Vignettes highlighting research supported by the NIDDK over the past year that have opened new avenues for research and have the potential to profoundly affect our understanding of human health and disease.
Genes that Contribute to Developing Type 2 Diabetes and Genes that Affect Treatment Outcomes
Recent studies have dramatically increased knowledge about the complex genetic underpinnings of type 2 diabetes, in which a great many genes are each thought to play a small role in promoting or preventing the disease. Two NIDDK-supported studies took a genome-wide association approach (discussed in detail in the Cross-Cutting Science chapter) to help locate genes involved. Although the method does not generally identify the precise genetic alterations that cause these disease effects, it greatly narrows their likely location in the genome. The researchers thus newly identified at least four such type 2 diabetes-affecting genomic neighborhoods and confirmed several others that had been found previously, bringing the total known to about 10. With this information, scientists can begin to dissect the actual genetic changes that increase or decrease likelihood of type 2 diabetes, and thus provide potential new targets in the quest to prevent or treat the disease. A third study builds on previously reported genetic findings in just that way. Following a group of patients from the landmark Diabetes Prevention Program (DPP) clinical trial, researchers examined the impact of different versions of two closely linked type 2 diabetes genes both on disease progression and on the effectiveness of the lifestyle and pharmacologic interventions studied in the trial. Interestingly, one of these genes, which encodes the Kir6.2 protein, is also implicated in the rare genetic disorder neonatal diabetes, as described elsewhere in this chapter. The research indicated a genetic variant associated with increased diabetes risk did not alter progression to diabetes in people who already had prediabetes, suggesting the increased risk occurred earlier in the course of disease. Lifestyle changes were equally effective in preventing diabetes in those with different versions of the gene, but the drug metformin was less effective in preventing progression to diabetes in people with a particular genetic risk variant. Because metformin is one of the most widely prescribed medications for diabetes, it will be important to confirm this finding in those at risk for diabetes and to determine whether genetic variation affects response to metformin in those who already have diabetes. Taken together these studies illustrate the dramatic advances in the fight against diabetes that have become possible in the genomic era.
Scott LJ, et al: A genome-wide association study of type 2 diabetes in Finns detects multiple susceptibility variants. Science 316: 1341-1345, 2007.
Diabetes Genetics Initiative of Broad Institute of Harvard and MIT, Lund University, and Novartis Institutes of BioMedical Research, Saxena R, et al: Genome-wide association analysis identifies loci for type 2 diabetes and triglyceride levels. Science 316: 1331-1336, 2007.
Florez JC, et al: Type 2 diabetes-associated missense polymorphisms KCNJ11 E23K and ABCC8 A1369S influence progression to diabetes and response to interventions in the Diabetes Prevention Program. Diabetes 56: 531-536, 2007.
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New Gene Associated with Type 1 Diabetes Susceptibility
Scientists have discovered that variation in a region of DNA that includes the KIAA0350 gene is associated with risk of developing type 1 diabetes. Using a genome-wide association approach, DNA from type 1 diabetes patients, unrelated controls, and family groupings including individuals with type 1 diabetes was examined to identify areas of variation potentially associated with the disease. In order to verify their results, the researchers conducted the genome-wide association study in two independent populations. Results from both populations indicate that three DNA variations, called single nucleotide polymorphisms (SNPs) in the region including the KIAA0350 gene are associated with decreased risk of developing type 1 diabetes. With the approach used in this study, it is difficult to determine conclusively that KIAA0350 is the gene that is influenced by these SNPs, and thus may confer protection from type 1 diabetes. However, KIAA0350 is the only known gene in the area and the protein product of this gene is found primarily in cells that are part of the immune system. It is possible that this gene product could play a role in mediating immunity, which may have significant implications for the way the immune system reacts to the pancreatic beta cells that are eventually destroyed in type 1 diabetes. The discovery of the association of the KIAA0350 region adds to the increasing understanding of the genetic basis of type 1 diabetes. Previously established genes associated with type 1 diabetes account for only half of the genetic risk of developing the disease; therefore, research into additional genetic markers is greatly needed. This discovery provides new avenues for exploration as researchers probe the function of this gene in the hope of establishing causes and developing new treatments for type 1 diabetes.
