Application of muscle biopsy in diabetes

Insulin resistance in skeletal muscle is a major hallmark of type 2 diabetes (Beck-Nielsen & Groop 1994; Beck-Nielsen 1998; Beck-Nielsen et al. 2003). During the past two decades, skeletal muscle biopsies have been increasingly applied in the search for biochemical and molecular abnormalities responsible for insulin resistance. It is evident that type 2 diabetes is caused by a complex interplay between genetic and environmental factors. The latter include intrauterine malnutrition and postnatal factors such as obesity, physical inactivity and modern Western lifestyle, as well as the metabolic milieu associated with type 2 diabetes and prediabetes, including glucose intolerance, hyperglycaemia, hyperlipidaemia and hyperinsulinaemia (Beck-Nielsen & Groop 1994; Beck-Nielsen 1998; Beck-Nielsen et al. 2003). The choice of study design is therefore extremely important for the interpretation of data obtained (Table 14.1).

Novel potential markers of insulin resistance and type 2 diabetes are usually studied first in muscle samples obtained from type 2 diabetic subjects and matched non-diabetic control subjects, preferably including both an obese and a lean control group. If abnormalities are found in these studies, the next step is to test for these defects in glucose-tolerant non-obese subjects with prediabetes such as first degree relatives of patients with type 2 diabetes (Vaag et al. 1992), or glucose-tolerant non-diabetic co-twins of monozygotic twin-pairs discordant for type 2 diabetes (Vaag et al. 1996). Abnormalities confirmed in prediabetic subjects are less likely to be due to environmental factors and provide support for early defects having genetic origin. It is possible to study in vivo abnormalities in cultured skeletal muscle cells

(myotubes) from patients with type 2 diabetes, which are assumed to provide evidence for early defects (see later).

Studies of the biochemical and molecular mechanisms of skeletal muscle insulin resistance in the fasting basal and insulin-stimulated states have been performed with or without intervention (such as endurance-training, treatment with drugs, lipid infusion etc.). Some methodological aspects may affect the data obtained. It is often useful to combine muscle sampling with the euglycaemic-hyperinsulinaemic clamp technique and indirect calorimetry (Table 14.1). Combined with tracer-technology, these techniques allow assessment of rates of glucose disposal, glycolytic flux, glucose storage, non-oxidative glucose metabolism and glucose and lipid oxidation, and hence allow us to investigate the relationship of these parameters to biochemical and molecular findings in muscle biopsies (Vaag et al. 1992, 1996; H0jlund et al. 2003). Drawing conclusions from such experiments, it should be recognised that whole-body indirect calorimetry only reflects glucose metabolism in skeletal muscle during insulin stimulation (Kelley & Mandarino 2000). It is possible to circumvent this problem by using limb-balance techniques, which allow assessment of substrate metabolism (indirect calorimetry) across a large bed of muscle (Kelley & Simoneau 1994; Mandarino et al. 1996).

Another important aspect to consider when studying the effects of insulin on muscle enzymes, genes and metabolites, is whether to stimulate with physiological (200-800 pmol/l) or supraphysiological (3,000-6,000 pmol/l) concentrations of insulin (H0jlund et al. 2003; Kim et al. 2003), and whether insulin-stimulated muscle samples should be obtained in the steady-state period of a clamp after hours of insulin stimulation (H0jlund et al. 2003; Kim et al. 2003), or after 30-40 min of insulin stimulation before euglycaemia or steady-state are obtained (Cusi et al. 2000). Sometimes insulin activation of enzymes reported in rodent muscle or cell lines using extreme insulin levels cannot be confirmed in human muscle biopsies using physiological concentrations of insulin. Moreover, in rodent muscle the response of enzymes to insulin may be transient, whereas in human skeletal muscle the effects of insulin on signaling enzymes are sustained for several hours (Wojtaszewski et al. 2000; Grimmsmann et al. 2002; Kim et al. 2003).

