abstract: This is the third in a series of articles, invited by the editors of *Diabetes*, that describes the research programs and aims of organizations committed to funding and fostering diabetes-related research. The first piece, contributed by the Juvenile Diabetes Research Foundation, appeared in the January 2012 issue of *Diabetes*. The second piece that describes the American Diabetes Association's research program appeared in the June 2012 issues of *Diabetes* and *Diabetes Care*.
author: Judith E. Fradkin; Griffin P. RodgersCorresponding author: Judith Fradkin, <email@example.com>.
date: 2013-02
references:
title: Diabetes Research: A Perspective From the National Institute of Diabetes and Digestive and Kidney Diseases

The growing human and economic toll of diabetes has caused consternation worldwide. Not only is the number of people affected increasing at an alarming rate, but onset of the major forms of the disease occurs at ever younger ages. We now know that the reach of diabetes extends far beyond the classic acute metabolic and chronic vascular complications to increased risk of an ever-increasing array of conditions including Alzheimer disease, cancer, liver failure, bone fractures, depression, and hearing loss. In the U.S. one in three Medicare dollars is spent on care of people with diabetes, and the proportion of cardiovascular disease (CVD) risk attributable to diabetes is rising. While complications of diabetes may develop slowly over decades, antecedents of diabetes may lay in utero or early life. Thus the breadth of meaningful research extends across the life span, ranging from studies of how the in utero environment alters diabetes risk to improved understanding of the special needs of older patients with diabetes.

Since the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) was established in 1950, we have seen huge progress in our ability to predict, classify, and treat diabetes and its complications as well as to prevent or delay type 2 diabetes. Landmark NIDDK-led clinical trials have demonstrated that glucose control can dramatically reduce diabetes complications, and lifestyle change producing modest weight loss, or the drug metformin, can substantially reduce development of type 2 diabetes. Were it not for this progress, the toll from rising rates of the major forms of diabetes would be much higher.

Despite a challenging fiscal climate, the National Institutes of Health (NIH) expends over \$1 billion on diabetes research annually. NIDDK accounts for about two-thirds of this total, and we are determined that these resources will be invested wisely and balanced among competing priorities. We must address the most compelling practical questions about clinical management and prevention of diabetes and its complications while also uncovering and exploiting novel pathways that will provide new targets and approaches to combat the disorder. Last year, a new strategic plan for diabetes research (1) was issued under NIDDK's leadership with input from over 100 scientists and multiple federal agencies and components of NIH. The plan highlights progress and opportunities in 10 key areas as well as resource and infrastructure needs. Conquering diabetes will require a wide range of expertise and talent from molecular and cell biology to behavioral and social sciences. Development and empowerment of this human capital is critical to this endeavor, including facilitating multidisciplinary collaborations and the application of new technologies to diabetes research. Here we will touch on highlights of our diabetes research priorities and initiatives, referring readers to the Diabetes Research Strategic Plan (1) for a more comprehensive analysis of advances and opportunities.

# ENHANCING THE DIABETES RESEARCH WORKFORCE

A well-trained and diverse scientific workforce is essential to our efforts to improve outcomes for people with or at risk for diabetes. To ensure a pipeline of new well-trained investigators in basic and clinical disciplines, NIDDK supports training grant, fellowship, and career award mechanisms to provide opportunities for investigators at all stages of the career trajectory. These are supplemented by programs targeted to specific needs, such as our medical student research program in diabetes, which allows medical students to conduct research under the direction of an established scientist at one of our seventeen NIDDK-funded Diabetes Research Centers; supplements to research and training grants to foster recruitment of underrepresented minority scientists to diabetes research; and institutional career development programs to attract pediatric endocrinologists to careers in childhood diabetes research. It is increasingly important to build multidisciplinary research teams and train multidisciplinary researchers. We are establishing interdisciplinary training grants to promote diabetes research training for bioengineers as well as career development programs in diabetes research for behavioral scientists. We will continue to foster the application of new expertise, for example in computational science and bioinformatics, to diabetes research problems. To help new investigators transition to independence, we also provide a less stringent pay line for early career investigators. We also invite new investigators with NIDDK research or career development grants to participate in NIDDK workshops designed to help them succeed as independent investigators.

# FUNDAMENTAL RESEARCH

To uncover new approaches to prevention and therapy of diabetes and its devastating complications, NIDDK will continue to support a robust portfolio of investigator-initiated basic research. NIDDK has recently developed data on application and funding trends to help our research community understand the application and funding dynamics over recent years. This information is available at <http://www2.niddk.nih.gov/Funding/Grants/FundingTrendsandValues.htm>. It shows that relative funding levels of most research categories have remained fairly stable since 2003, and demonstrates our continuing strong support of investigator-initiated research project grants or R01s and training and career development programs. Information on resources to empower researchers, such diabetes centers, human islets, mouse models, reagents, and databases, and on funding opportunities and staff to contact in specific program areas is available at <http://www2.niddk.nih.gov/Research/ScientificAreas/Diabetes/>.

# DIABETES PREVENTION

Diabetes prevention is a major public health challenge. For type 2 diabetes, the NIDDK-led Diabetes Prevention Program (DPP) demonstrated a dramatic effect of modest weight loss or the generic drug metformin in delaying or preventing type 2 diabetes (2). Ongoing studies are examining the durability of this risk reduction, the cost effectiveness of the interventions, and their impact in reducing diabetes complications. To facilitate translation of landmark clinical research into clinical practice and public health activities, NIDDK established a program to test practical, cost-effective approaches to deliver interventions proven efficacious in clinical trials for effectiveness in community and practice settings. One such NIDDK-supported study of a lifestyle change intervention delivered by YMCA fitness trainers (3) already is being rolled out nationwide by the YMCA with coverage from insurers such as United Health Group. In another promising approach, diabetes educators trained selected patients with well-controlled diabetes to serve as community health workers delivering a lifestyle intervention based on the DPP to community members with prediabetes (4). This NIDDK-funded research provides a basis for a new congressionally established National Diabetes Prevention Program at the Centers for Disease Control and Prevention (CDC) to foster delivery of evidence-based lifestyle change programs for people at high risk for type 2 diabetes. Given the sustained effort required to achieve and maintain lifestyle change, much additional research is needed to improve, disseminate, and evaluate type 2 diabetes prevention programs in the U.S. Approaches are also needed to reduce the development of risk factors for diabetes. Of particular importance are studies to reduce environmental exposures during pregnancy or childhood that may increase diabetes and obesity risk.

The incidence of type 1 diabetes is rising worldwide and the disease is occurring at younger ages suggesting an environmental trigger is responsible. Bold new programs aimed at preventing type 1 diabetes have been undertaken with support from the Special Statutory Funding Program for Type 1 Diabetes Research, which provides \$150 million per year through 2013 for type 1 diabetes research. These special funds are in addition to the regular NIH appropriation. One program established under the program, The Environmental Determinants of Diabetes in the Young (TEDDY), has screened nearly half a million neonates to establish a cohort of over 8,000 at high genetic risk for type 1 diabetes. Participants will be followed from birth through 15 years of age to identify dietary, infectious, microbiome, or other environmental triggers of autoimmunity and type 1 diabetes and to study the interaction between environmental factors and specific genetic variations associated with disease risk. Identification of an infectious agent or dietary factor that triggers or protects against the disease would have immense implications for prevention through the development of a vaccine or dietary change. Also with special program support, the Type 1 Diabetes TrialNet is identifying individuals recently diagnosed with type 1 diabetes or at high risk of developing the disease and testing interventions to prevent diabetes or to slow its progression. Selective immune modulation has been shown to preserve insulin secretion in newly diagnosed patients, and TrialNet is exploring the use of one such agent, teplizumab, to prevent type 1 diabetes in individuals at very high short-term risk of type 1 diabetes. In the future, combination therapies aimed at modulating multiple steps of the toxic immune response and restoring immunoregulation may produce a clinically significant delay in onset and ultimately the prevention of type 1 diabetes.

