Neuronal loss of TRPM8 leads to obesity and glucose intolerance in male mice.

OBJECTIVE: Mice with global deletion of the transient receptor potential channel melastatin family member 8 (TRPM8) are obese, and treatment of diet-induced obese (DIO) mice with TRPM8 agonists decrease body weight. Whether TRPM8 signaling regulates energy metabolism via central or peripheral effects is unknow. Here we assessed the metabolic phenotype of mice with either Nestin Cre-mediated neuronal loss of TRPM8, or with deletion of TRPM8 in Advillin Cre positive sensory neurons of the peripheral nervous system (PNS). METHODS: Nestin Cre- and Advillin Cre-Trpm8 knock-out (KO) mice were metabolically phenotyped under chronic exposure to either chow or high-fat diet (HFD), followed by assessment of energy and glucose metabolism. RESULTS: At room temperature, chow-fed neuronal Trpm8 KO are obese and show decreased energy expenditure when acutely treated with the TRPM8 selective agonist icilin. But body weight of neuronal Trpm8 KO mice is indistinguishable from wildtype controls at thermoneutrality, or when mice are chronically exposed to HFD-feeding. In contrast to previous studies, we show that the TRPM8 agonist icilin has no direct effect on brown adipocytes, but that icilin stimulates energy expenditure, at least in part, via neuronal TRPM8 signaling. We further show that lack of TRPM8 in sensory neurons of the PNS does not lead to a metabolically relevant phenotype. CONCLUSIONS: Our data indicate that obesity in TRPM8-deficient mice is centrally mediated and likely originates from alterations in energy expenditure and/or thermal conductance, but does not depend on TRPM8 signaling in brown adipocytes or sensory neurons of the PVN.

Hybridomas using athymic nude mouse injected with Crohn's disease (CD) tissue filtrate. Immunoreactivity of the hybridomas with CD sera.

Injections of Crohn's disease (CD) tissue filtrates produce lymphoma and hyperplastic lymph nodes from plasma cell hyperplasia (PCH) in athymic nude (nu/nu) mice; these lymphoid tissue contain an antigen(s) recognized by CD serum/gamma G immunoglobulin (IgG). To immortalize the "CD-reactive antigen(s)," the authors fused the lymphoid cells from a CD tissue filtrate primed nu/nu mouse with nonsecretory mouse myeloma cells. Hybrids were screened and selected based on their reactivity with CD serum IgG, but not with control serum IgG in an indirect immunofluorescence assay (IF). Two CD-positive hybridomas were examined by IF with sera from 47 CD, 38 ulcerative colitis (UC), 13 controls with other gastrointestinal diseases, 19 with autoimmune diseases, and 21 normal subjects. Sera from 16 CD patients (34%) reacted with the two hybridomas, but only one of 38 UC sera and none of the 53 other disease or normal control sera reacted. The immunoreactivity of CD sera was significantly higher than UC sera (P less than 0.01) and each of the other groups (P less than 0.007). Using immunoperoxidase techniques at light and electron microscopic levels, the authors localized CD-associated antigen(s) in the plasma membrane of the two hybridomas. Further characterization of these hybridomas and the immunoreactive protein(s) may provide an important probe(s) for the diagnosis and the understanding of the pathogenesis of CD.

Influence of cytosolic pH on receptor-mediated endocytosis of asialoorosomucoid.

Inhibitors of specific steps in the endocytosis of galactose-terminating glycoproteins (asialoglycoproteins) by cultured rat hepatocytes have been described (J. Cell Biol. 98: 375-381, 1984). In particular, substitution of K+ for Na+ in the culture medium results in reduced delivery of ligand to lysosomes; ligand-receptor internalization, dissociation, and segregation remain normal. We have now demonstrated by direct microelectrode measurement that incubation of hepatocytes in K+-substituted medium results in a reduction of intracellular pH by greater than or equal to 0.5 U. In addition, we have shown that reduced intracellular pH in these cells produced by either direct (CO2 diffusion) or indirect (K+ substitution) acidification inhibits ligand delivery to lysosomes. Return of internalized ligand-receptor complex to the cell surface (diacytosis) is also inhibited by these manipulations. These studies suggest that intracellular pH may modulate specific steps involving vesicle translocation and fusion in the receptor-mediated endocytosis of asialoglycoproteins. Similar effects of direct acidification of hepatocytes by CO2 diffusion and indirect acidification by K+ substitution for Na+, on diacytosis and ligand delivery to lysosomes, suggest that K+ substitution may influence these events by altering intracellular pH.

