C-reactive protein does not impair insulin suppression of glucose release in primary hepatocytes.

Recent studies have suggested that CRP may interfere with insulin signaling in skeletal muscle and endothelial cells. The aim of this study was to determine whether highly purified CRP increased the rate of glucose appearance in primary hepatocytes in the absence or presence of insulin. Primary rat hepatocytes were provided glucose-free media containing 10 mM lactate, 1 mM pyruvate, 0, 1 or 10 nM insulin, and 0 or 10 µg/ml of purified CRP for 6h. Purified CRP did not increase glucose release in the absence of insulin and did not reduce the ability of insulin to suppress glucose release.

Subcutaneous adipose tissue accumulation protects systemic glucose tolerance and muscle metabolism.

The protective effects of lower body subcutaneous adiposity are linked to the depot functioning as a "metabolic sink" receiving and sequestering excess lipid. This postulate, however, is based on indirect evidence. Mechanisms that mediate this protection are unknown. Here we directly examined this with progressive subcutaneous adipose tissue removal. Ad libitum chow fed mice underwent sham surgery, unilateral or bilateral removal of inguinal adipose tissue or bilateral removal of both inguinal and dorsal adipose tissue. Subsequently mice were separated into 5 week chow or 5 or 13 week HFD groups (N = 10 per group). Primary outcome measures included adipocyte distribution, muscle and liver triglycerides, glucose tolerance, circulating adipocytokines and muscle insulin sensitivity. Subcutaneous adipose tissue removal caused lipid accumulation in femoral muscle proximal to excision, however, lipid accumulation was not proportionally inverse to adipose tissue quantity excised. Accumulative adipose removal was associated with an incremental reduction in systemic glucose tolerance in 13 week HFD mice. Although insulin-stimulated pAkt/Akt did not progressively decrease among surgery groups following 13 weeks of HFD, there was a suppressed pAkt/Akt response in the non-insulin stimulated (saline-injected) 13 week HFD mice. Hence, increases in lower body subcutaneous adipose removal resulted in incremental decreases in the effectiveness of basal insulin sensitivity of femoral muscle. The current data supports that the subcutaneous depot protects systemic glucose homeostasis while also protecting proximal muscle from metabolic dysregulation and lipid accumulation. Removal of the "metabolic sink" likely leads to glucose intolerance because of decreased storage space for glucose and/or lipids.

Insulin protects liver cells from saturated fatty acid-induced apoptosis via inhibition of c-Jun NH2 terminal kinase activity.

Hepatocyte apoptosis is increased in patients with nonalcoholic steatohepatitis and correlates with disease severity. Long-chain saturated fatty acids, such as palmitate and stearate, induce apoptosis in liver cells. The present study examined insulin-mediated protection against saturated fatty acid-induced apoptosis in the rat hepatoma cell line, H4IIE, and primary rat hepatocytes. Cells were provided a control media (no fatty acids) or the same media containing 250 micromol/liter of albumin-bound oleate or palmitate for 16 h. Insulin concentrations were 0, 1, 10, or 100 nmol/liter (n=4-6/treatment). Palmitate, but not oleate, activated caspase-3 and induced DNA fragmentation in the absence of insulin. Insulin reduced palmitate-mediated activation of caspase-3 and DNA fragmentation in a dose-dependent manner. Phosphatidylinositol 3-kinase inhibitors abolished these effects of insulin. Insulin-mediated inhibition of palmitate-induced apoptosis was not due to an augmentation in the unfolded protein response or increased expression of genes encoding the inhibitor of apoptosis proteins, inhibitor of apoptosis protein-2 and X-linked mammalian inhibitor of apoptosis protein. Palmitate, but not oleate, increased c-Jun NH2 terminal kinase activity in the absence of insulin. Insulin or SP600125, a chemical inhibitor of c-Jun NH2 terminal kinase, blocked palmitate-mediated activation of c-Jun NH2 terminal kinase and reduced apoptosis. These data suggest that insulin is an important determinant of saturated fatty acid-induced apoptosis in liver cells and may have implications for fatty acid-mediated liver cell injury in insulin-deficient and/or -resistant states.

Comparison of the time courses of insulin and the portal signal on hepatic glucose and glycogen metabolism in the conscious dog.

