Mutation of the RIIbeta subunit of protein kinase A prevents diet-induced insulin resistance and dyslipidemia in mice.

The mechanisms by which obesity contributes to diabetic phenotypes remain unclear. We evaluated the role of protein kinase A (PKA) signaling events in mediating diabetes associated with obesity. PKA comprises two regulatory subunits and two catalytic subunits and is activated by cAMP. The RIIbeta regulatory subunit is abundantly expressed in adipose tissue and brain. Knockout mice lacking this subunit are lean and display remarkable resistance to diet-induced obesity. We investigated whether these mice were also resistant to diet-induced diabetes and whether this effect was dependent on reduced adiposity. Mice were fed a high-fat, high-carbohydrate diet and weight gain and diabetes phenotypes were examined. RIIbeta(-/-) mice displayed decreased body weights, reduced insulin levels, improved insulin sensitivity, and improved total-body glucose disposal as compared with wild-type controls. Plasma levels of VLDL and LDL cholesterol were also reduced in high fat-fed RIIbeta(-/-) mice compared with wild-type mice. Taken together, these data demonstrate that loss of RIIbeta protects mice from diet-induced obesity, insulin resistance, and dyslipidemia.

Validation of whole-body magnetic resonance spectroscopy as a tool to assess murine body composition.

OBJECTIVE: To evaluate proton magnetic resonance spectroscopy (MRS) as a tool for the non-invasive assessment of murine body composition. DESIGN: Twenty C57/BL6 male mice with a wide range of body adiposities underwent both pre- and post-mortem whole-body MRS to assess body composition. MRS measures were compared to the results obtained by chemical carcass analysis, the current 'gold standard' for determination of body composition. MEASUREMENTS: Areas under the curve (AUC) for lipid and water peaks of whole body MRS spectra (AUClipid and AUCH2O, respectively) were used to determine percentages of body fat (%FATMRS) and fat free mass by MRS (%FFMMRS). Total body fat, total body water, fat free mass, and total lean mass were determined by chloroform/methanol extraction of lipid from dessicated whole carcass and compared to MRS measures (%FATMRS, %FFMMRS, AUClipid, and AUCH2O). The variability of the MRS technique was assessed by determining the coefficients of variation (COV) associated with %FATMRS, AUClipid, and AUCH2O for mice of three different adiposities. RESULTS: %FATMRS in live mice was highly correlated with body fat percentage (r=0.994, P<0.001) and total body fat (r=0.980, P<0.001) derived from chemical carcass analysis over a broad range of adiposities (7-48% body fat content by carcass analysis). There was no difference in %FATMRS measured pre- vs post-mortem (r=1.00, P<0.001). AUClipid was highly correlated with chemically derived total fat mass (r=0.996, P<0.001) and body fat percentage (r=0.981, P<0.001), while %FFMMRS was strongly correlated to chemical determinations of percentage body water (r=0.994, P<0. 001), percentage fat free mass (r=0.993, P<0.001), and percentage lean mass (r=0.792, P<0.001). AUCH2O was strongly associated with carcass analysis determinations of total body water (r=0.964, P<0. 001), total fat free mass (r=0.953, P<0.001), and total lean mass (r=0.89, P<0.001). In mice of 6%, 12%, and 43% body fat, COVs determined for %FATMRS and AUClipid were less than 10%. The COVs for AUCH2O were less than 2%. CONCLUSIONS: MRS provides precise, accurate, rapid, and non-invasive measures of body fat, body water, fat free mass, and lean mass in living mice with a broad range of adiposities.

Increased glycogen synthase kinase-3 activity in diabetes- and obesity-prone C57BL/6J mice.

Although the precise mechanisms contributing to insulin resistance and type 2 diabetes are unknown, it is believed that defects in downstream components of the insulin signaling pathway may be involved. In this work, we hypothesize that a serine/threonine kinase, glycogen synthase kinase-3 (GSK-3), may be pertinent in this regard. To test this hypothesis, we examined GSK-3 activity in two inbred mouse strains known to be susceptible (C57BL/6J) or resistant (A/J) to diet-induced obesity and diabetes. Examination of GSK-3 in fat, liver, and muscle tissues of C57BL/6J mice revealed that GSK-3 activity increased twofold in the epididymal fat tissue and remained unchanged in muscle and liver of mice fed a high-fat diet, compared with their low-fat diet-fed counterparts. In contrast, GSK-3 activity did not change in the epididymal fat tissue of A/J mice, regardless of the type of diet they were fed. In addition, both basal and diet-induced GSK-3 activity was higher (2.3- and 3.2-fold, respectively) in the adipose tissue of C57BL/6J mice compared with that in A/J mice. Taken together, our studies suggest an unsuspected link between increased GSK-3 activity and development of insulin resistance and type 2 diabetes in fat tissue of C57BL/6J mice, and implicate GSK-3 as a potential factor contributing to susceptibility of C57BL/6J mice to diet-induced diabetes.

Obesity and diabetes in TNF-alpha receptor- deficient mice.

