metallothionein and Obesity

The development of obesity consists of 3 distinct processes: differentiation of new adipocytes from precursor preadipocytic cells, formation of mature adipocytes, and excess accumulation of lipids in the adipocytes resulting in cell enlargement. This review aims to discuss the possible role of metallothionein (MT) in adipose tissue in the development of obesity. MT is induced in adipose tissue during the development of obesity. In addition, MT plays a preventive role against obesity in female mice. Although the detailed mechanism is presently unknown, one of the factors involved is the mesoderm-specific transcript (MEST/Peg1), adipocyte enlargement factor. MT has a potential to prevent obesity-related diseases, at least in part, through suppression of disease-induced generation of superoxide and endoplasmic reticulum stress and associated damage. Studies on the development of a MT inducer will encourage potential clinical use for obesity- related diseases.

Topics: Adipocytes; Adipose Tissue; Animals; Humans; Metallothionein; Obesity

Much attention has been paid to lifestyle-related diseases including type 2 diabetes mellitus, cardiovascular disease, hypertension, and hyperlipidemia because the incidence rates of these diseases are increasing in developed countries. Elucidation of factors contributing to the development of obesity and insulin resistance is needed. Metallothionein (MT), a ubiquitous metal-binding protein, is induced not only by heavy metals but also by various kinds of stresses. Endoplasmic reticulum (ER) stress is caused by accumulation of misfolded proteins in ER. Recently, increased ER stress by obesity and impairment of insulin action by ER stress have been reported. Exposure to ER stress increased induction of MT synthesis, and an enhanced response to ER stress evaluated as expression of Bip/GRP78mRNA was observed in the liver of MT-null mice, suggesting that MT attenuates expression of ER stress. MT may prevent ER stress and thereby modulate the development of obesity and insulin resistance. A possible role of metallothionein in response reaction for ER stress is discussed.

Topics: Animals; Diabetes Mellitus, Type 2; Endoplasmic Reticulum; Humans; Insulin Resistance; Metallothionein; Mice; Obesity; Stress, Physiological; Zinc

Pathogenesis of diabetic cardiomyopathy (DCM) is a complicate and chronic process that is secondary to acute cardiac responses to diabetes. One of the acute responses is cardiac cell death that plays a critical role in the initiation and development of DCM. Besides hyperglycemia, inflammatory response in the diabetic heart is also a major cause for cardiac cell death. Diabetes or obesity often causes systemic and cardiac increases in tumor necrosis factor-alpha, interleukin-18 and plasminogen activator inhibitor-1. However, how these cytokines cause cardiac cell death remains unclear. It has been considered to relate to oxidative and/or nitrosative stress. We have demonstrated that metallothionein as a potent antioxidant or stress protein significantly protected the heart from oxidative damage and cell death caused by these cytokines, leading to effective prevention of DCM. The direct link of the inhibition of oxidative stress and damage to the prevention of cardiac cell death was defined by addition of superoxide or peroxynitrite specific inhibitor to completely prevent cytokine-induced cardiac cell death. Cardiac cell death is induced by the inflammatory cytokines that is increased in response to diabetes. Inflammatory cytokine-induced cardiac cell death is mediated by oxidative stress and is also the major initiator for DCM development.

Topics: Animals; Cardiomyopathies; Cell Death; Diabetes Mellitus; Humans; Inflammation; Interleukin-18; Metallothionein; Myocardium; Obesity; Oxidative Stress; Tumor Necrosis Factor-alpha