To investigate the temporal response of the liver to insulin and portal glucose delivery, somatostatin was infused into four groups of 42-h-fasted, conscious dogs (n = 6/group), basal insulin and glucagon were replaced intraportally, and hyperglycemia was created via a peripheral glucose infusion for 90 min (period 1). This was followed by a 240-min experimental period (period 2) in which hyperglycemia was matched to period 1 and either no changes were made (CON), a fourfold rise in insulin was created (INS), a portion of the glucose (22.4 mumol.kg-1.min-1) was infused via the portal vein (Po), or a fourfold rise in insulin was created in combination with portal glucose infusion (INSPo). Arterial insulin levels were similar in all groups during period 1 (approximately 45 pM) and were 45 +/- 9, 154 +/- 20, 43 +/- 7, and 128 +/- 14 pM during period 2 in CON, INS, Po, and INSPo, respectively. The hepatic glucose load was similar between periods and among groups (approximately 278 mumol.kg-1.min-1). Net hepatic glucose output was similar among groups during period 1 (approximately 0.1 mumol.kg-1.min-1) and did not change significantly in CON during period 2. In INS net hepatic glucose uptake (NHGU; mumol.kg-1.min-1) was -3.8 +/- 3.3 at 15 min of period 2 and did not reach a maximum (-15.9 +/- 6.6) until 90 min. In contrast, NHGU reached a maximum of -13.0 +/- 3.7 in Po after only 15 min of period 2. In INSPo, NHGU reached a maximum (-23.6 +/- 3.5) at 60 min. Liver glycogen accumulation during period 2 was 21 +/- 10, 84 +/- 17, 65 +/- 16, and 134 +/- 17 mumol/gram in CON, INS, Po, and INSPo, respectively. The increment (period 1 to period 2) in the active form of liver glycogen synthase was 0.7 +/- 0.4, 6.5 +/- 1.2, 2.8 +/- 1.0, and 8.5 +/- 1.3% in CON, INS, Po, and INSPo, respectively. Thus, in contrast to insulin, the portal signal rapidly activates NHGU. In addition, the portal signal independent of a rise in insulin, can cause glycogen accumulation in the liver.

Sources of carbon for hepatic glycogen synthesis in the conscious dog.

To identify the source(s) of carbon for the indirect pathway of hepatic glycogen synthesis, we studied nine 42-h fasted conscious dogs given a continuous intraduodenal infusion of glucose, labeled with [1-13C]glucose and [3-3H]glucose, at 8 mg.kg-1.min-1 for 240 min. Glycogen formation by the direct pathway was measured by 13C-NMR. Net hepatic balances of glucose, gluconeogenic amino acids, lactate, and glycerol were determined using the arteriovenous difference technique. During the steady-state period (the final hour of the infusion), 81% of the glucose infused was absorbed as glucose. Net gut output of lactate and alanine accounted for 5% and 3% of the glucose infused, respectively. The cumulative net hepatic uptakes were: glucose, 15.5 +/- 3.8 g; gluconeogenic amino acids, 32.2 +/- 2.2 mmol (2.9 +/- 0.2 g of glucose equivalents); and glycerol, 6.1 +/- 0.9 mmol (0.6 +/- 0.1 g of glucose equivalents). The liver produced a net of 29.2 +/- 9.6 mmol of lactate (2.6 +/- 0.8 g of glucose equivalents). Net hepatic glycogen synthesis totaled 9.3 +/- 2.5 g (1.8 +/- 0.4 g/100 g liver), with the direct pathway being responsible for 57 +/- 10%. Thus, net hepatic glucose uptake was sufficient to account for all glycogen formed by both the direct and indirect pathways. Total net hepatic uptake of gluconeogenic precursors (gluconeogenic amino acids, glycerol, and lactate) was able to account for only 20% of net glycogen synthesis by the indirect pathway. In a net sense, our data are consistent with an intrahepatic origin for most of the three-carbon precursors used for indirect glycogen synthesis.

Inhibition of adipose tissue PPARγ prevents increased adipocyte expansion after lipectomy and exacerbates a glucose-intolerant phenotype.