TNF-alpha may play a role in mediating insulin resistance associated with obesity. This concept is based on studies of obese rodents and humans, and cell culture models. TNF elicits cellular responses via two receptors called p55 and p75. Our purpose was to test the involvement of TNF in glucose homeostasis using mice lacking one or both TNF receptors. C57BL/6 mice lacking p55 (p55(-)/-), p75, (p75(-)/-), or both receptors (p55(-)/-p75(-)/-) were fed a high-fat diet to induce obesity. Marked fasting hyperinsulinemia was seen for p55(-)/-p75(-)/- males between 12 and 16 wk of feeding the high-fat diet. Insulin levels were four times greater than wild-type mice. In contrast, p55(-)/- and p75(-)/- mice exhibited insulin levels that were similar or reduced, respectively, as compared with wild-type mice. In addition, high-fat diet-fed p75(-)/- mice had the lowest body weights and leptin levels, and improved insulin sensitivity. Obese (db/db) mice, which are not responsive to leptin, were used to study the role of p55 in severe obesity. Male p55(-)/-db/db mice exhibited threefold higher insulin levels and twofold lower glucose levels at 20 wk of age than control db/db expressing p55. All db/db mice remained severely insulin resistant based on fasting plasma glucose and insulin levels, and glucose and insulin tolerance tests. Our data do not support the concept that TNF, acting via its receptors, is a major contributor to obesity-associated insulin resistance. In fact, data suggest that the two TNF receptors work in concert to protect against diabetes.

C57BL/6 mice fed high fat diets as models for diabetes-accelerated atherosclerosis.

Non-insulin-dependent diabetes mellitus (NIDDM) is a major risk factor for the development of atherosclerosis in humans. The development of an animal model that displays accelerated atherosclerosis associated with NIDDM will aid in elucidating the mechanisms that associate these disorders. C57BL/6 mice may provide such a model system. This strain becomes obese, hyperglycemic and insulin resistant when fed a high fat diet (diabetogenic diet) and is susceptible to atherosclerotic lesion development when fed a separate high fat diet containing cholesterol and bile acids (atherogenic diet). This report tests whether a diet commonly used to induce atherosclerosis also provokes a diabetic phenotype and whether a diet used to induce diabetes provokes the development of aortic fatty streak lesions. Mice of strains C57BL/6, C3H/He, BALB/c and seven recombinant inbred (RI) strains were fed an atherogenic diet for 14 weeks and glucose parameters were measured. No correlation was observed between atherosclerosis susceptibility and fasting insulin or glucose levels, or glucose clearance following short-term insulin or glucose treatment. Analysis of the RI strains suggested that multiple genes control these glucose metabolic parameters. Feeding the diabetogenic diet for 14 weeks to C57BL/6 mice induced obesity and diabetes and 2-fold increases in plasma lipoprotein concentrations. Also, small aortic sinus lipid deposits were observed in 40% of the mice. Thus, analysis of the diabetogenic diet fed C57BL/6 mouse may provide an important tool for further studies of diabetes accelerated vascular disease.

The Role of Tumor Necrosis Factor-α Receptors in Atherosclerosis.

Tumor necrosis factor (TNF-α)-α is a cytokine exhibiting a plethora of activities involved in inflammation, immune regulation, and energy metabolism. TNF is produced by many cell types, including cells found in atherosclerotic lesions, such as activated monocytes or macrophages, T and B lymphocytes, mast cells, and smooth muscle cells. Two receptors mediate the functions of TNF, and both receptors are also present on cells of the artery wall and on cells involved in lesion development. Mice genetically engineered to lack expression of TNF and each of its receptors are now available and are being used to dissect the role of these molecules in protection from or development of atherosclerosis. The role of TNF receptors in atherosclerosis is the primary focus of this review.

Accelerated atherosclerosis in mice lacking tumor necrosis factor receptor p55.

TNF-alpha (TNF) is produced primarily from macrophages and promotes numerous inflammatory reactions associated with atherosclerosis including the induction of vascular adhesion molecules and the recruitment and proliferation of monocyte/macrophages. There are two receptors known to elicit TNF responses, termed p55 and p75. Since p55 is thought to play the primary role in inflammatory processes, we postulated that the absence of p55 in mice would protect against atherosclerosis. In contrast, C57BL/6 mice lacking p55 had aortic sinus lesion sizes 2.3-fold larger than C57BL/6 wild type mice when fed an atherogenic diet (37,123 +/- 3485 microm2 versus 16, 688 +/- 2887 microm2, respectively, p < 0.0004). Plasma lipid levels were not different between strains. A 3-fold increase in the uptake and degradation of acetylated low density lipoprotein for p55-null as compared with wild type mice was demonstrated in cultured peritoneal macrophages. Immunohistochemical staining for scavenger receptor protein in the aortic sinus was more intense in lesions from the p55-null mice as compared with wild type controls. Our results support the concept that increased scavenger receptor activity contributes to excessive fatty streak formation. We conclude that TNF p55 receptors protect against atherosclerotic lesion development in the mouse.

Hypercatabolism of lipoprotein-free apolipoprotein A-I in HDL-deficient mutant chickens.

