Caspase 3 activity is low in primary rat islets in response to ER stress and DNA damage.

<p>832/13 cells (<b>A</b>, <b>C</b>) and primary rat islets (<b>B</b>, <b>D</b>) were treated with DMSO (control), thapsigargin (100 nM or 1 μM, respectively), or etoposide (100 μM or 200 μM, respectively) for the indicated times. Clarified lysates were examined by immunoblot analysis (<b>A</b>, <b>B</b>) and caspase 3/7 colorimetric activity assay (<b>C</b>, <b>D</b>). Data represent the mean +S.E.M of 3 independent experiments. * p ≤ 0.05 as compared to DMSO treated cells.</p

Early and late apoptotic marker analysis demonstrates low levels of apoptosis in primary rat islets.

<p>832/13 cells (<b>A</b>, <b>C</b>) and primary rat islets (<b>B</b>, <b>D</b>) were treated with DMSO (control), thapsigargin (100 nM or 1 μM, respectively), or etoposide (100 μM or 200 μM, respectively) for the indicated times. (<b>A</b>, <b>B</b>) Cells were stained with Annexin V and counted by flow cytometry. (<b>C</b>, <b>D</b>) Cells were dispersed onto coverslips and stained with TUNEL and counterstained with DAPI. Positive cells were counted using Fiji. Data represent the mean +S.E.M. of 3 independent experiments. * p ≤ 0.05 as compared to DMSO treated cells.</p

Caspase 3 activity is low in primary rat islets in response to ER stress and DNA damage.

<p>832/13 cells (<b>A</b>, <b>C</b>) and primary rat islets (<b>B</b>, <b>D</b>) were treated with DMSO (control), thapsigargin (100 nM or 1 μM, respectively), or etoposide (100 μM or 200 μM, respectively) for the indicated times. Clarified lysates were examined by immunoblot analysis (<b>A</b>, <b>B</b>) and caspase 3/7 colorimetric activity assay (<b>C</b>, <b>D</b>). Data represent the mean +S.E.M of 3 independent experiments. * p ≤ 0.05 as compared to DMSO treated cells.</p

Delayed apoptosis allows islet β-cells to implement an autophagic mechanism to promote cell survival

<div><p>Increased β-cell death coupled with the inability to replicate existing β-cells drives the decline in β-cell mass observed in the progression of both major forms of diabetes. Understanding endogenous mechanisms of islet cell survival could have considerable value for the development of novel strategies to limit β-cell loss and thereby promote β-cell recovery. Insulinoma cells have provided useful insight into β-cell death pathways but observations made in cell lines sometimes fail to translate to primary islets. Here, we report dramatic differences in the temporal regulation and engagement of the apoptotic program in primary rodent islets relative to the INS-1 derived 832/13 cell line. As expected, 832/13 cells rapidly induced cell stress markers in response to ER stress or DNA damage and were fully committed to apoptosis, resulting in >80% cell death within 24 h. In contrast, primary rat islets were largely refractory to cell death in response to ER stress and DNA damage, despite rapid induction of stress markers, such as XBP-1(s), CHOP, and PUMA. Gene expression profiling revealed a general suppression of pro-apoptotic machinery, such as Apaf-1 and caspase 3, and sustained levels of pro-survival factors, such as cIAP-1, cIAP-2, and XIAP, in rat islets. Furthermore, we observed sustained induction of autophagy following chronic ER stress and found that inhibition of autophagy rendered islet β-cells highly vulnerable to ER stress-induced cell death. We propose that islet β-cells dampen the apoptotic response to delay the onset of cell death, providing a temporal window in which autophagy can be activated to limit cellular damage and promote survival.</p></div

Expression of pro- and anti-apoptotic mRNAs is muted in primary rat islets.

<p>(<b>A</b>) Relative mRNA expression levels in primary islet cells were evaluated by qRT-PCR and compared to levels in 832/13 cells. Data represent the mean +S.E.M. (n = 3–5). * p < 0.05 as compared to 832/13 cells. (<b>B</b>) Immunoblot analysis of whole cell lysates.</p

Autophagy inhibition unveils apoptotic response to ER stress.

<p>832/13 (<b>A</b>) and primary rat islets (<b>B</b>) were treated with DMSO (control) or thapsigargin (100 nM or 1 μM, respectively) for the indicated times. Clarified lysates were examined by immunoblot analysis. LC3-I indicates the full length protein and LC3-II indicates the cleaved form of the protein conjugated to phosphatidylethanolamine. (<b>B</b>) Primary rat islets were dispersed onto HTB9 coated 96-well plates and treated with DMSO (control; 96 h), chloroquine (CHQ; 5 μM), or bafilomycin A1 (BAF; 100 nM), alone or in combination with thapsigargin (Tg; 1 μM) for the indicated times. Islet cells were stained for TUNEL and insulin and counterstained with DAPI. Cells were imaged using a high content imager and analyzed using Cellomics software. Data represent the mean +S.E.M. of 3 independent experiments. * p < 0.05 as compared to the treatment with thapsigargin alone for the same time period. (<b>C</b>) Rat islets were treated with either DMSO (control) or thapsigargin (Tg; 1 μM) for 24 h and bafilomycin A1 (BAF; 10 nM) was added during the final 6 h. Clarified lysates were examined by immunoblot analysis.</p

Similar induction of stress markers following thapsigargin treatment in 832/13 cells and primary rat islets.