Hakonarson H, et al: A genome-wide association study identifies KIAA0350 as a type 1 diabetes gene. Nature 448: 591-594, 2007.
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First Year of the Look AHEAD Trial Yields Encouraging News for Patients with Type 2 Diabetes
After 1 year, individuals in the Look AHEAD (Action for Health in Diabetes) clinical trial who were assigned to an intensive lifestyle intervention had significantly greater improvement in health measures than did individuals receiving diabetes support and education alone. The Look AHEAD trial enrolled over 5,000 patients to determine whether the intensive lifestyle intervention could impact the long-term health of overweight and obese adults with type 2 diabetes. Half of the patients received this intervention, which consisted of weight loss through decreased calorie consumption and increased physical activity. This group of patients also received counseling through weekly individual or group meetings with a team of specialists. The diabetes support and education intervention consisted of three diabetes education sessions held throughout the year. Assessments of the use of diabetes, blood pressure, and cholesterol medication; weight; blood sugar (glucose) levels; blood pressure; cholesterol levels; kidney function; presence of the metabolic syndrome; and other measures were completed for all patients at the beginning of the trial and after 1 year of the interventions. The Look AHEAD trial was designed to follow the participants for up to 11.5 years, if specific criteria during the first year were met. These included a greater than 5 percent difference in average weight change between the two intervention groups and a greater than 5 percent loss in average weight over 1 year in the intensive lifestyle intervention group. Both of these criteria were exceeded. In addition to the greater weight loss in the patients receiving the intensive lifestyle intervention, these individuals also had lower blood glucose levels; decreased use of medications for diabetes, blood pressure, and cholesterol; and improved cholesterol and blood pressure levels. As the Look AHEAD trial continues over the next several years, it will provide valuable information regarding the use of an intensive lifestyle intervention for reaching and maintaining a weight loss goal in people with type 2 diabetes, and whether the promising results seen thus far lead to long-term health benefits associated with weight loss.
Look AHEAD Research Group, Pi-Sunyer X, et al: Reduction in weight and cardiovascular disease risk factors in individuals with type 2 diabetes: One-year results of the Look AHEAD trial. Diabetes Care 30: 1374-1383, 2007.
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New Insights on the Relationship Between Obesity and Gut Bacteria
While genetics and lifestyle can conspire to promote obesity, an additional potential accomplice has emerged from recent research: the bacteria and other tiny organisms (microbes) that normally reside in the gut. Studies are revealing how some of these trillions of microbes may not only contribute to a flood of extra calories, but also modulate the biologic pathways that regulate metabolism and whether calories are burned or stored as fat.
Able to digest many dietary components that the body’s own intestinal cells can’t, gut microbes offer extra energy from food in exchange for a home in the gut. However, some may provide more food energy than others in the form of calories. By analyzing the genomes of these microbes, referred to collectively as the “microbiome,” scientists discovered that the relative abundance of two major types of gut bacteria differ between lean and obese mice. They additionally found, through genomic and biochemical analyses, that these types of gut bacteria differ in their capacity to harvest energy from food. Those more prevalent in the obese mice are better able to extract calories—and potentially provide too many extra calories to their mouse “hosts.” In a parallel study in humans, the scientists found that the relative abundance of these types of gut bacteria also differs between lean and obese people. Additionally, when the obese study volunteers lost weight by dieting, the relative proportions of these bacteria in their guts also changed. To further explore these effects, the researchers turned again to mice. They raised several mice in “germ-free” conditions so the mice would lack normal gut bacteria and then gave them gut bacteria from other (donor) mice. No longer germ-free, the mice who received gut bacteria from obese donors gained significantly more body fat over the next two weeks than mice who received gut bacteria from lean donors.
