Genetic Background (C57BL/6J Versus FVB/N) Strongly Influences the ...

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Endocrinology 145(7):3258 –3264 Copyright © 2004 by The Endocrine Society doi: 10.1210/en.2004-0219

Genetic Background (C57BL/6J Versus FVB/N) Strongly Influences the Severity of Diabetes and Insulin Resistance in ob/ob Mice MARTIN HALUZIK, CARLO COLOMBO, OKSANA GAVRILOVA, STREAMSON CHUA, NICOLE WOLF, MIN CHEN, BETHEL STANNARD, KELLY R. DIETZ, DEREK LE ROITH, AND MARC L. REITMAN Diabetes Branch (M.H., C.C., O.G., B.S., K.R.D., D.L.R., M.L.R.), National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892; Third Department of Medicine (M.H.), First Faculty of Medicine, Charles University, 12808 Prague-2, Czech Republic; Division of Molecular Genetics and New York Obesity Research Center (S.C.), Department of Pediatrics, Columbia University, New York, New York 10032; and Metabolic Diseases Branch (N.W., M.C.), National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, Maryland 20892 We studied the effects of genetic background on the phenotype of ob/ob mice, a model of severe obesity, insulin resistance, and diabetes caused by leptin deficiency. Despite a comparable degree of obesity and hyperinsulinemia, C57BL/6J ob/ob mice had much milder hyperglycemia and, surprisingly, normal circulating adiponectin levels despite still-prominent signs of insulin resistance. Hyperinsulinemiceuglycemic clamp revealed relatively less whole-body and muscle insulin resistance in C57BL/6J ob/ob mice, whereas

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liver insulin resistance tended to be more severe than in FVB/N ob/ob mice. C57BL/6J ob/ob mice had also more rapid clearance of circulating triglycerides and more severe hepatic steatosis. We suggest that strain-related distinction in lipid handling is the most important player in the differences in diabetic phenotype and insulin sensitivity, whereas the impact of circulating adiponectin levels on the overall phenotype of ob/ob mice is less important. (Endocrinology 145: 3258 –3264, 2004)

We have recently demonstrated the importance of the genetic background on the phenotype of A-ZIP/F-1 mice, which lack adipose tissue (12). The lipoatrophic A-ZIP/F-1 mouse is an extreme example of insulin resistance with hepatic steatosis and severe hyperinsulinemia (13). A-ZIP/F-1 mice on the FVB background have severe hyperglycemia and high circulating triglyceride and free fatty acid levels. AZIP/F-1 mice on B6 background, with similarly severe hyperinsulinemia, have milder hyperglycemia but worse hepatic steatosis (12). It appears that the increased capacity of the liver of B6 A-ZIP/F-1 mice to store triglycerides leads to lower circulating lipid levels, less lipid deposition in muscle tissue, and thus milder muscle, but worse liver insulin resistance. Here, we test the hypothesis that the effects of B6 vs. FVB genetic background on glucose and lipid phenotypes are manifest in the ob/ob mouse, a diabetes model in which adipose tissue is in excess, rather than missing. We demonstrate that, like A-ZIP/F-1 mice, B6 ob/ob mice are less hyperglycemic and have less muscle insulin resistance than FVB ob/ob mice. Additionally, differences between B6 ob/ob and FVB ob/ob mice were observed in acute lipid clearance and circulating adiponectin levels.

IABETES AND OBESITY are complex genetic diseases caused by a combination of genetic predisposition and environmental exposure (1, 2). The genetic contribution can be either monogenic or polygenic, with polygenic inheritance being the predominant mode of inheritance in human type 2 diabetes and obesity. Although rare, single mutations causing monogenic obesity have been identified (3–5). Notable among these are mutations of the leptin gene, leading to a complete deficiency of the adipocyte-produced protein hormone leptin (6). The Lepob (hereafter, ob) mutation arose spontaneously in C57BL/6J (hereafter B6) mice; ob/ob mice on this genetic background have severe early-onset obesity, hyperphagia, hyperinsulinemia, and insulin resistance with modest hyperglycemia (7). Although leptin deficiency causes the major phenotypic features of ob/ob mice, studies of ob/ob mice with different genetic backgrounds demonstrate the importance of the strain-related modifier genes. For example, congenic ob/ob mice on the BLKS background are initially obese, but then lose weight and die prematurely due to severe diabetes with islet ␤-cell failure (8, 9). In comparison, ob/ob mice on the BALB/cJ genetic background have a more modest increase in white adipose tissue mass, more severe diabetes, improved fertility, and normal tolerance to cold (10). Finally, ob/ob mice on the FVB/N (hereafter FVB) background have persistent hyperinsulinemia with hyperglycemia but without any signs of premature ␤-cell failure (11).

