Prognostic impact and genomic backgrounds of renal parenchymal infiltration or micronodular spread in non-metastatic clear cell renal cell carcinoma.

A subset of clear cell renal cell carcinomas (ccRCCs) exhibits various growth patterns that infiltrate the normal renal parenchyma; however, our understanding of its association with cancer aggressiveness is incomplete. Here, we show that the morphology of the tumor interface with normal renal parenchyma is robustly associated with cancer recurrence after surgery, even when compared to the TNM staging system or the World Health Organization/International Society of Urological Pathology (WHO/ISUP) nuclear grade in non-metastatic ccRCC. Hematoxylin and eosin-stained slides of whole tissue sections from surgical specimens were analyzed using a cohort of 331 patients with non-metastatic ccRCC treated with radical nephrectomy. The patients were classified into 10 subgroups based on our classification algorithms for assessing the tumor interface with normal renal parenchyma. Among the 10 subgroups, four subgroups consisting of 40 patients (12%) were identified to have aggressive forms of non-metastatic ccRCC associated with poor prognosis and unified as renal parenchymal infiltration or micronodular spread (RPI/MNS) phenotypes. Multivariable analyses showed that RPI/MNS phenotypes were robustly associated with shorter disease-free survival, independently of existing pathological factors including the TNM staging system and WHO/ISUP nuclear grade. The hazard ratio was highest for RPI/MNS (4.62), followed by WHO/ISUP grades 3-4 (2.11) and ≥pT3a stage (2.05). In addition, we conducted genomic analyses using next-generation sequencing of infiltrative lesions in 18 patients with RPI/MNS and tumor lesions in 33 patients without RPI/MNS. Results showed that alterations in SETD2 and TSC1 might be associated with RPI/MNS phenotypes, whereas alterations in PBRM1 might be associated with non-RPI/MNS phenotypes. These data suggest that RPI/MNS may be associated with aggressive genomic backgrounds of ccRCC, although more comprehensive analyses with a larger sample size are required. Future studies may further elucidate the clinical implications of RPI/MNS, particularly for deciding the indication of adjuvant treatment after nephrectomy.

Effect of Ovariectomy and Ovarian Hormone Administration on Hepatic Autophagy in Female Rats.

Autophagy is a major intracellular proteolytic process that contributes to the maintenance of protein homeostasis. Recent studies reported the induction of autophagy in the uterus and proximal tibias of ovariectomized (OVX) rodents, which was blocked by the injection of ovarian hormones. However, whether OVX and ovarian hormone treatment can regulate autophagy in the liver has not been clarified. Recently, it was revealed that autophagy is closely involved in hepatic lipid metabolism. The purpose of this study was to investigate the influence of OVX on autophagy in the rat liver as a first step to clarify the relationship between hepatic fat accumulation and the change in autophagy caused by OVX. We found that hepatic autophagy tended to be lower in OVX rats than in sham-operated rats. In addition, hepatic autophagy in OVX rats was induced by short-term exposure to ovarian hormones, especially progesterone. This study suggests that autophagy in the rat liver is suppressed by OVX and activated by progesterone. However, the autophagy-promoting effect of ß-estradiol was not clarified.

Ingestion of potato starch containing esterified phosphorus increases alkaline phosphatase activity in the small intestine in rats.

Alkaline phosphatase (ALP) hydrolyzes a variety of monophosphate esters and plays an important role in phosphorus (P) metabolism. Several nutrients in food have been reported to affect intestinal ALP activity in animal models. Previous reports indicated that high levels of P or phosphate in diets decreased intestinal ALP activity in rats. Because potato starch contains considerable amounts of esterified P, unlike other starch-derived plants, we hypothesized that the feeding of potato starch would decrease ALP activity in the intestinal tract. Male Sprague-Dawley rats (7 weeks old) were fed 3 different types of diet containing 60% corn starch or 1 of 2 types of potato starch with different esterified P content for 1 or 5 weeks. Body weight and food intake of each rat were measured every day throughout the experimental periods. At the end of the feeding periods, the small intestine was removed to determine ALP activity in the mucosal tissues. Significant differences were observed in ALP activity in the small intestine between the 2 feeding periods, among the 4 segments of the small intestine, and among the 3 diet groups. Significant positive linear correlations between the amount of P derived from the starch and mucosal ALP activity were obtained in the jejunum and jejunoileum in rats after feeding for 5 weeks. We concluded, contrary to our hypotheses, that the ingestion of potato starch adaptively increases ALP activity in the upper part of the small intestine of growing rats in an esterified P content-dependent manner.

