Gross anatomy of the head and neck and neuroscience in an integrated first-year medical school curriculum.

The curriculum for first year medical students at the University of Cincinnati has changed. Beginning in the fall of 1998, material in the first year was presented in an Integrated Educational Program. The goal of this program was to provide students with an understanding of the normal structure, function, and development of the human body. The purpose of this report is to discuss the unique integration that occurs in a block offered during the Spring Quarter. The two components of this block are Gross Anatomy of the Head and Neck and Brain and Behavior I. Brain and Behavior I is a new offering combining neuroanatomy, neurophysiology, neurology, and a psychiatry/behavioral component. The unique combinations offered in this block are logical, educationally sound, and have been enthusiastically received by both the students and faculty.

Anatomy education in a changing medical curriculum.

How we educate students in the first two years of medical school is changing at many institutions. Effective medical education should be viewed as a continuum, integration of the basic sciences and clinical medicine should occur throughout the curriculum, and self-directed, life-long learning should be emphasized. Curricular revision may be appropriate if these fundamental concepts are absent. The principles of three curricular models are discussed: traditional, problem-based, and systems-oriented. The ideal curriculum may draw from each of these: A truly integrated curriculum. However, the curricular model chosen must meet the needs of the institution and its students. As anatomists we should not shy away from this process of change. With progressive educational approaches, we can be leaders in this climate of curricular reform. Anatomy courses are laboratory based and the laboratory is an outstanding small group, faculty/student interactive opportunity. However, we must show flexibility and innovation in our educational approaches whatever the curricular design being proposed.

Insulin replacement therapy in diabetic rats using an osmotic pump normalizes expression of enzymes key to hepatic carbohydrate metabolism.

Intensively treating type I diabetics with continuous subcutaneous insulin infusions or multiple daily insulin injections to normalize mean blood glucose concentrations significantly reduces the onset of secondary diabetic complications when compared to conventionally treated diabetics. Our studies focused on characterizing hepatic enzyme expression in intensively and conventionally treated diabetic rats. Alloxan-induced diabetic rats were conventionally treated with insulin injected twice daily or intensively treated with similar daily dosages of insulin administered via a surgically implanted osmotic pump. Our results demonstrate a significant difference in hepatic enzyme expression when these treatment regimes are compared. In conventionally treated diabetic rats, phosphoenolpyruvate carboxykinase (PEPCK) protein and mRNA levels remained slightly elevated when compared to normal animals, glycogen phosphorylase (GP) protein levels were still slightly decreased, and glycogen synthase (GS) protein and mRNA levels remained at the elevated levels observed in untreated diabetics. In contrast, the protein and mRNA levels of all three enzymes were normalized in the insulin pump-treated animals. These results suggest that intensive insulin therapy improves glycemia directly by normalizing hepatic gene expression while conventional insulin therapy normalizes plasma glucose concentrations indirectly.

Anatomy education in a changing medical curriculum.

How we educate students in the first two years of medical school is changing at many institutions. Effective medical education should be viewed as a continuum, integration of the basic sciences and clinical medicine should occur throughout the curriculum, and self-directed, life-long learning should be emphasized. Curricular revision may be appropriate if these fundamental concepts are absent. The principles of three curricular models are discussed: traditional, problem-based, and systems-oriented. The ideal curriculum may draw from each of these: A truly integrated curriculum. However, the curricular model chosen must meet the needs of the institution and its students. As anatomists we should not shy away from this process of change. With progressive educational approaches, we can be leaders in this climate of curricular reform. Anatomy courses are laboratory based and the laboratory is an outstanding small group, faculty/student interactive opportunity. However, we must show flexibility and innovation in our educational approaches whatever the curricular design being proposed.

Hepatic lobular patterns of phosphoenolpyruvate carboxykinase, glycogen synthase, and glycogen phosphorylase in fasted and fed rats.

The goal of this study was to localize phosphoenolpyruvate carboxykinase (PEPCK), glycogen synthase (GS), and glycogen phosphorylase (GP) in the liver lobule by immunocytochemical techniques and to describe the effects of feeding and fasting on the distribution and quantity of these enzymes. Livers from ad lib fed and overnight fasted normal adult male rats were frozen in liquid nitrogen after transcardial perfusion with 30% sucrose. Serial cryostat sections of tissue were collected on slides, fixed by immersion in 4% paraformaldehyde, and incubated with antibodies against PEPCK, GS, and GP. Antibodies to these enzymes were visualized with a gold-conjugated secondary antibody and a silver enhancement technique. Fed animals demonstrated a periportal to pericentral gradient of PEPCK. Fasting increased the periportal content of PEPCK, induced the midlobular and centrilobular cells to express the enzyme, and steepened the periportal to pericentral gradient. The increase of PEPCK was confirmed by Western blot analysis. GS and GP were distributed throughout the lobule in the fed animal but often showed a centrilobular pattern, and fasting did not alter the lobular distribution of either enzyme. Western blot analysis revealed no changes in the amount of these enzymes in the fed or fasted state. The cellular distribution of the three enzymes is similar to that of hepatic glycogen, in that the immunoreactive material has a clumped appearance in the periportal hepatocytes and is more dispersed in the pericentral cells. On fasting the periportal hepatocytes lose the dense compact localization of the enzymes and the protein becomes more homogeneously distributed throughout the cytosol. Further studies are needed to elucidate the functional significance of the regional heterogeneity of the glycogen-metabolizing enzymes and the molecular mechanisms regulating their gene expression.

