Skeletal Muscle Energetics and Mitochondrial Function Are Impaired Following 10 Days of Bed Rest in Older Adults.

BACKGROUND: Older adults exposed to periods of inactivity during hospitalization, illness, or injury lose muscle mass and strength. This, in turn, predisposes poor recovery of physical function upon reambulation and represents a significant health risk for older adults. Bed rest (BR) results in altered skeletal muscle fuel metabolism and loss of oxidative capacity that have recently been linked to the muscle atrophy program. Our primary objective was to explore the effects of BR on mitochondrial energetics in muscle from older adults. A secondary objective was to examine the effect of ß-hydroxy-ß-methylbuturate (HMB) supplementation on mitochondrial energetics. METHODS: We studied 20 older adults before and after a 10-day BR intervention, who consumed a complete oral nutritional supplement (ONS) with HMB (3.0 g/d HMB, n = 11) or without HMB (CON, n = 9). Percutaneous biopsies of the vastus lateralis were obtained to determine mitochondrial respiration and H2O2 emission in permeabilized muscle fibers along with markers of content. RNA sequencing and lipidomics analyses were also conducted. RESULTS: We found a significant up-regulation of collagen synthesis and down-regulation of ribosome, oxidative metabolism and mitochondrial gene transcripts following BR in the CON group. Alterations to these gene transcripts were significantly blunted in the HMB group. Mitochondrial respiration and markers of content were both reduced and H2O2 emission was elevated in both groups following BR. CONCLUSIONS: In summary, 10 days of BR in older adults causes a significant deterioration in mitochondrial energetics, while transcriptomic profiling revealed that some of these negative effects may be attenuated by an ONS containing HMB.

Characterization of Islet Leukocyte Populations in Human and Murine Islets by Flow Cytometry.

An increasing body of evidence indicates that a local islet immune response is not only limited to type 1 diabetes, but also is associated with islet dysfunction in type 2 diabetes. Recently, the presence of pancreatic CD68+ macrophages within islet tissues was demonstrated by RT-PCR and immunohistochemical methods. However, the precise profile and activation status of intraislet leukocytes, which are present in both murine and human islets, are poorly defined. Here, we describe a detailed flow cytometry protocol designed to analyze both human and murine islets for intraislet leukocytes and leukocyte subsets. This approach permits the simultaneous identification of multiple intraislet leukocyte subsets, as well as their activation statuses. The use of flow cytometry-based approaches will advance the field of islet biology and help to identify unique changes in the immune cell composition that accompanies pathological islet inflammation and dysfunction in type 2 diabetes.

Loss of mitochondrial energetics is associated with poor recovery of muscle function but not mass following disuse atrophy.

Skeletal muscle atrophy is a clinically important outcome of disuse because of injury, immobilization, or bed rest. Disuse atrophy is accompanied by mitochondrial dysfunction, which likely contributes to activation of the muscle atrophy program. However, the linkage of muscle mass and mitochondrial energetics during disuse atrophy and its recovery is incompletely understood. Transcriptomic analysis of muscle biopsies from healthy older adults subject to complete bed rest revealed marked inhibition of mitochondrial energy metabolic pathways. To determine the temporal sequence of muscle atrophy and changes in intramyocellular lipid and mitochondrial energetics, we conducted a time course of hind limb unloading-induced atrophy in adult mice. Mitochondrial respiration and calcium retention capacity were diminished, whereas H2O2 emission was increased within 3 days of unloading before significant muscle atrophy. These changes were associated with a decrease in total cardiolipin and profound changes in remodeled cardiolipin species. Hind limb unloading performed in muscle-specific peroxisome proliferator-activated receptor-γ coactivator-1α/ß knockout mice, a model of mitochondrial dysfunction, did not affect muscle atrophy but impacted muscle function. These data suggest early mitochondrial remodeling affects muscle function but not mass during disuse atrophy. Early alterations in mitochondrial energetics and lipid remodeling may represent novel targets to prevent muscle functional impairment caused by disuse and to enhance recovery from periods of muscle atrophy.

Impaired Mitochondrial Energetics Characterize Poor Early Recovery of Muscle Mass Following Hind Limb Unloading in Old Mice.