# CLINICAL TRIALS TO INFORM DIABETES MANAGEMENT

While information on how to prevent and treat type 2 diabetes has grown rapidly, adequate data from rigorous clinical trials are not available to inform many routine decisions on care for patients with diabetes. Current guidelines are moving away from a "one size fits all" approach to incorporate factors such as diabetes duration and the presence of complications or other comorbidities. However, we lack information to individualize therapy based on demographic, physiologic, or genetic variation. Improved understanding of the genetic, physiologic, and environmental factors that underlie diabetes mellitus are necessary for more individualized diabetes treatment.

Numerous drugs are approved for the treatment of type 2 diabetes, based largely on relatively short-term efficacy in glycemic reduction. However, it is not known whether particular drugs or drug combinations will have more durable effects in the maintenance of glucose control. NIDDK is supporting a large comparative effectiveness trial to inform the choice of second agent when metformin alone is inadequate for glycemic control. This multicenter randomized trial will provide information on health benefits as well as cost effectiveness of widely used treatments.

Small studies suggest it may be possible to preserve β-cell function during prediabetes and early in the course of type 2 diabetes. Major questions include the optimal timing of interventions, whether specific treatments have maximum benefit at different stages of the disease, and what patient characteristics influence the choice of initial therapy for individuals. A newly formed consortium will examine the approaches to the initial treatment of type 2 diabetes that may reverse or slow the decline in β-cell function over time.

While major trials have established the importance of blood pressure and lipid control in reducing CVD in type 2 diabetes, much less is known about how cardiovascular risk factors should be managed in type 1 diabetes. When blood pressure and lipid lowering should begin and optimal therapeutic targets remain to be established. Although type 1 diabetes increases the risk of CVD as much as 10-fold compared to an age-matched population, testing practical approaches to mitigate this risk is challenging due to the low incidence of CVD in the younger type 1 diabetes population. Such trials may require the development of the validated biomarkers discussed below.

Diabetes self-management training and promotion of effective self-care behaviors are vital to improving outcomes. The choices patients make daily about diet, physical activity, adherence to medications, self-monitoring, foot and dental care, and medical follow-up for early detection of complications are critical for improving diabetes outcomes. Research has established effective counseling and education strategies, including motivational interviewing, patient empowerment, and social and peer support. However, research is needed to expand the reach of such approaches to more patients and providers. NIDDK encourages research studying approaches such as group visits, telemedicine, and social media that may extend the impact of the limited workforce skilled in the provision of this care.

# DIABETES IN SPECIAL POPULATIONS

Diabetes spares no age, sex, racial or ethnic group, yet each such group faces special challenges. Intensive glycemic control in youth may afford lifelong protection from complications yet infancy and adolescence pose unique challenges in attaining such control. Treatment priorities and optimal glycemic, blood pressure, and cholesterol targets to prevent complications and maintain quality of life may differ for older adults or those with limited life expectancy. The *Eunice Kennedy Shriver* National Institute of Child Health and Human Development (NICHD)-led Hyperglycemia and Adverse Pregnancy Outcome (HAPO) study (5) showed that perinatal harm to mother and offspring occurs in pregnancy at lower levels of glycemia than previously appreciated. To identify gestational glycemic thresholds for longer-term effects on offspring and the risk of type 2 diabetes in mothers, NIDDK will support a follow-up study of this important cohort. NIDDK has also recently launched a study to explore approaches for the prevention of gestational diabetes mellitus. It also remains to be established what treatments during pregnancy mitigate perinatal and/or long-term complications of gestational diabetes mellitus.

The biologic and environmental risk factors that contribute to underlying racial and ethnic disparities are poorly understood. The alarming emergence of type 2 diabetes in youth overwhelmingly occurs in minority populations. Ominous data on poor risk factor control portends very poor outcomes for this vulnerable population. To address this threat, NIDDK recently completed a major multicenter trial comparing three approaches to the treatment of type 2 diabetes in youth and a trial conducted in schools serving poor and minority children to prevent the development of risk factors for type 2 diabetes.

As better therapy for HIV infection, organ transplantation, cystic fibrosis, and other conditions improve survival, diabetes has emerged as an increasingly important complication. Critical questions must be addressed about optimal strategies to prevent and treat diabetes. For example, early diabetes diagnosis and treatment helped improve survival in people with cystic fibrosis–related diabetes (CFRD). An NIDDK-funded trial showed that chronic weight loss can be reversed with the institution of insulin therapy but not with repaglinide early in the course of CFRD before fasting hyperglycemia develops (6). A current initiative will support research to understand the pathogenesis of CFRD, which affects half of adults with cystic fibrosis. NIDDK will also encourage research on treatment and prevention of diabetes in HIV patients.

Exceptional contributions to understanding diabetes in special populations have emerged from NIDDK's intramural research program. Spanning decades and generations, longitudinal studies of Pima Indians have uncovered physiologic, environmental, and genetic determinants of diabetes in the U.S. population with highest rates of type 2 diabetes. These studies have foreshadowed findings in the broader population, such as the contribution of intrauterine factors to childhood obesity and type 2 diabetes and the prognosis for these youth. Collaborations between intramural and extramural researchers studying individuals with severe insulin resistance and lipodystrophy have yielded important physiologic information and the emergence of leptin replacement therapy for lipodystrophy.

# EPIDEMIOLOGY AND SURVEILLANCE IN DIABETES

Population health data are essential to inform prevention strategies for diabetes and its complications, to identify trends in the development of diabetes and diabetes complications in the general population and in subpopulations, and to measure gaps in the translation of proven therapies into practice. NIDDK has partnered with CDC to support the SEARCH for Diabetes in Youth study. This multicenter study identifies cases of diabetes in people below 20 years of age in five geographically dispersed populations that encompass the ethnic diversity of the U.S.

SEARCH has found that 1 out of every 523 persons under 20 years of age has diabetes, and 15,000 children are diagnosed with type 1 diabetes and 3,700 diagnosed with type 2 diabetes annually (7). SEARCH also provides data on trends in incidence and risk factor control in the pediatric diabetes population.

So that information on diabetes can be obtained at lower cost than if an independent study were initiated, NIDDK provides substantial support for the diabetes components of major CDC-led efforts including the National Health and Nutrition Evaluation Survey and the National Health Information Survey; partners with other components of NIH to enhance diabetes measures in ongoing studies; and offers support for investigator-initiated ancillary studies focused on diabetes. Of particular importance are collaborations with the National Heart, Lung, and Blood Institute (NHLBI) to address the increasing proportion of CVD attributable to diabetes and issues such as balancing the cardiometabolic risks and benefits associated with statin use.

