Insulin Resistance In Animals Research Articles

Diabetes-related Changes in cAMP Response Element-binding Protein Content Enhance Smooth Muscle Cell Proliferation and Migration

We hypothesized that diabetes and glucose-induced reactive oxygen species lead to depletion of cAMP response element-binding protein (CREB) content in the vasculature. In primary cultures of smooth muscle cells (SMC) high medium glucose decreased CREB function but increased SMC chemokinesis and entry into the cell cycle. These effects were blocked by pretreatment with the antioxidants. High glucose increased intracellular reactive oxygen species detected by CM-H(2)DCFA. SMC exposed to oxidative stress (H(2)O(2)) demonstrated a 3.5-fold increase in chemokinesis (p < 0.05) and accelerated entry into cell cycle, accompanied by a significant decrease in CREB content. Chronic oxidative challenge similar to the microenvironment in diabetes (glucose oxidase treatment) decreases CREB content (40-50%). Adenoviral-mediated expression of constitutively active CREB abolished the increase in chemokinesis and cell cycle progression induced by either high glucose or oxidative stress. Analysis of vessels from insulin resistant or diabetic animals indicates that CREB content is decreased in the vascular stroma. Treatment of insulin-resistant animals with the insulin sensitizer rosiglitazone restores vessel wall CREB content toward that observed in normal animals. In summary, high glucose and oxidative stress decrease SMC CREB content increase chemokinesis and entry into the cell cycle, which is blocked by antioxidants or restoration of CREB content. Thus, decreased vascular CREB content could be one of the molecular mechanisms leading to increased atherosclerosis in diabetes.

EFFECT OF CARBOHYDRATE SUPPLEMENTATION ON THE TIME-COURSE OF POST-EXERCISE GLYCOGEN RESYNTHESIS IN INSULIN-RESISTANT ZUCKER RATS

Glucose transport is the rate-limiting step for glucose uptake and storage in skeletal muscle. We determined the effect of an acute bout of swimming (8 × 30 min) followed by either carboydrate administration (i.p. 0.5 mg/g weight of glucose and free access to chow; CHO) or fasting (FAST) on the time-course (0, 30 min, 6 h post-exercise) of glycogen resynthesis in skeletal muscle (soleus) and liver from lean (L) and insulin resistant (IR) obese Zucker rats. Muscle [glycogen] at rest was similar between L and IR (29 ± 2 vs. 38 ± 4 μmol/g wet weight; mean ± SEM) and was reduced by ∼65% after exercise (p < 0.05). Liver [glycogen] was greater in IR than L (334 ± 31 vs. 247 ± 16 μmol/g; p < 0.05) and fell by 44% and 94% after exercise (P < 0.05). Muscle and liver [glycogen] remained unchanged after 30 min in L for both FAST and CHO. Liver and muscle [glycogen] was also unaltered by FAST in IR after 30 min, but muscle [glycogen] increased 68% from 14 ± 1 to 24 ± 3 μmol/g (P < 0.05) with CHO. After 6 h FAST, muscle [glycogen] of IR was restored to pre-exercise levels, but liver [glycogen] did not change with FAST or CHO. Muscle [glycogen] for L remained 56% below rest after 6 h, while liver [glycogen] had not increased above post-exercise values. After 6 h CHO, liver [glycogen] in L had been restored to resting levels while muscle [glycogen] was supercompensated by 68% (50 ± 4 μmol/g; p < 0.05). At this time, muscle [glycogen] in IR was 44% higher than rest (55 ± 3 μmol/g; p < 0.05). We conclude that with carbohydrate administration, 1) the rapid (≤ 30 min post-exercise) phase of muscle glycogen resynthesis was greater in insulin-resistant than lean animals, 2) most of the muscle glycogen resynthesis in lean animals occurred in the slow (> 30 min) phase, 3) there was an additive effect on muscle glycogen resynthesis in insulin-resistant animals, and 4) muscle, but not liver, glycogen was supercompensated 6 h following an intense bout of exercise in both lean and insulin-resistant obese Zucker rats.

Beta cell adaptation to dexamethasone-induced insulin resistance in rats involves increased glucose responsiveness but not glucose effectiveness.

