Jack L. Leahy

Characterization of a novel metabolic strategy used by drug-resistant tumor cells

Acquired or inherent drug resistance is the major problem in achieving successful cancer treatment. However, the mechanism(s) of pleiotropic drug resistance remains obscure. We have identified and characterized a cellular metabolic strategy that differentiates drug‐resistant cells from drug‐sensitive cells. This strategy may serve to protect drug‐resistant cells from damage caused by chemotherapeutic agents and radiation. We show that drug‐resistant cells have low mitochondrial membrane potential, use nonglucose carbon sources (fatty acids) for mitochondrial oxygen consumption when glucose becomes limited, and are protected from exogenous stress such as radiation. In addition, drug‐resistant cells express high levels of mitochondrial uncoupling protein 2 (UCP2). The discovery of this metabolic strategy potentially facilitates the design of novel therapeutic approaches to drug resistance.—Harper, M.‐E., Antoniou, A., Villalobos‐Menuey, E., Russo, A., Trauger, R., Vendemelio, George, A. M., Bartholomew, R., Carlo, D., Shaikh, A., Kupperman, J., Newell, E. W., Bespalov, I. A., Wallace, S. S., Liu, Y., Rogers, J. R., Gibbs, G. L., Leahy, J. L., Camley, R. E., Melamede, R., Newell, M. K. Characterization of a novel metabolic strategy used by drug‐resistant tumor cells. FASEB J. 16, 1550–1557 (2002)

β-Cell Adaptation to Insulin Resistance INCREASED PYRUVATE CARBOXYLASE AND MALATE-PYRUVATE SHUTTLE ACTIVITY IN ISLETS OF NONDIABETIC ZUCKER FATTY RATS

The β-cell biochemical mechanisms that account for the compensatory hyperfunction with insulin resistance (so-calledβ-cell adaptation) are unknown. We investigated glucose metabolism in isolated islets from 10–12-week-old Zucker fatty (ZF) and Zucker lean (ZL) rats (results expressed per mg/islet of protein). ZF rats were obese, hyperlipidemic, and normoglycemic. They had a 3.8-fold increased β-cell mass along with 3–10-fold increases in insulin secretion to various stimuli during pancreas perfusion despite insulin content per milligram of β-cells being only one-third that of ZL rats. Islet glucose metabolism (utilization and oxidation) was 1.5–2-fold increased in the ZF islets despite pyruvate dehydrogenase activity being 30% lowered compared with the ZL islets. The reason was increased flux through pyruvate carboxylase (PC) and the malate-pyruvate and citrate-pyruvate shuttles based on the following observations (% ZL islets): increasedV max of PC (160%), malate dehydrogenase (170%), and malic enzyme (275%); elevated concentrations of oxaloacetate (150%), malate (250%), citrate (140%), and pyruvate (250%); and 2-fold increased release of malate from isolated mitochondria. Inhibition of PC by 5 mm phenylacetic acid markedly lowered glucose-induced insulin secretion in ZF and ZL islets. Thus, our results suggest that PC and the pyruvate shuttles are increased in ZF islets, and this accounts for glucose mitochondrial metabolism being increased when pyruvate dehydrogenase activity is reduced. As the anaplerosis pathways are implicated in glucose-induced insulin secretion and the synthesis of glucose-derived lipid and amino acids, our results highlight the potential importance of PC and the anaplerosis pathways in the enhanced insulin secretion and β-cell growth that characterize β-cell adaptation to insulin resistance.

Regulation of Pancreatic β-Cell Regeneration in the Normoglycemic 60% Partial-Pancreatectomy Mouse

β-Cell mass is determined by a dynamic balance of proliferation, neogenesis, and apoptosis. The precise mechanisms underlying compensatory β-cell mass (BCM) homeostasis are not fully understood. To evaluate the processes that maintain normoglycemia and regulate BCM during pancreatic regeneration, C57BL/6 mice were analyzed for 15 days following 60% partial pancreatectomy (Px). BCM increased in Px mice from 2 days onwards and was ∼68% of the shams by 15 days, partly due to enhanced β-cell proliferation. A transient ∼2.8-fold increase in the prevalence of β-cell clusters/small islets at 2 days post-Px contributed substantially to BCM augmentation, followed by an increase in the number of larger islets at 15 days. To evaluate the signaling mechanisms that may regulate this compensatory growth, we examined key intermediates of the insulin signaling pathway. We found insulin receptor substrate (IRS)2 and enhanced-activated Akt immunoreactivity in islets and ducts that correlated with increased pancreatic duodenal homeobox (PDX)1 expression. In contrast, forkhead box O1 expression was decreased in islets but increased in ducts, suggesting distinct PDX1 regulatory mechanisms in these tissues. Px animals acutely administered insulin exhibited further enhancement in insulin signaling activity. These data suggest that the IRS2-Akt pathway mediates compensatory β-cell growth by activating β-cell proliferation with an increase in the number of β-cell clusters/small islets.