A third edition of the NIDDK publication *Diabetes in America* is currently in preparation. *Diabetes in America* provides a compilation and assessment of epidemiologic, public health, and clinical data on diabetes and its complications in the U.S.

# RESEARCH TO PRACTICE TRANSLATION

There is a substantial gap between the results achieved in clinical trials and the outcomes in real-world settings. This is particularly true for the minority racial and ethnic groups and low socioeconomic status populations that suffer a disproportionate diabetes burden. Addressing this disparity is a major focus of our multipronged translation research program. Newly established Centers for Diabetes Translation Research will serve as a key component of our program to translate efficacious research findings into practice and the community to improve the health of Americans with—or at risk for—diabetes. In addition, R34 planning grants and R18 translational clinical trial grants offer a targeted mechanism to test strategies to improve the delivery of evidence-based therapies to improve diabetes management or prevention. These programs test innovative methods to improve clinical care and translate research findings into cost-effective and sustainable clinical treatment strategies, including community-based approaches to make preventive measures as widely accessible and practical as possible. NIDDK has also partnered with CDC to support the Natural Experiments for Translation in Diabetes (NEXT-D) Study, a research network designed to test the effectiveness and sustainability of population-targeted diabetes prevention and control policies emerging from health care systems, business and community organizations, and health care legislation. It includes large-scale natural experiments or effectiveness studies and rigorously designed prospective studies of diabetes prevention and control interventions. The National Diabetes Education Program, jointly sponsored by NIH and CDC, plays an important role in dissemination and translation of NIDDK-supported clinical research.

# GENETICS OF TYPE 1 AND TYPE 2 DIABETES

Because knowledge of genetic risk factors has the potential for disease prediction, patient stratification, and insights into pathogenesis that can generate new approaches to prevention and treatment, NIDDK has expended considerable resources to apply state-of-the-art molecular and computational science to identify diabetes risk genes. Perhaps the most profound impact of these genetic discoveries has been in children with neonatal diabetes, who were often wrongly diagnosed with type 1 diabetes and treated with insulin. Now children with mutations in the genes encoding the SUR1 and Kir6.2 subunits of the potassium ion channel that regulates insulin secretion are treated more safely and effectively with sulfonylurea drugs rather than insulin (8). Genetic testing for type 1 diabetes risk has made possible the TEDDY study and TrialNet prevention studies described above. The NIDDK-led international Type 1 Diabetes Genetics Consortium helped increase the number of identified risk genes and gene regions from 3 only a decade ago to over 50 today. The challenge now is to understand how this variation contributes to disease pathogenesis opening up new therapeutic targets.

For type 2 diabetes, we are encouraged by the DPP finding that relative risk reduction with lifestyle change was as great in those carrying the high-risk TCF7L2 mutation as in other participants, despite higher rates of progression to diabetes. DPP also provided important pharmacogenetic data showing participants with a specific *KCNJ11* variant or alterations in metformin transport genes were less protected by metformin. These observations offer the possibility of individualizing therapy based on genotype (9). However, despite substantial progress in identifying genetic variation contributing to type 2 diabetes risk in populations of European origin, there is a critical lack of information about genetic variation contributing to type 2 diabetes in disproportionately affected minority populations. NIDDK has established a consortium of investigators to identify genetic variation contributing to type 2 diabetes in minorities. With known risk genes explaining only a small fraction of the genetic risk for type 2 diabetes, the identification of epigenetic changes that may play a role in the transmission of diabetes risk across generations is assuming increasing importance.

# DIABETES COMPLICATIONS

Research has identified many pathways contributing to glucose-induced damage to endothelial cells including elevated flux through polyol and hexosamine pathways, accumulation of advanced glycation end products, activation of proinflammatory pathways, and inactivation of protein kinase C. Yet many questions remain about how these pathways may interact and converge to increase production of reactive oxygen species (ROS) in the mitochondria and how we can build on this new knowledge to develop therapies that can reduce ROS, reverse glycation, and lessen inflammation. Pathogenetic mechanisms, therapeutic targets, and biomarkers must be identified in specific tissues, including mechanisms of injury to specialized cells such as podocytes and pericytes. The relative importance of hyperglycemia and insulin resistance is of particular interest in understanding the link between diabetes and Alzheimer disease.

A finite period of glycemic control has profound and long-lasting effects on the development of complications, a phenomenon termed metabolic memory (10). It has been proposed that the interaction of epigenetic changes with other persistent effects of hyperglycemia, such as glycation and oxidation of long-lived macromolecules, may explain this finding. Understanding pathways that contribute to metabolic memory may yield therapies targeted at the underlying molecular mechanisms. It may also help us learn about whether treatments that prevent the development of complications also prevent progression.

The course and development of long-term complications cannot be solely explained by the extent and duration of exposure to hyperglycemia. Genetic variation may explain why some people develop complications despite good control and others with poor control are protected. This information could yield undiscovered disease pathways and therapeutic targets, improve disease prediction over currently available clinical markers, and identify individuals for whom intensive therapy would be more or less beneficial. However, we know relatively little about genetic variation that may contribute to or protect from complications, and this is an important area for investigation.

We are also elucidating mechanisms that impair tissue repair and regeneration in diabetes, including dysfunction of endothelial and other stem cell populations. Identification of specific populations of stem or progenitor cells affected by diabetes and the extent to which damage is reversible is vital for understanding how complications of diabetes might be reversed by stimulating formation of normal new vessels and regrowth of nerves. Differentiation of induced pluripotent stem cells also holds promise for the repair of damaged tissues.

While rates of blindness, kidney failure, amputation, and CVD have fallen substantially in those with diabetes, population-wide lowering of CVD has outpaced that in people with diabetes and the share of CVD in the U.S. attributable to diabetes is rising. To address this challenge, NIDDK and NHLBI have collaborated in a number of major multicenter trials to establish effective approaches to reducing CVD in those with type 2 diabetes, including Action for Health in Diabetes (Look AHEAD), Action to Control Cardiovascular Risk in Diabetes (ACCORD), and Bypass Angioplasty Revascularization Investigation in Type 2 Diabetes (BARI 2D). Results from ACCORD and other recent large clinical trials attempting to prevent CVD did not demonstrate a benefit of intensified near-normal glucose control on clinical CVD events in people with moderate to long-term diabetes duration and moderate to high CVD risk. More information is needed on the impact of diabetes duration and preexisting tissue damage on the ability to respond to therapies. Beyond the practical management questions addressed in clinical trials, new approaches to uncouple diabetes and CVD must be based on a mechanistic understanding of factors linking these conditions, including obesity, inflammation, insulin resistance, metabolic perturbations, altered coagulation, neuropathy, and nephropathy, and how the pathophysiology of atherosclerosis differs between people with type 1 and type 2 diabetes.

# THE β-CELL

Impaired insulin production is key to all forms of diabetes. The extent to which it is possible to preserve and/or restore β-cell function early in the course of diabetes and whether β-cell recovery is possible later in the disease remains to be established. Both the nature and optimal timing of interventions to preserve β-cell function and the impact on clinical care and outcomes must be addressed. NIDDK has recently established the Restore Insulin SEcretion (RISE) consortium to explore approaches to remission of insulin secretory function early in type 2 diabetes. Investigators will study both pharmacologic interventions and bariatric surgery. The Type 1 Diabetes TrialNet studies approaches to slow β-cell loss early in the course of type 1 diabetes. Specific immunomodulatory therapies have been shown to preserve C-peptide, and additional strategies including intense metabolic control with continuous glucose monitoring and pump therapy at onset of disease are being investigated.