Maternal malnutrition does not affect fetal hepatic glycogen synthase ontogeny.

Maternal malnutrition late in pregnancy results in the reduced storage of fetal hepatic glycogen in the final days of gestation and an accentuation of normal birth-related hypoglycemia. It was of interest to determine whether or not low glycogen levels resulted when maternal malnutrition disrupted the normal ontogeny of fetal hepatic glycogen synthase, an important glycogenic enzyme. A defect in this enzyme would be expected to seriously affect prenatal and postnatal glycogen synthesis. For this study, livers were removed from fetuses from malnourished (50% of normal dietary intake) mice, as well as from ad libitum-fed mice, and used for the determination of hepatic glycogen, glycogen synthase activity, and glycogen synthase protein levels. In this paper we report that maternal dietary restriction late in pregnancy produces growth-retarded fetuses with severely reduced hepatic glycogen levels, but the normal ontogenic changes in the quantity and activity of hepatic glycogen synthase were not affected. It is especially significant that the accumulation of glycogen synthase occurred despite the minimal level of natural substrate available for the enzyme. These results suggest that the accumulation and activity of hepatic glycogen synthase during late gestation is related to developmental events rather than levels of substrate or glycogen.

Appearance of a nonfunctional isozyme of hepatic glycogen synthase in late gestation.

Glycogen levels, glycogen synthase activities, and glycogen synthase protein levels were determined in liver tissues obtained from 14- to 19-day-old fetal mice, newborn mice, and adult mice. The results of these experiments demonstrate a significant increase in the quantity of hepatic glycogen synthase beginning at Day 17 of gestation and reaching adult levels at birth. However, during the same time period, there is a dramatic decrease in total glycogen synthase activity suggesting that the accumulating glycogen synthase molecules are unable to transfer UDP-glucose to glycogen. These inversely coordinated changes in the quantity and activity of glycogen synthase are consistent with the suggestion that glycogen synthesis in the near-term fetal mouse is being maintained by preexisting enzyme, while accumulating enzyme molecules may represent a quiescent isozyme.

Insulin-mediated regulation of epididymal fat pad malic enzyme.

The complex nature of insulin-mediated biological responses has made it difficult to interpret such data. Prior studies in our laboratory had characterized the insulin-mediated increases in hepatic malic enzyme activity in normal and diabetic rats (Drake et al., 1983; Drake and Mucenski, 1985). However, since insulin-mediated regulatory processes have been shown to be tissue specific, we decided to examine malic enzyme activity in the epididymal fat pads of normal, diabetic, and insulin-treated normal and diabetic rats. This data revealed that in direct contrast to the hepatic studies, the normal epididymal fat pad contained the low specific activity malic enzyme molecule. Insulin treatment of both normal and diabetic rats resulted in an increase in epididymal fat pad malic enzyme activity due to increases in both enzyme quantity and specific activity. In order to quantitate epididymal fat pad malic enzyme mRNA levels, we isolated a 2.4 kb malic enzyme specific cDNA which was designated pR ME 1. In both normal and diabetic rats, the observed increases in malic enzyme quantity were directly paralleled by increases in malic enzyme mRNA content. The Northern blot data revealed an apparent differential expression of the malic enzyme mRNA doublet between insulin-treated normal and diabetic epididymal fat pads. This study demonstrates that insulin modulates epididymal fat pad and hepatic malic enzyme activity in a tissue-specific manner utilizing a defined subset of insulin-sensitive parameters involving alterations in enzyme specific activity and/or quantity.

A cadaveric study of the anatomic variations of the recurrent motor branch of the median nerve.

Anatomic dissections were performed in 72 cadaveric upper extremities from 36 cadavers to determine the incidence of anomalous variations in the course of the median nerve and its branches. The classic recurrent motor branch anatomy was demonstrated in 86% of the dissections (62/72). Of the 10 variations noted (14% of all upper extremities), all were transretinacular branches that pierced the transverse carpal ligament 2 to 6 mm proximal to the distal edge. Of the six cadavers with anomalous branching, four (67%) had bilateral anomalies and two (33%) had unilateral branching.