Animals

Abbreviation: ScD-1, Stearoyl-coenzyme A desaturase-1. Endocrinology is published monthly by The Endocrine Society (http:// www.endo-society.org), the foremost professional society serving the endocrine community.

Female B6 ob/ob mice and their wild-type littermates were purchased from the Jackson Laboratory (Bar Harbor, ME). The FVB-ob/ob congenic strain was developed by backcrossing the Lepob allele from the C57BL/ 6J-ob strain for a minimum of 10 generations as described previously (11). Wild-type littermates of FVB ob/ob mice were used as controls.

Materials and Methods

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Experiments were performed using mice at 10 wk of age, except for the clamp study, which used 7-wk-old mice. The blood glucose, serum insulin, and adiponectin concentrations measurements were repeated in an independent set of animals, with results similar to those described bellow. Animals were maintained on a 12-h light, 12-h dark cycle (0600 h, 1800 h) and a standard pellet diet (NIH-07, 5% fat by weight). Animal experiments were approved by the National Institute of Diabetes and Digestive and Kidney Diseases Animal Care and Use Committee.

account for the majority of insulin-stimulated glucose uptake (18). Muscle and white and brown adipose tissue glucose uptake was calculated from the plasma 2-deoxy-d-[1-14C] glucose concentration profile (using plasma 14C counts at 80 –120 min, the area under the curve was calculated by trapezoidal approximation) and tissue 2-deoxy-d-[1-14C] glucose-6-phosphate content as described previously (17).

Hyperinsulinemic-euglycemic clamp

Glucose was measured using a Glucometer Elite (Bayer, Elkhart, IN). Insulin and adiponectin (no. SRI-13K and no. MADP-60HK, respectively, Linco Research, St. Charles, MO), triglycerides (no. 337-B, Sigma, St. Louis, MO), and nonesterified fatty acids (no. 13831175, Roche Molecular Biochemicals, Indianapolis, IN) were quantitated with the indicated kits. Liver triglycerides were measured by solvent extraction followed by a radiometric assay for glycerol as previously described (19 –21).

Catheters were implanted under ketamine and xylazine anesthesia. The SILASTIC catheter (inside diameter, 0.30 mm; outside diameter, 0.64 mm; no. 508 – 001; Dow Corning Corp., Midland, MI), filled with heparin solution (100 USP U/ml in 0.9% NaCl), was inserted via a right lateral neck incision, advanced into the superior vena cava via the right internal jugular vein, and sutured in place [procedure adapted from MacLeod and Shapiro (14)]. The distal end of the catheter was knotted, tunneled sc, exteriorized first at the dorsal cervical midline, and then further tunneled sc and exteriorized in the dorsal midline, 2 cm above the tail. A silk suture was fastened around the catheter at the neck site. The clamps were then performed 4 –5 d later, after complete recovery of the animals from the operation. On the day of the clamp, the catheter was externalized by pulling the suture through the dorsal cervical incision site. The clamps were performed after 16 h of fasting in conscious mice, as described in detail previously (15, 16), using [3-3H]glucose and 2-deoxy-d-[1-14C] glucose (both from NEN Life Science Products, Boston, MA) for the estimation of whole-body glucose fluxes and tissue glucose uptake, respectively.

Biochemical and hormonal assays

Triglyceride clearance test Clearance of triglycerides (400 ␮l peanut oil, delivered by gavage) from the circulation was measured in mice previously fasted for 4 h. Blood was taken before gavage and hourly for 6 h after gavage, and plasma triglycerides were measured as described above.

RNA analysis Total RNA extraction, Northern blots, and quantitation by phosphorimager were done as previously described, using probes excised from plasmids (22).

