Arthur Jenkins

Skeletal Muscle Triglyceride Levels Are Inversely Related to Insulin Action

In animal studies, increased amounts of triglyceride associated with skeletal muscle (mTG) correlate with reduced skeletal muscle and whole body insulin action. The aim of this study was to test this relationship in humans. Subjects were 38 nondiabetic male Pima Indians (mean age 28 ± 1 years). Insulin sensitivity at physiological (M) and supraphysiological (MZ) insulin levels was assessed by the euglycemic clamp. Lipid and carbohydrate oxidation were determined by indirect calorimetry before and during insulin administration. mTG was determined in vastus lateralis muscles obtained by percutaneous biopsy. Percentage of body fat (mean 29 ± 1%, range 14–44%) was measured by underwater weighing. In simple regressions, negative relationships were found between mTG (mean 5.4 ± 0.3 μmol/g, range 1.3–1.9 μmol/g) and log10M (r = −0.53, P ≤ 0.001), MZ (r = −0.44, P = 0.006), and nonoxidative glucose disposal (r = −0.48 and −0.47 at physiological and supraphysiological insulin levels, respectively, both P = 0.005) but not glucose or lipid oxidation. mTG was not related to any measure of adiposity. In multiple regressions, measures of insulin resistance (log10M, MZ, log10[fasting insulin]) were significantly related to mTG independent of all measures of obesity (percentage of body fat, BMI, waist-to-thigh ratio). In turn, all measures of obesity were related to the insulin resistance measures independent of mTG. The obesity measures and mTG accounted for similar proportions of the variance in insulin resistance in these relationships. The results suggest that in this human population, as in animal models, skeletal muscle insulin sensitivity is strongly influenced by local supplies of triglycerides, as well as by remote depots and circulating lipids. The mechanism(s) underlying the relationship between mTG and insulin action on skeletal muscle glycogen synthesis may be central to an understanding of insulin resistance.

Influence of dietary fat composition on development of insulin resistance in rats. Relationship to muscle triglyceride and omega-3 fatty acids in muscle phospholipid.

High levels of some but not all dietary fats lead to insulin resistance in rats. The aim of this study was to investigate the important determinants underlying this observation. Insulin action was assessed with the euglycemic clamp. Diets high in saturated, monounsaturated (ω-9), or polyunsaturated (ω-6) fatty acids led to severe insulin resistance; glucose infusion rates [GIR] to maintain euglycemia at ∼1000 pM insulin were 6.2 ± 0.9, 8.9 ± 0.9, and 9.7 ± 0.4 mg · kg−1 · min−1, respectively, versus 16.1 ± 1.0 mg · kg−1 · min−1 in chow-fed controls. Substituting 11% of fatty acids in the polyunsaturated fat diet with long-chain ω-3 fatty acids from fish oils normalized insulin action (GIR 15.0 ± 1.3 mg · kg−1 · min−1). Similar replacement with short-chain ω-3 (α-linolenic acid, 18:3ω3) was ineffective in the polyunsaturated diet (GIR 9.9 ± 0.5 mg · kg−1 · min−1) but completely prevented the insulin resistance induced by a saturated-fat diet (GIR 16.0 ± 1.5 mg · kg−1 · min−1) and did so in both the liver and peripheral tissues. Insulin sensitivity in skeletal muscle was inversely correlated with mean muscle triglyceride accumulation (r = 0.95 and 0.86 for soleus and red quadriceps, respectively; both P = 0.01). Furthermore, percentage of long-chain ω-3 fatty acid in phospholipid measured in red quadriceps correlated highly with insulin action in that muscle (r = 0.97). We conclude that 1) the particular fatty acids and the lipid environment in which they are presented in high-fat diets determine insulin sensitivity in rats; 2) impaired insulin action in skeletal muscle relates to triglyceride accumulation, suggesting intracellular glucose–fatty acid cycle involvement; and 3) long-chain ω-3 fatty acids in phospholipid of skeletal muscle may be important for efficient insulin action.