OBJECTIVES: Adipose tissue plays a fundamental role in glucose homeostasis. For example, fat removal (lipectomy, LipX) in lean mice, resulting in a compensatory 50% increase in total fat mass, is associated with significant improvement in glucose tolerance. This study was designed to further examine the link between fat removal, adipose tissue compensation and glucose homeostasis using a peroxisome proliferator-activated receptor γ (PPAR γ; activator of adipogenesis) knockout mouse. MATERIAL AND METHODS: The study involved PPARγ knockout (FKOγ) or control mice (CON), subdivided into groups that received LipX or Sham surgery. We reasoned that as the ability of adipose tissue to expand in response to LipX would be compromised in FKOγ mice, so would improvements in glucose homeostasis. RESULTS: In CON mice, LipX increased total adipose depot mass (~60%), adipocyte number (~45%) and changed adipocyte distribution to smaller cells. Glucose tolerance was improved (~30%) in LipX CON mice compared to Shams. In FKOγ mice, LipX did not result in any significant changes in adipose depot mass, adipocyte number or distribution. LipX FKOγ mice were also characterized by reduction of glucose tolerance (~30%) compared to shams. CONCLUSIONS: Inhibition of adipose tissue PPARγ prevented LipX-induced increases in adipocyte expansion and produced a glucose-intolerant phenotype. These data support the notion that adipose tissue expansion is critical to maintain and/or improvement in glucose homeostasis.

Insulin is required for the liver to respond to intraportal glucose delivery in the conscious dog.

To determine whether insulin is essential for the augmented hepatic glucose uptake observed in the presence of intraportal glucose delivery, SRIF was used to induce acute insulin deficiency in 5 conscious dogs, and glucose was infused into the portal vein or a peripheral vein in two sequential, randomized periods. Insulin and C-peptide levels were below the limits of detection after SRIF infusion, and the load of glucose presented to the liver was approximately doubled and equivalent during the portal and peripheral periods. Net hepatic glucose output was 2.9 +/- 0.9 and 2.1 +/- 1.1 mumol.kg-1.min-1 during portal and peripheral glucose delivery, respectively. In an additional set of protocols, pancreatectomized dogs were used to investigate the effects of prolonged insulin deficiency (n = 5) and acute insulin replacement (n = 4) on the hepatic response to intraportal glucose delivery. In the prolonged insulin deficiency protocol, SRIF was used to lower glucagon and thereby reduce circulating glucose levels, and glucose was infused into the portal or peripheral circulations in two sequential, randomized periods. As with acute insulin deficiency, net hepatic glucose output was still evident and similar (3.6 +/- 1.1 and 4.2 +/- 1.3 mumol.kg-1.min-1) during portal and peripheral glucose delivery, respectively. When the pancreatectomized dogs were restudied using a similar protocol, but one in which insulin was replaced (4X-basal), and the glucose load to the liver was matched to that which occurred in the prolonged insulin deficiency protocol, net hepatic glucose uptake was 23.6 +/- 6.1 mumol.kg-1.min-1 during portal glucose delivery but only 10.3 +/- 3.5 mumol.kg-1.min-1 during peripheral glucose delivery. These results suggest that the induction of net hepatic glucose uptake and the augmented hepatic response to intraportal glucose delivery require the presence of insulin.

Magnitude of negative arterial-portal glucose gradient alters net hepatic glucose balance in conscious dogs.

To examine the relationship between the magnitude of the negative arterial-portal glucose gradient and net hepatic glucose uptake, two groups of 42-h fasted, conscious dogs were infused with somatostatin, to suppress endogenous insulin and glucagon secretion, and the hormones were replaced intraportally to create hyperinsulinemia (3- to 4-fold basal) and basal glucagon levels. The hepatic glucose load to the liver was doubled and different negative arterial-portal glucose gradients were established by altering the ratio between portal and peripheral vein glucose infusions. In protocol 1 (n = 6) net hepatic glucose uptake was 42.2 +/- 6.7, 35.0 +/- 3.9, and 33.3 +/- 4.4 mumol.kg-1.min-1 at arterial-portal plasma glucose gradients of -4.1 +/- 0.9, -1.8 +/- 0.4, and -0.8 +/- 0.1 mM, respectively. In protocol 2 (n = 6) net hepatic glucose uptake was 26.1 +/- 2.8 and 12.2 +/- 1.7 mumol.kg-1.min-1 at arterial-portal plasma glucose gradients of -0.9 +/- 0.2 and -0.4 +/- 0.1 mM, respectively. No changes in the hepatic insulin or glucose loads were evident within a given protocol. Although net hepatic glucose uptake was lower in protocol 2 when compared with protocol 1 (26.1 +/- 2.8 vs. 33.3 +/- 4.4 mumol.kg-1.min-1) in the presence of a similar arterial-portal plasma glucose gradient (-0.9 vs. -0.8 mM) the difference could be attributed to the hepatic glucose load being lower in protocol 2 (i.e., hepatic fractional glucose extraction was not significantly different) primarily as a result of lower hepatic blood flow. In conclusion, in the presence of fixed hepatic glucose and insulin loads, the magnitude of the negative arterial-portal glucose gradient can modify net hepatic glucose uptake in vivo.