In vivo glucose flux analysis The determination of plasma [3-3H]glucose and 2-deoxy-d-[1-14C] glucose concentrations and tissue 2-deoxy-d-[1-14C] glucose-6-phosphate were performed as described previously (17).

Statistical analysis Data are expressed means ⫾ se. Statistical significance between the groups was determined with SigmaStat (SPSS Inc, Chicago, IL) using two-way ANOVA or t test, as appropriate.

Calculations Basal endogenous glucose production was calculated as the ratio of the preclamp [3-3H]glucose infusion rate (dpm/min) to the specific activity of the plasma glucose (mean of the values in the 90 and 120 min of basal preclamp period, in dpm/␮mol). Clamp whole-body glucose uptake was calculated as the ratio of the [3-3H]glucose infusion rate (dpm/min) to the specific activity of plasma glucose (dpm/␮mol) during the last 30 min of the clamp (mean of the 90 –120 min samples). Whole-body glycolysis was determined from the rate of increase in plasma 3H2O determined by linear regression using the 80 –120 min points. Plasma 3H2O concentrations were measured from the difference between nondried vs. dried plasma 3H counts. Clamp endogenous glucose production was determined by subtracting the average glucose infusion rate in the last 30 min of clamp from the whole-body glucose uptake. Whole-body glycogen and lipid synthesis were estimated by subtracting the whole-body glycolysis from the whole-body glucose uptake, which assumes that glycolysis and glycogen/lipid synthesis

Results Anatomical and biochemical characteristics of ob/ob mice on B6 and FVB background

At 10 wk of age, ob/ob mice on both B6 and FVB genetic background were morbidly obese, with body weights 2.4fold higher than wild-type littermates (Table 1). FVB ob/ob mice were 3 g heavier than B6 ob/ob mice, the same as the difference between wild-type mice of B6 and FVB strains. The body length of ob/ob mice of both strains was higher relative to respective wild-type controls. FVB mice were significantly longer relative to respective B6 groups. Liver weights in ob/ob mice were 3.1- and 2.5-fold greater than wild-type B6 and FVB controls, respectively (Table 1). The difference in liver

TABLE 1. Metabolic and anatomical parameters of female ob/ob and wild-type mice on B6 and FVB genetic background

Body weight (g) Body length (cm) Liver weight (g) Liver weight (% of body weight) Liver triglycerides ␮mol/g ␮mol/liver Inguinal fat pad weight (g) Gonadal fat pad weight (g) Brown adipose tissue (mg)

Wild type, B6

Ob/ob, B6

Wild type, FVB

ob/ob, FVB

23.3 ⫾ 0.3 8.6 ⫾ 0.2 1.24 ⫾ 0.01 5.39 ⫾ 0.11

56.9 ⫾ 1.5a 9.4 ⫾ 0.1a 3.83 ⫾ 0.21a 6.73 ⫾ 0.28a

26.2 ⫾ 0.4b 9.1 ⫾ 0.1 1.23 ⫾ 0.05 4.69 ⫾ 0.20b

60.4 ⫾ 2.4a 10.1 ⫾ 0.1ac 3.03 ⫾ 0.09ac 5.02 ⫾ 0.10c

7.45 ⫾ 0.51 9.28 ⫾ 0.62 0.21 ⫾ 0.02 0.20 ⫾ 0.02 77 ⫾ 2

19.5 ⫾ 1.0a 75.3 ⫾ 5.82a 4.57 ⫾ 0.22a 4.69 ⫾ 0.14a 381 ⫾ 36a

8.43 ⫾ 0.82 10.4 ⫾ 1.4 0.47 ⫾ 0.04 0.64 ⫾ 0.06b 118 ⫾ 5

15.9 ⫾ 1.1ac 48.3 ⫾ 4.4ac 5.98 ⫾ 0.40ac 2.84 ⫾ 0.20ac 474 ⫾ 61a

Samples are from nonfasting mice anesthetized with avertin and bled between 0900 and 1200 h. Values are mean ⫾ SE with n ⫽ 6/group. Statistical significance is from two-way ANOVA. a P ⬍ 0.05 vs. wild-type group of the same genotype; b P ⬍ 0.05 for FVB wild-type vs. B6 wild-type group; c P ⬍ 0.05 for the interaction between ob mutation and background genotype.