The progression of age-related sarcopenia can be accelerated by impaired recovery of muscle mass following periods of disuse due to illness or immobilization. However, the mechanisms underlying poor recovery of aged muscle following disuse remain to be delineated. Recent evidence suggests that mitochondrial energetics play an important role in regulation of muscle mass. Here, we report that 22- to 24-month-old mice with low muscle mass and low glucose clearance rate also display poor early recovery of muscle mass following 10 days of hind limb unloading. We used unbiased and targeted approaches to identify changes in energy metabolism gene expression, metabolite pools and mitochondrial phenotype, and show for the first time that persistent mitochondrial dysfunction, dysregulated fatty acid ß-oxidation, and elevated H2O2 emission occur concomitantly with poor early recovery of muscle mass following a period of disuse in old mice. Importantly, this is linked to more severe whole-body insulin resistance, as determined by insulin tolerance test. The findings suggest that muscle fuel metabolism and mitochondrial energetics could be a focus for mining therapeutic targets to improve recovery of muscle mass following periods of disuse in older animals.

Host Factors Alter Effects of Angiopoietin-Like Protein 8 on Glucose Homeostasis in Diabetic Mice.

Recovery of functional beta cell mass offers a biological cure for type 1 diabetes. However, beta cell mass is difficult to regain once lost since the proliferation rate of beta cells after youth is very low. Angiopoietin like-protein 8 (ANGPTL8), a peptide that has a role in the regulation of lipoprotein lipase activity, was reported to increase beta cell proliferation in mice in 2013. Subsequent studies of human ANGPTL8 for short term (3 to 8 days) in non-diabetic mice showed little or no increase in beta cell proliferation. Here, we examined the effect of ANGPTL8 on glucose homeostasis in models that have not been examined previously. We expressed mouse ANGPTL8 using adenovirus in 2 mouse models of diabetes (streptozotocin and Non-Obese Diabetic (NOD) mice) over 2 weeks. Also, we tested ANGPTL8 in NOD mice deficient in leukocyte 12-lipoxygenase (12LO), an enzyme that contributes to insulitis and loss of beta cell function in NOD, in an effort to determine whether 12LO deficiency alters the response to ANGPTL8. Adenovirus-mediated expression of ANGPTL8 lowered blood glucose levels in streptozotocin treated mice without an increase in beta cell proliferation or serum insulin concentration. While ANGPTL8 did not reverse hyperglycemia in overtly hyperglycemic NOD mice or alter glucose homeostasis of non-diabetic NOD mice, ANGPTL8 reduced blood glucose levels in 12LOKO NOD mice. However, the lower glucose levels in 12LOKO NOD were not associated with higher serum insulin levels or beta cell proliferation. In summary, while mouse ANGPTL8 does not increase beta cell proliferation in NOD mice or streptozotocin treated mice in agreement with studies in non-diabetic mice, it lowers blood glucose levels in multiple low-dose streptozotocin induced diabetes and 12LO deficiency indicating that host factors influence the impact of ANGPTL8 on glucose homeostasis.

Liver Perilipin 5 Expression Worsens Hepatosteatosis But Not Insulin Resistance in High Fat-Fed Mice.

Perilipin 5 (PLIN5) is a lipid droplet (LD) protein highly expressed in oxidative tissues, including the fasted liver. However, its expression also increases in nonalcoholic fatty liver. To determine whether PLIN5 regulates metabolic phenotypes of hepatosteatosis under nutritional excess, liver targeted overexpression of PLIN5 was achieved using adenoviral vector (Ad-PLIN5) in male C57BL/6J mice fed high-fat diet. Mice treated with adenovirus expressing green fluorescent protein (GFP) (Ad-GFP) served as control. Ad-PLIN5 livers increased LD in the liver section, and liquid chromatography with tandem mass spectrometry revealed increases in lipid classes associated with LD, including triacylglycerol, cholesterol ester, and phospholipid classes, compared with Ad-GFP liver. Lipids commonly associated with hepatic lipotoxicity, diacylglycerol, and ceramides, were also increased in Ad-PLIN5 liver. The expression of genes in lipid metabolism regulated by peroxisome proliferator-activated receptor-α was reduced suggestive of slower mobilization of stored lipids in Ad-PLIN5 mice. However, the increase of hepatosteatosis by PLIN5 overexpression did not worsen glucose homeostasis. Rather, serum insulin levels were decreased, indicating better insulin sensitivity in Ad-PLIN5 mice. Moreover, genes associated with liver injury were unaltered in Ad-PLIN5 steatotic liver compared with Ad-GFP control. Phosphorylation of protein kinase B was increased in Ad-PLIN5-transduced AML12 hepatocyte despite of the promotion of fatty acid incorporation to triacylglycerol as well. Collectively, our data indicates that the increase in liver PLIN5 during hepatosteatosis drives further lipid accumulation but does not adversely affect hepatic health or insulin sensitivity.