Islet beta cell adaptation to dexamethasone-induced insulin resistance was characterized with respect to glucose-stimulated insulin secretion and islet innervation. Male Sprague-Dawley rats were injected daily with dexamethasone (2 mg/kg for 12 days), which resulted in hyperinsulinemia and hyperglycemia compared with controls (which were injected with sodium chloride). Insulin secretion was characterized in collagenase-isolated islets. Islet innervation was examined by immunocytochemical analysis of tyrosine hydroxylase, neuropeptide Y (sympathetic nerves), and vasoactive intestinal polypeptide (cholinergic nerves). In islets isolated from the insulin-resistant animals, the insulin response to 3.3 or 8.3 mM glucose was three times greater during perifusion compared with controls (p < 0.001). Incubation of islets at 0 to 20 mM glucose revealed a marked leftward shift of the glucose dose-response relation after dexamethasone treatment (potency ratio, 1.78; p < 0.01), with no difference at 0 or 20 mM glucose. Thus, the potency but not the efficacy of glucose was increased. The number of islet nerves did not differ between dexamethasone-treated rats and controls. Dexamethasone-induced insulin resistance leads to adaptively increased glucose responsiveness of the islet beta cells, with increased potency, but not increased efficacy, of glucose to stimulate insulin secretion without any evidence of altered islet innervation.

Reduced glucose uptake precedes insulin signaling defects in adipocytes from heterozygous GLUT4 knockout mice.

Decreased GLUT4 expression, impaired insulin receptor (IR), IRS-1, and pp60/IRS-3 tyrosine phosphorylation are characteristics of adipocytes from insulin-resistant animal models and obese NIDDM humans. However, the sequence of events leading to the development of insulin signaling defects and the significance of decreased GLUT4 expression in causing adipocyte insulin resistance are unknown. The present study used male heterozygous GLUT4 knockout mice (GLUT4(+/-)) as a novel model of diabetes to study the development of insulin signaling defects in adipocytes with the progression of whole body insulin resistance and diabetes. Male GLUT4(+/-) mice with normal fed glycemia and insulinemia (N/N), normal fed glycemia and hyperinsulinemia (N/H), and fed hyperglycemia with hyperinsulinemia (H/H) exist at all ages. The expression of GLUT4 protein and the maximal insulin-stimulated glucose transport was 50% decreased in adipocytes from all three groups. Insulin signaling was normal in N/N adipose cells. From 35 to 70% reductions in insulin-stimulated tyrosine phosphorylation of IR, IRS-1, and pp60/IRS-3 were noted with no changes in the cellular content of IR, IRS-1, and p85 in N/H adipocytes. Insulin-stimulated protein tyrosine phosphorylation was further decreased to 12-23% in H/H adipose cells accompanied by 42% decreased IR and 80% increased p85 expression. Insulin-stimulated, IRS-1-associated PI3 kinase activity was decreased by 20% in N/H and 68% reduced in H/H GLUT4(+/-) adipocytes. However, total insulin-stimulated PI3 kinase activity was normal in H/H GLUT4(+/-) adipocytes. Taken together, these results strongly suggest that hyperinsulinemia triggers a reduction of IR tyrosine kinase activity that is further exacerbated by the appearance of hyperglycemia. However, the insulin signaling cascade has sufficient plasticity to accommodate significant changes in specific components without further reducing glucose uptake. Furthermore, the data indicate that the cellular content of GLUT4 is the rate-limiting factor in mediating maximal insulin-stimulated glucose uptake in GLUT4(+/-) adipocytes.

Pioglitazone attenuates basal and postprandial insulin concentrations and blood pressure in the spontaneously hypertensive rat

Subjects with hypertension are hyperinsulinemic and resistant to insulin-stimulated glucose uptake. A similar paradigm is found in the spontaneously hypertensive rat (SHR). These findings suggest the possibility that insulin resistance and hyperinsulinemia may play an important role in blood pressure regulation. Pioglitazone, a thiazolidinedione derivative, sensitizes target tissues to insulin and decreases hyperglycemia and hyperinsulinemia in various insulin-resistant animals. The purpose of this study was to assess the influence of pioglitazone administration on pre- and postprandial glucose and insulin concentrations and determine whether changes in β-cell secretion resulted in any change in blood pressure measurements. Twelve SHR were fed custom diets ad libitum, six with and six without pioglitazone (20 mg/kg chow). Fasting and postprandial glucose levels were unaltered by pioglitazone treatment. Fasting insulin concentrations were similar at week 1, but were significantly lower ( P < .01) in the pioglitazone group at weeks 3 (1.89 ± 0.3 v7.94 ± 1.5 ng/mL) and 4 (4.5 ± 1.4 v9.1 ± 0.7 ng/mL), compared with the control group. Pioglitazone also significantly ( P < .01) lowered postprandial insulin concentrations after an oral glucose challenge. Systolic, mean, and diastolic blood pressures were significantly lower ( P < .01), 177 ± 3 v190 ± 4.7 mm Hg, 162 ± 2.1 v175 ± 5.9 mm Hg, and 156 ± 2.1 v168 ± 6.2 mm Hg, respectively, in the animals receiving pioglitazone versus the control group. Heart rate, body weight, serum cholesterol, and triglyceride levels were comparable between the two groups. In conclusion, pioglitazone significantly decreased fasting and postprandial insulin concentrations and effectively lowered blood pressure in the SHR.