Increased secretory demand rather than a defect in the proinsulin conversion mechanism causes hyperproinsulinemia in a glucose-infusion rat model of non-insulin-dependent diabetes mellitus.

Hyperproinsulinemia in non-insulin-dependent diabetes mellitus (NIDDM) is due to an increased release of proinsulin from pancreatic beta cells. This could reside in increased secretory demand placed on the beta cell by hyperglycemia or in the proinsulin conversion mechanism. In this study, biosynthesis of the proinsulin conversion enzymes (PC2, PC3, and carboxypeptidase-H [CP-H]) and proinsulin, were examined in islets isolated from 48-h infused rats with 50% (wt/vol) glucose (hyperglycemic, hyperinsulinemic, and increased pancreatic proinsulin to insulin ratio), 20% (wt/vol) glucose (normoglycemic but hyperinsulinemic), and 0.45% (wt/vol) saline (controls). A decrease in the islet content of PC2, PC3, and CP-H from hyperglycemic rats was observed. This reduction did not correlate with any deficiency in mRNA levels or biosynthesis of PC2, PC3, CP-H, or proinsulin. Furthermore, proinsulin conversion rate was comparable in islets from hyperglycemic and control rats. However, in islets from hyperglycemic rats an abnormal increased proportion of proinsulin was secreted, that was accompanied by an augmented release of PC2, PC3 and CP-H. Stimulation of the beta cells secretory pathway by hyperglycemia, resulted in proinsulin being prematurely secreted from islets before its conversion could be completed. Thus, hyperproinsulinemia induced by chronic hyperglycemia likely results from increased beta cell secretory demand, rather than a defect in the proinsulin processing enzymes per se.

Targeting β-Cell Function Early in the Course of Therapy for Type 2 Diabetes Mellitus

OBJECTIVE This report examines current perspectives regarding likely mechanisms of beta-cell failure in type 2 diabetes and their clinical implications for protecting or sparing beta-cells early in the disease progression. In addition, it considers translation strategies to incorporate relevant scientific findings into educational initiatives targeting clinical practice behavior. PARTICIPANTS On January 10, 2009, a working group of basic researchers, clinical endocrinologists, and primary care physicians met to consider whether current knowledge regarding pancreatic beta-cell defects justifies retargeting and retiming treatment for clinical practice. Based on this meeting, a writing group comprised of four meeting participants subsequently prepared this consensus statement. The conference was convened by The Endocrine Society and funded by an unrestricted educational grant from Novo Nordisk. EVIDENCE Participants reviewed and discussed published literature, plus their own unpublished data. CONSENSUS PROCESS The summary and recommendations were supported unanimously by the writing group as representing the consensus opinions of the working group. CONCLUSIONS Workshop participants strongly advocated developing new systems to address common barriers to glycemic control and recommended several initial steps toward this goal. These recommendations included further studies to establish the clinical value of pharmacological therapies, continuing basic research to elucidate the nature and mechanisms of beta-cell failure in type 2 diabetes mellitus, and exploring new educational approaches to promote pathophysiology-based clinical practices. The Endocrine Society has launched a new website to continue the discussion between endocrinologists and primary care physicians on beta-cell pathophysiology in type 2 diabetes and its clinical implications. Join the conversation at http://www.betacellsindiabetes.org

Peroxisome proliferator-activated receptor-γ regulates expression of PDX-1 and NKX6.1 in INS-1 cells