Perilipin 5 regulates islet lipid metabolism and insulin secretion in a cAMP-dependent manner: implication of its role in the postprandial insulin secretion.

Elevation of circulating fatty acids (FA) during fasting supports postprandial (PP) insulin secretion that is critical for glucose homeostasis and is impaired in diabetes. We tested our hypothesis that lipid droplet (LD) protein perilipin 5 (PLIN5) in ß-cells aids PP insulin secretion by regulating intracellular lipid metabolism. We demonstrated that PLIN5 serves as an LD protein in human islets. In vivo, Plin5 and triglycerides were increased by fasting in mouse islets. MIN6 cells expressing PLIN5 (adenovirus [Ad]-PLIN5) and those expressing perilipin 2 (PLIN2) (Ad-PLIN2) had higher [(3)H]FA incorporation into triglycerides than Ad-GFP control, which support their roles as LD proteins. However, Ad-PLIN5 cells had higher lipolysis than Ad-PLIN2 cells, which increased further by 8-Br-cAMP, indicating that PLIN5 facilitates FA mobilization upon cAMP stimulation as seen postprandially. Ad-PLIN5 in islets enhanced the augmentation of glucose-stimulated insulin secretion by FA and 8-Br-cAMP in G-protein-coupled receptor 40 (GPR40)- and cAMP-activated protein kinase-dependent manners, respectively. When PLIN5 was increased in mouse ß-cells in vivo, glucose tolerance after an acute exenatide challenge was improved. Therefore, the elevation of islet PLIN5 during fasting allows partitioning of FA into LD that is released upon refeeding to support PP insulin secretion in cAMP- and GPR40-dependent manners.

Pancreatic islet neuropeptide Y overexpression has minimal effect on islet morphology and ß-cell adaptation to high-fat diet.

Neuropeptide Y (NPY) is highly expressed in the hypothalamus, where it regulates feeding and energy homeostasis. Interestingly, NPY and its receptors are also expressed in peripheral tissues with roles in metabolism, including pancreatic islets. In islets, NPY is known to suppress insulin secretion acutely. In addition, the role of NPY in ß-cell de-differentiation has been postulated recently. Therefore, we studied transgenic mice expressing NPY under rat insulin promoter (TG) to determine the effects of chronic up-regulation of NPY on islet morphology and function. NPY levels were 25 times higher in islets of TG mice compared with wild-type (WT) littermates, whereas no differences in NPY expression were noted in the brains of TG and WT mice. Islet NPY secretion was 2.3-fold higher in TG compared with WT mice. There were no significant changes in body weight, glucose tolerance, or insulin sensitivity in TG mice fed regular rodent diet or high-fat diet (HF). Islet ß-cell area was comparable between TG and WT mice both on regular rodent and HF diets, indicating that NPY overexpression is insufficient to alter ß-cell maturation or the compensatory increase of ß-cell area on HF. One abnormality noted was that the glucose-stimulated insulin secretion in islets isolated from TG was reduced compared with those from WT mice on HF diet. Overall, an increase in islet NPY level has little impact on islet function and is insufficient to affect glucose homeostasis in mice.

Understanding factors influencing Latina women's screening behavior: a qualitative approach.

The purpose of this qualitative study was to understand influential factors associated with decisions to obtain breast or cervical cancer screening by diverse Latinas after attending a community-based educational program. Forty-five interviews were conducted in Arkansas, New York City and Buffalo, New York. Thematic data analyses were conducted to understand influential factors following from the intervention. Four major themes emerged from the interviews: Social Capital, Screening Utilization, Health Care Provider (HCP) Communication and Social Networks. Social Capital included resources, access or screening knowledge women had prior to participation in the program and new resources and contacts gained through the program that influenced care seeking. Screening Utilization factors included past health experiences and participation in the program. HCP Communication included perceptions of quality of care and communication issues with HCPs that positively and negatively impacted screening. Social Networks included women's networks regarding emotional support, encouragement to discuss health issues, overall family network and how these may influence health-seeking behaviors. These findings suggest that participating in group-based health outreach programs empowered women by increasing their knowledge and awareness about the health care system and enabled them, with the tools provided, to become proactive in their health care-seeking behaviors.

Cancer screening behaviors among Latina women: the role of the Latino male.