The effects of an angiotensin-converting enzyme inhibitor and an angiotensin II receptor antagonist on insulin resistance in fructose-fed rats

The aim of this study was to compare the effects of an angiotensin-converting enzyme (ACE) inhibitor and an angiotensin II receptor (AT) antagonist on insulin resistance, especially on muscle fiber composition in fructose-induced insulin-resistant and hypertensive rats. Six-week-old male Sprague-Dawley rats were fed either normal rat chow (control) or a fructose-rich diet (FFR). For the last two weeks of a six-week period of either diet, the rats were treated with gum arabic solution as a vehicle (control or FFR), angiotensin-converting enzyme inhibitor (FFR+ACE), temocapril (1 mg/kg/day) or an angiotensin II receptor antagonist (FFR+AT), CS-866 (0.3 mg/kg/day), by gavage, and then the euglycemic hyperinsulinemic glucose clamp technique was performed to evaluate insulin sensitivity. At the end of the glucose clamp, the soleus muscle was dissected for determination of the muscle fiber composition by ATPase methods. Blood pressure at the glucose clamp in the FFR group was significantly higher than that of the control group, and both temocapril and CS-866 significantly lowered the blood pressure of the FFR group. The average rate of glucose infusion during the glucose clamp, as a measure of insulin sensitivity (M value), was significantly lower in the FFR rats compared to the controls (15.4 ± 0.4, 10.9 ± 0.6 mg/kg/min, for control and FFR, respectively, P < .01). Both temocapril and CS-866 partially improved the M values compared to FFR (13.2 ± 0.7, 12.8 ± 0.5 mg/kg/min, for FFR+ACE, FFR+AT, respectively, P < .01 compared with FFR, P< .05 compared with control). The composite ratio of type I fibers of the soleus muscle was decreased significantly in the FFR rats compared with the controls (82% ± 2%, 75% ± 2%, for control and FFR, respectively, P < .01), and both temocapril and CS-866 restored a composite ratio of type I fibers to the same level as that of the controls (81% ± 1%, 80% ± 1% for FFR+ACE and FFR+AT, respectively). The M value was significantly correlated with the composition of type I and type II fibers. These results suggest that the fiber composition of skeletal muscle is correlated to insulin resistance, and that both ACE inhibitors and AT antagonists may modulate the muscle fiber composition in a hypertensive and insulin-resistant animal model, fructose-fed rats, to the same extent.

Chronic infusion of norepinephrine into the VMH of normal rats induces the obese glucose-intolerant state.

Increases in ventromedial hypothalamic (VMH) norepinephrine (NE) levels and/or activities have been observed in a variety of animal models of the obese insulin-resistant condition. This study examined the metabolic effects of chronic NE infusion (25 nmol/h) into the unilateral VMH of normal rats. Within 4 days, VMH NE infusion significantly increased plasma insulin (140%), glucagon (45%), leptin (300%), triglyceride (100%), abdominal fat pad weight (50%), and white adipocyte lipogenic (100%) and lipolytic (100%) activities relative to vehicle-infused rats. Furthermore, isolated islet insulin secretory response to glucose (15 mM) within 4 days of such treatment was increased over twofold (P < 0.05). Among treated animals, fat stores continued to increase over time and plateaued at approximately 2 wk (3-fold increase), remaining elevated to the end of the study (5 wk). By week 4 of treatment, NE infusion induced glucose intolerance as evidenced by a 32% increase in plasma glucose total area under the glucose tolerance test curve (P < 0.01). Whole body fat oxidation rate measured after 5 wk of infusion was significantly increased among treated animals as evidenced by a reduced respiratory quotient (0.87 +/- 0.01) relative to controls (0. 90 +/- 0.01). VMH NE infusion induced hyperphagia (30%) only during the first week and did not affect body weight over the 5-wk period. Increases in VMH NE activity that are common among obese insulin-resistant animal models can cause the development of this obese glucose-intolerant (metabolic) syndrome.

Amelioration by KRP-297, a new thiazolidinedione, of impaired glucose uptake in skeletal muscle from obese insulin-resistant animals.