In another set of experiments in mice, scientists found that the bacteria that live in the gut also engage in a form of home remodeling, at the molecular level. They reduce the amounts of some native mouse proteins that would otherwise keep body weight down. The researchers first found that germ-free mice do not gain as much weight on a high-fat “Western diet” as do mice that have normal gut bacteria. They then showed that mice raised germfree have increased activity of a protein called AMPK, which helps burn fat in muscles and the liver. Building on previous research, they found that gut microbes may also contribute to diet-induced obesity by reducing levels of another mouse protein, called Fiaf, as well as mouse proteins that are regulated by Fiaf, which results in enhanced fat storage. Finally, using an implantable device in the mice to track locomotion, the scientists observed that mice raised without gut microbes move more, and thus may be burning more calories than mice with gut bacteria.
These studies show that the gut’s resident microbes affect not only the amount of calories obtained from food, but also whether the calories are stored or burned. Manipulation of the composition of gut bacteria may one day be a novel approach to obesity prevention or treatment.
Bäckhed F, et al: Mechanisms underlying the resistance to diet-induced obesity in germ-free mice. Proc Natl Acad Sci USA 104: 979-984, 2007.
Turnbaugh PJ, et al: An obesity-associated gut microbiome with increased capacity for energy harvest. Nature 444: 1027-1031, 2006 .
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Autophagy Is Implicated in Crohn’s Disease
Scientists have recently identified a gene that increases susceptibility for Crohn’s disease (CD), a major form of inflammatory bowel disease (IBD). The gene is involved in autophagy, a process cells use to eliminate unwanted cellular components by capturing and degrading them into molecules that can be recycled by the cell. This exciting discovery was made in a two-phase study supported by the NIDDK IBD Genetics Consortium.
As described in the Cross-Cutting Science chapter, genome-wide association scans are now possible which can screen individuals’ complete genomes for hundreds-of-thousands of small mutations, or gene variants. In the first phase of the study, members of the IBD Genetics Consortium conducted a screen using this state-of-the-art technology to identify genetic variants that contribute to CD. The scans were performed using the DNA of 547 CD patients and 548 healthy volunteers. Surprisingly, when the genetic variants of the two groups were compared, a relatively rare variant of the IL-23 receptor gene was identified that protects against CD. (See highlights from Dr. Judy Cho’s Scientific Presentation, which appears in the Digestive Diseases and Nutrition chapter.)
CD is a complex genetic disease caused by inappropriate immune responses to bacteria that naturally reside in the intestine. Because CD is a complex disease involving several genetic and environmental factors, the scientists conducting this study believed that some of the genes contributing to CD may have only a modest effect on disease risk and that larger study cohorts would be required if these subtle gene variants were to be detected. Therefore, a second phase of the study was conducted, which increased the sizes of the study cohorts to a total of 946 CD patients and 977 healthy volunteers. Analyses of these larger scans identified the new CD autophagy gene. Several other significant CD-associated genetic variants were identified in these scans as well. The discovery of an autophagy gene associated with CD is an important step in understanding this complex disease. Additionally, autophagy and the signaling pathways used to initiate this process can now be explored as targets for novel drugs designed to prevent and treat CD.
Rioux JD, et al: Genome-wide association study identifies new susceptibility loci for Crohn disease and implicates autophagy in disease pathogenesis. Nat Genet 39: 596-604, 2007.
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Of Mice, Fish, and Men—Multi-Species Studies Explore Impact of Intestinal Microbes
Three recent studies using animal models and human samples yield new insights and renewed curiosity about the impact of the trillions of microbes that inhabit the human intestine. These studies combine new genetic knowledge and techniques with the most useful animal and bacterial models available to shine a light on the interior world of intestinal microbes.
