2126 DIABETES, VOL. 49, DECEMBER 2000 A Model to Explore the Interaction Between Muscle Insulin Resistance and -Cell Dysfunction in the Development of Type 2 Diabetes Franck Mauvais-Jarvis, Antti Virkamaki, M. Dodson Michael, Jonathon N. Winnay, Ariel Zisman, Rohit N. Kulkarni, and C. Ronald Kahn Type 2 diabetes is a polygenic disease characterized by defects in both insulin secretion and insulin action. We have previously reported that isolated insulin resis- tance in muscle by a tissue-specific insulin receptor knockout (MIRKO mouse) is not sufficient to alter glu- cose homeostasis, whereas -cell–specific insulin receptor knockout (IRKO) mice manifest severe pro- gressive glucose intolerance due to loss of glucose- stimulated acute-phase insulin release. To explore the interaction between insulin resistance in muscle and altered insulin secretion, we created a double tissue- specific insulin receptor knockout in these tissues. Sur- prisingly, IRKO-MIRKO mice show an improvement rather than a deterioration of glucose tolerance when compared to IRKO mice. This is due to improved glu- cose-stimulated acute insulin release and redistribu- tion of substrates with increased glucose uptake in adi- pose tissue and liver in vivo, without a significant decrease in muscle glucose uptake. Thus, insulin resis- tance in muscle leads to improved glucose-stimulated first-phase insulin secretion from -cells and shunting of substrates to nonmuscle tissues, collectively leading to improved glucose tolerance. These data suggest that muscle, either via changes in substrate availability or by acting as an endocrine tissue, communicates with and regulates insulin sensitivity in other tissues. Diabetes 49:2126–2134, 2000 T ype 2 diabetes is the most common endocrine disorder characterized by impaired insulin-stimu- lated glucose uptake in skeletal muscle and adi- pose tissue, increased hepatic glucose produc- tion, and inadequate compensation by the pancreatic -cells, ultimately leading to fasting hyperglycemia (1,2). To clarify the pathogenesis of type 2 diabetes, we and others have dis- rupted genes for proteins involved in the insulin-signaling cascade in mice, including the insulin receptor (IR) (3,4), insulin receptor substrate (IRS)-1 (5,6) and IRS-2 (7), and the insulin-sensitive glucose transporter GLUT4 (8). This research has provided important insights into the role of each of these proteins in insulin action. More recently, we have developed genetic models to assess the contribution of individual insulin-sensitive tis- sues to glucose homeostasis and to the development of type 2 diabetes using the Cre-loxP–mediated recombination strategy to inactivate the IR gene in a tissue-specific fashion. Although insulin resistance in muscle is an early defect in the pathogenesis of type 2 diabetes, muscle-specific insulin receptor knockout (MIRKO) mice show no alteration in glu- cose homeostasis, but rather manifest alterations in circu- lating triglycerides, free fatty acid (FFA) levels, and fat mass (9). In contrast, the pancreatic -cell insulin receptor knock- out (IRKO) mice exhibit a selective loss of acute insulin release in response to glucose, resulting in a progressive impairment of glucose tolerance (10). Because type 2 diabetes is characterized by a combination of insulin resistance in skeletal muscle and impaired insulin secretion from pancre- atic -cells, we have generated a double tissue-specific knockout of the insulin receptor in the pancreatic -cell and in skeletal muscle (IRKO-MIRKO) to determine if these defects might synergize or alter the phenotype of the indi- vidual tissue defects. RESEARCH DESIGN AND METHODS Creation of mutant mice and polymerase chain reaction genotyping. Creation of MIRKO mice and IRKO mice (i.e., mice homozygous for the IR lox sites, but carrying either the MCK-Cre or RIP-Cre transgene) has been described previously (9,10). The IR(lox/lox) mouse (carrying the IR allele with loxP sites flanking exon 4) was bred with the MCK-Cre transgenic mouse and the RIP-Cre transgenic mouse to generate MIRKO and IRKO mice respec- tively. To generate the double knockout (IRKO-MIRKO) mouse, IRKO mice were bred with MIRKO mice. The resulting F1 generation led to four dif- ferent genotypes on a similar mixed genetic background: control mice [IR(lox/lox)], MIRKO mice [IR(lox/lox): MCK-Cre (+/–)], IRKO mice [IR(lox/lox: RIP-Cre (+/–)], and IRKO -MIRKO mice [IR(lox/lox): MCK-Cre (+/–): RIP-Cre (+/–)]. To homogenize the genetic background, the F1 gener- ation was used for multiple breedings to obtain the F2 generation. Among breeders, a given male was never bred with the same female twice. No other mouse was introduced in the breeding scheme. All genotyping was per- formed by polymerase chain reaction (PCR) using genomic DNA from tail biopsies of 3- to 4-week-old mice. The sequence of the 5' primer used to iden- tify the presence of the MCK-Cre transgene is AGA TGA CCT TGA ACT GCT GG. The sequence of the 5' primer used to identify the presence of the RIP- Cre transgene is CTC TGG CCA TCT GCT GAT CC. The sequence of the 3' primer, designed from the Cre sequence, is common for both: CGC GCC TGA AGA TAT AGA AG. From the Research Division, Joslin Diabetes Center and Department of Med- icine, Harvard Medical School, Boston, Massachusetts. Address correspondence and reprint requests to C. Ronald Kahn, Research Division, Joslin Diabetes Center, One Joslin Place, Boston, MA 02215. E-mail: c.ronald.kahn@joslin.harvard.edu. Received for publication 8 March 2000 and accepted in revised form 30 August 2000. 2-DG, 2-[ 3 H]deoxyglucose; 2-DG-6-P, [ 3 H]-deoxyglucose-6-phosphate; AUC, area under the curve; ELISA, enzyme-linked immunosorbent assay; FFA, free fatty acid; GSA, glucose specific activity; GSIS, glucose stimula- tion of insulin secretion; GTT, glucose tolerance test; IPGTT, intraperi- toneal glucose tolerance test; IR, insulin receptor; IRS, insulin receptor sub- strate; ITT, insulin tolerance test; PCR, polymerase chain reaction. Downloaded from http://diabetesjournals.org/diabetes/article-pdf/49/12/2126/365623/11118016.pdf by guest on 04 November 2022