We examined the effect of KRP-297, a new thiazolidinedione derivative, on glucose uptake in the soleus muscle of two animal models of insulin resistance that show moderate (ob/ob mice) and severe (db/db mice) hyperglycemia. Insulin-stimulated 2-deoxyglucose (2DG) uptake in soleus muscle was 53.8% lower in ob/ob mice versus lean mice (P < .05). When administered to ob/ob mice, KRP-297 (0.3 to 10 mg/kg) decreased plasma glucose and insulin levels and improved the impaired insulin-stimulated 2DG uptake in soleus muscle in a dose-dependent manner. Soleus muscle from db/db mice exhibited defects in both basal (35.0% decrease, P < .01) and insulin-stimulated (50.5% decrease, P < .01) 2DG uptake. These defects were improved by treatment with KRP-297 (0.3 to 10 mg/kg). Moreover, KRP-297 prevented severe hyperglycemia and the marked decrease in pancreatic insulin content in db/db mice. These results suggest that KRP-297 treatment is useful to prevent the development of diabetic syndromes in addition to ameliorating the impaired glucose transport in skeletal muscle.

Long-term infusion of norepinephrine plus serotonin into the ventromedial hypothalamus impairs pancreatic islet function.

To examine the possibility of a cause-effect relationship between enhanced monoamine content in the ventromedial hypothalamus ([VMH] a characteristic of hyperinsulinemic and insulin-resistant animals) and islet dysfunction, we infused norepinephrine ([NE] 25 nmol/h) and/or serotonin ([5-HT] 2.5 nmol/h) into the VMH of normal hamsters for 5 weeks and then examined insulin release from the isolated pancreatic islets. VMH infusion of NE + 5-HT, but not of either neurotransmitter alone, produced a marked leftward shift in the dose-response curve of glucose-induced insulin release (twofold to sixfold increase at 5 to 7.5 mmol/L glucose v vehicle-treated animals). In addition, the islet responsiveness to 1 micromol/L NE and 10 micromol/L acetylcholine was abolished in these NE + 5-HT VMH-infused hamsters. These findings indicate that an increase of NE and 5-HT content in the VMH can induce dysregulation of islet insulin release in response to glucose and neurotransmitters. Inasmuch as VMH NE and 5-HT levels are elevated in hyperinsulinemic and insulin-resistant animals, the present findings suggest that an endogenous increase in these hypothalamic monoamines may contribute to islet dysfunction, which is one of the characteristics of type 2 diabetes.

Insulin-induced tyrosine phosphorylation of Shc in liver, muscle and adipose tissue of insulin resistant rats

Insulin stimulates rapid tyrosine phosphorylation of the protein Shc, which subsequently binds to Grb2, resulting in the activation of a complex mitogenic signaling network. In this study, we examined the levels of Shc protein, its phosphorylation state and Shc–Grb2 association in liver, muscle and adipose tissue before and after insulin administration in three animal models of insulin resistance (chronic dexamethasone treatment, 72-h starvation and aging). There were no differences in Shc protein expression between tissues from control and insulin resistant animals. In fasted hypoinsulinemic rats, there was a decrease in insulin-induced Shc phosphorylation in liver and adipose tissue. However, a significant increase in Shc phosphorylation was observed in liver and muscle from dexamethasone-treated hyperinsulinemic rats and in liver, muscle and adipose tissue of hyperinsulinemic 20-month-old rats. Alterations in Shc phosphorylation correlated well with the level of Shc–Grb2 association. These results indicate that Shc tyrosyl phosphorylation and Shc–Grb2 association are regulated in the different types of insulin resistance and that this regulation is apparently related to the animals’ plasma insulin levels. The Shc–Grb2 association is directly related to the insulin-induced tyrosyl phosphorylation of Shc.

Glucose tolerance and insulin resistance in the JCR:LA-corpulent rat: Effect of miglitol (Bay m1099)

A standardized meal tolerance test (MTT) using 5 g rat chow provides a sensitive index of insulin and glucose metabolism in the insulin-resistant, hyperinsulinemic, hypertriglyceridemic, and atherosclerosis-prone JCR:LA-corpulent (cp) strain of rats. The MTT revealed differences in insulin/glucose metabolism that were not evident in either an intravenous (IVGTT) or intraperitoneal (IPGTT) glucose tolerance test. The glycemic response of control rats to a 5-g carbohydrate test meal containing miglitol (Bay m 1099) was sharply reduced, with a 50% effective dose (ED 50) of 36.4 ± 7.5 mg/100 g food. At a dose of 60 mg/100 g food, the plasma glucose curve was flat and indistinguishable from that found in the nonfed state. The plasma insulin response was similarly reduced, with an ED 50 of 42.8 ± 14.8 mg/100 g food. Obese male rats were treated with miglitol at 60 mg/100 g food from 6 to 12 weeks of age. Treated rats had a significantly reduced food consumption and lower body weight at 12 weeks of age ( P < .05). The treatment resulted in no significant changes in serum lipid concentrations. When subjected to the MTT using control (non—miglitol-containing) food, treated rats demonstrated markedly improved insulin sensitivity, with a greatly reduced insulin response, which may reflect an improved hepatic glucose metabolism. The results confirm that miglitol is highly effective in this obese insulin-resistant animal model. It reduced postprandial glycemic and insulin responses, and on long-term treatment, it improved glucose and insulin metabolism. These beneficial metabolic changes suggest that miglitol may have vascular protective effects in insulin-resistant states.