Ingestion of potato starch containing high levels of esterified phosphorus reduces calcium and magnesium absorption and their femoral retention in rats.

Many studies have shown that esterified phosphorus (P) in diets has a favorable effect on mineral absorption in humans and animals. Phosphorylated oligosaccharides derived from potato starch increase calcium (Ca) absorption from the rat intestine both in situ and in vitro. We hypothesized that the feeding of potato starch has a potential to increase Ca or magnesium (Mg) absorption. Male Sprague-Dawley rats at 7 weeks were fed 4 types of diet containing either 60% sucrose, cornstarch, or 1 of 2 types of potato starch with different P contents for 1, 3, or 5 weeks. A balance test for Ca, Mg, and P was undertaken, and these mineral contents in the femur were determined for the 4 diet groups at each feeding period in vivo. Ingestion of potato starch increased Ca, Mg, and P excretion into feces and decreased the absorption rate of Ca and Mg. Femoral Ca contents were also decreased in the rats fed the potato starch diets compared with those in rats fed the sucrose or cornstarch diet. In vitro experiment in Ca absorption was undertaken using everted jejunal and ileal sacs of the small intestine in male Sprague-Dawley rats (7 weeks old). The potato starch application did not induce significant increase in Ca absorption compared with nonstarch (control) or cornstarch application. In conclusion, the ingestion of potato starch does not increase Ca and Mg absorption and rather accelerates their excretion, inducing the decrease in mineral absorption and retention in growing rats.

Ingestion of gelatinized potato starch containing a high level of phosphorus decreases serum and liver lipids in rats.

Potato starch is known to have a higher concentration of phosphate than other starches. The presence of phosphate groups in amylopectin results in resistance to digestion by amylase. Therefore, there is a possibility that potato starch is slowly digested, inducing a physiological effect similar to that of resistant starch and indigestible oligosaccharides. The amount of phosphate group in starch differs with potato cultivar. In the present study, we investigated the effects of gelatinized potato starch containing a high level of phosphorus on lipid metabolism in rats. For this purpose, we determined lipid levels in the serum and liver in rats fed two kinds of gelatinized potato starches with different phosphorus contents. Four groups of male Sprague-Dawley rats were fed a 60% sucrose diet (control) or one of three diets containing cornstarch (CS), Benimaru (BM) potato starch or Hokkaikogane (HK) potato starch. Fat pad weight was slightly decreased in the HK diet group compared with that in the other groups. Free fatty acids in serum were significantly lowered by dietary HK starch compared with control, and serum triglyceride in rats fed the HK diet was also decreased. In the BM and HK diet groups, triglyceride levels in the liver were decreased compared with that in the control and CS groups. As for hepatic total cholesterol level, there were no significant differences among three starch diet groups. Fecal bile acid excretion was greater in the two potato starch groups than in the control group. On the other hand, there were no significant differences in cecal short-chain fatty acid content or pH. Thus, we conclude that dietary gelatinized potato starch reduces free fatty acid and triglyceride in serum and hepatic triglyceride, but does not affect cecal fermentation.

Cytosolic LC3 ratio as a sensitive index of macroautophagy in isolated rat hepatocytes and H4-II-E cells.