The mechanisms responsible for beta cell compensation in obesity and for beta cell failure in type 2 diabetes are poorly defined. The mRNA levels of several metallothionein (MT) genes are upregulated in islets from individuals with type 2 diabetes, but their role in beta cells is not clear. Here we examined: (1) the temporal changes of islet Mt1 and Mt2 gene expression in mouse models of beta cell compensation and failure; and (2) the role of Mt1 and Mt2 in beta cell function and glucose homeostasis in mice.. Mt1 and Mt2 expression was assessed in islets from: (1) control lean (chow diet-fed) and diet-induced obese (high-fat diet-fed for 6 weeks) mice; (2) mouse models of diabetes (db/db mice) at 6 weeks old (prediabetes) and 16 weeks old (after diabetes onset) and age-matched db/+ (control) mice; and (3) obese non-diabetic ob/ob mice (16-week-old) and age-matched ob/+ (control) mice. MT1E, MT1X and MT2A expression was assessed in islets from humans with and without type 2 diabetes. Mt1-Mt2 double-knockout (KO) mice, transgenic mice overexpressing Mt1 under the control of its natural promoter (Tg-Mt1) and corresponding control mice were also studied. In MIN6 cells, MT1 and MT2 were inhibited by small interfering RNAs. mRNA levels were assessed by real-time RT-PCR, plasma insulin and islet MT levels by ELISA, glucose tolerance by i.p. glucose tolerance tests and overnight fasting-1 h refeeding tests, insulin tolerance by i.p. insulin tolerance tests, insulin secretion by RIA, cytosolic free Ca. Mt1 and Mt2 mRNA levels were reduced in islets of murine models of beta cell compensation, whereas they were increased in diabetic db/db mice. In humans, MT1X mRNA levels were significantly upregulated in islets from individuals with type 2 diabetes in comparison with non-diabetic donors, while MT1E and MT2A mRNA levels were unchanged. Ex vivo, islet Mt1 and Mt2 mRNA and MT1 and MT2 protein levels were downregulated after culture with glucose at 10-30 mmol/l vs 2-5 mmol/l, in association with increased insulin secretion. In human islets, mRNA levels of MT1E, MT1X and MT2A were downregulated by stimulation with physiological and supraphysiological levels of glucose. In comparison with wild-type (WT) mice, Mt1-Mt2 double-KO mice displayed improved glucose tolerance in association with increased insulin levels and enhanced insulin release from isolated islets. In contrast, isolated islets from Tg-Mt1 mice displayed impaired glucose-stimulated insulin secretion (GSIS). In both Mt1-Mt2 double-KO and Tg-Mt1 models, the changes in GSIS occurred despite similar islet insulin content, rises in cytosolic free Ca. These findings implicate Mt1 as a negative regulator of insulin secretion. The downregulation of Mt1 is associated with beta cell compensation in obesity, whereas increased Mt1 accompanies beta cell failure and type 2 diabetes.

Topics: Acrylates; Animals; Blood Glucose; Cell Line; Diabetes Mellitus, Type 2; Diet, High-Fat; Female; Gene Expression; Glucose; Glucose Tolerance Test; Humans; Insulin; Insulin Secretion; Insulin-Secreting Cells; Islets of Langerhans; Metallothionein; Mice; Obesity; Phenyl Ethers; Prediabetic State

To evaluate the role of metallothionein (MT) in sex differences of obesity, we examined the effect of MT on regulation of lipid accumulation in female and male wild type (WT) and MT1/MT2-null (MT-KO) mice.. Male and female WT and MT-KO mice fed standard diet (SD) or high-fat diet (HFD) for 35 weeks. Surgical castration in male mice was also performed to examine the effects of androgen on fat accumulation under HFD condition.. The fat mass and size of adipocytes in white adipose tissue (WAT) was greater in adult MT-KO mice than in WT mice after 35 weeks of SD feeding without gender differences, suggesting a role of MT in limiting WAT development during normal growth in both sexes. In female mice fed HFD, weights of WAT and body were greater in MT-KO mice than in WT mice, indicating that MT had a preventive role against excess fat accumulation. In male mice fed HFD, WAT weight hardly increased in MT-KO mice compared to the increase in WT mice. Surgically castrated WT males fed HFD had lower WAT weight compared with sham-treated mice, although castrated MT-KO males fed HFD had greater increases in WAT weight compared with sham-treated mice and castrated WT males.. These data suggest that MT could enhance the preventive action of estrogen against excess fat accumulation, on the contrary, MT augmented the ability of androgen to increase fat accumulation. MT may act to modify the susceptibility to obesity under sex hormones.