BM 17.0744: A structurally new antidiabetic compound with insulin-sensitizing and lipid-lowering activity

BM 17.0744 (2,2-dichloro-12-( p-chlorophenyl)-dodecanoic acid) is a substance from a group of ω-substituted alkyl carboxylic acids with the general formula, ring-spacer-carboxylic acid. With BM 17.0744—a compound structurally unrelated to thiazolidinediones—antihyperglycemic and antihyperinsulinemic potency has been demonstrated in various animal models of type II diabetes. The antidiabetic effect is independent of the genetic background of the disease, gender, and animal species. The 24-hour blood glucose profile was dose- and time-dependently improved in ob ob mice after a single and fourth oral administration of 0.3, 1, and 3 mg/kg/d. A dose-dependent reduction of hyperglycemia (10%, 15%, 28%, and 66%) was found in rmdb db mice after the fifth oral administration of 3, 10, 30, and 100 mg/kg/d. Hyperinsulinemia was reduced dose-dependently in yellow KK mice by 1%, 24%, 34%, and 66% after the fifth oral administration of 0.3, 1, 3, and 10 mg/kg/d. Overall glucose metabolism was predominantly higher in euglycemic-hyperinsulinemic clamp studies in obese rmfa fa rats pretreated for 14 days with 10 mg/kg/d BM 17.0744. The data in diabetic and insulin-resistant animals suggest an improvement of insulin action that is supported by enhancement of insulin effects in vitro. There is no evidence of a risk for hypoglycemia in diabetic and metabolically healthy animals. Triglyceride (TG) and cholesterol were reduced in the serum of metabolically healthy rats, as well as serum lipids in db db mice, which suggests this effect is independent of amelioration of the diabetic status. Lipid-lowering effects in diabetic and healthy animals show an additional property of BM 17.0744. Because of its antidiabetic and lipid-lowering potency, the substance is of great interest in treating the metabolic syndrome. Lipid decreases in rats are associated with a dose-dependent increase in carnitine acetyltransferase activity in the liver to about 100-fold (12.5 mg/kg/dl). This together with hepatomegaly in small rodents may indicate peroxisomal proliferation, a phenomenon considered species-specific. Its relevance for humans is well documented for other classes of compounds including fibrates. Specific side effects of insulin sensitizers of the thiazolidinedione type, such as an increase in body weight and heart weight, could not be observed after 4-week oral application of BM 17.0744 in rats. In general, BM 17.0744 was well tolerated in the pharmacological dose range in all species tested.

Synergistic actions of insulin and troglitazone on contractility in endothelium-denuded rat aortic rings.

Insulin attenuates vascular contraction via inhibition of voltage-operated Ca2+ channels and by enhancement of endothelium-dependent vasodilation. Thus it has been suggested that hypertension-associated insulin resistance results from an insensitivity to the hormone's effects on vascular reactivity. This hypothesis has been strengthened by reports that thiazolidinediones, a class of insulin-sensitizing agents, lower blood pressure and improve insulin responsiveness in hypertensive, insulin-resistant animal models. We tested the hypothesis that troglitazone enhances the vasodilating effect of insulin via inhibition of voltage-operated Ca2+ channels in vascular smooth muscle cells. Rat thoracic aortic rings (no endothelium) were suspended in tissue baths for isometric force measurement. Rings were incubated with 0.1 DMSO vehicle (control), troglitazone (10(-5) M), insulin (10(-7) U/l), or both troglitazone and insulin (1 h) and then contracted with phenylephrine (PE), KCl, or BAY K 8644. Troglitazone increased the EC50 values for PE and KCl. Contractions to BAY K 8644 in troglitazone-treated rings were virtually abolished. Insulin alone had no effect on contraction. However, when insulin was combined with troglitazone, the EC50 values for PE and KCl were further increased. Additionally, the maximum contractions to both PE (14 +/- 4% of control) and KCl (12 +/- 2% of control) were reduced. Measurement of Ca2+ concentration ([Ca2+]) with fura 2-AM in dispersed vascular smooth muscle cells indicated that neither insulin nor troglitazone alone altered PE-induced increases in intracellular [Ca2+]. However, troglitazone and insulin together caused a significant reduction in PE-induced increases in intracellular [Ca2+] (expressed as percentage of preincubation stimulation to PE: 47 +/- 10%, treated; 102 +/- 13%, vehicle). These results demonstrate that troglitazone inhibits Ca2+ influx and that it acts synergistically with insulin to attenuate further vascular contraction via inhibition of voltage-operated Ca2+ channels.