One study characterized the functions and evolutionary adaptations of a type of bacterium that is abundant in the human intestine and known to influence host nutrient digestion. The bacterium is called Methanobrevibacter smithii , or M. smithii . This bacterium was shown in previous experiments to increase the efficiency of host digestion. The result was more calories absorbed and a heightened risk for obesity. (Additional information on how gut microbes influence obesity development is presented in the chapter on “Obesity.”) Building upon these findings, researchers sequenced the bacterium’s genome and compared it to other bacterial strains to identify genes that are specific to M. smithii and point to its unique functions inside the human intestine. They then used a mouse model that had been raised in a sterile environment to keep its intestine completely free of bacteria. In this model, they identified the unique RNAs expressed and metabolic functions performed when M. smithii was introduced into the mouse gut. The researchers concluded this set of investigations by exploring some potential targets in the M. smithii genome for future drug discovery, specifically inhibitors of the bacterial genes that enhance their host’s ability to harvest energy from the diet.
In another study, resourceful researchers used advanced genomic tools and data, some swimming bacteria, and a see-through fish to track the movements and host impacts of intestinal bacteria. Their findings contrasted with previous research, which presented a static view of bacterial behavior in the gut. When watching “live” through a microscope, researchers could see the bacteria traveling through the intestine of the naturally transparent zebrafish, Danio rerio . Similarly to the mouse model in the M. smithii study, the zebrafish were raised in a sterile environment until the introduction of the intestinal bacteria Pseudomonas aeruginosa . This type of bacteria is usually thought of as harmful, in part because of its presence in patients with inflammatory bowel disease and cystic fibrosis. However, recent research has challenged this belief by showing beneficial effects in fish. For these experiments, the bacteria were made to glow by introducing a fluorescent gene for easier visualization. Using this simplified, highly visible system, scientists were able to observe how the bacteria navigated the intestine, swimming with their flagellum—a whip-like tail. To test the impact of this bacterial colonization on the fish, some of the bacteria were hobbled by disabling their flagellum. Immobilization of the bacteria correlated with a drop in immune responses, demonstrating the bacteria’s beneficial effects on fish immunity.
A third study combined both the mouse and zebrafish models in a reciprocal transplant experiment to answer fundamental questions about the evolutionary origins and inter-species differences of intestinal microbial communities. Mammals and fish typically host very different types of microbes in their intestines. By swapping intestinal contents between these species, scientists hoped to determine how the host habitat shapes the microbial community. In these experiments, both animals were raised either under sterile conditions to keep their intestines free of microbes, or in a normal environment where microbes naturally colonize the intestine. Then, intestinal contents from a normally raised zebrafish were transplanted into a sterile mouse, and vice versa (sterile zebrafish were colonized with mouse intestinal contents). Days after this initial colonization, the intestinal contents were sampled from the transplanted animals, and DNA was sequenced to identify the bacterial species present. Remarkably, the researchers found that each animal shaped the foreign microbial community to more closely resemble its native mix of intestinal microbes. For example, in the transplanted mouse intestine, bacteria that are abundant in the native mouse intestinal community, such as the Firmicutes, were amplified, while the zebrafish’s dominant bacteria, known as Proteobacteria, were diminished. A similar phenomenon was observed in the transplanted zebrafish intestine, where the Proteobacteria, a minor type of species in the mouse’s intestine, was substantially amplified in the zebrafish, while other species were reduced or absent.
Each of these studies reveals a fascinating facet of intestinal microbes, from their origins to their impact on the host’s everyday functions, such as absorbing nutrients and maintaining a healthy immune system. Future studies will continue these explorations of bacterial-host interactions in the intestine and their relationship to human health.
Rawls JF, et al: Reciprocal gut microbiota transplants from zebrafish and mice to germ-free recipients reveal host habitat selection. Cell 127: 423-433, 2006.
Rawls JF, et al: In vivo imaging and genetic analysis link bacterial motility and symbiosis in the zebrafish gut. Proc Natl Acad Sci USA 104: 7622-7627, 2007.
Samuel BS, et al: Genomic and metabolic adaptations of Methanobrevibacter smithii to the human gut. Proc Natl Acad Sci USA 104: 1064310648, 2007.