Lower body adipose tissue removal decreases glucose tolerance and insulin sensitivity in mice with exposure to high fat diet.

It has been postulated that the protective effects of lower body subcutaneous adipose tissue (LBSAT) occur via its ability to sequester surplus lipid and thus serve as a "metabolic sink." However, the mechanisms that mediate this protective function are unknown thus this study addresses this postulate. Ad libitum, chow-fed mice underwent Sham-surgery or LBSAT removal (IngX, inguinal depot removal) and were subsequently provided chow (Chow; typical adipocyte expansion) or high fat diet (HFD; enhanced adipocyte expansion) for 5 weeks. Primary outcome measures included glucose tolerance and subsequent insulin response, muscle insulin sensitivity, liver and muscle triglycerides, adipose tissue gene expression, and circulating lipids and adipokines. In a follow up study the consequences of extended experiment length post-surgery (13 wks) or pre-existing glucose intolerance were examined. At 5 wks post-surgery IngX in HFD-fed mice reduced glucose tolerance and muscle insulin sensitivity and increased circulating insulin compared with HFD Sham. In Chow-fed mice, muscle insulin sensitivity was the only measurement reduced following IngX. At 13 wks circulating insulin concentration of HFD IngX mice continued to be higher than HFD Sham. Surgery did not induce changes in mice with pre-existing glucose intolerance. IngX also increased muscle, but not liver, triglyceride concentration in Chow- and HFD-fed mice 5 wks post-surgery, but chow group only at 13 wks. These data suggest that the presence of LBSAT protects against triglyceride accumulation in the muscle and HFD-induced glucose intolerance and muscle insulin resistance. These data suggest that lower body subcutaneous adipose tissue can function as a "metabolic sink."

The role of visceral and subcutaneous adipose tissue fatty acid composition in liver pathophysiology associated with NAFLD.

Visceral adiposity is associated with type-2-diabetes, inflammation, dyslipidemia and non-alcoholic fatty liver disease (NAFLD), whereas subcutaneous adiposity is not. We hypothesized that the link between visceral adiposity and liver pathophysiology involves inherent or diet-derived differences between visceral and subcutaneous adipose tissue to store and mobilize saturated fatty acids. The goal of the present study was to characterize the fatty acid composition of adipose tissue triglyceride and portal vein fatty acids in relation to indices of liver dysregulation. For 8 weeks rats had free access to control (CON; 12.9% corn/safflower oil; 3.6 Kcal/g), high saturated fat (SAT; 45.2% cocoa butter; 4.5 Kcal/g) or high polyunsaturated fat (PUFA; 45.2% safflower oil; 4.5 Kcal/g) diets. Outcome measures included glucose tolerance, visceral and subcutaneous adipose tissue triglyceride, liver phospholipids and plasma (portal and systemic) free fatty acid composition, indices of inflammation and endoplasmic reticulum stress in the liver and adipose tissue depots and circulating adipo/cytokines. Hepatic triglycerides were significantly increased in both high fat diet groups compared to control and were significantly higher in PUFA compared to SAT. Although glucose tolerance was not different among diet groups, SAT increased markers of inflammation and ER stress in the liver and both adipose tissue depots. Fatty acid composition did not differ among adipose depots or portal blood in any dietary group. Overall, these data suggest that diets enriched in saturated fatty acids are associated with liver inflammation, ER stress and injury, but that any link between visceral adipose tissue and these liver indices does not involve selective changes to fatty acid composition in this depot or the portal vein.

High fat diets elevate adipose tissue-derived tumor necrosis factor-alpha activity.