Mechanisms of insulin resistance in experimental hyperinsulinemic dogs.

This study was undertaken to characterize the insulin resistance and the mechanism thereof caused by chronic hyperinsulinemia produced in dogs by surgically diverting the veins of the pancreas from the portal vein to the vena cava. Pancreatic venous diversion (PVD, n = 8) caused a sustained increase in arterial insulin and decrease in portal insulin concentration compared with the control group (n = 6). Hyperinsulinemic euglycemic clamps were conducted 4 wk after surgery. The increase in the glucose disposal rate (GDR) was significantly less in the PVD group (39.0+/-5.0 vs. 27.9+/-3.2 micromol/kg/min, P < 0.01) compared with the control group, but the suppression of hepatic glucose production by insulin was similar for both groups. Muscle insulin receptor tyrosine kinase activity (IR-TKA) increased from 6.2+/-0.4 to 20.3+/-2.7 in the control group, but from 5.8+/-0.5 to only 12.7+/-1.7 fmol P/fmol IR in the PVD group (P < 0.01). With respect to the periphery, the time to half-maximum response (t1/2a) for arterial insulin was the same for both groups, whereas the t1/2a for lymph insulin (30+/-3 vs. 40+/-4 min, P < 0.05) and GDR (29+/-3 vs. 66+/-10 min, P < 0.01) were greater for the PVD group. Chronic hyperinsulinemia led to marked peripheral insulin resistance characterized by decreased insulin-stimulated GDR, and impaired activation of GDR kinetics due, in part, to reduced IR-TKA. Transendothelial insulin transport was impeded and was responsible for one third of the kinetic defect in insulin-resistant animals, while slower intracellular mechanisms of GDR were responsible for the remaining two thirds.

Thiazolidinediones and their effects on glucose transporters.

Recently, considerable attention has been paid to a promising new family of antidiabetic therapeutic agents, the thiazolidinediones. Several members of this family have been shown to have potent effects to lower hyperglycemia, hyperinsulinemia and hypertriglyceridemia in insulin resistant humans and animals. Even as one member of this family, troglitazone (Rezulin), has become available for clinical use in the US and Japan, we still do not fully understand the mechanisms by which these agents exert their effects. Shimaya and colleagues (1) have employed an insulin resistant model system, the obese Zucker rat, to investigate potential mechanisms of thiazolidinedione action. Treatment of obese animals with a new thiazolidinedione analog, YM268, greatly reduced fasting glucose and insulin levels and improved glucose tolerance. These workers confirmed the reduced glucose transporter 4 (GLUT4) protein expression in adipocytes of obese animals compared with lean littermates and found that YM268 treatment resulted in large increases in GLUT4 protein, along with a generalized increase in membrane protein. They suggest that this change can account, at least in part, for the demonstrated amelioration of insulin resistance after treatment. This, and related work, give rise to two questions. First, what is the ultimate significance of changes in GLUT4 expression in adipose tissue? Secondly, how might thiazolidinediones be acting to regulate glucose transporter expression? Effects of thiazolidinediones on adipose tissue glucose transport activity and transporter expression have been reported since the earliest papers on these agents. A common observation, regardless of the animal model or agent tested, is that glucose transport activity is increased, as is GLUT4 expression (2–6) following treatment. These changes occur together with reductions in fasting glucose and insulin levels, so it is uncertain if the effects on transport/transporters are direct or due to the change in the metabolic environment. Considering the demonstrated abilities of glucose and insulin to regulate glucose transport, it is difficult to separate the cause from the effect. Studies in cultured cell systems, where glucose and insulin levels can be tightly controlled, could help to resolve this issue. 3T3-L1 and 3T3-F442A adipocytes have been treated with a variety of thiazolidinediones, resulting in increases in transport activity and transporter expression (7–11). However, these treatments were performed during fibroblast differentiation into adipocytes and, since the appearance of GLUT4 is one marker of adipocyte differentiation, it is uncertain whether this result is due to augmented differentiation, or a more specific effect on transport which would have greater relevance to the therapeutic situation where adipocytes are already differentiated. Given the results showing that thiazolidinediones alone can drive adipocyte differentiation (7, 11), or augment that due to insulin (7), it is even more difficult to draw definitive conclusions from the majority of these studies. However, it has been shown that troglitazone treatment of 3T3-L1 cells following differentiation into adipocytes resulted in upregulation of basal glucose transport and GLUT1 expression (11). GLUT4 expression was unaltered, further suggesting that changes in GLUT4 may be indirect responses in adipose cells. In even the most obese individual (or animal), the portion of a glucose load utilized in adipose tissue is a modest fraction of the total. It is glucose uptake into muscle that represents the major portion of insulinstimulated glucose uptake and is the most significant site of insulin resistance. Thiazolidinedione treatment of insulin resistant rodents has also been shown to augment glucose uptake into muscle as well as increase GLUT4 expression (2–4, 6, 8, 12–14). Such changes could account, to a greater extent, for increases in whole body glucose disposal and would represent the most significant action of thiazolidinediones. Again, the question remains whether these are direct effects of thiazolidinediones, or due to changes in insulin and glucose levels. Fortunately, data in cultured muscle cells are helpful in resolving this issue. Troglitazone treatment of L6 myocytes (15) and isolated rat cardiac myocytes (16) increased glucose transport and the expression of both GLUT1 and GLUT4. This effect was independent of differentiation because it occurred even in L6 cells that were not differentiated into myotubes (15) or in BC3H-1 myocytes (with pioglitazone) (17) that are incapable of differentiation. It should be noted that the non-differentiated myocytes express only GLUT1 and it is this isoform that is upregulated by thiazolidinedione treatment. We have performed similar European Journal of Endocrinology (1997) 137 610–612 ISSN 0804-4643