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weight was paralleled by a difference in liver triglycerides, significantly higher in the B6 ob/ob mice, indicating greater liver steatosis in the B6 ob/ob mice. No significant difference in liver triglycerides was observed between B6 and FVB wild-type groups (Table 1). Inguinal and gonadal fat pad weights were 10- to 20-fold higher in ob/ob mice compared with wild-type controls. Inguinal fat pad weight was higher in FVB ob/ob than in the B6 ob/ob mice, whereas the opposite was observed for gonadal fat pad weight (Table 1). Brown adipose tissue weight was increased in ob/ob mice, with no significant strain-related differences (Table 1). Blood glucose was moderately (1.7-fold) elevated in B6 ob/ob mice as compared with wild-type littermates, whereas FVB ob/ob mice were severely diabetic, with blood glucose levels 4-fold higher than in wild-type mice and 2-fold higher than in B6 ob/ob mice (Table 2). Insulin levels were comparably increased in ob/ob mice of both strains, 40-fold higher than in wild-type mice (Table 2). Triglyceride levels were significantly elevated in ob/ob mice and by the FVB background (in both wild-type and ob/ob mice, Table 2). Circulating adiponectin levels were 2.5-fold higher in FVB wild-type mice compared with FVB ob/ob animals (Table 2).

Interestingly, no difference in adiponectin levels was found between B6 ob/ob and wild-type mice or between B6 and FVB wild-type mice. Adiponectin mRNA expression in white adipose tissue of B6 ob/ob mice was 3-fold lower than in wildtype mice; whereas in FVB ob/ob mice, it was further reduced, being 12-fold decreased relative to wild-type mice. Resistin mRNA expression in white adipose tissue followed the same pattern as that of adiponectin, with a 4-fold reduction in B6 ob/ob and 14-fold reduction in FVB ob/ob mice relative to wild-type controls. Triglyceride clearance is strongly affected by genetic background

A possible contribution to the greater liver triglycerides in the B6 ob/ob mice is increased hepatic triglyceride clearance. Because we have observed that B6 vs. FVB genetic background significantly affects lipid handling (12), we studied triglyceride clearance after an oral lipid load in the ob/ob mice. Two profound effects were seen. We confirmed that wildtype B6 mice have much more rapid triglyceride clearance than do wild-type FVB mice. Triglyceride clearance was also

TABLE 2. Blood glucose, serum biochemical and hormonal parameters, and adipose tissue adiponectin, and resistin mRNA expression of female ob/ob and wild-type mice on B6 and FVB genetic background

Blood glucose (mmol/liter) Serum triglycerides (mmol/liter) Serum insulin (ng/ml) Serum adiponectin (ng/ml) Adipose tissue adiponectin mRNA (%) Adipose tissue resistin mRNA (%)

Wild-type, B6

Ob/ob, B6

Wild-type, FVB

ob/ob, FVB

6.3 ⫾ 0.2 0.34 ⫾ 0.04 0.88 ⫾ 0.16 2177 ⫾ 174 100 ⫾ 4 100 ⫾ 21

11.5 ⫾ 1.6a 0.59 ⫾ 0.10a 37.83 ⫾ 7.99a 2687 ⫾ 396 32 ⫾ 6a 26 ⫾ 5a

6.8 ⫾ 0.2 0.50 ⫾ 0.05b 0.97 ⫾ 0.35 2518 ⫾ 226 96 ⫾ 23 91 ⫾ 3

26.6 ⫾ 1.6ac 0.72 ⫾ 0.07ac 43.99 ⫾ 5.35a 1034 ⫾ 138ac 9 ⫾ 3ac 8 ⫾ 2ac

Samples are from nonfasting mice anesthetized with avertin and bled between 0900 and 1200 h. Values are mean ⫾ SE with n ⫽ 6/group. Adiponectin and resistin mRNA amount is expressed as a percentage of wild-type B6 group (wild-type B6 ⫽ 100%). Statistical significance is from two-way ANOVA. a P ⬍ 0.05 vs. wild-type group of the same genotype; b P ⬍ 0.05 for FVB wild-type vs. B6 wild-type group; c P ⬍ 0.05 for the interaction between ob mutation and background genotype.