Macroautophagy, an intracellular bulk degradation process in eukaryotes, is sensitive to nutrient supply and deprivation. Microtubule-associated protein 1 light chain 3 (LC3), a mammalian homologue of yeast Atg8, plays an indispensable role in macroautophagy formation and is a suitable marker for this process. Through analysis of the subcellular distribution of LC3, we determined that the cytosolic fraction contained not only a precursor form (LC3-I), but also an apparent active form (LC3-IIs). Both cytosolic LC3-I and LC3-IIs were more responsive to amino acids than those of total homogenate. Moreover, changes in the LC3-IIs/I ratio reflected those in the total proteolytic flux remarkably in both fresh rat hepatocytes and H4-II-E cell lines. Thus, in addition to a sensitive index of macroautophagy, calculating the cytosolic LC3 ratio became an easy and quick quantitative method for monitoring its regulation in hepatocytes and H4-II-E cells.

Amino acids as regulators of proteolysis.

Proteolysis, as well as protein synthesis, is a major process that contributes to the body protein turnover. Despite the huge variety of proteases in the body, there are very few proteolytic systems contributing to the complete hydrolysis of proteins to amino acids. The autophagic-lysosomal pathway is responsible for bulk proteolysis, whereas the ubiquitin-proteasome pathway plays a significant role in the fine control of the degradation of specific proteins. Both systems can produce free amino acids as a final product, but only the autophagy system is physiologically controlled by plasma amino acids. Recently, the study of amino acids as regulators of macromolecular turnover has been focused on for their signal transduction mechanism. In autophagic proteolysis, several amino acids have a direct regulatory potential: Leu, Gln, Tyr, Phe, Pro, Met, Trp and His in the liver, and Leu in the skeletal muscle. These amino acids are recognized at the plasma membrane, indicating the possible existence of an amino acid receptor/sensor for their recognition and subsequent intracellular signaling. Another line of evidence has emerged that protein kinase cascades such as mTOR, Erk, eIF2alpha etc. may be involved in the regulation of autophagy, and that amino acids, in combination with insulin, may exert their effects through these pathways. From the viewpoint of amino acid safety, the contribution of proteolysis to possible adverse effects caused by excessive amino acid intake is not clear. At present, there is one report that excess glutamine at 10-fold the plasma level has an abnormal inhibitory effect on hepatic proteolysis, due to a lysosomotropic toxicity of ammonia derived from glutamine degradation. Whether this may lead to an adverse effect in humans remains to be clarified.

Amino acids and insulin control autophagic proteolysis through different signaling pathways in relation to mTOR in isolated rat hepatocytes.

Autophagy, a major bulk proteolytic pathway, contributes to intracellular protein turnover, together with protein synthesis. Both are subject to dynamic control by amino acids and insulin. The mechanisms of signaling and cross-talk of their physiological anabolic effects remain elusive. Recent studies established that amino acids and insulin induce p70 S6 kinase (p70(S6k)) phosphorylation by mTOR, involved in translational control of protein synthesis. Here, the signaling mechanisms of amino acids and insulin in macroautophagy in relation to mTOR were investigated. In isolated rat hepatocytes, both regulatory amino acids (RegAA) and insulin coordinately activated p70(S6k) phosphorylation, which was completely blocked by rapamycin, an mTOR inhibitor. However, rapamycin blocked proteolytic suppression by insulin, but did not block inhibition by RegAA. These contrasting results suggest that insulin controls autophagy through the mTOR pathway, but amino acids do not. Furthermore, micropermeabilization with Saccharomyces aureus alpha-toxin completely deprived hepatocytes of proteolytic responsiveness to RegAA and insulin, but still maintained p70(S6k) phosphorylation by RegAA. In contrast, Leu(8)-MAP, a non-transportable leucine analogue, did not mimic the effect of leucine on p70(S6k) phosphorylation, but maintained the activity on proteolysis. Finally, BCH, a System L-specific amino acid, did not affect proteolytic suppression or mTOR activation by leucine. All the results indicate that mTOR is not common to the signaling mechanisms of amino acids and insulin in autophagy, and that the amino acid signaling starts extracellularly with their "receptor(s)," probably other than transporters, and is mediated through a novel route distinct from the mTOR pathway employed by insulin.