The Wisconsin Hypoalpha Mutant (WHAM) chicken has a sex-linked mutation associated with a 90% reduction in high-density lipoprotein (HDL) cholesterol and apolipoprotein A-I (apoA-I). In the present studies, we did not detect a defect in apoA-I synthesis or secretion in liver or intestine. We tested the hypothesis that apoA-I is not binding properly to lipoprotein particles and is undergoing hypercatabolism. We therefore studied the in vivo turnover of lipid-free 125I-apoA-I. Its turnover was fourfold faster in WHAM chickens than in normal chickens. The 125I-apoA-I equilibrated more slowly with HDL in the WHAM chickens, and these animals had a much larger steady-state pool of lipid-free apoA-I than did control chickens. To determine the tissue sites of degradation of apoA- I, the tissue distribution of 125I-tyramine cellobiose apoA-I was assessed. The liver and kidneys were the major sites of apoA-I degradation, but in the WHAM chickens, the kidney made a twofold larger contribution to apoA-I degradation than in normal chickens. Total plasma phospholipid levels are reduced by 44% to 78% in the WHAM chickens. A phospholipid deficit might explain the elevated lipid-free apoA-I pool and, secondarily, the HDL deficiency of the WHAM chickens.

Spontaneous high density lipoprotein deficiency syndrome associated with a Z-linked mutation in chickens.

A mutant strain of chicken previously identified by a "recessive white skin" phenotype was found to have a profound deficiency in high density lipoprotein (HDL) and apolipoprotein A-I (apoA-I). ApoA-I levels in the mutant chickens were reduced by greater than 90%. Since HDL is the predominant cholesterol transporter in chickens, the HDL deficiency was associated with a greater than 80% decrease in total plasma cholesterol. The mutation segregates with markers linked to the Z-chromosome. The structure of the apoA-I produced by the mutant chickens appeared normal as judged by two-dimensional gel electrophoresis. The genetic and biochemical evidence, therefore, suggests that the mutation is not in the apoA-I structural gene. Turnover studies were performed on labeled HDL or on labeled apoA-I preincubated with HDL prior to intravenous injection. Both types of experiments showed that both defective apoA-I production and hypercatabolism contributed to the HDL deficiency, although defective production made a much larger contribution.

The biological characteristics of a water soluble porphyrin in rat lymph nodes.

The biological characteristics of a radiolabeled metalloporphyrin, 5,10,15,20-tetrakis(4-carboxyphenyl)-porphinato [67Cu]copper (II) [( 67Cu]TCPP), in rat lymph nodes, surrounding muscle, fat, and blood were determined. Lymphatic tissue localized greater amounts of [67Cu]TCPP than did surrounding muscle and fat. Inflamed lymph nodes localized greater amounts of [67Cu]TCPP than did noninflamed lymph nodes. Time course studies suggest that the uptake of [67Cu]TCPP in noninflamed and in inflamed lymph nodes may involve different biological processes. The affinity of [67Cu]TCPP for inflamed lymph nodes may be influenced by the degree of inflammation. If further studies confirm these results, [67Cu]TCPP may be useful as a potential radiopharmaceutical for imaging inflamed lymph nodes.

Labeling antibodies with copper radionuclides using N-4-nitrobenzyl-5-(4-carboxyphenyl)-10,15,20-tris(4-sulfophenyl) porphine.

Antibody conjugates labeled with copper-64 and -67 (64Cu and 67Cu) were prepared using the porphyrin chelator N-4-nitrobenzyl-5-(4-carboxyphenyl)-10,15,20-tris(4-sulfophenyl) porphine (N-bzHCS3P). N-bzHCS3P was chosen because it has only one carboxylate group available for activation and coupling to antibody. The conjugates were characterized with respect to (1) the location of the porphyrin on the antibody, (2) the retention of immunoreactivity, and (3) the serum stability of the amide bond linking porphyrin to antibody. These studies showed that porphyrin attachment on the antibody surface is random. The conjugates exhibited high retention of immunoreactivity and reasonable serum stability for potential application in nuclear medicine.

Copper-67 labeled porphyrin localization in inflamed tissue.

A series of experiments compared the uptake of 5,10,15,20 tetrakis(4-carboxyphenyl) porphinato [67Cu] copper (II), 67CuTCPP, by the lymph nodes of inflamed and two sets or control rats. The results demonstrate that 67CuTCPP localizes in greater concentration in inflamed lymph nodes than in noninflamed control lymph nodes. This enhanced uptake of 67CuTCPP by inflamed lymph nodes was 3.6 times greater than was the uptake by control lymph nodes. A time course study demonstrated that the uptake of 67CuTCPP by inflamed lymph nodes reached the maximum level by 24 hours post-injection of 67CuTCPP and remained constant throughout the 96 hours examined. It was also found that the uptake of 67CuTCPP by inflamed lymph nodes was not exclusively dependent upon an increase in the weight of inflamed lymph nodes. These studies show that 67CuTCPP has potential as a lymphoscintigraphy agent.