FIG. 1. Triglyceride clearance after peanut oil gavage in female B6 WT (wild-type) (F), B6 ob/ob (E), FVB WT (), and FVB ob/ob (ƒ) mice. Values are means ⫾ SE, with n ⫽ 5– 6/group. The areas under curves (mean ⫾ SE) are 790 ⫾ 80 for B6 WT, 1296 ⫾ 130 for B6 ob/ob, 1915 ⫾ 234 for FVB WT, and 3515 ⫾ 640 for FVB ob/ob mice.



reduced by the ob/ob mutation but with an effect smaller in magnitude than that of background genotype (Fig. 1).

mice, whereas no strain difference was found in white or brown adipose tissue glucose uptake.

Increased liver insulin resistance and decreased muscle insulin resistance in B6 vs. FVB ob/ob mice

The changes in the liver mRNA expression of the genes involved in glucose and lipid metabolism

Although B6 and FVB ob/ob mice have a similar degree of obesity and increase in circulating insulin levels, FVB ob/ob mice are severely diabetic, whereas B6 ob/ob mice are only moderately hyperglycemic. This indicates a difference in insulin sensitivity caused by genetic background. To study whole-body and tissue insulin sensitivity in detail, we performed euglycemic-hyperinsulinemic clamps. Basal plasma glucose levels, after 16-h fasting in B6 ob/ob mice, were not significantly different from wild-type mice on either genetic background, whereas basal plasma glucose of FVB ob/ob mice was 2-fold higher than in FVB controls (Table 3). Fasting insulin levels were 4-fold higher in ob/ob mice of both strains as compared with wild-type controls (Table 3). Basal endogenous glucose production was significantly decreased in B6 ob/ob mice as compared with the rest of the groups (Fig. 2), possibly reflecting their decreased body temperature after fasting at the start of the clamp (data not shown). Whole-body glucose uptake during the clamp reflects predominantly muscle glucose uptake and thus insulin sensitivity. Although this variable was greatly reduced in ob/ob mice of both strains, it was 30% higher in B6 ob/ob mice than in FVB ob/ob mice, indicating relatively less insulin resistance in the B6 ob/ob compared with the FVB ob/ob mice (Fig. 3). Whole-body glycolysis was similarly reduced greatly in ob/ob mice but less so on the B6 than the FVB background. Wholebody glycogen/lipid synthesis was decreased to the same extent in B6 and FVB ob/ob mice (Fig. 3). Clamp endogenous glucose production is a measure of liver insulin sensitivity. Clamp endogenous glucose production in both B6 and FVB wild-type mice was appropriately low, due to suppression by the hyperinsulinemia of the clamp (Fig. 2). In ob/ob mice of both strains, the clamp endogenous glucose production remained high, reflecting severe liver insulin resistance (Fig. 2). However, when expressed as the percent suppression of basal endogenous glucose production, in FVB ob/ob mice this value was suppressed by 27%; whereas in B6 ob/ob mice, glucose production actually increased from the basal values (P ⫽ 0.06, B6 ob/ob vs. FVB ob/ob mice). Glucose uptake measured directly in muscle, white, and brown adipose tissue was severely reduced in ob/ob mice of both strains as compared with controls (Fig. 4). Muscle glucose uptake of B6 ob/ob mice was 40% higher than in FVB ob/ob

To further clarify the etiology of the differences between B6 and FVB strains, liver mRNA levels of selected genes involved in gluconeogenesis, lipogenesis, lipid transport, and oxidation were measured. Levels of gluconeogenic [glucose-6-phosphatase (G6P), phosphoenolpyruvate carboxykinase (PEPCK)], lipogenic [fatty acid synthase (FAS), stearoyl-coenzyme A desaturase-1 (SCD-1)], lipid oxidation [acyl-coenzyme A oxidase (AOX), carnitine palmitoyl transferase-1 (CPT-1)], and fatty acid transport (CD36) mRNAs were significantly increased in ob/ob mice of both strains (Fig. 5). The increase of lipogenic genes was generally more pronounced on B6 genetic background, with SCD-1 being 2.1fold higher in B6 wild-type compared with FVB wild-type, and 3.4-fold higher in B6 ob/ob compared with FVB ob/ob. Discussion