Using a diversity of study designs (Table 14.1), we have studied a number of enzymes, genes and metabolites in skeletal muscle samples obtained by the percutaneous needle biopsy technique. This includes biochemical determination of 1) glucose, glucose-6-phosphate, lactate and glycogen in muscle cells (Damsbo et al. 1991; Vaag et al. 1991, 1992); 2) activity, phosphorylation and/or protein content of insulin signaling enzymes including insulin receptor tyrosine kinase (IRTK), phosphotyrosine phosphatase (PTPase), insulin receptor substrate-1 and -2 (IRS-1 and -2), phosphoinositide-3-kinase (PI3K), phosphoinositide-dependent-kinase-1 (PDK-1), glucose transporter 4 (GLUT4), Akt/protein kinase B (PKB), glycogen synthase kinase-3 (GSK-3) and glycogen synthase (GS) (Mandarino et al. 1987; Wright et al. 1988; Handberg et al. 1990, 1993; Damsbo et al. 1991, 1998; Vaag et al. 1991, 1992a, 1992b, 1996; Worm et al. 1996; Meyer et al. 2002a, 2002b; H0jlund et al. 2003; Levin et al. 2004); as well as 3) enzymes in other pathways regulating glucose and lipid metabolism, such as pyruvate dehydrogenase (PDH), phosphofructokinase (PFK), protein phosphatase 2A (PP2A), AMP-activated protein kinase (AMPK) and acetyl-carboxylase CoA (ACC) (Mandarino et al. 1987; Wright et al. 1988; H0jlund et al. 2002, 2004). The most consistent abnormality associated with insulin resistance in subjects with type 2 diabetes and prediabetes is impaired insulin activation of GS (Wright et al. 1988; Vaag et al. 1992a; Damsbo et al. 1998; H0jlund et al. 2003). A recent study suggests that this may involve

Figure 14.3 Impaired Insulin Activation of Muscle GS in Type 2 Diabetes The effect of insulin on: A) glycogen synthase kinase-3a (GSK-3a); B) glycogen synthase (GS) activity, given as fractional velocity; C) phosphorylation of GS at sites 3a+3b; and D) phosphorylation of GS at sites 2+2a in skeletal muscle biopsies from 10 obese type 2 diabetic patients and 10 obese non-diabetic control subjects. Muscle biopsies were obtained from the vastus lateralis muscle before (white bars) and after (black bars) a 4h euglycaemic-hyperinsulinaemic clamp using insulin infusion rate of 40mU/min/m2. **P < 0.01 vs basal; ffP < 0.01 vs control.

Figure 14.3 Impaired Insulin Activation of Muscle GS in Type 2 Diabetes The effect of insulin on: A) glycogen synthase kinase-3a (GSK-3a); B) glycogen synthase (GS) activity, given as fractional velocity; C) phosphorylation of GS at sites 3a+3b; and D) phosphorylation of GS at sites 2+2a in skeletal muscle biopsies from 10 obese type 2 diabetic patients and 10 obese non-diabetic control subjects. Muscle biopsies were obtained from the vastus lateralis muscle before (white bars) and after (black bars) a 4h euglycaemic-hyperinsulinaemic clamp using insulin infusion rate of 40mU/min/m2. **P < 0.01 vs basal; ffP < 0.01 vs control.

hyperphosphorylation of GS at sites 2+2a. This seems to counteract a normal insulinmediated dephosphorylation of GS at sites 3a+3b in patients with type 2 diabetes (H0jlund et al. 2003) (Figure 14.3).

An increasing number of enzymes either directly involved in insulin signaling or working as modulators of insulin signaling, metabolic fuel regulators or nutrient sensors, have been suggested to be involved in the pathogenesis of skeletal muscle insulin resistance (Shulman 2000, 2004; Evans et al. 2002; Schmitz-Peiffer 2002; Wells et al. 2003; Krebs & Roden 2004; Pirola et al. 2004). Therefore, there has been a growing demand for global approaches capable of evaluating these multiple genes and proteins, and their modifications, simultaneously. Indeed, two such techniques - gene expression profiling using cDNA microarray and quantitative proteomics - emerged in the mid 90s. Recently, the application of these novel global approaches has revealed a coordinated down-regulation of genes and proteins involved in mitochondrial oxidative phosphorylation and increased cellular stress in skeletal muscle biopsies on subjects with type 2 diabetes and prediabetes (Sreekumar et al. 2002; H0jlund et al. 2003; Mootha et al. 2003; Patti et al. 2003). Together with recent morphological studies of muscle mitochondria (see later) and the use of nuclear magnetic resonance spectroscopy, they have provided evidence for perturbations in skeletal muscle mitochondrial metabolism in the pathogenesis of type 2 diabetes (Petersen et al. 2004, 2005; Lowell & Shulman 2005). These abnormalities have been hypothesised to be responsible for lipid accumulation and increased oxidative stress, which may cause activation of lipid- and stress-activated serine/threonine protein kinases, with subsequent inhibitory modulation of insulin signaling (Kelley & Mandarino 2000; Evans et al. 2002; Schmitz-Peiffer 2002; Shulman 2004). Taken together, these novel insights into the molecular mechanisms underlying insulin resistance will inevitably cause an even higher interest in studying skeletal muscle biopsies in the future.

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