Dawn Belt Davis

A gene expression network model of type 2 diabetes links cell cycle regulation in islets with diabetes susceptibility

Insulin resistance is necessary but not sufficient for the development of type 2 diabetes. Diabetes results when pancreatic beta-cells fail to compensate for insulin resistance by increasing insulin production through an expansion of beta-cell mass or increased insulin secretion. Communication between insulin target tissues and beta-cells may initiate this compensatory response. Correlated changes in gene expression between tissues can provide evidence for such intercellular communication. We profiled gene expression in six tissues of mice from an obesity-induced diabetes-resistant and a diabetes-susceptible strain before and after the onset of diabetes. We studied the correlation structure of mRNA abundance and identified 105 co-expression gene modules. We provide an interactive gene network model showing the correlation structure between the expression modules within and among the six tissues. This resource also provides a searchable database of gene expression profiles for all genes in six tissues in lean and obese diabetes-resistant and diabetes-susceptible mice, at 4 and 10 wk of age. A cell cycle regulatory module in islets predicts diabetes susceptibility. The module predicts islet replication; we found a strong correlation between (2)H(2)O incorporation into islet DNA in vivo and the expression pattern of the cell cycle module. This pattern is highly correlated with that of several individual genes in insulin target tissues, including Igf2, which has been shown to promote beta-cell proliferation, suggesting that these genes may provide a link between insulin resistance and beta-cell proliferation.

Normal myoblast fusion requires myoferlin

Muscle growth occurs during embryonic development and continues in adult life as regeneration. During embryonic muscle growth and regeneration in mature muscle, singly nucleated myoblasts fuse to each other to form myotubes. In muscle growth, singly nucleated myoblasts can also fuse to existing large, syncytial myofibers as a mechanism of increasing muscle mass without increasing myofiber number. Myoblast fusion requires the alignment and fusion of two apposed lipid bilayers. The repair of muscle plasma membrane disruptions also relies on the fusion of two apposed lipid bilayers. The protein dysferlin, the product of the Limb Girdle Muscular Dystrophy type 2 locus, has been shown to be necessary for efficient, calcium-sensitive, membrane resealing. We now show that the related protein myoferlin is highly expressed in myoblasts undergoing fusion, and is expressed at the site of myoblasts fusing to myotubes. Like dysferlin, we found that myoferlin binds phospholipids in a calcium-sensitive manner that requires the first C2A domain. We generated mice with a null allele of myoferlin. Myoferlin null myoblasts undergo initial fusion events, but they form large myotubes less efficiently in vitro, consistent with a defect in a later stage of myogenesis. In vivo, myoferlin null mice have smaller muscles than controls do, and myoferlin null muscle lacks large diameter myofibers. Additionally, myoferlin null muscle does not regenerate as well as wild-type muscle does, and instead displays a dystrophic phenotype. These data support a role for myoferlin in the maturation of myotubes and the formation of large myotubes that arise from the fusion of myoblasts to multinucleate myotubes.

Thioredoxin-interacting protein deficiency induces Akt/Bcl-xL signaling and pancreatic beta-cell mass and protects against diabetes

Pancreatic beta‐cell loss through apoptosis represents a key factor in the pathogenesis of diabetes;however, no effective approaches to block this process and preserve endogenous beta‐cell mass are currently available. To study the role of thioredoxin‐interacting protein (TXNIP), a proapoptotic beta‐cell factor we recently identified, we used HcB‐19 (TXNIP nonsense mutation) and beta‐cell‐specific TXNIP knockout (bTKO) mice. Interestingly, HcB‐19 mice demonstrate increased adiposity, but have lower blood glucose levels and increased pancreatic beta‐cell mass (as assessed by morphometry). Moreover, HcB‐19 mice are resistant to streptozotocin‐induced diabetes. When intercrossed with obese, insulin‐resistant, and diabetic mice, double‐mutant BTBRlepob/obtxniphcb/hcb are even more obese, but are protected against diabetes and beta‐cell apoptosis, resulting in a 3‐fold increase in beta‐cell mass. Beta‐cell‐specific TXNIP deletion also enhanced beta‐cell mass (P< 0.005) and protected against diabetes, and terminal deoxynucleotidyl transferase‐mediated nick end labeling (TUNEL) revealed a ~50‐fold reduction in beta‐cell apoptosis in streptozotocin‐treated bTKO mice. We further discovered that TXNIP deficiency induces Akt/Bcl‐xL signaling and inhibits mitochondrial beta‐cell death, suggesting that these mechanisms may mediate the beta‐cell protective effects of TXNIP deficiency. These results suggest that lowering beta‐cell TXNIP expression could serve as a novel strategy for the treatment of type 1 and type 2 diabetes by promoting endogenous beta‐cell survival.—Chen, J., Hui, S. T., Couto, F. M., Mungrue, I. N., Davis, D. B., Attie, A. D., Lusis, A. J., Davis, R. A., Shalev, A. Thioredoxin‐interacting protein deficiency induces Akt/ Bcl‐xL signaling and pancreatic beta‐cell mass and protects against diabetes. FASEB J. 22, 3581–3594 (2008)