Despite a similar degree of obesity and circulating insulin levels, B6 ob/ob mice have only modest diabetes in contrast to severely hyperglycemic FVB ob/ob mice. The euglycemichyperinsulinemic clamp studies revealed that this difference

FIG. 2. Basal (black bars) and clamp (white bars) endogenous glucose production in female B6 wild-type, B6 ob/ob, FVB wild-type, and FVB ob/ob mice. Values are means ⫾ SE, n ⫽ 6/group. *, P ⬍ 0.05 vs. clamp EGP of the control group of the same genotype; ⫹, P ⬍ 0.05 for basal EGP of ob/ob groups vs. basal endogenous glucose production (EGP) of wild-type group of the same genotype. BW, Body weight.

TABLE 3. Metabolic parameters during basal (16-h fasted) period and hyperinsulinemic-euglycemic clamp in female wild type and ob/ob mice on FVB and B6 genetic backgrounds

Body weight (g) Basal plasma glucose (mmol/liter) Basal plasma insulin (ng/ml) Clamp plasma glucose (mmol/liter) Clamp plasma insulin (ng/ml)

Wild type, B6

ob/ob, B6

Wild type, FVB

ob/ob, FVB

15.6 ⫾ 0.6 7.8 ⫾ 0.2 0.42 ⫾ 0.05 6.4 ⫾ 0.2 2.8 ⫾ 0.5

29.7 ⫾ 1.3 8.9 ⫾ 1.3 2.60 ⫾ 0.56a 7.3 ⫾ 0.7 4.4 ⫾ 0.2a

19.4 ⫾ 0.5 7.1 ⫾ 0.5 0.44 ⫾ 0.05 6.5 ⫾ 0.2 3.24 ⫾ 1.0

38.2 ⫾ 1.9a 13.6 ⫾ 0.8a 1.99 ⫾ 0.22a 8.0 ⫾ 0.7 4.6 ⫾ 0.5a

a

Values are mean ⫾ SE, with n ⫽ 5– 6/group. Statistical significance is from two-way ANOVA. a P ⬍ 0.05 vs. wild-type group of the same genotype.

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FIG. 3. Whole-body glucose fluxes during hyperinsulinemic-euglycemic clamps in female B6 wild-type, B6 ob/ob, FVB wild-type, and FVB ob/ob mice. Values are means ⫾ SE, n ⫽ 6/group. *, Statistical significance is from two-way ANOVA; *, P ⬍ 0.05 vs. wild-type group of the same genotype; ⫹, P ⬍ 0.05 for the interaction between ob mutation and background genotype.

FIG. 4. Muscle (gastrocnemius), white adipose tissue (WAT), and brown adipose tissue (BAT) glucose uptake in female B6 wild-type, B6 ob/ob, FVB wild-type, and FVB ob/ob mice. Values are means ⫾ SE, n ⫽ 6/group. Statistical significance is from two-way ANOVA; *, P ⬍ 0.05 vs. wildtype group of the same genotype; ⫹, P ⬍ 0.05 for the interaction between ob mutation and background genotype.

is due to milder muscle insulin resistance in B6 ob/ob mice. What is the cause of less muscle insulin resistance on B6 relative to FVB ob/ob mice? Obesity can cause insulin resis-

tance by accumulation of triglycerides and/or other lipid metabolites in nonadipose tissues, by imbalance of hormones or other metabolic signals produced by adipose tissue



FIG. 5. Liver mRNA levels of the genes involved in the metabolism of glucose, triglycerides, and fatty acids. In each set, the order of the bars is B6 wild-type, B6 ob/ob, FVB wild-type, and FVB ob/ob. Data are expressed as percentage of B6 wild-type levels. Data are from female mice (mean ⫾ SE, n ⫽ 5– 6/group). Statistical significance is from two-way ANOVA; *, P ⬍ 0.05 for ob/ob vs. wild-type; ⫹, P ⬍ 0.05 for B6 vs. FVB; ˆ, P ⬍ 0.05 for background genotype and ob mutation interaction. G6P, Glucose-6-phosphatase; PEPCK, phosphoenolpyruvate carboxykinase; FAS, fatty acid synthase; CPT-1, carnitine palmitoyltransferase-1; AOX, acyl-coenzyme A oxidase; SREBP1c, sterol regulatory element binding protein lc.

