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Volume 4, Issue 5 EXPERIMENTAL BIOLOGY 2000Access to full-text is available with a subscription to Obesity-news. To subscribe please fill out our subscription information form. Subscribing is easy using your American Express, VISA or Mastercard on our secure server. We also take credit card orders by fax at 703/960-7462. Contents
Most obesity treatments being developed suppress appetite, increase energy expenditure or cause the malabsorbtion of food, but recently a new gene has been identified that may actually inhibit the body's ability to make new fat cells when suppressed. Mice lacking the gene high mobility group 1C (HMGIC) develop only 10 percent of the body fat of normal mice no matter what they eat. The gene was discovered while investigating an effective treatment for lipomas. Lipomas are benign tumors that develop in fat tissue, but they can become malignant as they grow. In a letter published in the April 24 issue of Cell, Kiran Chada and Ashim Anand of the Robert Wood Johnson Medical School in Camden, New Jersey, report HMGIC deficient mice that eat a high fat diet gain no more weight than littermates or control mice eating a standard diet. They also found that a disruption of HMGIC reduces obesity caused by leptin deficiency. Chada and Anand first became interested in HMGIC when they observed that the fat depots of mice fed a high fat diet expressed HMGIC, whereas mice fed a standard diet did not. After developing HMGIC deficient mouse models, they tested their hypothesis that the mice would be resistant to obesity in the following two experiments.
Why are HMGIC mice resistant to weight gain?While the actual mechanism is unknown, Chada and Anand propose that HMGIC prevents obesity by reducing the proliferation of preadipocytes. Under obesity inducing conditions the number of fat cells increases in normal animals. The absence of HMGIC results in mice with fewer mature adipocytes and hence less fat. This completely prevents weight gain produced by a high-fat diet, and reduces weight gain induced by leptin deficiency. Chada and Anand suggest that the reason obesity is not completely eliminated in ob/ob mice is that leptin and HMGIC function via independent genetic pathways. A novel obesity drug?HMGIC has many advantages over appetite suppressants that stimulate the central nervous system. First it is specific to the tissue responsible for the obese phenotype, and second the gene-dosage effect suggests titratable pharmacological action for HMGIC inhibitors. This means that HMGIC inhibitors would be available to patients who cannot take CNS acting drugs, and that dosage could be easily adjusted to accommodate for varying levels of obesity. Dr. Rudy Leibel, director of the Molecular Genetics Lab at Columbia University, expressed concern that restricting the number of fat cells might result in fat building up in the liver. However, Chada says that he has seen no sign of fatty liver buildup in his experiments.
Suppression of an enzyme responsible for triglyceride synthesis produces thin and healthy mice, according to a study published in the May issue of Nature Genetics. The enzyme, diacylglycerol o-acyltransferase (Dgat), is a protein that catalyzes the final step in triglyceride synthesis. At one time Dgat was thought to be the fundamental regulator of triglyceride, and that animals without it would die. But not only are Dgat deficient mice lean, they have an increased metabolic rate and are resistant to diet induced obesity. The only abnormality seen in the mice was that the females do not lactate.
Feeding experiment.Researchers had hypothesized that Dgat deficiency would result in lower body weight. This turned out to be untrue. Dgat and control mice fed a standard diet had similar body weights, although the Dgat -/- mice had less fat and lower triglyceride levels. But when control mice were fed a high-fat diet they became obese (45-50 g), whereas Dgat -/- mice remained at a comparable weight to chow-fed mice (30-35 g). Even while on a high fat diet, total triglycerides were lower in Dgat -/- mice (see table). Why are Dgat -/- mice obesity resistant?The mutant mice stay thin because they expend more energy. To test energy expenditure, control and Dgat -/- mice were placed in metabolic cages for two days on a standard diet and then for seven days on a high-fat diet. During chow feeding Dgat -/- mice had a higher metabolic rate. The increase in energy expenditure was unexpected and researchers are unsure of the cause. On the high-fat diet, Dgat -/- mice were twice as active as wild-type mice, indicating that their activity levels contributed to the higher energy expenditure. But Dgat -/- mice did not move more when fed a standard diet, nor did the increased energy expenditure result from greater lean body mass, or increased thermogenesis measured by core body temperature. Researchers also tested for evidence of fat malabsorbtion, by measuring fecal output and caloric content, but found no evidence that this was the cause of the lower weight.