The purpose of this article is to determine, through a community-based breast and cervical cancer intervention program, the impact Latino males may have on Latinas and their cancer screening behaviors. This report includes data collected from 163 Latino males recruited throughout rural Arkansas and four New York City boroughs for the Esperanza y Vida program, designed to evaluate cancer screening outcomes among Latinas and address their health care needs and cancer control challenges. Basic demographics and identical pre- and post-program knowledge surveys were collected and analyzed using SPSS 15.0 and SAS 9.2. Results from this study suggest Latino men have little knowledge about breast or cervical cancer screening and are unfamiliar with their partners' screening histories. Male participants were also less likely to complete program assessment forms (pre, post, demographic questionnaires) and more likely to commit response errors (i.e. multiple answers, illegible responses). These findings suggest that including males in education programs for Latinas may be a crucial component in decreasing cancers among this segment of the population. The further development of programs such as Esperanza y Vida, that empowers Latino males, will be important in reducing the unequal burden of breast and cervical cancers for Latinas. It is important to continue including Latino men in these types of studies because the impact of their role on Latina's health remains understudied, unknown, and misunderstood.

Esperanza y Vida: a culturally and linguistically customized breast and cervical education program for diverse Latinas at three different United States sites.

Breast cancer is the most common cause of cancer and the leading cause of cancer death among Latinas in the United States. In addition, Latinas experience a disproportionate burden of cervical cancer incidence, morbidity, and mortality compared with non-Hispanic White women. Lower use of breast and cervical cancer screening services may contribute to these disparities. To address the underutilization of breast and cervical cancer screening among diverse subgroups of Latinas, a peer-led education program called Esperanza y Vida ("Hope and Life") was developed and administered at 3 sites (2 in New York and 1 in Arkansas). Immigrant Latina women and their partners were educated about the importance of breast and cervical cancer screening, with the goals of increasing their knowledge about these cancers and their screening behavior. An analysis of the intervention's findings at baseline among female participants demonstrated significant sociodemographic, interpersonal, cultural, health care system, and program variability in 3 distinct geographic regions in the United States. These data indicate the need for and feasibility of customizing cancer outreach and educational programs for diverse Latina subgroups living in various U.S. regions, with implications for informing the expansion and replication of the program in other regions of the country.

Esperanza y Vida: training lay health advisors and cancer survivors to promote breast and cervical cancer screening in Latinas.

The use of lay health advisors (LHAs) to promote community-based health education programs is well documented and is considered an effective way to reach underserved communities. Esperanza y Vida (Hope & Life) is an educational outreach program to increase breast and cervical cancer screening for diverse Latinas. It incorporates Latino LHAs (men and women) and cancer survivor role models, sobrevivientes, in the program delivery. An interactive training program, conducted by bilingual staff across three sites (Little Rock, Arkansas; Buffalo, New York and New York City) included 74 sobrevivientes and LHAs who were recruited and trained. All training attendees completed an initial application assessing socio-demographics, experience and availability as well as, true/false surveys at the beginning (pre-) and end of the training (post-) measuring knowledge levels of breast and cervical cancer health. Data analysis indicated a significant increase of both breast and cervical cancer knowledge for attendees trained as LHAs (pre = 60%; post = 80%; p = 0.000), whereas sobrevivientes had a higher baseline knowledge of breast health (74%), and therefore did not show a significant increase following training (79%). However, sobrevivientes did display a significant increase in cervical cancer knowledge (p = 0.003). These findings demonstrate the impact of training and how LHAs may be recruited at different levels of knowledge and experience and be successfully trained in key program elements. Moreover, results indicate that sobrevivientes may be impacted differently, or require variations in training approaches. This information can be useful in developing and customizing curriculum for future lay health training programs.

Contextualizing diversity and culture within cancer control interventions for Latinas: changing interventions, not cultures.

While there is a growing interest in the development of cancer control intervention initiatives, there continues to be a need to understand how the nuances of different Latino cultures translate to opportunities and barriers for access to cancer screening and care. The diversity by country of origin for Latinas in the United States is often overlooked in cancer control initiatives, and the application of qualitative research can expose processes of inequity and cultural variation to improve these initiatives. This paper presents an interpretation of diverse Latina immigrants' perceptions, experiences and knowledge about breast and cervical cancer screening and demonstrates the use of the PEN-3 model to analyze these data to develop an effective outreach intervention. We conducted 13 focus groups consisting of a total of 112 Latinas in New York City (nine groups) and rural and urban sites in Arkansas (four groups) in 2003 through 2004. Through nonprobability theoretical sampling, we included women from Puerto Rico, the Dominican Republic and Mexico in New York and recent Mexican immigrants in Arkansas. Findings demonstrated that country of origin and current geographic residency in the U.S. were significant determinants of women's perspectives on community-based religious organizations, knowledge of anatomy, experiences with the medical system, and access to services which are essential factors to consider in developing effective cancer control interventions. Although breast and cervical cancer are considered women's health issues, they cannot be addressed outside the sociopolitical structures of local communities, especially for the most recent immigrant women. Applying the PEN-3 framework to these data demonstrated a valuable method to interpret and transform qualitative data into intervention content and structure that responds to characteristics and perspectives within diverse Latino communities, such as gender relations, religious affiliations and experiences.