Development of a monoclonal antibody specifically reactive to gastrointestinal goblet cells.

A mouse monoclonal antibody (7E6A5) of IgG isotype, reacting specifically with mucin-producing goblet cells of the human gastrointestinal tract, has been developed. 7E6A5 reacts by an ELISA with colonic protein eluted from a DEAE column. A screening by immunoperoxidase assay of 76 specimens from 19 different human tissues showed that the immunoreactivity of 7E6A5 was confined exclusively in the globules of goblet cells in the colon, the appendix, and the small intestine. Nongoblet small and large intestinal epithelial cells did not react. Immunoelectron microscopy demonstrated the reactivity with mucin droplets in a homogeneous granular pattern inside the globules of goblet cells. Mucus-secreting cells from remaining parts of the gastrointestinal tract and other mucus-secreting organs such as respiratory, genitourinary tracts, salivary and mammary glands did not show any reactivity to 7E6A5. These findings indicate that the antigen recognized by 7E6A5 is shared by the goblet cells of both the small and large intestines and is unique to them. The monoclonal antibody may be useful in the study of function of mucus-secreting goblet cells and may represent an important tool in the evaluation of diseases such as ulcerative colitis, colon cancer, and intestinal metaplasia in gastric mucosa that are associated with quantitative changes in goblet cell numbers or with qualitative differences in mucin secretion.

Survival, organization, and function of microcarrier-attached hepatocytes transplanted in rats.

Hepatocytes harvested by collagenase perfusion of rat liver were attached to collagen-coated microcarriers and injected intraperitoneally into congeneic or allogeneic bilirubin-UDP-glucuronosyltransferase (EC 2.4.1.17)-deficient (Gunn) rats or allogeneic analbuminemic (NAR) rats. Five days later, the microcarriers were observed to have formed conglomerates chiefly on the anterior surface of the pancreas. Scanning electron microscopy showed hepatocytes attached to the granular collagen-coated surface of the microcarriers and newly formed connective tissue. Light microscopy revealed that the microcarriers formed a lattice with the collagen tissue; hepatocytes were seen within this lattice or on the surface of the microcarriers. Hepatocyte plasma membranes were nucleoside-diphosphatase (NDPase)-positive. Newly formed blood islands, blood vessels containing erythrocytes and leukocytes and NDPase-positive endothelium were observed in close proximity to the hepatocytes and fibroblasts. Transmission electron microscopic examination showed hepatocytes with microvilli and nucleoid-containing peroxisomes with catalase activity. Hepatocytes were present for up to 2 months in congeneic recipients, the longest period of observation after transplantation. After normal microcarrier-attached hepatocytes were transplanted into allogeneic Gunn rats, bilirubin glucuronides were present in bile for 6 days. When congeneic Gunn rat recipients were used, bilirubin glucuronides were present in bile throughout the study (28 days); this was accompanied by reduction of serum bilirubin concentrations to nearly normal levels. After injection of normal hepatocytes into allogeneic NAR rats, plasma albumin concentration progressively increased for 6 days and then declined. In NAR recipients which were immunosuppressed with cyclosporin A, peak plasma albumin levels were reached in 14 days and persisted nearly at that level throughout the study (28 days).

Effects of swainsonine on rat liver and kidney: biochemical and morphological studies.

Among the reported effects of the plant toxin swainsonine in animals are a decreased level of Golgi mannosidase II activity, an increase in lysosomal alpha-D-mannosidase activity, oligosaccharide accumulation, vacuolization of cells, and neurological changes. We now find that, in the rat, the alkaloid rapidly induces vacuolization of both liver and kidney cells, but oligosaccharides accumulate only in the latter. We demonstrate by enzyme- and immunocytochemistry that the induced pleomorphic vacuoles are lysosomal in nature. The vacuoles do not appear to be derived from the Golgi apparatus, which retains its typical ultrastructural appearance, but are formed by autophagy. In swainsonine-fed rats, the lysosomal system is highly developed in hepatocytes, Kupffer cells, and cells of the proximal convoluted tubules. The relation of this hypertrophy of the lysosomal system to the known effects of swainsonine on glycoprotein biosynthesis and on Golgi and lysosomal alpha-mannosidases is not clear. In addition, in liver there occurs a marked increase in mitotic figures in the hepatocytes. This occurred in the absence of both cell death and increased liver size as estimated by gross morphology.

Distribution of UDPglucuronosyltransferase in rat tissue.