Other measurements were taken, including thyroid hormone and leptin. thyroxine levels were normal in Dgat -/- mice, but leptin levels were lower, probably reflecting the difference in white fat between the groups. Although mouse models with reduced white adipose tissue are known to exhibit insulin resistance, Dgat -/- mice had lower glucose and insulin levels after a glucose tolerance test, suggesting increased insulin sensitivity. Dgat deficiency altered triglyceride metabolism in other tissues, notably in the mammary tissue of female mice. Females with the deletion did not lactate, and pups had to be transferred to foster mothers for survival. In addition, Dgat -/- mice fed a high-fat diet accumulated less fat in the liver. The mutation also had effects on the skin and fur of the mice. Conclusions.Researchers conclude that Dgat activity is not the only mechanism for triglyceride synthesis, although they do not know what other mechanisms may exist. In an accompanying editorial, Dr. C. Ronald Kahn pointed out that this discovery is just one more example of the redundancies that exist in energy regulation. The fact that Dgat -/- mice are viable, lean and resistant to diet induced obesity, suggests that disrupting triglyceride synthesis is a target for obesity treatment.
Several pharmaceutical companies have announced that they are developing protein tyrosine phosphatase-1B (PTP1B) inhibitors to treat type-2 diabetes and obesity. In presentations at the annual meeting of the American Chemical Society, researchers from Wyeth-Ayerst Research, Pharmacia & Upjohn, and Ontogen Corporation described compounds under investigation.
Last year Merck Frosst researchers reported that mice lacking the PTP1B gene were resistant to diet-induced obesity, remained insulin sensitive and had significantly lower triglyceride levels. The fact that an absence of PTPIB protects against obesity was unexpected. Increasing insulin sensitivity drives glucose into cells, which has been shown to increase adiposity. Thiazolidinediones like Avandia and Rezulin increase insulin sensitivity by increasing adipocyte differentiation via PPAR-gamma, often resulting in weight gain. The reason that the mutant mice do not gain weight is unknown, but the results suggest that they are burning more calories. If the companies are successful in producing a bioavailable PTP1B inhibitor, it is likely to be safer than existing diabetes and obesity drugs, as the mice in the study were healthy and showed no signs of toxicity. Wyeth-Ayerst is looking at four classes of compounds that appear effective: terphenyls, tricycles, biphenyls and salicylates. The company has already tested a PTP1B inhibiting biphenyl compound, known as compound 68, on ob/ob mice. Mice treated with compound 68 for 4 days experienced a significant decrease in glucose, insulin and triglyceride levels. Pharmacia & Upjohn is investigating mimetics and Ontogen small molecule drugs that inhibit PTP1B.
Small-molecule inhibitors of PTP1B. Jones TK, et al. Ontogen Corporation. 219th ACS National Meeting, March 26-30, 2000, San Francisco, California. Abstract. Through high throughput screening, we have identified classes of low molecular weight compounds that are active site inhibitors of the protein tyrosine phosphatase enzyme PTP1B. In knock-out mice studies reported in the literature this enzyme has been demonstrated to play an important role in blood glucose levels, validating PTP1B as a target for diabetes. Our experience in this area, including synthesis, docking studies of our molecules with the X-ray structure, selectivity and other biological activity will be reported.
Summary. Pramlintide (Symlin), a synthetic amylin analog currently in Phase 3 human trials, was found to delay gastric emptying in both type-1 and type-2 diabetics in a dose dependant manner. In other studies, patients taking Symlin and insulin lost weight, while patients taking insulin alone gained weight. Patients also experienced a reduction in hemoglobin A1c (HbA1c) and an improvement in lipid profiles. Symlin, an injectable medication, is being developed as an adjutant therapy for type-1 and type-2 diabetics who are insulin dependent.
Amylin and gastric emptying.Abstract. Pramlintide an amylin analog, delays gastric emptying lowering postprandial glucose concentrations. It is unknown if people with type 1 diabetes (DM) who are amylin deficient are more sensitive to pramlintide than people with type 2 DM who have normal/elevated levels of amylin. Also, the dose-related effects of pramlintide, and its mechanism of action are unknown. To address these questions, we utilized a crossover 3 dose, randomized, placebo-controlled design to study 6 people with type 1 and 6 with type 2 DM. Subjects received 0, 30 or 60g tid sc of pramlintide. Gastric emptying of solids was measured by 13C-Spirulina breath test. Pramlintide delayed gastric emptying in a dose-related manner, with greater prolongation with 30 vs 60g (p=0.03); there was no difference in gastric-emptying in type 1 or type 2 DM (p>0.6). Postprandial pancreatic polypeptide (PP) concentrations were lower with pramlintide than placebo (p<0.05). The reduction in PP was more pronounced in people with type 1 DM (p<0.01). An inverse correlation was observed between mean PP concentration in the first postprandial hour and gastric emptying t½ (rs=-0.67, p=0.05, adjusted for 3 correlation tests). Pramlintide equally delays gastric emptying in type 1 and type 2 DM.
Summary. Studies indicate that zinc increases glucose sensitivity and food intake. When animals are fed a zinc deficient diet they eat less. The relationship between zinc and glucose transport is unclear, but zinc may stimulate neuropeptide y and thyroid hormone.