Adipose tissue-derived tumor necrosis factor-alpha (AT-TNF) has been associated with genetic models of insulin resistance and obesity. It is presently unknown if secreted AT-TNF protein is bioactive or whether it can be increased by environmentally induced obesity. In this study, male Wistar rats were fed either a low fat (LF; 12% of energy from corn oil) or a high fat (HF; 45% of energy from corn oil) diet for 5 weeks. From previous data, it is known that after 3 weeks, HF fed animals are obese and insulin resistant compared with the LF group. Hence, animals were killed at 1 week of HF feeding, during the acute response to the diet, and at 5 weeks, when differences in body fat are manifest. Weight gain was significantly increased by diet (P = 0.03) and time (P < 0.0001). AT-TNF bioactivity was measured on secreted protein collected from medium of minced, incubated epididymal (EPI), mesenteric (MES), and retroperitoneal (RETRO) fat pads. AT-TNF bioactivity was significantly increased by diet (P = 0.003) in the RETRO pad and tended to increase (P = 0.07) in EPI. AT-TNF activity was unaffected by diet or time in the MES pad. In the RETRO pad, TNF activity correlated negatively with RETRO fat cell number (r = -0.46, P = 0.002). Secreted AT-TNF protein did not correlate with AT-TNF activity but instead decreased in RETRO with time but not diet. In EPI, secreted AT-TNF protein decreased with the HF diet. Thus, these data suggest that high fat diets and obesity can influence AT-TNF bioactivity and secretion but in an apparent fat pad-specific manner.

Adipose tissue-derived tumor necrosis factor activity correlates with fat cell size but not insulin action in aging rats.

Adipose tissue-derived tumor necrosis factor (AT-TNF) protein and messenger RNA (mRNA) has been shown to correlate with insulin resistance in some studies. However, in a study using different aged Fischer 344 rats, AT-TNF activity correlated more strongly with cell size than with fasting plasma insulin. The present study was undertaken to more carefully examine the relationship among AT-TNF, adipose cell size, and insulin action using more precise measures of insulin action. Basal and hyperinsulinemic, euglycemic clamps were performed in male Sprague Dawley rats at four different ages (8, 13, 21, and 61 weeks old). [3-(3)H]glucose and 2-deoxy-D-[1-(14)C]glucose were used to assess glucose kinetics and tissue-specific glucose uptake. Because TNF activity represents the summation of TNF synthesis, secretion, and the amount of soluble inhibitors present, TNF activity was measured using a bioassay, in addition to measuring TNF protein and mRNA levels. AT-TNF activity increased significantly with age, as did the glucose infusion rate, a measure of whole body insulin resistance. However, AT-TNF activity did not correlate with any parameter of insulin action measured during the hyperinsulinemic, euglycemic clamps. In epididymal fat, AT-TNF activity correlated with: glucose infusion rate: r = -0.50, P = 0.17; rate of appearance: r = -0.19, P = 0.35; rate of disappearance: r = 0.08, P = 0.69. As was noted before, AT-TNF activity correlated well with fat cell size (r = 0.76, P < 0.001 in epididymal fat; r = 0.58, P = 0.007 in SUB fat). These data suggest that although AT-TNF activity and insulin resistance increase with age, the two are not functionally related. These data do not eliminate the potential role of nonadipose TNF in the regulation of insulin action.

Influence of endurance training on the age-related decline in hepatic glyconeogenesis.

Hepatic gluconeogenic and glyconeogenic capabilities were investigated in Fischer 344 rat livers (ages 7, 15 and 25 months; n = 66) to determine if endurance training affected age related decrements in these hepatic functions. Animals were trained 1 h/day, 5 days/week for 10 weeks at treadmill speeds of 75% of age-specific maximal capacity. After training, rats were injected (300 mg/kg) with a known gluconeogenic inhibitor, 3-mercaptopicolinic acid (MPA). Two endurance tests were performed to help assess the contribution of gluconeogenesis to exercise performance, an initial test 4 days prior to injection and a final test immediately post-injection. MPA significantly (P < 0.05) reduced running times in all trained groups compared to their control test: 89%, 81%, and 51% in the young, middle-aged, and old, respectively. MPA reduced running times in the untrained animals 19%, 11%, and 8%, respectively. Three days after the last exercise bout, the animals were anesthetized and liver sections were sliced and incubated in [14C]lactic acid or [14C]fructose. An age-related decline was found in [14C]lactate incorporation (middle-aged decreases 66%, old decreases 54%) and in [14C]fructose incorporation (middle-aged decreases 51%, old decreases 48%) into glycogen. Differences existed in lactate incorporation in trained compared to untrained animals for the young, middle-aged, and old groups: 150.1 +/- 11.3 vs. 102.1 +/- 10.0; 75.3 +/- 6.2 vs. 34.9 +/- 6.4; and 69.3 +/- 14.9 vs. 47.0 +/- 4.7 nmol/g/h, respectively. No differences were found with training in any of the age groups for fructose. Phosphoenolpyruvate carboxykinase (PEPCK) activity and messenger RNA (mRNA) were significantly reduced in the old compared to the young rats (decreases 64% and decreases 58%, respectively). No training effects were found for either PEPCK activity or mRNA for any age group. These results suggest that hepatic gluconeogenic and glyconeogenic capabilities declined with age. Training had an effect in attenuating the glyconeogenic decline but had a minimal effect in offsetting the age-related decline in PEPCK.