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Novel Gene Implicated in Early-Onset Kidney Disease
Scientists have recently identified a role for the phospholipase C epsilon gene ( PLCE1 ) in early-onset nephrotic syndrome. Nephrotic syndrome is a kidney disease characterized by elevated levels of protein in the urine, diminished levels of protein in the blood, and fluid retention and tissue swelling. Abnormal function of podocytes, specialized cells within the kidneys’ filtering units, appears to be at the center of nephrotic syndrome. Widespread scarring of the kidney in this syndrome can result in diminished kidney function and end-stage renal disease, in which case dialysis or a kidney transplant is required. There is no effective long-term treatment.
At least six genes have previously been implicated in nephrotic syndrome. Researchers performed genome-wide scans of several extended families with nephrotic syndrome who did not carry any of the previously described mutations, to search for additional genetic clues to this condition. Seven families or individuals were found to carry mutations in their PLCE1 gene .
In six, the mutations resulted in a truncated protein; in the seventh, it produced a full-length but defective protein.
In the kidney, PLCE1 protein initiates a cascade of intracellular signaling events resulting in changes in gene expression, cell growth, and differentiation. Using cultured cells and tissue analysis, the researchers demonstrated that PLCE1 protein is expressed in normal podocytes. When PLCE1 protein expression was experimentally reduced during development in zebrafish, the kidneys showed anatomic changes consistent with nephrotic syndrome.
Some of the children in the study had been treated previously with steroids or the drug cyclosporine A, two drugs used to suppress the immune system. Most were not responsive to therapy, as are most patients with nephrotic syndrome. Surprisingly, two of them—now 6 and 13 years of age—retained near-normal kidney function. To explain this exception, the researchers hypothesize that PLCE1 protein may be necessary for the kidneys to complete a particular developmental phase, and that drug therapy somehow compensated for the absence of PLCE1 signaling during this critical window. Future studies will examine additional individuals with nephrotic syndrome to test these intriguing hypotheses, as well as characterize a recently-described mouse that lacks the PLCE1 gene as a potentially valuable model system to study this condition.
Hinkes B, et al: Positional cloning uncovers mutations in PLCE1 responsible for a nephrotic syndrome variant that may be reversible. Nat Genet 38: 1397-1405, 2006.
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Identification of New Genetic Cause of Kidney Disease
Researchers have identified a gene that, when mutated, leads to an inherited form of kidney disease known as nephronophthisis (NPHP) and may explain why kidney size is significantly decreased in this disease. Characterized by kidney degeneration during childhood, NPHP leads to renal failure and ultimately the need for kidney transplantation. By conducting a genome-wide scan of three related children with early onset NPHP and their family members, researchers identified a single mutation in the gene encoding GLIS2 that is linked to the disease. To gain insight into its functional role, the scientists studied GLIS2 in normal mice and in mice containing a mutated form of the protein. The GLIS2 protein is expressed in normal adult mouse kidney, specifically in the cilia—microscopic hair-like projections on the cell surface. When the GLIS2 gene is mutated in mice, researchers observed that the kidneys demonstrated several of the hallmarks of NPHP, including fibrosis, smaller kidney size, and loss of tissue organization.
To understand the mechanism by which the diseased kidneys are decreased in size in NPHP, the researchers looked for genes that were specifically switched on in the mice lacking functional GLIS2. The researchers examined genes with a specific DNA sequence to which GLIS2 is known to bind, indicating that these genes are directly regulated by GLIS2. They found that the absence of GLIS2 resulted in greater expression of genes involved in such processes as change in cell type, cell signaling, fibrosis, and cell death. Notably, the activated genes indicated a high level of cell death in the diseased kidney without the cell proliferation necessary to maintain organ size. By discovering how GLIS2 regulates genes involved in a change in kidney cell type to one that is typically involved in fibrosis, the researchers may have uncovered an explanation for the loss of normal kidney structure and progressive fibrosis that occur in NPHP. These results provide insight into the cause of the reduced kidney size seen in NPHP and may guide future approaches to treating and preventing this inherited form of kidney disease.
Attanasio M, et al: Loss of GLIS2 causes nephronophthisis in humans and mice by increased apoptosis and fibrosis. Nat Genet 39: 1018-1024, 2007.
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