UDPglucuronosyltransferase [UDPglucuronate beta-D-glucuronosyltransferase (acceptor-unspecific), EC 2.4.1.17] is a group of enzymes with distinct but partially overlapping substrate specificity. A rabbit antiserum raised against one purified rat liver UDPglycuronosyltransferase isoform was specific for UDPglucuronosyltransferase and recognized all transferase isoforms by immunodiffusion or immunotransblot analysis. The transferase activity toward all substrates was immunoabsorbed from solubilized rat liver microsomes by IgG purified from the antiserum. The purified IgG was used for immunocytochemical localization of UDP-glucuronosyltransferase in rat liver, jejunum, kidney, and adrenal gland. In the liver, UDPglucuronosyltransferase was present exclusively in hepatocytes and was uniformly distributed within all zones of the hepatic lobule. In the jejunum, the transferase was present exclusively in the epithelial cells and showed a progressive increase in concentration from the crypt to the villar tip. In the kidney, the greatest concentration of the transferase was observed in the epithelial cells of the proximal convoluted tubule. Adrenal medullary cells showed intense immunocytochemical staining; the zona glomerulosa and the zona reticularis of the adrenal cortex were more intensely stained than the zona fasciculata. By light microscopy, UDPglucuronosyltransferase was found in the endoplasmic reticulum and nuclear envelope of all the four organs; this was confirmed in the hepatocyte by electron microscopy. The transferase was not observed in mitochondria, Golgi apparatus, lysosomes, peroxisomes, and plasma membrane, even after 3- to 4-fold induction of various substrate-specific UDPglucuronosyltransferase activities.

Immunocytochemical localization of lysosomal beta-galactosidase in rat liver.

beta-galactosidase is a ubiquitous lysosomal hydrolase that specifically cleaves terminal beta-galactosyl residues from glycoproteins, glycosaminoglycans, oligosaccharides, and glycolipids. To study the intracellular distribution of this enzyme, we prepared a specific polyclonal antibody to lysosomal beta-galactosidase by immunizing rabbits with a highly purified preparation of beta-galactosidase from rat liver. Using this antibody we employed an immunocytochemical technique (protein A coupled to horseradish peroxidase and diaminobenzidine cytochemistry) and showed that beta-galactosidase is present in all hepatocytes of the rat liver. All types of lysosomes, the rough endoplasmic reticulum, and the specialized region of smooth endoplasmic reticulum known as GERL showed immunoreactivity. This in situ distribution suggests that these organelles are involved in the biosynthesis and intracellular sorting of this lysosomal enzyme.

Immunocytochemical localization of gamma-glutamyltransferase in induced hyperplastic nodules of rat liver.

The immunocytochemical localization of gamma-glutamyltransferase [(5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2; gamma-GluTase] was demonstrated in hyperplastic liver induced by the carcinogen 2-acetylaminofluorene (2-AAF). The method used a specific antiserum and protein A-horseradish peroxidase and permitted visualization of antigenic sites at both the light and electron microscopic levels. Electron microscopy revealed deposits of 3,3'-diaminobenzidine (DAB) reaction product in the plasma membranes of (i) hyperplastic cells, (ii) bile canaliculi, (iii) endothelial cell membranes, and (iv) lymphocytes. The so-called ATPase activity was localized in the plasma membrane in bile canaliculi and in endothelial cells; the hyperplastic cells show marked variability in the levels of this activity.

Immunocytochemical localization of alpha-D-mannosidase II in the Golgi apparatus of rat liver.

Mannosidase II is involved in the trimming of alpha-1,6-mannosyl residues during the biosynthesis of glycoproteins containing N-linked oligosaccharides of the complex type. A highly specific polyclonal antibody (IgG) was isolated from rabbits immunized with a homogeneous preparation of mannosidase II prepared from rat liver. With this antibody, light and electron microscopic immunocytochemical studies on rat liver reveal that essentially all mannosidase II in hepatocytes is localized in the Golgi apparatus, the only other site with reaction product being the endoplasmic reticulum. The indirect immunocytochemical method used in this study involved three major steps: exposure of aldehyde-fixed tissue to immune and nonimmune IgG, treatment with staphylococcal protein A labeled with horseradish peroxidase, and incubation in diaminobenzidine to reveal sites of peroxidase activity. The procedures described overcome major problems in immunocytochemistry, allowing preservation of antigenic sites and maintaining adequate ultrastructural integrity. The in situ localization of other carbohydrate-processing enzymes, involved in either trimming or attachment of sugar residues, should be possible with this procedure. Because biosynthetic precursors of the processing enzymes may be revealed by an immunocytochemical approach, it is potentially significant that mannosidase II reaction product is present in areas of the endoplasmic reticulum as well as in the Golgi apparatus.