In the 60% pancreatectomy (Px) rat model of β-cell adaptation, normoglycemia is maintained by an initial week of β-cell hyperplasia that ceases and is followed by enhanced β-cell function. It is unknown how this complex series of events is regulated. We studied isolated islets and pancreas sections from 14-day post-Px versus sham-operated rats and observed a doubling of β-cell nuclear peroxisome proliferator–activated receptor (PPAR)-γ protein, along with a 2-fold increase in nuclear pancreatic duodenal homeobox (Pdx)-1 protein and a 1.4-fold increase in β-cell nuclear Nkx6.1 immunostaining. As PPAR-γ activation is known to both lower proliferation and have prodifferentiation effects in many tissues, we studied PPAR-γ actions in INS-1 cells. A 3-day incubation with the PPAR-γ agonist troglitazone reduced proliferation and increased Pdx-1 and Nkx6.1 immunostaining, along with glucokinase and GLUT2. Also, a 75% knockdown of PPAR-γ using RNA interference lowered the mRNA levels of Pdx-1, glucokinase, GLUT2, and proinsulin II by more than half. Our results show a dual effect of PPAR-γ in INS-1 cells: to curtail proliferation and promote maturation, the latter via enhanced expression of Pdx-1 and Nkx6.1. Additional studies are needed to determine whether there is a regulatory role for PPAR-γ signaling in the β-cell adaptation following a 60% Px in rats.

In Vivo and in Vitro Studies of a Functional Peroxisome Proliferator-activated Receptor γ Response Element in the Mouse pdx-1 Promoter

We reported that peroxisome proliferator-activated receptor γ (PPARγ) transcriptionally regulates the β-cell differentiation factor pancreatic duodenal homeobox (PDX)-1 based on in vitro RNA interference studies. We have now studied mice depleted of PPARγ within the pancreas (PANC PPARγ-/-) created by a Cre/loxP recombinase system, with Cre driven by the pdx-1 promoter. Male PANC PPARγ-/- mice were hyperglycemic at 8 weeks of age (8.1 ± 0.2 mm versus 6.4 ± 0.3 mm, p = 0.009) with islet cytoarchitecture and pancreatic mass of islet β-cells that were indistinguishable from the controls. Islet PDX-1 mRNA (p = 0.001) and protein levels (p = 0.003) were lowered 60 and 40%, respectively, in tandem with impaired glucose-induced insulin secretion and loss of thiazolidinedione-induced increase in PDX-1 expression. We next identified a putative PPAR-response element (PPRE) in the mouse pdx-1 promoter with substantial homology to the corresponding region of the human PDX-1 promoter. Electrophoretic mobility supershift assays with nuclear extracts from β-cell lines and mouse islets, also in vitro translated PPARγ and retinoid X receptor, and chromatin immunoprecipitation analysis demonstrated specific binding of PPARγ and retinoid X receptor to the human and mouse pdx-1 × PPREs. Transient transfection assays of β-cells with reporter constructs of mutated PPREs showed dramatically reduced pdx-1 promoter activity. In summary, we have presented in vivo and in vitro evidence showing PPARγ regulation of pdx-1 transcription in β-cells, plus our results support an important regulatory role for PPARγ in β-cell physiology and thiazolidinedione pharmacology of type 2 diabetes.

Enhanced expression of insulin receptor substrate–2 and activation of protein kinase B/Akt in regenerating pancreatic duct epithelium of 60 %-partial pancreatectomy rats

Abstract.Aims/hypothesis: Early compensatory mechanisms of regeneration following partial pancreatectomy involve ductal proliferation and, subsequently, differentiation into acinar and endocrine cell types, although it is not clear how these processes are regulated. We investigated the expression and roles of insulin receptor substrate-2 (IRS-2) and protein kinase B/Akt (Akt) in pancreatic regeneration that starts with the common duct epithelium using a non-diabetic model of beta cell adaptation and mass expansion, 60 %-pancreatectomy rats. Methods: We used confocal immunofluorescence microscopy to study IRS-2 and Akt expression and activation in pancreatic common ducts at intervals after surgery. These proteins were studied in relation to proliferation markers and insulin immunostaining. Results: In pancreatectomized rats, a short-term increase in proliferation was observed in the common duct epithelial lining ( ∼ 4-fold) compared with sham-operated control rats which correlated with about a 1.8-fold increase in IRS-2 immunoreactivity 2 days after surgery. Interspersed with proliferating cells of the common duct, evaginations were rare single and clustered insulin immunopositive cells which expressed high levels of IRS-2 immunoreactivity. Epithelium of duct evaginations from 2-day post-Px rats exhibited striking phospho-Akt staining ( ∼ 3.5-fold above control rats) without any detectable changes in total Akt staining. Conclusion/interpretation: Our data suggest that IRS-2 plays an important role in pancreatic regeneration and growth by mediating duct proliferation and by maintaining the differentiated beta cell. The restricted staining pattern of phospho-Akt to cells of the common duct evaginations suggests that it has a role in regulating post-mitotic events related to cell-specific gene expression or survival or both. [Diabetologia (2001) 44: 2056–2065]