The Relation between Insulin Sensitivity and the Fatty-Acid Composition of Skeletal-Muscle Phospholipids

BACKGROUND Insulin resistance and hyperinsulinemia are features of obesity, non-insulin-dependent diabetes mellitus, and other disorders. Skeletal muscle is a major site of insulin action, and insulin sensitivity may be related to the fatty-acid composition of the phospholipids within the muscle membranes involved in the action of insulin. METHODS We determined the relation between the fatty-acid composition of skeletal-muscle phospholipids and insulin sensitivity in two groups of subjects. In one study, we obtained samples of the rectus abdominis muscle from 27 patients undergoing coronary artery surgery; fasting serum insulin levels provided an index of insulin sensitivity. In the second study, a biopsy of the vastus lateralis muscle was performed in 13 normal men, and insulin sensitivity was assessed by euglycemic-clamp studies. RESULTS In the patients undergoing surgery, the fasting serum insulin concentration (a measure of insulin resistance) was negatively correlated with the percentage of individual long-chain polyunsaturated fatty acids in the phospholipid fraction of muscle, particularly arachidonic acid (r = -0.63, P < 0.001); the total percentage of C20-22 polyunsaturated fatty acids (r = -0.68, P < 0.001); the average degree of fatty-acid unsaturation (r = -0.61, P < 0.001); and the ratio of the percentage of C20:4 n-6 fatty acids to the percentage of C20:3 n-6 fatty acids (r = -0.55, P < 0.01), an index of fatty-acid desaturase activity. In the normal men, insulin sensitivity was positively correlated with the percentage of arachidonic acid in muscle (r = 0.76, P < 0.01), the total percentage of C20-22 polyunsaturated fatty acids (r = 0.76, P < 0.01), the average degree of fatty-acid unsaturation (r = 0.62, P < 0.05), and the ratio of C20:4 n-6 to C20:3 n-6 (rho = 0.76, P = 0.007). CONCLUSIONS Decreased insulin sensitivity is associated with decreased concentrations of polyunsaturated fatty acids in skeletal-muscle phospholipids, raising the possibility that changes in the fatty-acid composition of muscles modulate the action of insulin.

Abdominal Fat and Insulin Resistance in Normal and Overweight Women: Direct Measurements Reveal a Strong Relationship in Subjects at Both Low and High Risk of NIDDM

Insulin resistance appears to be central to obesity, NIDDM, hyperlipidemia, and cardiovascular disease. While obese women with abdominal (android) fat distribution are more insulin resistant than those with peripheral (gynecoid) obesity, in nonobese women, the relationship between abdominal fat and insulin resistance is unknown. By measuring regional adiposity with dual-energy X-ray absorptiometry and insulin sensitivity by euglycemic-hyperinsulinemic clamp in 22 healthy women, with a mean ± SE body BMI of 26.7 ± 0.9 kg/m2 and differing risk factors for NIDDM, we found a strong negative relationship between central abdominal (intra-abdominal plus abdominal subcutaneous) fat and whole-body insulin sensitivity (r = −0.89, P < 0.0001) and nonoxidative glucose disposal (r = −0.77, P < 0.001), independent of total adiposity, family history of NIDDM, and past gestational diabetes. There was a large variation in insulin sensitivity, with a similar variation in central fat, even in those whose BMI was <25 kg/m2. Abdominal fat had a significantly stronger relationship with insulin sensitivity than peripheral nonabdominal fat (r2 = 0.79 vs. 0.44), and higher levels were associated with increased fasting nonesterified fatty acids, lipid oxidation, and hepatic glucose output. Because 79% of the variance in insulin sensitivity in this heterogeneous population was accounted for by central fat, abdominal adiposity appears to be a strong marker and may be a major determinant of insulin resistance in women.

Development of Muscle Insulin Resistance After Liver Insulin Resistance in High-Fat–Fed Rats

Muscle and hepatic insulin resistance are two major defects of non-insulin-dependent diabetes mellitus. Dietary factors may be important in the etiology of insulin resistance. We studied progressive changes in the development of high-fat–diet–induced insulin resistance in tissues of the adult male Wistar rat. In vivo insulin action was compared 3 days and 3 wk after isocaloric synthetic high-fat or high-starch feeding (59 and 10% cal as fat, respectively). Basal and insulin-stimulated glucose metabolism were assessed in the conscious 5- to 7-h fasted state with the euglycemic clamp (600 pM insulin) with a [3-3H]-glucose infusion. Fat feeding significantly reduced suppressibility of hepatic glucose output by insulin after both 3 days and 3 wk of diet (P < 0.01). However, a significant impairment of insulin-mediated peripheral glucose disposal was only present after 3 wk of diet. Further in vivo [3H]-2-deoxyglucose uptake studies supported this finding and demonstrated adipose but not muscle insulin resistance after 3 days of high-fat feeding. Muscle triglyceride accumulation due to fat feeding was not significant at 3 days but had doubled by 3 wk in red muscle (P < 0.001) compared with starch-fed controls. By 3 wk, high-fat—fed animals had developed significant glucose intolerance. We concludethat fat feeding induces insulin resistance in liver and adipose tissue before skeletal muscle with early metabolic changes favoring an oversupply of energy substrate to skeletal muscle relative to metabolic needs. This may generate later muscle insulin resistance.