dcDegenerate oligonucleotide primed-PCR for multilocus, genome-wide analysis from limited quantities of DNA.

This study modified the degenerate oligonucleotide primed-polymerase chain reaction (DOP-PCR)-based whole genome amplification method for improvement of downstream genome-wide analysis of low copy number DNA samples ( 0.50 intralocus heterozygote peak ratios were observed for most DNA input quantities examined. These results show that modifications of the traditional DOP-PCR reaction (dcDOP-PCR) to include the use of a more degenerate primer (10 N), 12 nonspecific cycles, and a proofreading enzyme allows for a more complete, balanced chromosome amplification from limited and/or compromised clinical and biological samples.

Breast and cervical cancer screening among Latinas attending culturally specific educational programs.

BACKGROUND: Latinas in the United States have higher morbidity and mortality rates for breast and cervical cancers (compared with non-Latina Whites), often due to lower screening rates. A community-based participatory research (CBPR) approach could help to improve screening rates by creating a culturally customized educational program for Latino men and women addressing low knowledge, gender roles, and spirituality. OBJECTIVES: This study was designed to assess the effectiveness of a culturally customized program (Esperanza y Vida [Hope and Life]) in increasing breast and cervical cancer screening among Latinas, and to examine how screening rates related to changes in cancer knowledge, differences in ethnic origins, and geographic location. METHODS: Participants were recruited to attend either a breast and cervical (intervention) or diabetes (control) education program, within a randomized plan. Sixty-nine programs (44 intervention; 25 control) were conducted in Arkansas (AR; n = 39) and New York City (NYC; n = 30) with a total of 847 Latino men and women. Telephone follow-up data were collected on 49% of the women who consented to being contacted 2 months postintervention. RESULTS: At the 2-month follow-up call, screening rates were significantly higher for the intervention versus the control group for clinical breast examination (CBE; 48% vs. 31%; adjusted odds ratio [aOR], 2.2; 95% confidence interval [CI], 1.1-4.2), breast self-examination (45% vs. 27%; aOR, 2.3; 95% CI, 1.1-5.0), and Pap testing (51% vs. 30%; aOR, 3.9; 95% CI, 1.1-14.1), but not for mammography (67% vs. 58%; aOR, 0.7; 95% CI, 0.1-3.6). The aORs accounted for the significant effects of study site (AR vs. NYC) and marital status. CONCLUSIONS: Esperanza y Vida has the potential to reduce health disparities in breast and cervical cancer morbidity and mortality rates through increasing cancer screening and thereby increasing early detection.

A comparison of African American and Latina social networks as indicators for culturally tailoring a breast and cervical cancer education intervention.

As similar cancer health disparities have been documented for African American (AA) women and Latinas, it would be important to determine whether comparable interventions could be used to increase screening among these 2 culturally different populations. This paper reports research findings comparing cultural dimensions of breast and cervical cancer as they impact Latino and AA social networks and explore the feasibility of creating outreach models that may serve both groups. An existing intervention that integrates the social roles and relationships of AA women, The Witness Project(R), is used as a framework for tailoring an intervention for Latino communities. Findings and data from focus groups and key informant interviews were collected from more than 120 Latinos in Arkansas and New York City. These findings are analyzed using the Pen-3 Model, categorized, and compared with previous social role and network information from AA women as reflected in the Witness Project(R) intervention model. The findings from this study demonstrated variations between AA women and Latinas with regard to roles and gender relationships while demonstrating similarities with regard to spiritual beliefs and attitudes toward cancer. We applied our results to culturally tailor and develop a breast and cervical cancer intervention, Esperanza y Vidatrade mark (Hope and Life), that incorporates Latino values and social relationships. This study demonstrates that a proven education and outreach model for AA women may provide a framework for creating a culturally appropriate intervention for Latinas. Further research is needed to study the efficacy of the new model. Cancer 2007. (c) 2006 American Cancer Society.