Physiologic and Pharmacologic Modulation of Glucose-Dependent Insulinotropic Polypeptide (GIP) Receptor Expression in β-Cells by Peroxisome Proliferator–Activated Receptor (PPAR)-γ Signaling Possible Mechanism for the GIP Resistance in Type 2 Diabetes

OBJECTIVE We previously showed that peroxisome proliferator–activated receptor (PPAR)-γ in β-cells regulates pdx-1 transcription through a functional PPAR response element (PPRE). Gene Bank blast for a homologous nucleotide sequence revealed the same PPRE within the rat glucose-dependent insulinotropic polypeptide receptor (GIP-R) promoter sequence. We investigated the role of PPARγ in GIP-R transcription. RESEARCH DESIGN AND METHODS Chromatin immunoprecipitation assay, siRNA, and luciferase gene transcription assay in INS-1 cells were performed. Islet GIP-R expression and immunohistochemistry studies were performed in pancreas-specific PPARγ knockout mice (PANC PPARγ−/−), normoglycemic 60% pancreatectomy rats (Px), normoglycemic and hyperglycemic Zucker fatty (ZF) rats, and mouse islets incubated with troglitazone. RESULTS In vitro studies of INS-1 cells confirmed that PPAR-γ binds to the putative PPRE sequence and regulates GIP-R transcription. In vivo verification was shown by a 70% reduction in GIP-R protein expression in islets from PANC PPARγ−/− mice and a twofold increase in islets of 14-day post-60% Px Sprague-Dawley rats that hyperexpress β-cell PPARγ. Thiazolidinedione activation (72 h) of this pathway in normal mouse islets caused a threefold increase of GIP-R protein and a doubling of insulin secretion to 16.7 mmol/l glucose/10 nmol/l GIP. Islets from obese normoglycemic ZF rats had twofold increased PPARγ and GIP-R protein levels versus lean rats, with both lowered by two-thirds in ZF rats made hyperglycemic by 60% Px. CONCLUSIONS Our studies have shown physiologic and pharmacologic regulation of GIP-R expression in β-cells by PPARγ signaling. Also disruption of this signaling pathway may account for the lowered β-cell GIP-R expression and resulting GIP resistance in type 2 diabetes.

Enhanced β-cell mass without increased proliferation following chronic mild glucose infusion

The physiological mechanisms underlying pancreatic beta-cell mass (BCM) homeostasis are complex and not fully resolved. Here we examined the factors contributing to the increased BCM following a mild glucose infusion (GI) whereby normoglycemia was maintained through 96 h. We used morphometric and immunochemical methods to investigate enhanced beta-cell growth and survival in Sprague-Dawley rats. BCM was elevated >2.5-fold over saline-infused control rats by 48 h and increased modestly thereafter. Unexpectedly, increases in beta-cell proliferation were not observed at any time point through 4 days. Instead, enhanced numbers of insulin(+) cell clusters and small islets (400-12,000 microm(2); approximately 23- to 124-microm diameter), mostly scattered among the acini, were observed in the GI rats by 48 h despite no difference in the numbers of medium to large islets. We previously showed that increased beta-cell growth in rodent models of insulin resistance and pancreatic regeneration involves increased activated Akt/PKB, a key beta-cell signaling intermediate, in both islets and endocrine cell clusters. GI in normal rats also leads to increased Akt activation in islet beta-cells, as well as in insulin(+) and insulin(-) cells in the common duct epithelium and endocrine clusters. This correlated with strong Pdx1 expression in these same cells. These results suggest that mechanisms other than proliferation underlie the rapid beta-cell growth response following a mild GI in the normal rat and involve Akt-regulated enhanced beta-cell survival potential and neogenesis from epithelial precursors.