole-body glucose disposal and glucose uptake into muscle in vivo were reduced by 39–50%. Insulin injection resulted in normal tyrosyl phosphorylation of the insulin receptor and insulin receptor substrate 1 (IRS-1) in muscle of transgenic mice. However, phosphorylation of IRS-2 was reduced by 62%, PI3′ kinase activity associated with phosphotyrosine, IRS-1, or IRS-2 was reduced by 34–57%, and association of p85α with both IRS proteins was reduced by 39–52%. Thus, overexpression of LAR in muscle causes whole-body insulin resistance, most likely due to dephosphorylation of specific regulatory phosphotyrosines on IRS proteins. Our data suggest that increased expression and/or activity of LAR or related PTPs in insulin target tissues of obese humans may contribute to the pathogenesis of insulin resistance.

The genetic mediation of individual differences in sensitivity to pain and its inhibition

The underlying bases of the considerable interindividual variability in pain-related traits are starting to be revealed. Although the relative importance of genes versus experience in human pain perception remains unclear, rodent populations display large and heritable differences in both nociceptive and analgesic sensitivity. The identification and characterization of particularly divergent populations provides a powerful initial step in the genetic analysis of pain, because these models can be exploited to identify genes contributing to the behavior-level variability. Ultimately, DNA sequence differences representing the differential alleles at pain-relevant genes can be identified. Thus, by using a combination of “top-down” and “bottom-up” strategies, we are now able to genetically dissect even complex biological traits like pain. The present review summarizes the current progress toward these ends in both humans and rodents.

Tissue-specific overexpression of lipoprotein lipase causes tissue-specific insulin resistance

Insulin resistance in skeletal muscle and liver may play a primary role in the development of type 2 diabetes mellitus, and the mechanism by which insulin resistance occurs may be related to alterations in fat metabolism. Transgenic mice with muscle- and liver-specific overexpression of lipoprotein lipase were studied during a 2-h hyperinsulinemic–euglycemic clamp to determine the effect of tissue-specific increase in fat on insulin action and signaling. Muscle–lipoprotein lipase mice had a 3-fold increase in muscle triglyceride content and were insulin resistant because of decreases in insulin-stimulated glucose uptake in skeletal muscle and insulin activation of insulin receptor substrate-1-associated phosphatidylinositol 3-kinase activity. In contrast, liver–lipoprotein lipase mice had a 2-fold increase in liver triglyceride content and were insulin resistant because of impaired ability of insulin to suppress endogenous glucose production associated with defects in insulin activation of insulin receptor substrate-2-associated phosphatidylinositol 3-kinase activity. These defects in insulin action and signaling were associated with increases in intracellular fatty acid-derived metabolites (i.e., diacylglycerol, fatty acyl CoA, ceramides). Our findings suggest a direct and causative relationship between the accumulation of intracellular fatty acid-derived metabolites and insulin resistance mediated via alterations in the insulin signaling pathway, independent of circulating adipocyte-derived hormones.

Autocrine/paracrine role of insulin-related growth factors in neurogenesis: local expression and effects on cell proliferation and differentiation in retina.

Early neurogenesis progresses by an initial massive proliferation of neuroepithelial cells followed by a sequential differentiation of the various mature neural cell types. The regulation of these processes by growth factors is poorly understood. We intend to understand, in a well-defined biological system, the embryonic chicken retina, the role of the insulin-related growth factors in neurogenesis. We demonstrate the local presence of signaling elements together with a biological response to the factors. Neuroretina at days 6-8 of embryonic development (E6-E8) expressed proinsulin/insulin and insulin-like growth factor I (IGF-I) mRNAs as well as insulin receptor and IGF type I receptor mRNAs. In parallel with this in vivo gene expression, E5 cultured neuroretinas synthesized and released to the medium a metabolically radiolabeled immunoprecipitable insulin-related peptide. Furthermore, insulin-related immunoreactive material with a HPLC mobility close to that of proinsulin was found in the E6-E8 vitreous humor. Exogenous chicken IGF-I, human insulin, and human proinsulin added to E6 cultured neuroretinas showed relatively close potencies stimulating proliferation, as determined by [methyl-3H]thymidine incorporation, with a plateau reached at 10(-8) M. These factors also stimulated neuronal differentiation, indicated by the expression of the neuron-specific antigen G4. Thus, insulin-related growth factors, interestingly including proinsulin, are present in the developing chicken retina and appear to play an autocrine/paracrine stimulatory role in the progression of neurogenesis.

Insulin-like growth factor I receptors of fetal brain are enriched in nerve growth cones and contain a beta-subunit variant.

Nerve growth cones isolated from fetal rat brain are highly enriched in a 97-kDa glycoprotein, termed beta gc, that comigrates with the beta subunit of the IGF-I receptor upon two-dimensional PAGE and is disulfide-linked to this receptor's alpha subunit. Antibodies prepared to a conserved domain shared by the insulin and IGF-I receptor beta subunits (AbP2) or to beta gc were used to study receptor distribution further. Subcellular fractionation of the fetal brain segregated most AbP2 immunoreactivity away from growth cones, whereas most beta gc immunoreactivity copurified with growth cones. Experiments involving ligand-activated receptor autophosphorylation confirmed the concentration of IGF-I but not of insulin receptors in growth cone fractions. These results indicate the enrichment of IGF-I receptors in (presumably axonal) growth cones of the differentiating neuron. Furthermore, the segregation of beta gc from AbP2 immunoreactivity suggests that such neurons express an immunochemically distinct variant of the IGF-I receptor beta subunit at the growth cone.

High fat diet-induced hyperglycemia: prevention by low level expression of a glucose transporter (GLUT4) minigene in transgenic mice.

High-fat intake leading to obesity contributes to the development of non-insulin-dependent diabetes mellitus (NIDDM, type 2). Similarly, mice fed a high-fat (safflower oil) diet develop defective glycemic control, hyperglycemia, and obesity. To assess the effect of a modest increase in the expression of GLUT4 (the insulin-responsive glucose transporter) on impaired glycemic control caused by fat feeding, transgenic mice harboring a GLUT4 minigene were fed a high-fat diet. Low-level tissue-specific (heart, skeletal muscle, and adipose tissue) expression of the GLUT4 minigene in transgenic mice prevented the impairment of glycemic control and accompanying hyperglycemia, but not obesity, caused by fat feeding. Thus, a small increase (< or = 2-fold) in the tissue level of GLUT4 prevents a primary symptom of the diabetic state in a mouse model, suggesting a possible target for intervention in the treatment of NIDDM.