Dysferlin protein analysis in limb-girdle muscular dystrophies

Dysferlin is the protein product of the DYSF gene mapped at 2p31, which mutations cause limb-girdle muscular dystrophy type 2B (LGMD2B) and Miyoshi myopathy. To date, nine autosomal recessive forms (AR-LGMD) have been identified: four genes, which code for the sarcoglycan glycoproteins, are associated with both mild and severe forms, the sarcoglycanopathies (LGMD2C, 2D, 2E and 2F). The other five forms, usually causing a milder phenotype are LGMD2A (calpain 3), LGMD2B (dysferlin), LGMD2G (telethonin), LGMD2H (9q31-11), and LGMD2I (19q13.3).We studied dysferlin expression in a total of 176 patients, from 166 LGMD families: 12 LGMD2B patients, 70 with other known forms of muscular dystrophies (LGMD2A, sarcoglycanopathies, LGMD2G), in an attempt to assess the effect of the primary gene-product deficiency on dysferlin. In addition, 94 still unclassified LGMD families were screened for dysferlin deficiency.In eight LGMD2B patients from five families, no dysferlin was observed in muscle biopsies, both through immunofluorescence (IF) and Western blot methodologies, while in two families, a very faint band was detected. Both patterns, negative or very faint bands, were concordant in patients belonging to the same families, suggesting that dysferlin deficiency is specific to LGMD2B.Myoferlin, the newly identified homologue of dysferlin was studied for the first time in LGMD2B patients. Since no difference was observed between patients mildly and severely affected, this protein do not seem to modify the phenotype in the present dysferlin-deficient patients.Dystrophin, sarcoglycans, and telethonin were normal in all LGMD2B patients, while patients with sarcoglycanopathies (2C, 2D, and 2E), LGMD2A, LGMD2G, and DMD showed the presence of a normal dysferlin band by Western blot and a positive pattern on IF. These data suggest that there is no interaction between dysferlin and these proteins. However, calpain analysis showed a weaker band in four patients from two families with intra-familial concordance. Therefore, this secondary deficiency of calpain in LGMD2B families, may indicate an interaction between dysferlin and calpain in muscle.Dysferlin was also present in cultured myotubes, in chorionic villus, and in the skin.Dysferlin deficiency was found in 24 out of a total of 166 Brazilian AR-LGMD families screened for muscle proteins (∼14%), thus representing the second most frequent known LGMD form, after calpainopathy, in our population.

Laparoscopic reversal of Roux-en-Y gastric bypass: Technique and utility for treatment of endocrine complications

BACKGROUND The anatomic and physiologic changes with Roux-en-Y gastric bypass (RYGB) may lead to uncommon but occasionally difficult to treat complications such as hyperinsulinemic hypoglycemia with neuroglycopenia and recalcitrant hypocalcemia associated to hypoparathyroidism. Medical management of these complications is challenging. Laparoscopic reversal of RYGB anatomy with restoration of pyloric function and duodenal continuity is a potential treatment. The objective of this study was to present the indications, surgical technique, and clinical outcomes of laparoscopic reversal of RYGB. METHODS Prospective study of consecutive patients offered laparoscopic reversal of RYGB. RESULTS Five patients with remote laparoscopic RYGB underwent laparoscopic reversal of RYGB to normal anatomy (n = 2) or modified sleeve gastrectomy (n = 3). Indications were medically refractory hyperinsulinemic hypoglycemia with neuroglycopenia (n = 3), recalcitrant hypocalcemia with hypoparathyroidism (n = 1), and both conditions simultaneously (n = 1). Before reversal, all patients had a gastrostomy tube placed in the excluded stomach to document improvement of symptoms. Laparoscopic reversal was accomplished successfully in all patients. Three postoperative complications occurred: bleeding that required transfusion, gallstone pancreatitis, and a superficial trocar site infection. Average length of stay was 3 days. At a mean follow-up of 12 months (range 3 to 22), no additional episodes of neuroglycopenia occurred, average number of hypoglycemic episodes per week decreased from 18.5 ± 12.4 to 1.5 ± 1.9 (P = .05), and hypocalcemia became responsive to oral replacement therapy in both patients. CONCLUSIONS Laparoscopic reversal of RYGB to normal anatomy or modified sleeve gastrectomy is feasible and may be a therapeutic option for selected patients with medically refractory hyperinsulinemic hypoglycemia and/or recalcitrant hypocalcemia associated with hypoparathyroidism.

Attention to Background Strain Is Essential for Metabolic Research: C57BL/6 and the International Knockout Mouse Consortium

The International Knockout Mouse Consortium (IKMC) introduces its targeted constructs into C57BL/6N embryonic stem cells. However, breeding with a Cre-recombinase and/or Flp-recombinase mouse is required for the generation of a null allele with the IKMC cassette. Many recombinase strains are in the C57BL/6J background, resulting in knockout animals on a mixed strain background. This can lead to variability in metabolic data and the use of improper control groups. While C57BL/6N and C57BL/6J are derived from the same parental C57BL/6 strain, there are key genotypic and phenotypic differences between these substrains. Many researchers may not even be aware of these differences, as the shorthand C57BL/6 is often used to describe both substrains. We found that 58% of articles involving genetically modified mouse models did not completely address background strain. This review will describe these two substrains and highlight the importance of separate consideration in mouse model development. Our aim is to increase awareness of this issue in the diabetes research community and to provide practical strategies to enable researchers to avoid mixed strain animals when using IKMC knockout mice.