Fat and energy balance.

In summary, an imbalance between energy intake and energy expenditure can explain approximately 80% of the variance in body weight gain in this dietary model of obesity. Several metabolic variables appear to contribute to differences in energy balance. A high RQ and an inappropriate suppression of glucose production by insulin appear to be linked to the increase in energy intake that occurs when obesity-prone rats are provided with the high-fat diet. In addition, early tissue enzymatic differences in obesity-prone versus obesity-resistant rats may contribute to differences in energy expenditure and/or to differences in nutrient partitioning. In this dietary model, susceptibility to dietary obesity involves a metabolic environment that includes a high RQ and a reduced ability of insulin to suppress glucose appearance (FIG. 9). However, this environment does not lead to obesity nor to a measurable difference in body weight gain when the susceptible rats are eating a low-fat diet. The high-fat diet is a necessary catalyst for the observed variability in body weight gain and the development of obesity. As a catalyst, the high-fat diet results in an imbalance between energy intake and energy expenditure in some, but not all, rats. This imbalance interacts with the permissive metabolic environment (tissue enzymatic profile favoring carbohydrate utilization and lipid storage) to produce obesity on the high-fat diet. Later, in the HFD feeding period, the rate of weight gain is not significantly different between OP and OR rats, although net fat accumulation remains greater in the former group. It is interesting that this later period is characterized by a reduction in the difference in both RQ and energy intake between OP and OR rats. Thus, during the later stages of HFD feeding, the discrepancy in both energy balance and nutrient balance between OP and OR rats is reduced. This dietary model of obesity is relevant to human obesity. While the prevalence of obesity is high, the majority of people are not obese. The high prevalence of obesity may be due to environmental catalysts that interact with inherent behavioral and metabolic characteristics that favor nutrient retention. Resistance to obesity can be achieved by avoiding these environmental catalysts, by having inherent characteristics that prevent nutrient retention, or both. Our work suggests that the complete understanding of obesity will require not only the identification and functional significance of the genes that determine the inherent capacity of the behavioral and metabolic systems, but also the role of environmental catalysts in determining where and how these systems operate.

Adipose tissue-derived tumor necrosis factor-alpha activity is elevated in older rats.

High levels of adipose tissue-derived tumor necrosis factor-alpha (AT-TNF) mRNA and protein have previously been associated with genetic models of obesity and insulin resistance. Because there are endogenous TNF inhibitors it is unknown if AT-TNF activity is also increased. We hypothesized that AT-TNF activity would increase in older animals because of an accumulation of fat mass. We chose to study 2 different-aged male Fischer 344 rats, 3-month-old (young) and 14-month-old (mature) because fat mass should be quite different but insulin action on glucose metabolism similar. Indeed, mature rats had over 1.5-fold more fat mass, but whole body insulin resistance, as estimated by fasting plasma insulin, was similar to young rats. Mature rats had twice as much AT-TNF activity as the young in both the epididymal (EPI) and retroperitoneal (Retro) fat pads (p < .0005). AT-TNF correlated with fasting plasma insulin in Retro only (r = .48, p = .04). AT-TNF activity strongly correlated with cell size in both EPI and Retro (r = .79 and .81, respectively, p < .0001). Because cytokines can be regulated at several levels, AT-TNF activity, protein, and mRNA were measured. AT-TNF protein levels were higher in young rats, suggesting that these animals may secrete an inhibitor that reduces AT-TNF activity. There were no significant differences in AT-TNF mRNA between groups. Since TNF has been shown to affect several key genes in tissue culture, mRNA for lipoprotein lipase, hormone-sensitive lipase, and Glut4 were measured. No differences were found between groups. In summary, AT-TNF activity increased in mature animals in relation to adipose cell size.