Mechanistic studies are necessary to understand why β-cells lose their ability to secrete insulin as well as the physiology underlying recovery and preservation of endogenous insulin secretion. Particularly encouraging is the finding of some residual C-peptide production in a substantial proportion of patients with long-established type 1 diabetes. Such individuals might be amenable to novel strategies under development to stimulate islet neogenesis with the potential to improve β-cell mass and function. The intriguing observation of diabetes remission after some forms of bariatric surgery must be investigated to establish durability of the effect and the characteristics of patients and procedures associated with remission. Elucidation of mechanisms underlying improved β-cell function after bariatric surgery is being pursued in both human studies and animal models potentially generating new approaches to restore β-cell function. Moreover, the recent identification of platelet-derived growth factor as a factor involved in the replication of β-cells in early life that are lost over time offers a potential pathway to β-cell regeneration (11).

Clinical studies of approaches to mitigate β-cell loss and/or restore function could be accomplished much more efficiently if more reliable biomarkers or methods to image β-cell mass or function were available. The development of such tools has been and continues to be a high priority of NIDDK. It may be facilitated by the identification of proteins expressed in β-cells and β-cell surface markers and antibodies through the Beta Cell Biology Consortium (<http://www.betacell.org>). This consortium is pursuing a multifaceted approach to correct the loss of β-cell mass in diabetes, including cell reprogramming, regeneration, and replacement. It is also supporting research to develop mouse models in which development and function of human islets can be studied. Also, because of differences between murine and human islets, NIDDK has established a resource, the Integrated Islet Distribution Program (<http://iidp.coh.org>), to make human cadaveric islets available to the research community.

The Clinical Islet Transplantation Consortium, a joint effort of NIDDK and the National Institute of Allergy and Infectious Diseases (NIAID), is fostering development of islet transplantation as a cure for type 1 diabetic patients whose disease cannot be effectively managed with current methods of insulin administration or who have received a successful kidney transplant. Its ongoing trials aim to improve the methods of isolating and administering islets and minimizing the toxic effects of immunosuppressive drugs required for transplantation. NIDDK also supports collection, analysis, and communication of comprehensive and current data on all islet/β-cell transplants performed in North America, as well as some European and Australian centers through the Collaborative Islet Transplant Registry. This clinical islet transplantation research is entirely supported through the special appropriation for type 1 diabetes research.

# OBESITY

A trans-NIH Task Force cochaired by NIDDK, NHLBI, and NICHD coordinates NIH efforts to identify genetic, behavioral, and environmental causes of obesity to understand how obesity leads to type 2 diabetes, CVD, and other serious health problems and to build on basic and clinical research findings to develop and study innovative prevention and treatment strategies. One key goal is to understand how biologic, cognitive, behavioral, social, and physical environmental factors interact to influence the development of obesity. For example, how do diet, exercise, and other factors influence reprogramming of neural circuits involved in regulating food intake and thermogenesis. Another goal is to identify distinct strategies that may be needed for achieving weight loss, maintaining weight loss, and preventing weight gain. Understanding responses to weight change that contribute to the very high recidivism to obesity may lead to effective strategies for maintenance of reduced body weight. These therapies might be quite different from those used to induce weight loss per se.

Childhood obesity has fueled the rise of type 2 diabetes in teens and young adults. An NIDDK-led randomized trial testing an intervention to improve nutrition and physical activity in middle schools serving high-risk children demonstrated efficacy in secondary outcomes, reducing BMI *z*-score and other indices of adiposity. However, the primary outcome of the combined prevalence of overweight and obesity decreased in both the intervention and control schools perhaps due to information about the participating children's health, which was sent to all families (12). Family-based interventions have been successful for childhood weight control, but strategies for translation and widespread implementation of such interventions in high-risk populations remain to be developed. The roles of technologies such as smartphones and social networking and of community organizations must be evaluated for their potential to support individualized and tailored delivery of interventions outside of the clinical setting.

At the molecular level, there has been an explosion of knowledge about the mechanisms linking obesity to insulin resistance and excitement about potential therapeutic manipulation of adipose tissues based on the understanding of white adipose tissue heterogeneity, persistence of brown adipose tissue into adulthood, and metabolic flexibility of adipocytes. Tools and techniques have enabled researchers not only to define adipose anatomy and morphology but also to examine its dynamic function. Yet key questions remain about the mechanisms that determine adipocyte number and size, govern development and distribution, and link variation in body fat deposition to metabolic sequelae and macrophage recruitment and activation.

Particularly challenging but of utmost importance are studies to understand mechanisms by which obesity, hyperglycemia, or other metabolic factors in pregnant women may predispose their offspring to obesity or diabetes. Research is needed on how placental biology and the intrauterine environment shape neural circuits, adipose tissue, and islet development in the fetus. Studies relating differences in energy homeostasis and body composition to genetic variation and defining the critical developmental periods for imprinting maladaptive metabolic changes will contribute to understanding how metabolic fate may be programmed early in development.

The finding that Roux-en-Y gastric bypass not only causes profound weight loss but can also restore euglycemia through mechanisms that appear independent of weight loss makes identification of these mechanisms a very high priority with the potential to uncover new therapeutic pathways to treat and possibly reverse type 2 diabetes. NIDDK is pursuing this through clinical research on the response to various bariatric surgical procedures and through murine studies in which examination of defined procedures in genetically altered mice enables examination of the roles of specific pathways in the metabolic outcomes of these surgeries. Understanding the hormonal and neural controllers of energy balance will be key to designing potential drug combinations targeting multiple components of the regulatory system with additive or synergistic effects.

# DIABETES RESOURCES

NIDDK supports numerous resources to improve the quality and multidisciplinary nature of research on diabetes by providing shared access to specialized resources. The NIDDK-supported Diabetes Research Centers, formerly known as Diabetes Endocrinology Research Centers and Diabetes Research and Training Centers, provide increased and cost-effective collaboration among multidisciplinary groups of investigators at institutions with an established, comprehensive research base in diabetes and related areas of endocrinology and metabolism. The National Mouse Metabolic Phenotyping Centers (<http://www.mmpc.org>) provide a range of complex exams used to characterize mouse metabolism, hormones, energy balance, eating and exercise, organ function and morphology, physiology, and histology. NIDDK supported research consortia, such as the Beta Cell Biology Consortium (<http://www.betacell.org/>) and the Nuclear Receptor Signaling Atlas (<http://www.nursa.org/>), provide data and reagents to the broader scientific community. Other important diabetes resources supported by NIDDK include a type 1 diabetes mouse repository at The Jackson Laboratory (<http://www.jax.org/t1dr/>) and Islet Cell Resource Centers (<http://icr.coh.org/>) that provide human islets for research.