Pancreatic β-Cell Proliferation in Obesity

Because obesity rates have increased dramatically over the past 3 decades, type 2 diabetes has become increasingly prevalent as well. Type 2 diabetes is associated with decreased pancreatic β-cell mass and function, resulting in inadequate insulin production. Conversely, in nondiabetic obesity, an expansion in β-cell mass occurs to provide sufficient insulin and to prevent hyperglycemia. This expansion is at least in part due to β-cell proliferation. This review focuses on the mechanisms regulating obesity-induced β-cell proliferation in humans and mice. Many factors have potential roles in the regulation of obesity-driven β-cell proliferation, including nutrients, insulin, incretins, hepatocyte growth factor, and recently identified liver-derived secreted factors. Much is still unknown about the regulation of β-cell replication, especially in humans. The extracellular signals that activate proliferative pathways in obesity, the relative importance of each of these pathways, and the extent of cross-talk between these pathways are important areas of future study.

Cholecystokinin Is Up-Regulated in Obese Mouse Islets and Expands β-Cell Mass by Increasing β-Cell Survival

An absolute or functional deficit in beta-cell mass is a key factor in the pathogenesis of diabetes. We model obesity-driven beta-cell mass expansion by studying the diabetes-resistant C57BL/6-Leptin(ob/ob) mouse. We previously reported that cholecystokinin (Cck) was the most up-regulated gene in obese pancreatic islets. We now show that islet cholecystokinin (CCK) is up-regulated 500-fold by obesity and expressed in both alpha- and beta-cells. We bred a null Cck allele into the C57BL/6-Leptin(ob/ob) background and investigated beta-cell mass and metabolic parameters of Cck-deficient obese mice. Loss of CCK resulted in decreased islet size and reduced beta-cell mass through increased beta-cell death. CCK deficiency and decreased beta-cell mass exacerbated fasting hyperglycemia and reduced hyperinsulinemia. We further investigated whether CCK can directly affect beta-cell death in cell culture and isolated islets. CCK was able to directly reduce cytokine- and endoplasmic reticulum stress-induced cell death. In summary, CCK is up-regulated by islet cells during obesity and functions as a paracrine or autocrine factor to increase beta-cell survival and expand beta-cell mass to compensate for obesity-induced insulin resistance.

Contamination with E1A-Positive Wild-Type Adenovirus Accounts for Species-Specific Stimulation of Islet Cell Proliferation by CCK: A Cautionary Note

We have previously reported that adenovirus-mediated expression of preprocholecystokin (CCK) stimulates human and mouse islet cell proliferation. In follow-up studies, we became concerned that the CCK adenovirus might have been contaminated with a wild-type E1A-containing adenovirus. Here we show conclusively that the proliferative effects reported in the original paper in mouse and human islets were not due to CCK expression but rather to a contaminating E1A-expressing wild-type adenovirus. We also show, however, that CCK expression does have a proliferative effect in rat islets. We hope that our report of the steps taken to detect the wild-type virus contamination, and purification of the contributing viral stocks, will be helpful to other investigators, and that our experience will serve as a cautionary tale for use of adenovirus vectors, especially for studies on cellular replication.

Glucagon-Like Peptide-1 Regulates Cholecystokinin Production in β-Cells to Protect From Apoptosis

Cholecystokinin (CCK) is a classic gut hormone that is also expressed in the pancreatic islet, where it is highly up-regulated with obesity. Loss of CCK results in increased β-cell apoptosis in obese mice. Similarly, islet α-cells produce increased amounts of another gut peptide, glucagon-like peptide 1 (GLP-1), in response to cytokine and nutrient stimulation. GLP-1 also protects β-cells from apoptosis via cAMP-mediated mechanisms. Therefore, we hypothesized that the activation of islet-derived CCK and GLP-1 may be linked. We show here that both human and mouse islets secrete active GLP-1 as a function of body mass index/obesity. Furthermore, GLP-1 can rapidly stimulate β-cell CCK production and secretion through direct targeting by the cAMP-modulated transcription factor, cAMP response element binding protein (CREB). We find that cAMP-mediated signaling is required for Cck expression, but CCK regulation by cAMP does not require stimulatory levels of glucose or insulin secretion. We also show that CREB directly targets the Cck promoter in islets from obese (Leptin(ob/ob)) mice. Finally, we demonstrate that the ability of GLP-1 to protect β-cells from cytokine-induced apoptosis is partially dependent on CCK receptor signaling. Taken together, our work suggests that in obesity, active GLP-1 produced in the islet stimulates CCK production and secretion in a paracrine manner via cAMP and CREB. This intraislet incretin loop may be one mechanism whereby GLP-1 protects β-cells from apoptosis.