Enhanced efficiency of lactate removal after endurance training.

The effects of endurance training (running 1 h/day at 40 m/min, 10% grade) on net lactate removal at various lactate concentrations were assessed in resting rats by use of constant exogenous lactate infusion (0, 69.3, 123.6, and 175.0 mumol.kg-1.min-1). No consistent difference in resting lactate concentrations, 1.17 +/- 0.09 mM, was observed between control and trained animals with no exogenous infusion of lactate. With increasing lactate infusion rates, control animals demonstrated a twofold greater increase in blood lactate concentration (range 1.2-11.4 mM) compared with trained animals (range 1.0-5.5 mM). This response resulted from a more rapid rise in net lactate removal with changes in blood lactate concentration for trained animals. The estimated maximal reaction velocity for net lactate removal in trained animals was 19% lower than in control animals; however, the Michaelis-Menten constant was greater than 66% lower in trained animals (4 mM) compared with controls (12 mM). Control animals also demonstrated a twofold greater increase in lactate concentration as a function of the tracer-estimated lactate turnover. The ratio of 14CO2 yield to lactate specific activity as a function of total tracer removal was not significantly different between groups, suggesting that the relative contributions of oxidation and gluconeogenesis to lactate removal were similar for both groups. At blood concentrations greater than 1 mM, trained animals achieve higher rates of lactate removal for any given lactate concentration.

Effects of a high-fat diet and voluntary wheel running on gluconeogenesis and lipolysis in rats.

The purpose of the present study was to determine the effects of diet composition and exercise on glycerol and glucose appearance rate (Ra) and on nonglycerol gluconeogenesis (Gneo) in vivo. Male Wistar rats were fed a high-starch diet (St, 68% of energy as cornstarch, 12% corn oil) for a 2-wk baseline period and then were randomly assigned to one of four experimental groups: St (n = 7), high-fat (HF; 35% cornstarch, 45% corn oil; n = 8), St with free access to exercise wheels (StEx; n = 7), and HF with free access to exercise wheels (HFEx; n = 7). After 8 wk, glucose Ra when using [3-3H]glucose, glycerol Ra when using [2H5]glycerol (estimate of whole body lipolysis), and [3-13C]alanine incorporation into glucose (estimate of alanine Gneo) were determined. Body weight and fat pad mass were significantly (P < 0.05) decreased in exercise vs. sedentary animals only. The average amount of exercise was not significantly different between StEx (3,212 +/- 659 m/day) and HFEx (3,581 +/- 765 m/day). The ratio of glucose to alanine enrichment and absolute glycerol Ra (micromol/min) were higher (P < 0.05) in HF and HFEx compared with St and StEx rats. In separate experiments, the ratio of 3H in C-2 to C-6 of glucose from 3H2O (estimate of Gneo from pyruvate) was also higher (P < 0.05) in HF (n = 5) and HFEx (n = 5), compared with St (n = 5) and StEx (n = 5) rats. Voluntary wheel running did not significantly increase estimated alanine or pyruvate Gneo or absolute glycerol Ra. Voluntary wheel running increased (P < 0.05) glycerol Ra when normalized to fat pad mass. These data suggest that a high-fat diet can increase in vivo Gneo from precursors that pass through pyruvate. They also suggest that changes in the absolute rate of glycerol Ra may contribute to the high-fat diet-induced increase in Gneo.

Fuel metabolism in men and women during and after long-duration exercise.

This study aimed to determine gender-based differences in fuel metabolism in response to long-duration exercise. Fuel oxidation and the metabolic response to exercise were compared in men (n = 14) and women (n = 13) during 2 h (40% of maximal O2 uptake) of cycling and 2 h of postexercise recovery. In addition, subjects completed a separate control day on which no exercise was performed. Fuel oxidation was measured using indirect calorimetry, and blood samples were drawn for the determination of circulating substrate and hormone levels. During exercise, women derived proportionally more of the total energy expended from fat oxidation (50.9 +/- 1.8 and 43. 7 +/- 2.1% for women and men, respectively, P < 0.02), whereas men derived proportionally more energy from carbohydrate oxidation (53.1 +/- 2.1 and 45.7 +/- 1.8% for men and women, respectively, P < 0.01). These gender-based differences were not observed before exercise, after exercise, or on the control day. Epinephrine (P < 0.007) and norepinephrine (P < 0.0009) levels were significantly greater during exercise in men than in women (peak epinephrine concentrations: 208 +/- 36 and 121 +/- 15 pg/ml in men and women, respectively; peak norepinephrine concentrations: 924 +/- 125 and 659 +/- 68 pg/ml in men and women, respectively). As circulating glycerol levels were not different between the two groups, this suggests that women may be more sensitive to the lipolytic action of the catecholamines. In conclusion, these data support the view that different priorities are placed on lipid and carbohydrate oxidation during exercise in men and women and that these gender-based differences extend to the catecholamine response to exercise.