Topics: Adipocytes; Adipogenesis; Adipose Tissue, White; Androgens; Animals; Body Weight; Diet, High-Fat; Dietary Fats; Estrogens; Female; Lipid Accumulation Product; Male; Metallothionein; Mice; Mice, 129 Strain; Mice, Knockout; Obesity; Sex Characteristics; Sex Factors

Obesity often leads to obesity-related cardiac hypertrophy (ORCH), which is suppressed by zinc-induced inactivation of p38 mitogen-activated protein kinase (p38 MAPK). In this study, we investigated the mechanisms by which zinc inactivates p38 MAPK to prevent ORCH. Mice (4-week old) were fed either high fat diet (HFD, 60% kcal fat) or normal diet (ND, 10% kcal fat) containing variable amounts of zinc (deficiency, normal and supplement) for 3 and 6 months. P38 MAPK siRNA and the p38 MAPK inhibitor SB203580 were used to suppress p38 MAPK activity in vitro and in vivo, respectively. HFD activated p38 MAPK and increased expression of B-cell lymphoma/CLL 10 (BCL10) and caspase recruitment domain family member 9 (CARD9). These responses were enhanced by zinc deficiency and attenuated by zinc supplement. Administration of SB203580 to HFD mice or specific siRNA in palmitate-treated cardiomyocytes eliminated the HFD and zinc deficiency activation of p38 MAPK, but did not significantly impact the expression of BCL10 and CARD9. In cultured cardiomyocytes, inhibition of BCL10 expression by siRNA prevented palmitate-induced increased p38 MAPK activation and atrial natriuretic peptide (ANP) expression. In contrast, inhibition of p38 MAPK prevented ANP expression, but did not affect BCL10 expression. Deletion of metallothionein abolished the protective effect of zinc on palmitate-induced up-regulation of BCL10 and phospho-p38 MAPK. HFD and zinc deficiency synergistically induce ORCH by increasing oxidative stress-mediated activation of BCL10/CARD9/p38 MAPK signalling. Zinc supplement ameliorates ORCH through activation of metallothionein to repress oxidative stress-activated BCL10 expression and p38 MAPK activation.

Topics: Animals; B-Cell CLL-Lymphoma 10 Protein; CARD Signaling Adaptor Proteins; Cardiomegaly; Diet, High-Fat; Dietary Supplements; Gene Expression Regulation; Humans; Imidazoles; Metallothionein; Mice; Myocytes, Cardiac; Obesity; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Pyridines; RNA, Small Interfering; Signal Transduction; Zinc

Obesity continues to rise as an alarming global epidemic. System level mechanisms, diagnostics, and therapeutics are sorely needed so as to identify at risk individuals and design appropriate population scale interventions. The present study evaluated the protective role of metallothioneins (MTs) against obesity and high-fat diet-induced effects such as insulin resistance in both male and female MT-1+2 knockout and MT-3 knockout mice. As the metabolome is closest to the functional phenotype, changes in metabolite levels were also evaluated, and the direct or indirect involvement of MTs in metabolism examined. MT-1+2-, MT-3 knockout, and wild-type mice were given a high-fat diet for 2 months. Variation in body weight gain, tissue weight, and response to oral glucose tolerance test and insulin tolerance test were determined and compared to mice that received the control diet. Effect of the high-fat diet on the knockout mice were investigated on the metabolome level in specific tissues using metabolomics. Both knockout mice strains were more susceptible to high-fat diet-induced effects, such as weight gain and moderate insulin resistance, with the MT-3 knockout mice most susceptible. Brain tissue of the knockout mice showed most metabolic variation and pointed to possible impairment of mitochondrial function. The protective effect of MTs against high-fat diet and obesity-induced effects such as insulin resistance was evident from our observations. The putative role MTs play in mitochondrial function is possibly the main contributor to the lack of these effects in wild-type mice. Considering the expression profiles of the MT isoforms and similarity in brain metabolic variation in the knockout strains, it appears that they promote mitochondrial function in the hypothalamus, thereby limiting weight gain and insulin resistance. Furthermore, metabolomics research in preclinical models of obesity and in the clinic is warranted in the near future.

Topics: Adipose Tissue; Animals; Blood Glucose; Body Weight; Cerebral Cortex; Diet, High-Fat; Disease Models, Animal; Female; Glucose Tolerance Test; Insulin; Insulin Resistance; Male; Metabolome; Metabolomics; Metallothionein; Metallothionein 3; Mice; Mice, Knockout; Obesity