Samples and data from the limited number of cohorts from large diabetes studies with well-characterized phenotypes at baseline and with longitudinal measurement of characteristics of interest are highly prized. To expand the usefulness of these major clinical studies by allowing the wider research community to access study materials beyond the end of the study or after a limited proprietary interval for ongoing studies, NIDDK has established biosample, genetics, and data repositories. Recently NIDDK has gone beyond the repository concept to create a living biobank, which provides investigators with the opportunity to obtain "on-demand" biological samples from selected individuals. This effort builds on the unique population of individuals at risk for the development of type 1 diabetes ascertained and monitored through TrialNet. The unprecedented availability of such samples and data may allow immunologists to understand early inciting events in type 1 pathogenesis.

# APPLYING NEW TOOLS AND TECHNOLOGIES TO DIABETES RESEARCH AND PATIENT CARE

Large clinical trials have established the long-term benefits of intensive blood glucose control in lowing the risk of diabetes complications. However, insulin therapy is burdensome and limited by hypoglycemia. NIDDK has devoted considerable resources to develop technologies for accurate and rapid detection of glucose levels and appropriate adjustment of insulin delivery to create an artificial pancreas that simulates the functions of β-cells. Achieving this goal will require more accurate and robust glucose-sensing devices; more effective and rapidly acting insulin preparations; algorithms that align real-time glucose measurements with adjustment in insulin delivery; infusion devices that deliver insulin more effectively, conveniently, and physiologically; and fail-safe mechanisms to avoid hyper- or hypoglycemia. It will also be important to determine the benefit of combining insulin delivery with the delivery of the counterbalancing hormone glucagon to reduce hyperglycemia, and how timely transmission and remote interpretation of patient data may contribute to safety and efficacy. Research is ongoing to assess the capacity of current artificial pancreas technology to improve overall metabolism, increase patient well-being, restore hypoglycemia awareness, and preserve existing pancreatic β-cell function, as well as to understand the factors affecting its use and acceptance in different age-groups.

Advances in sensors, processors, memory storage, wireless communication, and Web-based data transport, processing, and sharing have applications not only to new therapies but also to many facets of diabetes research in free-living populations. These range from instruments that measure energy intake and physical activity to the application of continuous monitoring of blood glucose to enable exploration of questions about the impact on human health of glycemic excursions, which may not be captured by HbA~1c~ measurement. Such studies could address the question of whether and how hypoglycemia may contribute to CVD events. Studies of energy balance would benefit from tools to define the neural circuits and molecular mediators that regulate energy balance by sensing and responding to signals of energy status and tools to quantitate mitochondrial biogenesis and turnover and assess mitochondrial function. To assess the progression of diabetes, measures that directly measure β-cell mass are particularly important because current methods are all linked to β-cell function, which may have both reversible and irreversible components. Complications research could benefit from tools for the study of extracellular matrix proteins and their interactions with growth factors and circulating stem cells or for the study of epigenetic change or glycation and lipoxidation of proteins. Appropriate systems biology and computational tools are needed to facilitate the integration of sequencing, expression, proteome, and metabolome profiles to identify key biologic processes and their interactions. NIDDK seeks to foster development of paradigm-shifting technology and truly transformative tools through workshops, targeted funding opportunity announcements, and interdisciplinary research grants.

# DIABETES AS A GLOBAL HEALTH ISSUE

Diabetes is a universal problem. The impact of diabetes is rapidly growing among populations in developing and middle-income countries; without action, deaths and disability due to diabetes will continue to increase substantially. NIDDK promotes international collaboration between investigators in the U.S. and scientists in other countries to develop and test strategies to stem the epidemic of diabetes at home and globally. Many other countries have health care and medical records systems that are particularly useful for clinical research on diabetes. NIDDK has collaborated globally on type 1 diabetes research through networks such as TEDDY and TrialNet to expand access to research participants and gain insight from research collaborators. Genetic research on diabetes also knows no boundaries and our research efforts have benefited from combined analysis of international cohorts. Global collaboration on type 2 diabetes is particularly relevant to understanding diabetes in immigrant and minority populations in the U.S. International collaborations offer unique opportunities to compare effects of different environmental exposures and understand why specific populations may be particularly vulnerable to diabetes.

# CONCLUSION

Daunting as the challenge of diabetes appears, research has tremendously improved outcomes for people with the disorder. Were it not for declining rates of kidney disease, amputation, and CVD in people with diabetes, the burden associated with increased diabetes prevalence would be much greater. NIDDK recognizes the importance of collaboration with other components of NIH, other government agencies, and the diabetes voluntary organizations to realize the research progress. Our challenge is to stem the growing tide of diabetes through research ranging from understanding the fundamental processes underlying diabetes and its complications to studies of practical approaches to combat diabetes in medical and community settings.

## ACKNOWLEDGMENTS

No potential conflicts of interest relevant to this article were reported.

# REFERENCES


author: Eugene J. Barrett; Stephen RattiganCorresponding author: Eugene J. Barrett, <firstname.lastname@example.com>.
date: 2012-11
references:
subtitle: : Its Measurement and Role in Metabolic Regulation
title: Muscle Perfusion

Methods for measuring muscle blood flow have been evolving over the past 120 years (1,2). Studies of hormonal regulation of muscle flow and metabolism began with the classical work by Andres et al. (3). Numerous diabetes investigators interested in muscle metabolism in vivo have estimated the net balance of glucose and other metabolites across a skeletal muscle bed or limb from the product of the arterial-venous concentration difference and the blood flow. In this review, drawing upon early studies, we will emphasize some of the principles and limitations of various techniques for measuring flow to estimate net exchange or rates of production or consumption of metabolites. Table 1 summarizes pertinent strengths and limitations of the most commonly used methods for estimating either muscle blood flow or perfusion. From later studies, we will deal more directly with the issue of how flow is hormonally regulated and the relationship between skeletal muscle flow regulation and metabolic regulation. That discussion will extend beyond flow alone as an important regulated variable, emphasizing instead perfusion, which encompasses both the rate and distribution of blood flow in a tissue. We will highlight some of the new methodologies that have helped clarify further the linkage between the regulation of skeletal muscle perfusion and metabolic function.

Methods for measurement of limb and muscle blood flow

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# Limb balance measurements identify sites of insulin action and resistance

It is appropriate to begin this discussion with the development of the forearm balance technique by investigators at Johns Hopkins in the early 1950s. These investigators put forward the hypothesis that through continuous infusion of a dye "tracer" (in this case Evans blue dye) that binds tightly and rapidly to serum proteins into the brachial artery and sampling from an ipsilateral antecubital vein, blood flow to the forearm could be quantified using simple spectrophotometric methods (3). They pointed out several advantages to the forearm for such studies, including *1*) that skeletal muscle makes up the preponderance (\~80%) of the tissue mass of the forearm; *2*) the forearm's relatively small mass and slow blood flow allow infusion of very small amounts of dye, which minimizes the contribution of recirculating dye; and *3*) the vascular anatomy of the forearm is well understood and in \>80% of individuals bifurcation of the brachial artery occurs below the antecubital crease, and therefore infusion of dye above the elbow should distribute to both the radial and ulnar vessels. Their measurements of flow corresponded well with the plethysmographic measurements that were available at that time. Plethysmography measures blood flow from the time-dependent increase in volume of a segment of a limb after venous outflow occlusion using either a strain gauge or other detection device. The development and application of plethysmographic limb flow measurements have recently been excellently reviewed (4). As there is no gold standard for measuring flow in clinical studies, cross-validation between methods provides needed assurance.