Dietary fats and insulin action

The history of research into the relationship between dietary fat intake and impaired insulin action has its origin in the work of Himsworth approximately 60 years ago [1, 2]. In a series of pioneering studies using crude indices of insulin action and limited subject numbers (only one in an often quoted paper!), Himsworth linked high levels of fat intake with insulin resistance, and conversely, improved insulin action with predominantly carbohydrate diet. However, the link was tenuous and really apparent only at the extreme ends of the dietary spectrum (<20 or > 80 % of calories as fat). These studies, flawed as they were, influenced the field enormously in the absence of any significant work in the area for a remarkable period of time. The conclusions reached by Himsworth were reinforced to some extent by misuse of glucose tolerance data that showed deteriorations following periods on diets extremely low in carbohydrate (< 50 g/day) and improvement on liquid formula diets providing a remarkably high percentage (75-85 %) of calories from carbohydrate [3-5]. It was really not until the 1980 s that the development of acceptable techniques for the measurement of insulin action in vivo allowed the relationship between dietary fat intake and insulin action to be accurately assessed. These studies have essentially been confined to investigations in rodents and humans.

Important role of hypothalamic Y2 receptors in body weight regulation revealed in conditional knockout mice

Neuropeptide Y is implicated in energy homeostasis, and contributes to obesity when hypothalamic levels remain chronically elevated. To investigate the specific role of hypothalamic Y2 receptors in this process, we used a conditional Y2 knockout model, using the Cre-lox system and adenoviral delivery of Cre-recombinase. Hypothalamus-specific Y2-deleted mice showed a significant decrease in body weight and a significant increase in food intake that was associated with increased mRNA levels for the orexigenic NPY and AgRP, as well as the anorexic proopiomelanocortin (POMC) and cocaine- and amphetamine-regulated transcript (CART) in the arcuate nucleus. These hypothalamic changes persisted until at least 34 days after Y2 deletion, yet the effect on body weight and food intake subsided within this time. Plasma concentrations of pancreatic polypeptide and corticosterone were 3- to 5-fold increased in hypothalamus-specific Y2 knockout mice. Germ-line Y2 receptor knockout also produced a significant increase in plasma levels of pancreatic polypeptide. However, these mice differed from conditional knockout mice in that they showed a sustained reduction in body weight and adiposity associated with increased NPY and AgRP but decreased POMC and CART mRNA levels in the arcuate nucleus. The transience of the observed effects on food intake and body weight in the hypothalamus-specific Y2 knockout mice, and the difference of this model from germ-line Y2 knockout mice, underline the importance of conditional models of gene deletion, because developmental, secondary, or extrahypothalamic mechanisms may mask such effects in germ-line knockouts.

A New Antidiabetic Agent, BRL 49653, Reduces Lipid Availability and Improves Insulin Action and Glucoregulation in the Rat