Research has investigated the participation of zinc transport proteins and metallothionein in the metabolism of this mineral. However, studies about the genetic expression of these proteins in obese patients are scarce. The study determined the expression of zinc transporter protein codifying genes (ZnT-1, Zip-1 and Zip-3) and of metallothionein in 55 obese women, aged between 20 and 56 years. The assessment of body composition was carried out using anthropometric measurements and bioelectrical impedance. Zinc intake was obtained by recording diet over a 3-day period, and the nutritional analysis was carried out using NutWin software version 1.5. The plasmatic and erythrocytary zinc were analyzed by atomic absorption spectrophotometry (λ=213. 9 nm). The determination of mRNA expression of the zinc transporter proteins and metallothionein was carried out using blood, using the RT-PCR method. The mean values of body mass index were 37.9±5.5 kg/m2. The average intake of zinc was 9.4±2.3 mg/day. The analysis of the zinc plasma concentrations showed values of 58.4±10.9 μg/dL. The mean values of zinc in the erythroytes were 38.7±9.1 μg/g Hb. The metallothionein gene had a higher expression in the blood, when compared to zinc transporters ZnT-1, Zip-1, and Zip-3 (p=0.01). The study shows that there are alterations in the biochemical parameters of zinc in obese patients assessed, as well as higher expression of the codifying gene metallothionein, when compared to the investigated zinc transporters.

Topics: Adult; Body Composition; Cation Transport Proteins; Female; Humans; Metallothionein; Middle Aged; Obesity; Reverse Transcriptase Polymerase Chain Reaction; Spectrophotometry, Atomic; Young Adult; Zinc

Oxidative stress accelerates adipocyte differentiation and lipid accumulation, leading to endoplasmic reticulum (ER) stress, which causes insulin resistance. Because metallothionein (MT) has a role in prevention of oxidative and ER stress, we examined the effects of MT on the development of obesity induced by 27 wk of a high-fat diet (HFD) in female MT-I- and MT-II-null (MT(-/-)) and wild-type (MT(+/+)) mice. Body weight, fat mass, and plasma cholesterol increased at a greater rate in MT(-/-) mice fed an HFD than in MT(-/-) mice fed a control diet (CD) and MT(+/+) mice fed an HFD, indicating that MT(-/-) mice fed an HFD became obese and hypercholesterolemic and that MT could prevent HFD-induced obesity. The observed increases in the levels of plasma leptin and leptin mRNA in the white adipose tissue of MT(-/-) mice fed the HFD suggested a leptin-resistant state. Enhanced expression of a mesoderm-specific transcript, which regulates the enlargement of fat cells, was accompanied by enlarged adipocytes in the white adipose tissue of young MT(-/-) mice before obesity developed after 3 and 8 wk of feeding the HFD. Thus, MT may have a preventive role against HFD-induced obesity by regulating adipocyte enlargement and leptin signaling.

Topics: Adipocytes; Animals; Cell Size; Dietary Fats; Female; Hypercholesterolemia; Leptin; Metallothionein; Mice; Mice, Knockout; Obesity; RNA, Messenger

Critical shortening of telomeres, likely associated with a considerable increase of senescent cells, can be observed in PBMC of individuals aged 80 and older. We investigated the relationship between critical telomere shortening and zinc status in healthy or hypertensive participants with or without cardiovascular disease in old and very old participants. Telomere shortening and accumulation of cells with short telomeres (percent of cells with short telomeres) in advancing age was evident in patients and healthy controls, but exacerbated in those patients aged 80 and older. Moreover, in very old patients, the accumulation of % CST may impair intracellular zinc homeostasis and metallothioneins expression, which itself is linked to an increased number of inflammatory agents, thereby suggesting the existence of a possible causal relationship between % CST and zinc homeostasis. The determination of % CST could be a more reliable means than the simple measure of telomere length as fundamental parameter in ageing to determine whether individuals are still able to respond to stress.

Topics: Aged; Aged, 80 and over; Aging; Body Mass Index; Cardiovascular Diseases; Case-Control Studies; Cellular Senescence; Down-Regulation; Homeostasis; Humans; Hypertension; Inflammation; Metallothionein; Middle Aged; Obesity; Risk Factors; Smoking; Surveys and Questionnaires; Telomere; Zinc

It has been reported that the expression of certain genes was altered in rodent cells lacking metallothioneins (MTs). To further explore the effects of MT deficiency, we screened genes differentially expressed in the liver and kidney of MT-null mice by cDNA microarray analysis. In the liver, 29 of 8737 genes analyzed were altered in their expression levels: 19 and 10 genes were up-regulated and down-regulated, respectively. Particularly, 14 of the 29 genes were related to energy metabolism, and some of these suggested that loss of MTs might lead to obesity and irregular ATP synthesis. In the kidney, 41 differentially expressed genes were observed: 27 and 14 genes were up-regulated and down-regulated, respectively. Eleven of the 41 genes were also related to energy metabolism. Microarray results were confirmed by Northern blot analysis for five of the energy metabolism-related genes.