Zinc and glucose sensitivity.Abstract. Zinc (Zn) has some insulin-like effects, such as promoting lipogenesis and glucose transport. The relationship between Zn and stimulation of glucose transport is unclear. We hypothesize that Zn affects the insulin (Ins) signaling pathway. In the present study, 3T3-L1-preadipocytes and -adipocytes were used. Glucose transport assays showed that Zn enhanced glucose transport ± Ins. 200uM Zn increased glucose uptake, while lower concentrations did not. The effect of Zn on adipocytes was greater than on preadipocytes. The effect of Zn plus Ins was more than either Ins or Zn alone. At 10-100nM, Wortmannin, the PI 3-kinase inhibitor, decreased basal glucose transport and blocked Zn-stimulated glucose transport in both cell types. H7, an inhibitor of PKC, did not reduce basal glucose transport but decreased Zn-induced glucose transport in a dose-dependent manner. 200uM Zn increased Tyr phosphorylation of the Ins receptor beta subunit of preadipocytes and adipocytes 5 to 20 min after treatment. This effect, however, was not as great as the effect of Ins. Tyr phosphorylation of IRS-1 and -2 was also studied. Ins increased tyrosine phosphorylation of IRS-1 and -2 within 5 min, however, 200 uM Zn did not. Zn did not have an effect on the association of the p85 subunit of PI 3-kinase and IRS-1 or -2, consistent with our data showing no zinc-dependent phosphorylation of the IRS proteins. Hence, it appears that Zn can induce an increase in glucose transport into cells, likely acting through the Ins signaling pathway. It is possible that zinc deficiency may exacerbate Ins resistance, a characteristic of Type II diabetes.
Zinc deficiency and food intake.Abstract. Rats fed a zinc deficient diet demonstrate decreased serum zinc within 2 d and voluntarily reduce food intake within 4 d. Previously, we observed rats fed a zinc-deficient diet for 4 d had a moderate increase in 4 h food intake when stimulated by an intracerebroventricular injection (ICVI) of neuropeptide Y (NPY) into the right lateral ventricle of the hypothalamus when administered at the end of the dark feeding cycle. But the magnitude of the response to NPY was less than in zinc-adequate rats. To further characterize the neuropeptide response in zinc-deficient animals we used another modulator of food intake, orexin A. Zinc-deficient rats had a similar slight increase in 4 h food intake in response to ICVI of orexin A. In contrast, the zinc-adequate rats did not increase food intake in response to orexin A. To determine if zinc depletion in the cerebrospinal fluid could explain the impaired food intake in zinc-deficient rats, we injected a zinc solution directly into the right lateral ventricle of the hypothalamus. ICVI of zinc stimulated food intake in zinc-deficient, but not zinc-adequate rats. ICVI of zinc and NPY consecutively to zinc-deficient rats stimulated a still larger increase in food intake which was greater than was produced by either compound alone. This suggests a synergistic relationship between zinc and NPY in food intake stimulation, and demonstrates a role for zinc in the regulation of food intake by the hypothalamus. Funded by USDA-CSREES grant 95-37200.
Zinc deficiency and thyroid hormone.Abstract. To determine the effects of Zinc (Zn) availability on thyroid hormone (T3) function, 28 d old male Sprague-Dawley rats were assigned to treatment in a 3X3 factorial with main effects of Zn status [Zn-deficient (Zn-D), pair-fed (PF) to Zn-D and controls (C)] and T3 status (Hypo, Eu and Hyper thyroid). Rats were made Zn-D by feeding a Zn-D diet. C and Zn-D rats had free access to food. PF were fed an identical amount of feed on a metabolic BW basis as Zn-D rats. On d 5 of dietary treatment, 5 rats/diet were made hypothyroid by inclusion of 0.025% methimazole in drinking water. On d 18, 5 rats/diet were made hyperthyroid by daily injection of T3 (15 g/100g BW i.p) and 5 rats/diet remained Eu. At slaughter (d 25 or 26) serum was harvested from trunk blood and T3, GH (growth hormone) and Insulin-like Growth Factor I (IGF) were quantified by RIA, IGF Binding Protein (BP) 2 and 3 were quantified by ligand blot and Zn was determined by atomic absorption spectroscopy. Zn in PF and C were greater than in Zn-D (31 and 29 vs 11 M). T3 was lower in Hypo and greater in Hyper compared with Eu rats. There was an interaction between T3 and Zn status on serum T3, such that in Hyper rats, T3 in Zn-D and PF (480 and 489 ng/dL) rats was greater than C (339 ng/dL). Concentrations of IGF averaged 705, 1072 and 1445 ng/mL and 752, 1509 and 989 ng/mL in Zn-D, PF and C and Hypo, Eu and Hyper animals, respectively. BP 3 in Zn-D and PF was less than C (106 and 126 vs 172 /% of standard) but T3 had no effect. Neither Zn or T3 affected GH or BP 2. Thus, Zn and T3 status altered T3 and IGF concentrations and Zn status affected BP 3 levels.
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