Sucrose diets increase glucose-6-phosphatase and glucose release and decrease glucokinase in hepatocytes.

A high-sucrose diet (SU) decreases insulin action in the liver (Pagliassotti MJ, Shahrokhi KA, and Moscarello M. Am J Physiol Regulatory Integrative Comp Physiol 266: R1637-R1644, 1994). The present study was conducted to characterize the effect of SU on glucagon action in isolated periportal (PP) and perivenous (PV) hepatocytes by measuring glucagon-stimulated glycogenolysis and glucose release. Male rats were fed a SU (68% sucrose) or starch diet (ST, 68% starch) for 1 wk, and hepatocytes were isolated from PP or PV regions (n = 4/diet/cell population). Hepatocytes were incubated for 1 h in the presence of varying concentrations of glucagon (0-100 nM). In PP and PV cells, glucagon stimulation of glucose release and glycogenolysis (sum of glucose release and lactate accumulation) was not significantly different between SU and ST cells. However, in the SU PP cells, glucose release was increased compared with ST PP cells, both in the absence of glucagon (76.1 +/- 4 vs. 54.8 +/- 3 nmol x h(-1) x mg cell wet x wt(-1)) and at all glucagon concentrations. In SU-fed PV cells, glucose release was increased compared with ST PV cells in the absence of glucagon (79.3 +/- 5 vs. 56.4 +/- 5 nmol x h(-1) x mg cell wet x wt(-1)) and at low glucagon concentrations. Maximal glucose-6-phosphatase activity (in nmol x min(-1) x mg protein(-1)) was elevated in SU compared with ST cells (61.4 +/- 3 vs. 37.5 +/- 4 in PP and 37.5 +/- 4 vs. 29.5 +/- 3 in PV cells). In contrast, maximal glucokinase activity (in nmol x min(-1) x mg protein(-1)) was elevated in ST compared with SU cells (15.9 +/- 2 vs. 12.1 +/- 1 in PP and 19.4 +/- 2 vs. 14.2 +/- 1 in PV cells). These data demonstrate that SU increases the capacity for glucose release in both PP and PV hepatocytes, in part because of reciprocal changes in glucose-6-phosphatase and glucokinase.

Quantitative assessment of pathways for lactate disposal in skeletal muscle fiber types.

Quantifying the contribution of the various skeletal muscle fiber types toward lactate disposal has proven elusive. In part, this can be attributed to the lack of adequate preparations for the study of all potential metabolic pathways involved. Toward this end our laboratory developed several perfused muscle preparations that are homogeneous for specific fiber types. This paper briefly reviews our findings regarding the influence of fiber type on lactate disposal in resting skeletal muscle and the metabolic pathways involved. Perfusing over a range of lactate concentrations, 1-12 mM, all fiber types were shown to switch from net production at low lactate concentrations to net consumption at higher concentrations. This transition occurred at lower lactate concentrations for Type I and IIa fibers, when compared with IIb fibers. For Type I and IIa fibers oxidation was observed to be the primary route of disposal accounting for approximately 50% of the lactate removed. For all fiber types, transamination was a significant pathway for the disposal of lactate carbon, whereas glyconeogenesis was the primary pathway for disposal in Type IIb fibers. The glyconeogenic capacity was quantitatively similar for Type IIa and IIb fibers but was negligible for Type I fibers. The pathway for glyconeogenesis in skeletal muscle was shown to be substantially different from that employed in hepatic glyconeogenesis. Results indicated that neither the TCA cycle nor phosphoenolpyruvate carboxykinase is involved in skeletal muscle glyconeogenesis. Our findings suggested that PEP formation in skeletal muscle glyconeogenesis occurs by "reversal" of the pyruvate kinase reaction.