Topics: Adenosine Triphosphate; Animals; Blotting, Northern; DNA, Complementary; Down-Regulation; Gene Expression Regulation; Gene Library; Kidney; Liver; Metallothionein; Mice; Mice, Transgenic; Models, Biological; Obesity; Oligonucleotide Array Sequence Analysis; RNA; Up-Regulation

Expression of the gene encoding metallothionein, a low molecular-weight cysteine-rich, stress-response and metal-binding protein was examined in human adipose tissue. The mRNA for MT-2A, a major metallothionein isoform in humans, was detected in subcutaneous fat using a specific antisense oligonucleotide probe. The level of MT-2A mRNA was significantly higher in a group of obese subjects than in a lean group, paralleling a similar increase in ob mRNA. A two-week period on a diet of 800 calories/day did not lead to any significant change in MT-2 mRNA levels. Separation of mature adipocytes from the cells of the stromal vascular fraction indicated that in human adipose tissue the metallothionein (MT-2A) gene is expressed both in adipocytes and in other cells of the tissue.

Topics: Adipocytes; Adipose Tissue; Adult; Blotting, Northern; Body Mass Index; Energy Intake; Female; Gene Expression; Humans; Metallothionein; Middle Aged; Obesity; Oligonucleotides, Antisense; RNA, Messenger

White adipose tissue (WAT) has been examined to determine whether the gene encoding metallothionein (MT), a low-molecular-weight stress response protein, is expressed in the tissue and whether MT may be a secretory product of adipocytes. The MT-1 gene was expressed in epididymal WAT, with MT-1 mRNA levels being similar in lean and obese (ob/ob) mice. MT-1 mRNA was found in each of the main adipose tissue sites (epididymal, perirenal, omental, subcutaneous), and there was no major difference between depots. Separation of adipocytes from the stromal-vascular fraction of WAT indicated that the MT gene (MT-1 and MT-2) was expressed in adipocytes themselves. Treatment of mice with zinc had no effect on MT-1 mRNA levels in WAT, despite strong induction of MT-1 expression in the liver. MT-1 gene expression in WAT was also unaltered by fasting or norepinephrine. However, administration of a beta(3)-adrenoceptor agonist, BRL-35153A, led to a significant increase in MT-1 mRNA. On differentiation of fibroblastic preadipocytes to adipocytes in primary culture, MT was detected in the medium, suggesting that the protein may be secreted from WAT. It is concluded that WAT may be a significant site of MT production; within adipocytes, MT could play an antioxidant role in protecting fatty acids from damage.

Topics: Adipocytes; Adipose Tissue; Animals; Cell Differentiation; Cells, Cultured; Epididymis; Gene Expression Regulation; Heterozygote; Leptin; Male; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Obese; Obesity; RNA, Messenger; Thinness; Transcription, Genetic

Metallothionein (MT) has several putative roles in metal detoxification, in Zn and Cu homeostasis, in scavenging free radicals, and in the acute phase response. Mice of mixed 129/Ola and C57BL/6J background with targeted disruption of MT-I and MT-II genes are more sensitive to toxic metals and oxidative stress. We noted that these animals were larger than most strains of mice, and we systematically studied aspects of their physiology and biochemistry relating to energy metabolism. During the first 2 weeks after weaning, the growth rates of MT-null and C57BL/6J mice were similar, but the transgenic mice became significantly heavier at age 5-6 weeks. At age 14 weeks, the body weight and food intake of MT-null mice was 16 and 30% higher, respectively, compared with C57BL/6J mice. Most 22- to 39-week-old male MT-null mice were obese, as shown by increased fat accretion, elevated obese (ob) gene expression, and high plasma leptin levels, similar to those recorded in Zucker fatty (fa/fa) rats. Seven-week-old MT-null mice also had significantly higher levels of plasma leptin and elevated expression of ob, lipoprotein lipase, and CCAAT enhancer binding protein alpha genes as compared with age-matched C57BL/6J mice. These observations indicate that abnormal accretion of body fat and adipocyte maturation is initiated at 5-7 weeks of age, possibly coincident with sexual maturation. Targeted disruption of MT-I and MT-II genes seems to induce moderate obesity, providing a new obese animal model. A link between MT and the regulation of energy balance is implied.