In these dye dilution studies, the issue of dye mixing in the brachial artery was extensively examined (3), as adequate mixing is clearly required for accurate blood flow measurements. Despite the finding that dye streaming occurred at the infusion rate used, it was observed by simultaneously sampling from multiple forearm veins that adequate mixing had occurred in most subjects. Interestingly, use of a jet injector to promote mixing of the infusate at the arterial injection site provoked downstream vasodilation (perhaps secondary to ATP or adenosine released by the endothelium traumatized by the jet shear) and was abandoned. Traction on the arterial catheter also altered downstream arterial resistance and flow, underscoring that care must be taken with this method.

Combining this dye dilution method with arterial-venous (A-V) metabolite sampling allowed estimation of the substrate balance across the forearm (Fig. 1). These "limb balance" studies took advantage of the fact that the forearm receives only approximately one-fiftieth of the cardiac output (5). As a result, infusion of low doses of insulin (e.g., 0.05 mU/min/kg body wt) into the brachial artery provoked physiologically significant increases in plasma insulin concentrations bathing the forearm musculature, but when diluted in the whole-body plasma pool it had minimal or no effect on plasma glucose, potassium, or other metabolite concentrations. The same circumstance does not pertain for infusion of insulin into the femoral artery when leg balance measurements are made. The leg's greater mass and blood flow require higher rates of insulin infusion, and the insulin recirculates and affects plasma glucose and other metabolites. Using this forearm balance method a decade before they developed the insulin clamp (6), these investigators demonstrated that physiologic doses of insulin stimulated skeletal muscle glucose uptake under euglycemic conditions in humans (5,7) and that this action of insulin was impaired in obese adults (7).

Within a few years, other laboratories had begun to apply this limb balance method. In particular, the Cahill laboratory (8–10) at the Joslin Clinic and its diaspora (11–13) used limb balance measurements very effectively to study the actions of glucoregulatory hormones and exercise. Limb blood flow was measured by indocyanine green dye dilution or plethysmography. It is worthwhile noting that while these studies frequently ascribed the observed substrate use or production to muscle, other tissues within the limb can contribute to this measurement by either adding or removing metabolites and by contributing to the flow measurement. The former is perhaps less of an issue for the forearm where the deep antecubital vein cannulated in a retrograde fashion drains predominantly skeletal muscle, while the femoral vein is draining all muscle, adipose, and osseous tissues of the leg. However, to the extent that the A-V difference measurement selectively samples blood draining muscle (as it does in the forearm with retrograde deep forearm vein cannulation), multiplying the A-V difference for a metabolite by total forearm flow may not give an accurate balance if muscle and fat do not handle the metabolite in an identical fashion. It is important to recognize that when either forearm or leg metabolism is studied using the limb balance method, results are typically expressed as millimoles of substrate per minute per 100 mL of forearm or leg. As sex (14), age, and body habitus (15) influence the relative proportion of skeletal muscle versus adipose tissue present in the limb, caution needs to be used regarding ascribing metabolite exchange across the forearm or leg to a particular tissue type. In this regard, it bears keeping in mind that estimated rates of blood flow to subcutaneous adipose tissue and resting skeletal muscle are comparable (∼3–5 mL/min/100 g) (16) and glucose uptake to subcutaneous adipose tissue contributes significantly to body glucose disposal (17,18). Along this line, leg blood flow (milliliters per kilogram of leg) is not different between men and women, despite greater fat-free mass in males (19). These cautions and limitations pertain whether one is studying forearm or leg balances. With exercise, muscle blood flow can increase by \>20-fold.

Using the leg balance method combined with the euglycemic insulin clamp, investigators demonstrated that leg glucose uptake could account for ∼40% of body glucose disposal and suggested that muscle was principally responsible. Extrapolating from leg muscle mass to total body muscle, they concluded that during steady-state hyperinsulinemia, muscle accounted for 80–90% of glucose disposal (20). Leg glucose uptake was also found to be a major site of insulin resistance in both obesity and diabetes. The combined measurements of bulk blood flow together with the A-V substrate concentration differences proved to be a powerful tool for quantifying the metabolism of carbohydrates (21), fats (22), amino acids (23), and oxygen (24) by the limb tissues. Either dye dilution or plethysmographic methods were used. Whereas only modest data are available in the literature comparing these two methods for measurement of bulk blood flow, they do appear to yield comparable values for bulk limb blood flow (25).

The introduction of simultaneous A-V measurement of radiolabeled substrate with limb blood flow added yet another dimension to the limb balance technique. These methods were initially used to measure fatty acid uptake and oxidation by exercising muscle (26) and later to quantify both the transport and net uptake of glucose (27), free fatty acid (FFA) (28), and amino acids and subsequently to quantify rates of protein synthesis and degradation (29), of lipolysis and lipogenesis (22), and of oxidative and nonoxidative glucose disposal (30). Once again, the quantitative accuracy of these measurements hinges entirely on the measurement of blood flow. In aggregate, these studies demonstrated the great utility of the limb balance method to quantify metabolic events in peripheral tissue and to discover how they are regulated in health and disease.

With improvements in ultrasound methodology, Doppler flow measurements have become a third widely used method for measuring bulk limb blood flow, which has increasingly displaced the more invasive dye dilution technique. Doppler flow measurements were observed to correlate well with plethysmographic measurements (31).

Muscle perfusion can be evaluated by various nuclear magnetic resonance (NMR) techniques. One of the most useful, because of its ability to determine both blood flow and flow distribution noninvasively, is arterial spin labeling (ASL). This technique can be used in humans and experimental animals (32). It has the potential to be directly compared with metabolism determined by other NMR techniques in the same region of tissue but not simultaneously (32). The ASL technique has been shown to correlate reasonably well (*r*^2^ = 0.85) with leg blood flow measured by venous plethysmography in humans (33) and well (*r*^2^ = 0.95) with limb blood flow in a rat-perfused hindlimb model (34). However, due to the expense of specialized NMR equipment and the substantial complexity of postacquisition data processing required, this technique has not been widely used by physiologists or clinical researchers.

Muscle perfusion has also been measured by microdialysis where a semipermeable membrane is placed in the tissue and perfused with dialysate that contains markers such as ethanol or radiolabeled water. The exchange and recovery of these markers can be used to estimate total blood flow (35) and distribution of nutritive and nonnutritive blood flow (36). Combined with other tracers, the microdialysis technique can also provide information on the interstitial concentrations of metabolic and vasoactive moieties such as glucose, glycerol, lactate, amino acids, insulin, nitric oxide (NO), and adenosine (37). The technique, however, has several limitations that include its invasive nature; lack of spatial resolution, as it is difficult to determine the amount of tissue being sampled; and temporal resolution because of the long sampling times (minutes) required to collect sufficient dialysate for measurement.

# Perfusion as A regulator of muscle metabolism

A conceptual shift in the consideration of the role of blood flow in muscle metabolism occurred with studies from the laboratory of Alain Baron. He modified a method first used by Andersen and Saltin (38) for quantifying leg blood flow, which relied on a use of a thermodilution catheter introduced into the femoral vein (39). Thermodilution methods had previously been used extensively to measure blood flow in vascular beds other than skeletal muscle. While invasive, this methodology allowed very frequent measurements of blood flow during the course of prolonged metabolic balance studies. The design of the catheter allowed spraying of the chilled saline infusate to assure good mixing and nonlaminar flow prior to the temperature-sensing probe. With this technique, Baron observed that insulin not only increased glucose uptake by leg muscle in healthy individuals but also simultaneously increased blood flow. Further increasing flow with cholinergic stimulation during hyperinsulinemia significantly augmented leg glucose uptake (40). He and collaborators subsequently demonstrated that insulin increased flow by stimulation of NO production and that this flow increase was inhibited in type 2 and type 1 diabetes and obesity (39) and by experimentally induced insulin resistance (rev. in 41).