Ob (obese) gene expression and leptin levels in Psammomys obesus

In this study we investigated ob gene expression and plasma leptin levels in Psammomys obesus (the Israeli Sand Rat), a polygenic animal model of obesity and non-insulin-dependent diabetes mellitus. The ob gene was expressed exclusively in adipocytes of Psammomys obesus. DNA sequencing revealed a high degree of homology with other species (90% with mouse, 88% with rat and 79% with human). No ob gene sequence differences were found between lean and obese Psammomys obesus, and the codon 105 mutation found in ob/ ob mice was not detected. Ob gene expression in Psammomys obesus correlated with body weight ( r=0.436, p<0.001), percent body fat ( r=0.645, p<0.001) and plasma insulin concentration ( r=0.651, p<0.001). This is the first time that ob gene expression has been shown to increase steadily over a continuous wide range of body weight or plasma insulin in an animal model of obesity. Ob gene expression was significantly elevated in obese compared with lean Psammomys obesus ( p<0.05). No significant difference in ob gene expression was found between the four adipose tissue depots tested. Psammomys obesus plasma leptin levels correlated with body weight ( r=0.36, p<0.05), percent body fat ( r=0.702, p<0.01) and plasma insulin concentration ( r=0.735, p<0.001). Plasma leptin concentrations were significantly increased in insulin-resistant animals independent of body weight. These results show that Psammomys obesus is an excellent animal model in which to study the ob gene and leptin, and confirm the importance of insulin as a significant factor in the regulation of leptin and ob gene expression.

Changes in pancreatic islet glucokinase and hexokinase activities with increasing age, obesity, and the onset of diabetes.

We examined changes in high- and low-Km glucose phosphorylating activity in pancreatic islet extracts from the prediabetic Zucker diabetic fatty (ZDF) rat between 5-6 weeks and 12 weeks of age (after the onset of diabetes). Comparisons were made between the activity observed in the ZDF rat and that seen in the ZDF lean control (ZLC) rat and the obese nondiabetic Zucker fatty (ZF) rat. At 5-6 weeks of age, insulin resistant ZDF and ZF rats were hyperinsulinemic, compared with the ZLC rat, but had normal plasma glucose levels. Kinetic parameters (Vmax and Km for glucose) of hexokinase (HK) and Km of glucokinase (GCK) did not differ between groups. Islet GCK activity for ZDF and ZF rats was 1.7-fold greater than in ZLC rats (P < 0.02 and P < 0.001, respectively). By 12 weeks of age, hypersecretion of insulin at 5.0 mmol/l glucose was observed in perifused islets from both obese groups relative to the ZLC rat. Islets from ZDF rats failed to increase insulin secretion in response to increased glucose concentration. Group differences in the kinetic parameters for GCK or in the Km values for HK were not significant. Islet HK activity for ZDF and ZF rats was 1.9-fold (P < 0.05) and 1.7-fold (P < 0.05) greater, respectively, than for ZLC rats. Compared with the 5- to 6-week-old animals, HK activity increased 3.1-fold (P < 0.001), 2.5-fold (P < 0.002), and 2.0-fold (P < 0.05) for ZDF, ZF, and ZLC rats, respectively. Differences in GCK activity between 5- to 6- and 12-week-old rats were not significant for any of the groups. We conclude: 1) increased islet glucose phosphorylating activity is present in insulin resistant and hyperinsulinemic ZF and ZDF rats, relative to the ZLC rat; 2) at 12 weeks of age, hyperinsulinemic ZDF and ZF rats demonstrated significant increases in HK activity, compared with lean controls; and 3) deficiency in GCK activity does not explain failure of diabetic ZDF islets to respond to glucose, since differences between diabetic ZDF and nondiabetic ZF rats were not statistically significant. Increases in pancreatic islet phosphorylating activity seem to be important in maintaining basal hyperinsulinemia in insulin-resistant animals, but do not appear to play a role in the progression to glucose intolerance and diabetes.

