The aim of this study was to determine whether phenotypes associated with type 2 diabetes are altered in dyslipidemic obese mice. C57BL/6 wild-type, low-density lipoprotein (LDL) receptor-deficient (LDLR-/-), and apolipoprotein E-deficient (apoE-/-) mice were fed a high-fat, high-carbohydrate diet (diabetogenic diet), and the development of obesity, diabetes, and hypertriglyceridemia was examined. Wild-type mice became obese and developed hyperglycemia, but not hypertriglyceridemia, in response to this diet. LDLR-/- mice fed the diabetogenic diet became more obese than wild-type mice and developed severe hypertriglyceridemia and hyperleptinemia. Surprisingly, glucose levels were only modestly higher and insulin levels and insulin-to-glucose ratios were not strikingly different from those of wild-type mice. In contrast, diabetogenic diet-fed apoE-/- mice were resistant to changes in glucose and lipid homeostasis despite becoming obese. These data suggest that modifications in lipoprotein profiles associated with loss of the LDL receptor or apoE function have profound and unique consequences on susceptibility to diet-induced obesity and type 2 diabetic phenotypes.

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.

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TNF-␣ 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 Ϫ / Ϫ ), p75, (p75 Ϫ / Ϫ ), or both receptors (p55 Ϫ / Ϫ p75 Ϫ / Ϫ ) were fed a high-fat diet to induce obesity. Marked fasting hyperinsulinemia was seen for p55 Ϫ / Ϫ p75 Ϫ / Ϫ males between 12 and 16 wk of feeding the high-fat diet. Insulin levels were four times greater than wild-type mice. In contrast, p55Ϫ / Ϫ and p75 Ϫ / Ϫ mice exhibited insulin levels that were similar or reduced, respectively, as compared with wild-type mice. In addition, high-fat dietfed p75 Ϫ / Ϫ 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 Ϫ / Ϫ 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. ( J. Clin. Invest . 1998. 102:402-411.)

Inflammatory processes are involved with all phases of atherosclerotic lesion growth. Tumor necrosis factor-␣ (TNF␣) is an inflammatory cytokine that is thought to contribute to lesion development. Lymphotoxin-␣ (LT␣) is also a proinflammatory cytokine with homology to TNF␣. However, its presence or function in lesion development has not been investigated. To study the role of these molecules in atherosclerosis, the expression of these cytokines in atherosclerotic lesions was examined. The presence of both cytokines was observed within aortic sinus fatty streak lesions. To determine the function of these molecules in regulating lesion growth, mice deficient for TNF␣ or LT␣ were examined for induction of atherosclerosis. Surprisingly, loss of TNF␣ did not alter lesion development compared with wild-type mice. This brings doubt to the generally held concept that TNF␣ is a "proatherogenic cytokine." However, LT␣ deficiency resulted in a 62% reduction in lesion size. This demonstrates an unexpected role for LT␣ in promoting lesion growth. The presence of LT␣ was observed in aortic sinus lesions suggesting a direct role of LT␣ in modulating lesion growth. To determine which receptor mediated these responses, diet-induced atherosclerosis in mice deficient for each of the TNF receptors, termed p55 and p75, was examined. Results demonstrated that loss of p55 resulted in increased lesion development, but loss of p75 did not alter lesion size. The disparity in results between ligand-and receptor-deficient mice suggests there are undefined members of the TNF ligand and receptor signaling pathway involved with regulating atherogenesis.Inflammatory processes are an integral component to atherosclerotic lesion development. Cytokine-mediated proinflammatory responses such as endothelial cell activation and leukocyte recruitment are thought to positively contribute to the atherogenic process. One of the best-studied proinflammatory cytokines is tumor necrosis factor-␣ (TNF␣) 1 that is expressed in both human and rodent atherosclerotic plaques (1-5). However, the physiological role of TNF ligand and receptor family members in the atherogenic process remains unclear.TNF␣ and lymphotoxin-␣ (LT␣) are two predominant members of the TNF ligand family. Their structural genes are located on human chromosome 6 within the major histocompatibility complex (6). TNF␣ and LT␣ proteins are structurally similar and display 50% amino acid homology (7). TNF␣ is first synthesized as a type II transmembrane protein and is subsequently cleaved to form circulating homotrimeric TNF␣ (8). TNF␣ is synthesized primarily by activated macrophages (9), although under appropriate stimulation other cells can express this cytokine (10 -12). TNF␣ influences the function of macrophages, smooth muscle cells, and endothelial cells (13), which are major cell types observed in plaques. LT␣ is synthesized primarily by activated T and B lymphocytes (6, 7) and is also found in the circulation as a homotrimer. Unlike TNF␣, membrane-bound homotrimeric LT␣ has not been o...