This work strongly suggested that insulin's action to promote the uptake of glucose, amino acids, and other substrates into muscle was aided by the concerted action of insulin on the vasculature to increase blood flow and presumably the delivery of insulin, glucose, and other metabolites to the muscle (41). The observation that this vascular action of insulin was blocked by inhibitors of NO synthase (NOS) (42–44) suggested that the endothelium was the target for insulin's vascular action. Subsequent studies in isolated endothelial cells demonstrated that insulin, acting via the phosphatidylinositol-3 kinase Akt pathway (45,46), specifically enhanced the activity of NOS (Fig. 3).

While these observations of insulin's effect of increasing total limb blood flow were confirmed by a number of laboratories (47,48), concerns were raised and controversy ensued around both the reported need for prolonged, relatively high steady-state insulin concentrations to provoke insulin's vascular action relative to effects on glucose disposal (49,50) and the lack of effect of increasing flow pharmacologically on limb glucose uptake in insulin-resistant (51,52) subjects.

Studies using positron emission tomography (PET) methods by investigators in Turku, Finland, were particularly informative, as PET allowed simultaneous measurements of perfusion, using labeled water (H~2~^15^O) and glucose (^18^F-deoxyglucose) within voxels of muscle, avoiding the issue of tissue heterogeneity and bulk flow distribution to multiple tissues in the limb (51,53).

Radiolabeled or fluorescent microspheres have also been used in a number of animal studies to examine the effect of insulin on blood flow (54–56). As the technique requires tissue removal, it has not been applicable to clinical studies. However, results of published studies confirm that insulin can enhance skeletal muscle blood flow. Inasmuch as the microspheres used typically measure 15 ± 3 µ in diameter, it is not clear that they have access to the capillary bed; they are more likely retained within the terminal arterioles. Multiple measurements can be made in a single animal provided different isotopes or fluorescent labels are available whose emissions can be distinguished by differences in energy spectrum. Of interest is the study by Liang et al. (55), which included measurements of the effects of insulin on multiple tissues. Insulin-induced increases in blood flow during euglycemia were most apparent in skeletal and cardiac muscle.

Since the various laboratories examining the relationship between insulin's effect on limb blood flow and metabolism used different methods for measuring flow (e.g., strain gauge plethysmography, dye- or thermodilution, or PET), some of the discordant findings may have arisen from these measurements. This issue was never fully settled. However, additional opportunities became available with the development of techniques to measure the distribution of flow within a tissue rather than simply bulk flow to the tissue. The observation that the volume of muscle microvasculature perfused was regulated dates back to August Krogh (57). Changes in the volume of muscle microvasculature perfused could influence hormone and nutrient exchange within the muscle by altering the endothelial surface area exposed.

As shown in Fig. 2, the muscle microvasculature consists of third- and fourth-order arterioles, the capillary network, and small venules. Each terminal arteriole supplies multiple capillaries in an orderly arcade. At any given moment, only approximately one-third of the capillaries appear to be actively perfused in resting muscle (58). There is no conclusive evidence for the presence of smooth muscle "sphincters" at the capillary origin. Instead, it appears likely that many capillaries are functionally at least transiently not perfused because of *1*) the residual tone limiting flow to the terminal arteriole, *2*) the position of a particular capillary relative to the inflow into the arteriole, *3*) the interstitial pressure around the capillary, *4*) the intrinsic resistance of the capillary, and *5*) the residual pressure within the lumen of the draining venule. These factors in aggregate likely determine whether an individual capillary will be perfused at a particular time. Relaxation of the terminal arteriole, with consequent increases in precapillary pressure, will lead to perfusion of previously under- or unperfused capillaries. Time variable perfusion of terminal arterioles, a process termed "vasomotion" or "flowmotion," appears to be due to the action of sympathetic nervous input, intrinsic myogenic responses in the vascular smooth muscle, and autoregulation by the endothelium within terminal aspects of the microvasculature conducting signals retrograde through gap junctions to signal to feed arterioles when some relaxation is needed to at least transiently restore perfusion (59). While intermittent perfusion of muscle microvasculature has been difficult to demonstrate in intravital microscopy studies of surgically exposed thin muscles (60), the use of noninvasive contrast ultrasound (see below) appears to have resolved this issue in larger muscle groups in both human and animal studies.

Just over a decade ago, our laboratories began collaborating to develop and apply two methodologies for assessing the volume of vasculature perfused within muscle. The first relied on measurement of the A-V concentration difference of intravenously infused 1-methyl-xanthine (1-MX). Xanthine oxidase in the endothelial cell converts 1-MX to 1-methylurate, and the extent of metabolism provides an index of the endothelial surface perfused. Insulin was found in rats to increase 1-MX metabolism (61), and this was inhibited by factors that acutely provoked metabolic insulin resistance, including α-methyl serotonin (62), tumor necrosis factor-α (63), elevated FFA concentrations (64), and NOS inhibition (65), and with chronic obesity (66). This method, however, was not found to be useful in humans because of the substantially lower extraction ratio for 1-MX across the skeletal muscle capillary bed.

We subsequently applied the technique of contrast-enhanced ultrasound (CEU) to the measurement of microvascular blood volume in both rodent and human studies. CEU was developed initially to image myocardial perfusion. The method relies on the insonation of intravenously infused, perfluorocarbon–filled lipid microbubbles, which enhance the video intensity of ultrasound images. These microbubbles are typically 1–4 µ in diameter, remain within the vasculature, and have rheologic properties similar to erythrocytes. Most importantly, the microbubbles oscillate or rupture (depending on the energy of the ultrasound signal) when exposed to the ultrasound beam. This behavior is responsible for an intense reflected sound wave that enhances the image intensity.

Unlike Doppler, plethysmography, PET, or tracer dilution methods, CEU does not measure volume flow, i.e., milliliters per minute per 100 mL tissue. Therefore, CEU cannot be used to provide an estimate of blood flow that could be coupled with A-V difference measurements to obtain a limb balance. Rather, it provides a sensitive index of the volume of microvasculature (and therefore the microvascular surface area available for nutrient exchange) that is perfused. It is most useful for comparing acute responses to interventions like increasing plasma insulin, exercise, feeding, etc. In this regard, it has proven quite useful for examining the action of insulin on the microvasculature. Similar to PET, CEU allows study of a region of interest within the muscle or adipose tissue. However, while PET signal intensity in muscle can be calibrated to the activity in blood measured simultaneously yielding a flow measurement, this is not available with CEU. However, CEU affords several advantages over PET scanning for clinical studies. It is less expensive, involves no radioisotopes, is portable, and the image analysis is substantially simpler. Perhaps most importantly, it allows separation of signals arising from larger conduit and feed arteries from signals arising from the microcirculation. This separation is based on capturing a "replenishment curve," i.e., a time series of images that is initiated by instantaneously destroying all microbubbles in the volume being studied and following the microbubble replenishment and recovery of videointensity until it reaches a plateau. The plateau signal intensity is reached when all vessels in the tissue that are being perfused have again filled with blood containing microbubbles. Since flow in conduit and feed arteries is rapid, these fill quickly, while the capillaries and venules refill slowly. The image intensity created by vessels that fill rapidly can straightforwardly be subtracted from that seen at the plateau, with the difference being due to microvasculature containing microbubbles. The flow velocity for the replenishment curve is also provided by the image analysis, and flow is the product of flow velocity and volume perfused. However, both velocity and volume are measured in videointensity units, and their product is not readily converted to units of volume flow.