Transepithelial endoplasmic reticulum in rat proximal convoluted tubule.

Inosine 5'-diphosphatase (IDPase) activity was demonstrated cytochemically in the endoplasmic reticulum of rat kidney proximal tubule cells in tissue fixed by perfusion with glutaraldehyde--formaldehyde. Incubation for IDPase activity at pH 7.2 was performed with and without 0.5 mM levamisole, a potent inhibitor of alkaline phosphatase (AlkPase) (M Borgers, J Histochem Cytochem 21:812, 1973). Levamisole treatment of sections eliminated all reaction product in the brush border, but did not affect the IDPase activity the endoplasmic reticulum (ER). The ER appears as a basilar-luminal-oriented transcellular structure, suggesting a possible cellular transport route. This study supports and extends earlier observations made by others that suggest a transport role for the ER in these cells. It also emphasizes the value of thick section cytochemistry.

Immunocytochemical localization of gamma-glutamyltransferase in rat kidney with protein A-horseradish peroxidase.

Immunocytochemistry with a specific antiserum and protein A-horseradish peroxidase permits visualization of the sites of gamma-glutamyltransferase [(5-glutamyl)-peptide:amino-acid 5-glutamyltransferase, EC 2.3.2.2] at both the light- and the electron-microscope levels. As seen by light microscopy, the enzyme is localized only in the proximal convolutions of the renal tubules. Electron microscopy reveals dense deposits of 3,3'-diaminobenzidine reaction product embedded in the glycocalyx along the entire luminal surface of the microvilli and in the basolateral membranes.

Carbohydrate-specified endocytosis: localization of ligand in the lysosomal compartment.

Carbohydrate-directed endocytosis is mediated by a receptor, the hepatic binding protein; it is responsible for the clearance of galactose-terminated glycoproteins from the circulation. This process was investigated by using lactosaminated ferritin which is recognized by this receptor. Ferritin was seen in elements of an extensive "lysosomal compartment" that includes secretory vacuoles, coated vesicles, and GERL. The compartment marked in hepatocytes by the distribution of ligand is similar to that previously described in Kupffer cells (marked by acid phosphatase reaction product and horseradish peroxidase reaction product).

Lysosomal compartment of macrophages: extending the definition of GERL.

"Professional phagocytes" such as Kupffer cells show an extensive network of interconnected residual bodies with cytochemically demonstrable acid phosphatase [orthophosphoric monoester phosphohydrolase (acid optimum), EC 3.1.3.2]. In such cells and in other (hepatocytes, etc.) this compartment may be considered a part of GERL [a hydrolase-rich region of endoplasmic reticulum (ER) situated at the trans face of the Golgi apparatus from which various Lysosomes appear to arise].

Human chorionic trophoblasts, decidual cells, and macrophages: a histochemical and electron microscopic study.

Human chorion, amnion, and adherent decidua were studied in term placentas obtained at spontaneous normal delivery and at cesarean section from women not in labor. There were no differences in morphologic features or in distribution of acid phosphatase reaction product in the two delivery groups--in trophoblasts, decidual cells, or macrophages of the decidual layer. No diffusion of acid phosphatase reaction product from lysosomes was evident. The significance of these findings is discussed in the context of current hypotheses concerning cellular event associated with labor.

Endocytosis of asialoglycoprotein-enzyme conjugates by hepatocytes.

Desialylated glycoproteins were covalently linked to two cytochemically detectable enzymes, horseradish peroxidase or tyrosinase, and injected intravenously in amounts of approximately 0.5 per cent of total plasma glycoproteins into rats. Comparative studies of the rates of disappearance and distribution of free enzyme and conjugates established that recognition of the conjugates by the plasma membranes of hepatocytes was due to the exposure of the terminal galactose residue of the desialylated glycoproteins. At 1 minute after injection, reaction products of the enzyme markers were seen in coated pits and vesicles, elongated pinocytic channels and pleomorphic vesicles, at or close to the sinusoidal surface of hepatocytes. Vesicles containing reaction products were also observed along the lateral surfaces of hepatocytes. By 10 minutes, reaction products were seen in residual bodies near the biliary poles of hepatocytes. These studies confirm the existence of hepatocellular channels previously seen only with large excess of hemoglobin or following partial hepatectomy. They also indicate that the specific receptor for asialoglycoproteins is not restricted to the sinusoidal surfaces of hepatocytes and that transport to the catabolic sites proceeds via cytoplasmic channels and vesicles.