<p>832/13 cells (<b>A</b>) and primary rat islets (<b>B</b>) were treated with DMSO (control) or thapsigargin (100 nM or 1 μM, respectively) for the indicated times. mRNA expression levels were determined via qRT-PCR. Data represent the mean +S.E.M. (n = 3–4). * p ≤ 0.05 as compared to DMSO treated cells. Primary rat islets were treated with DMSO control (<b>C</b>) or thapsigargin (1 μM; <b>D</b>) for 6 h and dispersed onto coverslips. Cells were stained for insulin (green) and CHOP (red).</p

SRC-2 Coactivator Deficiency Decreases Functional Reserve in Response to Pressure Overload of Mouse Heart

<div><p>A major component of the cardiac stress response is the simultaneous activation of several gene regulatory networks. Interestingly, the transcriptional regulator steroid receptor coactivator-2, SRC-2 is often decreased during cardiac failure in humans. We postulated that SRC-2 suppression plays a mechanistic role in the stress response and that SRC-2 activity is an important regulator of the adult heart gene expression profile. Genome-wide microarray analysis, confirmed with targeted gene expression analyses revealed that genetic ablation of SRC-2 activates the “fetal gene program” in adult mice as manifested by shifts in expression of a) metabolic and b) sarcomeric genes, as well as associated modulating transcription factors. While these gene expression changes were not accompanied by changes in left ventricular weight or cardiac function, imposition of transverse aortic constriction (TAC) predisposed SRC-2 knockout (KO) mice to stress-induced cardiac dysfunction. In addition, SRC-2 KO mice lacked the normal ventricular hypertrophic response as indicated through heart weight, left ventricular wall thickness, and blunted molecular signaling known to activate hypertrophy. Our results indicate that SRC-2 is involved in maintenance of the steady-state adult heart transcriptional profile, with its ablation inducing transcriptional changes that mimic a stressed heart. These results further suggest that SRC-2 deletion interferes with the timing and integration needed to respond efficiently to stress through disruption of metabolic and sarcomeric gene expression and hypertrophic signaling, the three key stress responsive pathways.</p> </div

Effects of HIV Infection on the Metabolic and Hormonal Status of Children with Severe Acute Malnutrition

<div><p>Background</p><p>HIV infection occurs in 30% of children with severe acute malnutrition in sub-Saharan Africa. Effects of HIV on the pathophysiology and recovery from malnutrition are poorly understood.</p><p>Methods</p><p>We conducted a prospective cohort study of 75 severely malnourished Ugandan children. HIV status/CD4 counts were assessed at baseline; auxologic data and blood samples were obtained at admission and after 14 days of inpatient treatment. We utilized metabolomic profiling to characterize effects of HIV infection on metabolic status and subsequent responses to nutritional therapy.</p><p>Findings</p><p>At admission, patients (mean age 16.3 mo) had growth failure (mean W/H z-score −4.27 in non-edematous patients) that improved with formula feeding (mean increase 1.00). 24% (18/75) were HIV-infected. Nine children died within the first 14 days of hospitalization; mortality was higher for HIV-infected patients (33% v. 5%, OR = 8.83). HIV-infected and HIV-negative children presented with elevated NEFA, ketones, and even-numbered acylcarnitines and reductions in albumin and amino acids. Leptin, adiponectin, insulin, and IGF-1 levels were low while growth hormone, cortisol, and ghrelin levels were high. At baseline, HIV-infected patients had higher triglycerides, ketones, and even-chain acylcarnitines and lower leptin and adiponectin levels than HIV-negative patients. Leptin levels rose in all patients following nutritional intervention, but adiponectin levels remained depressed in HIV-infected children. Baseline hypoleptinemia and hypoadiponectinemia were associated with increased mortality.</p><p>Conclusions</p><p>Our findings suggest a critical interplay between HIV infection and adipose tissue storage and function in the adaptation to malnutrition. Hypoleptinemia and hypoadiponectinemia may contribute to high mortality rates among malnourished, HIV-infected children.</p></div

SRC-2 KO mice have no impairment in cardiac function in unstressed conditions.

<p><i>A</i>, Total heart weight normalized to tibia length for WT and SRC-2 KO mice (n = 5). <i>B</i>, Diastolic left ventricle wall thickness measured by Echocardiography in WT and SRC-2 KO mice (WT n = 15, KO n = 12). <i>C</i>, Cardiac Doppler measurements of blood flow rates (peak velocity, mean acceleration, and fractional shortening) and diastolic left ventricle interior diameter measured by Echocardiography in WT and SRC-2 KO mice (WT n = 15, KO n = 12).</p