Dietary heme injures surface epithelium resulting in hyperproliferation, inhibition of apoptosis and crypt hyperplasia in rat colon.

Epidemiological and animal model studies suggest that a high intake of heme, present in red meat, is associated with an increased risk of colon cancer. The aim of this study was to elucidate the effects of dietary heme on colonic cell homeostasis in rats. Rats were fed a purified, humanized, control diet or a similar diet supplemented with 0.5 mmol heme/kg for 14 days. Fecal water cytolytic activity was determined with a bioassay, and colon epithelial cell proliferation was evaluated with (3)H-thymidine or 5-bromo-2'-deoxyuridine incorporation into DNA or by Ki-67 immunohistochemistry. Exfoliation of colonocytes was measured as the amount of rat DNA in feces, and caspase-3 expression and activity were measured to study colonic mucosal apoptosis. Dietary heme induced a >10-fold increased cytolytic activity of the fecal water and a 100-fold lower excretion of host DNA. Colons of heme-fed rats showed injured surface epithelium and an approximately 25% increase in crypt depth. Finally, dietary heme doubled colonocyte proliferation, shown by all three markers, but inhibited colonic mucosal apoptosis. In conclusion, our results demonstrate that dietary heme injures colonic surface epithelium, which is overcompensated by inhibition of apoptosis and hyperproliferation of cells in the crypts, resulting in crypt hyperplasia. This disturbed epithelial cell homeostasis might explain why a high intake of dietary heme is associated with an increased risk of colon cancer.

Mitochondrial function, content and ROS production in rat skeletal muscle: effect of high-fat feeding.

A high intake of dietary fat has been suggested to diminish mitochondrial functioning in skeletal muscle, possibly attributing to muscular fat accumulation. Here we show however, that an 8-week high-fat dietary intervention did not affect intrinsic functioning of rat skeletal muscle mitochondria assessed by respirometry, neither on a carbohydrate- nor on a lipid-substrate. Interestingly, PPARGC1A protein increased by approximately 2-fold upon high-fat feeding and we observed inconsistent results on different markers of mitochondrial density. Mitochondrial ROS production, assessed by electron spin resonance spectroscopy remained unaffected. Intramyocellular lipid levels increased significantly illustrating that a reduced innate mitochondrial function is not a prerequisite for intra-muscular fat accumulation.

Muscular diacylglycerol metabolism and insulin resistance.

Failure of insulin to elicit an increase in glucose uptake and metabolism in target tissues such as skeletal muscle is a major characteristic of non-insulin dependent type 2 diabetes mellitus. A strong correlation between intramyocellular triacylglycerol concentrations and the severity of insulin resistance has been found and led to the assumption that lipid oversupply to skeletal muscle contributes to reduced insulin action. However, the molecular mechanism that links intramyocellular lipid content with the generation of muscle insulin resistance is still unclear. It appears unlikely that the neutral lipid metabolite triacylglycerol directly impairs insulin action. Hence it is believed that intermediates in fatty acid metabolism, such as fatty acyl-CoA, ceramides or diacylglycerol (DAG) link fat deposition in the muscle to compromised insulin signaling. DAG is identified as a potential mediator of lipid-induced insulin resistance, as increased DAG levels are associated with protein kinase C activation and a reduction in both insulin-stimulated IRS-1 tyrosine phosphorylation and PI3 kinase activity. As DAG is an intermediate in the synthesis of triacylglycerol from fatty acids and glycerol, its level can be lowered by either improving the oxidation of cellular fatty acids or by accelerating the incorporation of fatty acids into triacylglycerol. This review discusses the evidence that implicates DAG being central in the development of muscular insulin resistance. Furthermore, we will discuss if and how modulation of skeletal muscle DAG levels could function as a possible therapeutic target for the treatment of type 2 diabetes mellitus.

Natural chlorophyll but not chlorophyllin prevents heme-induced cytotoxic and hyperproliferative effects in rat colon.

Diets high in red meat and low in green vegetables are associated with an increased risk of colon cancer. In rats, dietary heme, mimicking red meat, increases colonic cytotoxicity and proliferation of the colonocytes, whereas addition of chlorophyll from green vegetables inhibits these heme-induced effects. Chlorophyllin is a water-soluble hydrolysis product of chlorophyll that inhibits the toxicity of many planar aromatic compounds. The present study investigated whether chlorophyllins could inhibit the heme-induced luminal cytotoxicity and colonic hyperproliferation as natural chlorophyll does. Rats were fed a purified control diet, the control diet supplemented with heme, or a heme diet with 1.2 mmol/kg diet of chlorophyllin, copper chlorophyllin, or natural chlorophyll for 14 d (n = 8/group). The cytotoxicity of fecal water was determined with an erythrocyte bioassay and colonic epithelial cell proliferation was quantified in vivo by [methyl-(3)H]thymidine incorporation into newly synthesized DNA. Exfoliation of colonocytes was measured as the amount of rat DNA in feces using quantitative PCR analysis. Heme caused a >50-fold increase in the cytotoxicity of the fecal water, a nearly 100% increase in proliferation, and almost total inhibition of exfoliation of the colonocytes. Furthermore, the addition of heme increased TBARS in fecal water. Chlorophyll, but not the chlorophyllins, completely prevented these heme-induced effects. In conclusion, inhibition of the heme-induced colonic cytotoxicity and epithelial cell turnover is specific for natural chlorophyll and cannot be mimicked by water-soluble chlorophyllins.

Green vegetables, red meat and colon cancer: chlorophyll prevents the cytotoxic and hyperproliferative effects of haem in rat colon.

Diets high in red meat and low in green vegetables are associated with increased colon cancer risk. This association might be partly due to the haem content of red meat. In rats, dietary haem is metabolized in the gut to a cytotoxic factor that increases colonic cytotoxicity and epithelial proliferation. Green vegetables contain chlorophyll, a magnesium porphyrin structurally analogous to haem. We studied whether green vegetables inhibit the unfavourable colonic effects of haem. First, rats were fed a purified control diet or purified diets supplemented with 0.5 mmol haem/kg, spinach (chlorophyll concentration 1.2 mmol/kg) or haem plus spinach (n = 8/group) for 14 days. In a second experiment we also studied a group that received haem plus purified chlorophyll (1.2 mmol/kg). Cytotoxicity of faecal water was determined with a bioassay and colonic epithelial cell proliferation was quantified in vivo by [methyl-(3)H]thymidine incorporation into newly synthesized DNA. Exfoliation of colonocytes was measured as the amount of rat DNA in faeces. In both studies haem increased cytotoxicity of the colonic contents approximately 8-fold and proliferation of the colonocytes almost 2-fold. Spinach or an equimolar amount of chlorophyll supplement in the haem diet inhibited these haem effects completely. Haem clearly inhibited exfoliation of colonocytes, an effect counteracted by spinach and chlorophyll. Finally, size exclusion chromatography showed that chlorophyll prevented formation of the cytotoxic haem metabolite. We conclude that green vegetables may decrease colon cancer risk because chlorophyll prevents the detrimental, cytotoxic and hyperproliferative colonic effects of dietary haem.