Topics: Adipose Tissue; Animals; Body Weight; CCAAT-Enhancer-Binding Proteins; DNA-Binding Proteins; Eating; Energy Metabolism; Insulin; Leptin; Liver; Liver Glycogen; Male; Metallothionein; Mice; Mice, Inbred C57BL; Mice, Knockout; Nuclear Proteins; Obesity; Proteins; Rats; RNA, Messenger; Transcription Factors

Mice with a temporally regulatable ovine metallothionein 1a--ovine growth hormone transgene (oMT1a-oGH) were utilized to study the effects of withdrawal of elevated circulating levels of growth hormone (GH) on growth and body composition. The transgene was activated from 21-42 days of age by provision of zinc sulfate in the drinking water. At 42 days, mice were allocated to either activated transgenic (remain on zinc sulfate) or inactivated transgenic (removal of zinc sulfate) groups, and to receive either ad libitum or restricted (80-90% of ad libitum) access to feed. Non-transgenic control mice were treated similarly. Body weights and intakes were recorded weekly. Mice were killed at 70 d and epididymal and subcutaneous fat pads, trimmed hind carcass and various organs were weighed. The main findings of this study are: (1) food-restricted mice possessing an activated oMT1a-oGH transgene fail to demonstrate increased growth, but exhibit significantly reduced levels of fat (P < 0.05) relative to all other genotype x feed level combinations; and (2) inactivation of the oMT1a-oGH transgene, following a period of elevated GH levels, leads to development of obesity as evidenced by two to three fold increases in epididymal and subcutaneous fat pad weights (P < 0.01) relative to both activated transgenic and non-transgenic control mice. These large increases in fat deposition also occurred when intake was restricted to 80-90% of ad libitum levels, indicating that metabolic changes independent of intake occur in these inactivated transgenic mice. It is possible that highly elevated production of GH in activated oMT1a-oGH transgenic mice leads to (1) enhanced promotion of preadipocyte differentiation, leading to increased numbers of adipocytes that, upon cessation of oGH production, are available for lipid deposition resulting in obesity, or (2) alterations in production of or responsiveness to insulin, leading to increased fat deposition upon removal of the chronic anti-lipogenic actions of GH. The oMT1a-oGH transgenic mouse line should provide a new genetic model with which to investigate the mechanisms by which growth hormone affects obesity.

Topics: Adipose Tissue; Animal Feed; Animals; Body Composition; Body Weight; Diet; Energy Intake; Female; Gene Expression Regulation; Growth Hormone; Male; Metallothionein; Mice; Mice, Inbred ICR; Mice, Transgenic; Obesity; Organ Size; Recombinant Proteins; Sulfates; Time Factors; Zinc Compounds; Zinc Sulfate

Cadmium metallothionein (CdMT) was injected subcutaneously into obese hyperglycaemic Umeå ob/ob mice or their lean litter mates (normal mice) at doses of 0, 0.1 and 0.4 mg Cd/kg. Proteinuria and calciuria were induced in both types of mice, but in the ob/ob mice this condition developed at a lower dose of CdMT (0.1 mg Cd/kg) than in the normal mice (0.4 mg Cd/kg). These results show, therefore, that Umeå ob/ob mice are particularly susceptible to CdMT-induced nephrotoxicity. The mechanism underlying this phenomenon needs to be further investigated. After the administration of CdMT, a dose-related increase in glycosuria was observed in both types of mice, in spite of decreased levels of serum insulin and glucose. It is suggested that such glycosuria induced by CdMT could be one of the signs of cadmium nephrotoxicity. The results of the present study thus indicate that metabolic changes like those in diabetes may increase susceptibility to cadmium-induced renal tubular damage.

Topics: Animals; Blood Glucose; Calcium; Diabetes Mellitus, Type 2; Disease Models, Animal; Dose-Response Relationship, Drug; Injections, Subcutaneous; Insulin; Kidney Diseases; Kidney Tubules; Liver; Male; Metallothionein; Mice; Mice, Obese; Obesity; Pancreas; Proteinuria; Tissue Distribution