In a series of studies using either contrast ultrasound or the A-V concentration difference for 1-MX as an index of the endothelial surface perfused, we observed that insulin at physiologic concentrations enhanced the microvascular volume perfused within either human forearm (67) or rat hind leg muscle. This occurs within 15 min of onset of hyperinsulinemia (68,69) at modest physiologic insulin concentrations (70) and is blocked by inhibition of NOS (65). Importantly, loss of insulin's ability to enhance microvascular volume either by a blockade of NOS or as a result of endogenous insulin resistance secondary to obesity (71), diabetes (72), or experimental conditions (e.g., elevating FFA \[73\] or tumor necrosis factor-α concentrations) impedes skeletal muscle glucose disposal. This recruitment process is illustrated in Fig. 3.

The simplest explanation for this apparent relationship between the microvascular volume perfused within skeletal muscle and metabolic insulin resistance arises from understanding that the delivery of glucose and insulin to skeletal muscle appears to be a limiting process for insulin action within skeletal muscle (6,74). Increasing the endothelial surface area and thereby enhancing insulin delivery could facilitate both the time of onset and the magnitude of the metabolic response to insulin. There may in addition be factors within the endothelium that relate to insulin transendothelial transport that are adversely affected by insulin resistance and contribute further to the metabolic disarray.

There are two other potential techniques that could be used to measure the volume of vasculature perfused within skeletal muscle. The first is an adaptation of PET in which ^15^O-labeled carbon monoxide is inhaled and binds rapidly to hemoglobin. In this manner, the PET tracer becomes a vascular tracer. With this technique, investigators have observed that insulin increases the intensity of the PET signal consistent with an increase in the volume occupied by vasculature within a given voxel. While statistically significant effects were seen, the magnitude of the effect is substantially less than is seen with CEU. This may relate to the fact that much of the PET signal appears to arise from larger blood vessels and there is no straightforward way to subtract out this component. Additionally, in order to have sufficient signal, the PET image is collected over a substantially longer period of time, and this exposure is longer than the mean transit time for blood traversing skeletal muscle. As a result, flowmotion within the tissue slice may lead to capturing signal from vessels that are only open a portion of the time. Perhaps because of these difficulties, very little has been done with this technique to examine microvascular perfusion within skeletal muscle using carbon monoxide–labeled tracers.

Near-infrared spectroscopy provides yet another potential methodology. This technique relies on the deeper penetration of near-infrared electromagnetic radiation into tissue compared with visible or ultraviolet light. It has been used extensively with pulse oximetry, and it can measure the relative amounts of oxyhemoglobin and desoxyhemoglobin. When coupled with venous occlusion, it can be used to determine the total rate of inflow of oxygenated blood to a muscle and potentially the microvascular volume filled. To our knowledge, it has not been applied to studies of insulin or low-intensity exercise–mediated capillary recruitment within muscle. There is concern regarding how much contribution skin and subcutaneous adipose tissue make to the near-infrared spectroscopy signal, and this would be a particular issue in obese individuals and females.

# Parallel effects of insulin and contraction on microvascular recruitment

It has long been known that exercise acts synergistically with hyperinsulinemia to enhance glucose disposal. Multiple mechanisms within muscle account for this including increased glycolytic activity, recruitment of GLUT4 transporters to the plasma membrane, and increased tissue perfusion. The impact of expanding microvascular volume by recruitment of previously unperfused or underperfused capillaries on delivery of insulin to muscle tissue can be appreciated from studies looking at the effect of microvascular recruitment induced by muscular contraction. We recently demonstrated that in the rat hind limb, even very modest contraction enhances the delivery of both insulin and albumin to the muscle interstitium (75). We were particularly intrigued by the parallel between low-dose insulin and very light exercise, which both stimulated microvascular recruitment within muscle without affecting total blood flow to the tissue. In contrast, more intense exercise (75,76) and higher insulin concentrations increase both microvascular recruitment and total blood flow. This suggests that there is an orderly, staged vascular response to either stimulus, with the microvasculature being more sensitive to either stimulus than larger resistance vessels, which regulate total flow.

Both microvascular recruitment and enhanced blood flow could increase the delivery of insulin, glucose, and other metabolites to skeletal muscle, but there may be some advantage to having a staged response with very modest increases in insulin or exercise. Capillary recruitment would not require simultaneous increases in cardiac output to prevent a fall in systemic arterial pressure. For insulin, this may be helpful particularly in the postprandial setting when there is already a demand for increased blood flow to splanchnic tissues. However, when the stimulus is particularly strong (as occurs with high concentrations of insulin or intense exercise), other systemic vascular regulatory responses must be called into play to maintain vascular homeostasis.

Increases in microvascular volume secondary to microvascular recruitment are not restricted to skeletal muscle. With use of contrast ultrasound, insulin has been observed to increase microvascular volume within cardiac muscle (77) and subcutaneous adipose tissue of the leg (78). Using an entirely different approach, investigators in the Netherlands have demonstrated increased numbers of perfused capillaries within the nail fold as captured by video microscopy in human skin (79). This method is unique in allowing direct quantitation of the number of perfused capillaries. Insulin can regulate this recruitment, but its impact is diminished in obesity and hypertension and by insulin resistance (80).

## Closing remarks.

Accurate measurements of blood flow are critical for in vivo quantitation of skeletal muscle fuel metabolism and how it is affected by insulin resistance, diabetes, or other metabolic disorders. Multiple methodologies are currently available for use by clinical and basic scientists performing studies of muscle metabolism (Table 1). Plethsymography, Doppler ultrasound, and dye dilution measurements are widely available, inexpensive, portable and not technically demanding and are consequently most commonly used. Other methods such as PET and magnetic resonance imaging, while more complex and expensive and not portable, give more detailed information about specific blood flow within a region of interest of tissue. This latter information is not available with the more readily performed methods. Doubtless, with improving technology these types of measurements will be made more often. Contrast ultrasound can provide unique information with regard to the perfusion of the microvasculature and a measurement of microvascular volume. However, CEU does not provide a measure of bulk flow. Using a variety of different techniques, investigators have clearly realized the intimate relationship between regulation of perfusion and metabolic regulation within muscle. In coming years, more will be learned with regard to the chemical signals responsible for the coordination of these two functions.

## ACKNOWLEDGMENTS

This study was supported by National Institutes of Health grants DK-R01-057878 and R01 DK-073759 and American Diabetes Association Grant BS 06.

No potential conflicts of interest relevant to this article were reported.

E.J.B. and S.R. reviewed literature and composed and edited the manuscript. E.J.B. is the guarantor of this work and, as such, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis.

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