Postischemic cardiac performance in the insulin-resistant JCR:LA-cp rat

Hearts from hyperinsulinemic, insulin-resistant JCR:LA-cp rats do not properly regulate intracellular Ca2+ concentration. We hypothesized, therefore, that these hearts may be unusually sensitive to ischemic insults in which Ca2+ overload would be expected. We investigated the response to global ischemia of hearts from JCR: LA-cp animals at three different ages. At 3 mo of age, isolated hearts from insulin-resistant cp rats were mildly resistant to both mild and severe ischemic insults in comparison to the lean control rat hearts. However, at 6 and 9 mo of age, the cp rats demonstrated a poorer recovery of developed tension after ischemia and/or a higher level of resting tension during reperfusion than the lean controls. Postischemic glycogen and ATP contents were significantly lower and lactate content was higher in hearts from 6-mo-old cp rats compared with controls. The results demonstrate that an insulin-resistant animal model exhibits an increased sensitivity to ischemic myocardial injury that develops with advancing age. The mechanism responsible for the enhanced sensitivity may involve augmented glycolytic metabolism. The data also emphasize the importance of the type of diabetes when cardiac dysfunction is examined.

Effect of an insulin sensitizer, pioglitazone, on hypertension in fructose-drinking rats.

To determine whether insulin resistance is responsible for the development of hypertension, we examined whether blood pressure changes in an insulin-resistant animal that was given a fructose solution as their drinking water. Wistar Kyoto rats that drank a 10% fructose solution for 10 weeks showed significant increases not only in plasma triglyceride and insulin levels but also in systolic blood pressure. The decrease in blood glucose in response to the intraperitoneal injection of insulin (0.2-1.0 U/kg) was slight in these fructose-drinking rats. To confirm whether insulin resistance contributes to the observed hypertension, we examined the effect of pioglitazone, an insulin sensitizer, on blood pressure in rats given a 10% fructose solution. When pioglitazone was administered to the rats at a dose of 10 mg/kg/day for 4 weeks from 12 weeks of age, plasma triglyceride and insulin levels and systolic blood pressure decreased, and blood glucose reduction in response to insulin was normalized. These results suggest that insulin resistance is responsible for the development of hypertension in fructose-drinking rats.

Circulating TNF-alpha and leptin levels in offspring of NIDDM patients do not correlate to individual insulin sensitivity.

Obesity plays a central role in the development of skeletal muscle insulin resistance. The molecular mechanism causing skeletal muscle insulin resistance in obese people is still poorly understood. It has been speculated that circulating factors derived from adipose tissue impair insulin signalling in the skeletal muscle cell. TNF-alpha and leptin, which are overproduced in fat tissue of obese insulin resistant animal models and in obese humans, might mediate such an inhibitory effect on insulin signalling in skeletal muscle. The aim of the present study was to evaluate whether circulating TNF-alpha and leptin correlates to the individual skeletal muscle insulin sensitivity in individuals with different degrees of obesity and insulin resistance. We measured circulating TNF-alpha and leptin values in non diabetic offsprings of NIDDM patients. 36 German and 47 Finnish subjects participated in the study. The GDR of each participant was determined by the euglycemic hyperinsulinemic clamp technique, a range between 1.37 to 14.01 mg/kg LBM x min was observed. Percent of desirable body weight (PDW) covered also a wide range (87.58% to 197.06%). Although linear regression analysis suggested a dependence between TNF-alpha and GDR (Germany group: r = -0.37, p < 0.05, Finnish group: r = -0.32, p < 0.05) and a dependence between TNF and PDW (German group: r = 0.46, p < 0.05, Finnish group: r = 0.38, p < 0.05), in multiple linear regression analysis only the correlation with PDW was significant. Leptin levels were measured from 29 German and 36 Finnish subjects and a strong association was found between leptin and PDW (German group: r = 0.55, p < 0.05, Finnish group: r = 0.73, p < 0.05). In contrast, leptin levels did not correlate with GDR and TNF-alpha. In summary, even though, in a few insulin resistant subjects, higher circulating TNF-alpha or leptin levels with the individual insulin sensitivity can be demonstrated, the data suggest that the circulating pool of TNF-alpha and leptin in blood is unlikely to be a major contributing factor for obesity induced insulin resistance in the vast majority of individuals at high risk to develop NIDDM.