Thiazolidinediones offer promise as oral insulin-sensitizing agents. The effects of a new, high-potency compound (BRL 49653, SmithKline Beecham, Epsom, U.K.) were examined in insulin-resistant (high-fat–fed, HF) and control (high-starch–fed, HS) rats. The diet period was 3 weeks, with a BRL 49653 (10 μmol · kg−1 · day−1) or vehicle gavage on the last 4 days. Then basal or euglycemic clamp studies were performed on animals in the conscious fasted state. In the basal state, BRL 49653 produced many similar metabolic responses in HF and HS rats (reduced insulin, glycerol, ketone body, and nonesterified fatty acid levels, reduced whole body glucose turnover, reduced brown adipose tissue glucose metabolism, and increased cardiac glucose metabolism and GLUT4 levels). In contrast, under euglycemic clamp conditions (500 pmol/l insulin), BRL 49653 only induced changes in the HF group (increased glucose infusion rate from 12.2 ± 0.9 to 21.6 ± 1.1 mg · kg−1 · min−1 [P < 0.001], increased insulin suppressibility of hepatic glucose production [P < 0.01], and increased glucose uptake in muscle [P < 0.01]). BRL 49653 significantly reduced liver but not muscle triglyceride content in HF rats. We conclude that the agent has a general effect on lowering circulating lipid and insulin levels, manifested similarly in normal and insulin-resistant rats, but that enhancement of peripheral insulin action is confined to insulin-resistant rats. Therefore, the hypoinsulinemic action of the thiazolidinediones is probably not related simply to improved peripheral insulin sensitivity. The pattern of individual tissue response to BRL 49653 suggests that altered lipid availability is an important mediator of its effects on glucose metabolism.

Obesity Is an Important Determinant of Baseline Serum C-Reactive Protein Concentration in Monozygotic Twins, Independent of Genetic Influences

Background—C-reactive protein (CRP) values predict atherothrombotic cardiovascular disease and type 2 diabetes mellitus. Associations between CRP and obesity, predominantly assessed anthropometrically, may partly explain these observations. Previous studies have been unable to control for genetic influences on CRP and obesity. The aim of this study was to examine the relationship between CRP and accurately measured body fat, lipids, apolipoproteins, blood pressure, and environmental and behavioral factors, independent of genetic influences. Methods and Results—One hundred ninety-four healthy female twins (age 57.2±7 years) were studied after excluding pairs with CRP values >10 mg/L. Total body fat and central abdominal fat (CAF) were measured by dual-energy x-ray absorptiometry. CRP concentration was strongly related to surrogate and direct measures of body fat (r =0.31 to 0.54, P <0.001), diastolic blood pressure (r =0.20, P =0.003), and lipid and apolipoprotein levels (r =0.21 to 0.51, P <0.008). Light-to-moderate alcohol consumers and nonusers of hormone replacement therapy (HRT) had lower CRP levels than abstainers and HRT users, respectively. In stepwise multiple regression analysis, CAF, triglycerides, apolipoprotein B, and HRT use explained 46% of the variance in circulating CRP. In analyses controlling for genetic influences in monozygotic twins, within-pair differences in CRP were associated with within-pair differences in total body fat (r =0.39, P <0.001), CAF (r =0.34, P =0.002), diastolic blood pressure (r =0.24, P =0.03), apolipoprotein AI (r =−0.33, P =0.01), HDL cholesterol (r =−0.42, P =0.001), and triglycerides (r =0.35, P =0.007). Conclusions—CRP was strongly related to total and central abdominal obesity, blood pressure, and lipid levels, independent of genetic influences. These relationships are likely to contribute significantly to prospective associations between CRP and type 2 diabetes and coronary events.

SKELETAL MUSCLE MEMBRANE LIPIDS AND INSULIN RESISTANCE

Skeletal muscle plays a major role in insulin-stimulated glucose disposal. This paper reviews the range of evidence in humans and experimental animals demonstrating close associations between insulin action and two major aspects of muscle morphology: fatty acid composition of the major structural lipid (phospholipid) in muscle cell membranes and relative proportions of major muscle fiber types. Workin vitro andin vivo in both rats and humans has shown that incorporation of more unsaturated fatty acids into muscle membrane phospholipid is associated with improved insulin action. As the corollary, a higher proportion of saturated fats is linked to impairment of insulin action (insulin resistance). Studiesin vitro suggest a causal relationship. Among polyunsaturated fatty acids (PUFA) there is some, but not conclusive, evidence that ω-3 (n−3) PUFA may play a particular role in improving insulin action; certainly a high n−6/n−3 ratio appears deleterious. In relation to fiber type, the more highly oxidative, insulin-sensitive type 1 and type 2a fibers have a higher percentage of unsaturated fatty acids, particularly n−3, in their membrane phospholipid, compared to the insulin-resistant, glycolytic, type 2b fibers. These variables, however, can be separated and may act in synergy to modulate insulin action. It remains to establish whether lifestyle (e.g., dietary fatty acid profile and physical activity), genetic predisposition, or a combination are the prime determinants of muscle morphology (particularly membrane lipid profile) and hence insulin action.