(TNF-␣, resistin, adiponectin, and others), or by a combination of both (23–26). We showed previously that both wildtype B6 and lipoatrophic A-ZIP/F-1 B6 mice have much faster triglyceride clearance than wild-type and A-ZIP/F-1 mice of FVB background, with more pronounced lipid deposition into the liver (12). Repartitioning of triglycerides from the muscle to the liver worsened liver insulin resistance but improved muscle insulin resistance, leading to milder diabetes (12). The B6 vs. FVB strain differences in triglyceride clearance in ob/ob mice were similar to those previously observed in A-ZIP/F-1. The same was true for liver triglyceride accumulation, which was more pronounced in B6 relative to FVB ob/ob mice. Thus, triglyceride handling is strongly affected by genetic background under highly divergent conditions: a lack of fat in A-ZIP/F-1 mice and excessive fat accumulation in ob/ob mice. As a result, B6 mice have increased capacity to store triglyceride in the liver, partially protecting muscle from lipid overload and thus from development of more severe insulin resistance. We also studied the adipose-derived hormones, adiponectin and resistin, to examine their possible role in strainrelated differences in diabetes/insulin resistance of ob/ob mice. Circulating adiponectin levels are usually inversely related to obesity and insulin resistance (27–29). In mice, acute adiponectin administration decreased blood glucose and increased fatty acid oxidation in the skeletal muscle (30) and liver insulin sensitivity measured by euglycemic-hyperinsulinemic clamp (31). Moreover, transgenic mice lacking adiponectin are more prone to diet-induced insulin resistance relative to wild-type controls (29, 32). Here, we show that relatively less diabetic B6 ob/ob mice had normal circulating adiponectin levels in contrast to a 50% reduction in

FVB ob/ob mice. Normal circulating adiponectin levels in B6 ob/ob mice contrasted with clearly (3-fold) decreased adipose tissue adiponectin mRNA expression. Our data thus suggest that circulating adiponectin levels do not necessarily correlate with its adipose tissue mRNA expression. Moreover, circulating adiponectin in B6 ob/ob mice at the age of 10 wk did not behave as a precise measure of insulin sensitivity, because its concentrations in severely insulin-resistant B6 ob/ob mice did not differ from those of B6 wild-type mice with normal insulin sensitivity. Normal circulating adiponectin levels in B6 ob/ob mice could have contributed to the milder diabetes of this strain relative to FVB ob/ob mice. The importance of this contribution, however, remains questionable, in the light of the results of our previous study that compared lipoatrophic A-ZIP/F-1 mice on the same genetic backgrounds (12). A-ZIP/F-1 mice have almost complete lack of white adipose tissue and thus do not produce any adipose tissue-derived hormones, including adiponectin (12, 33). Despite this fact, strain-related differences in lipid handling and insulin sensitivity in A-ZIP/F-1 mice were very similar to those observed in ob/ob mice in this study. Increased circulating resistin levels were proposed to link obesity to insulin resistance (34). However, others reported decreased resistin mRNA expression in adipose tissue of obese mice and humans (35, 36). The role of resistin as a causal factor in the development of insulin resistance is thus controversial. Our data show that resistin adipose tissue mRNA levels were 4-fold decreased in B6 ob/ob mice and even more (14-fold) decreased in more diabetic and insulinresistant FVB ob/ob mice compared with wild-type mice. These results argue against an etiologic role for resistin in the strain-related differences studied herein.

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In summary, we show here that different genetic background strongly modifies the severity of diabetes, insulin resistance, and response to fasting in genetically leptin-deficient ob/ob mice. Identification of background modifier genes could bring further insight into the mechanism of development of diabetes and insulin resistance relevant for human biology. Acknowledgments Received February 18, 2004. Accepted March 25, 2004. Address all correspondence and requests for reprints to: Martin Haluzik, Third Department of Medicine, First Faculty of Medicine, Charles University, U nemocnice 1, 12808, Prague-2, Czech Republic. E-mail: [email protected]. This work was supported, in part, by Grant IGA MHCR 7429-3 (to M.H.). Present address for M.L.R.: Merck Research Laboratories, Rahway, New Jersey 07065

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