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.

Glycerol-3-phosphate acyltransferase (GPAT) catalyses the first committed step in glycerolipid biosynthesis. The mitochondrial isoform (mtGPAT) is mainly expressed in liver, where it is highly regulated, indicating that mtGPAT may have a unique role in hepatic fatty acid metabolism. Because both mtGPAT and carnitine palmitoyl transferase-1 are located on the outer mitochondrial membrane, we hypothesized that mtGPAT directs fatty acyl-CoA away from ␤ -oxidation and toward glycerolipid synthesis. Adenoviralmediated overexpression of murine mtGPAT in primary cultures of rat hepatocytes increased mtGPAT activity 2.7-fold with no compensatory effect on microsomal GPAT activity. MtGPAT overexpression resulted in a dramatic 80% reduction in fatty acid oxidation and a significant increase in hepatic diacylglycerol and phospholipid biosynthesis. Following lipid loading of the cells, intracellular triacylglycerol biosynthesis was also induced by mtGPAT overexpression. Changing an invariant aspartic acid residue to a glycine [D235G] in mtGPAT resulted in an inactive enzyme, which helps define the active site required for mammalian mtGPAT function. To determine if obesity increases hepatic mtGPAT activity, two models of rodent obesity were examined and shown to have Ͼ 2-fold increased enzyme activity.Overall, these results support the concept that increased hepatic mtGPAT activity associated with obesity positively contributes to lipid disorders by reducing oxidative processes and promoting de novo glycerolipid synthesis. -Lindén, D., L. William-Olsson, M. Rhedin, A-K. Asztély, J. C. Clapham, and S. Schreyer. Overexpression of mitochondrial GPAT in rat hepatocytes leads to decreased fatty acid oxidation and increased glycerolipid biosynthesis.

Glycerol-3-phosphate acyltransferase (GPAT) catalyzes the first committed step in triacylglycerol (TAG) and phospholipid biosynthesis. GPAT activity has been identified in both ER and mitochondrial subcellular fractions. The ER activity dominates in most tissues except in liver, where the mitochondrial isoform (mtGPAT) can constitute up to 50% of the total activity. To study the in vivo effects of hepatic mtGPAT overexpression, mice were transduced with adenoviruses expressing either murine mtGPAT or a catalytically inactive variant of the enzyme. Overexpressing mtGPAT resulted in massive 12- and 7-fold accumulation of liver TAG and diacylglycerol, respectively but had no effect on phospholipid or cholesterol ester content. Histological analysis showed extensive lipid accumulation in hepatocytes. Furthermore, mtGPAT transduction markedly increased adipocyte differentiation-related protein and stearoyl-CoA desaturase-1 (SCD-1) in the liver. In line with increased SCD-1 expression, 18:1 and 16:1 in the hepatic TAG fraction increased. In addition, mtGPAT overexpression decreased ex vivo fatty acid oxidation, increased liver TAG secretion rate 2-fold, and increased plasma TAG and cholesterol levels. These results support the hypothesis that increased hepatic mtGPAT activity associated with obesity and insulin resistance contributes to increased TAG biosynthesis and inhibition of fatty acid oxidation, responses that would promote hepatic steatosis and dyslipidemia.