Insulin-mediated post-transcriptional regulation of hepatic malic enzyme and albumin mRNAs.

Livers of insulin-treated diabetic rats accumulate albumin and malic enzyme mRNAs at very different rates. We now report that in normal rats insulin directs a specific increase in malic enzyme mRNA, while albumin mRNA levels remain unaltered. These studies support the contention that insulin regulates the accumulation of hepatic mRNAs in a highly specific manner. To evaluate whether or not albumin and malic enzyme mRNA levels are determined by altered rates of transcription, in vitro transcription assays were performed. The results of these studies demonstrate that increased malic enzyme mRNA levels in insulin-treated normal rats and increased malic enzyme and albumin mRNA levels in insulin-treated diabetic rats do not involve altered rates of transcription of the genetic sequences encoding these proteins. For these two specific proteins, insulin mediates changes in mRNA levels by a post-transcriptional mechanism.

Effects of various insulin dosages on hepatic lipogenesis.

Insulin treatment of diabetic rats fed a high-carbohydrate, fat-free diet produces a dramatic accumulation of hepatic lipids. However, this increase in hepatic lipids may only be a response to injections of exceptionally high doses of insulin. This study addresses this possibility. Alloxan-diabetic rats, fed a high-carbohydrate, fat-free diet, were given insulin every 12 h for 60 h at the following dosages: 1/2 unit each, 1 unit each, 2 units each, and 4 units each of regular and NPH insulins. At the end of the treatment period, liver samples were collected and used for morphological and biochemical analyses. Histologic examination revealed hepatic lipid accumulations at all insulin doses; the amount of lipid increased until maximal levels were reached at an insulin dosage of 1 + 1, which was maintained at doses of 2 + 2 and 4 + 4. Thus, hepatic lipid accumulation occurs regardless of the dosage of insulin administered to the diabetic animal. It is not simply an abnormal cellular response to excessive hormone levels. Similarly, the activity of the hepatic lipogenic enzyme, malic enzyme, increased at initial insulin dosages and reached maximal levels at 2 + 2. However, in contrast to lipid accumulation, enzyme activity decreased at the final insulin dosage of 4 + 4. Thus, there appears to be a direct relationship between increasing insulin levels and malic enzyme activity until an optimal insulin concentration is reached. After this point, excessive insulin levels do inhibit malic enzyme activity.

Normal and diabetic malic enzyme: a biochemical comparison.

The ability of insulin to influence the specific activity of hepatic malic enzyme was examined by comparing the biochemical properties of hepatic malic enzyme isolated from normal and diabetic rats. Even though these two enzyme species possess very different specific activities, they appeared biochemically identical upon two-dimensional gel electrophoresis, Western blot analysis, and in mixing experiments. The only major difference observed for the two enzyme molecules was in their respective Km and Vmax apparent values. These results appeared to indicate that the two enzyme molecules were structurally identical but functionally different. Suggestive evidence showed that this functional difference was the result of substrate inhibition of the diabetic malic enzyme molecule.

Determination of molecular forms of brain acetylcholinesterase: technical considerations.

Sucrose density centrifugation has been used to characterize the relative levels of AChE molecular forms in different parts of the brain, during development, or in various disease states. We have examined the influence of various tissue or sample storage and handling techniques on the abundance of the 4S and 10S molecular forms of AChE in rat forebrain. Our results demonstrate that freezing either a subcellular fraction or the intact tissue causes dramatic shifts in the level of the 4S and 10S molecular forms as compared to the values obtained in fresh tissue. Total AChE activity was unchanged suggesting that 4S and 10S forms are equally active and that 4S AChE is easily dissociated from 10S. These observations suggest that 4S and 10S molecular forms in brain are extremely labile and that great care should be taken when studying the factors that regulate these forms.

Regulation of hepatic malic enzyme by perfluorodecanoic acid.

Perfluorodecanoic acid (PFDA) administration to adult male rats increased both the activity of hepatic malic enzyme and liver weight in a dose-dependent manner. Hepatomegaly and augmented activity of malic enzyme in liver were apparent within one day following PFDA administration and reached a plateau by three days posttreatment. Malic enzyme quantity per liver in PFDA-treated rats was elevated within one day following dosing and increased continually throughout five days posttreatment. Administration of PFDA to rats in the fed state also led to an increase in the specific activity of hepatic malic enzyme that peaked at three days following dosing. When compared to the fed condition, rats fasted for 48 hours had a decrease in both relative liver weight and the quantity of supernatant protein per liver. The total activity (U/liver) and specific activity of malic enzyme in the liver were also reduced in the fasted state. During the 24 hours after treatment in rats fasted for 48 hours, the body weight as well as the absolute and relative liver weight of animals receiving vehicle declined continuously in the absence of feed. Following the administration of PFDA to fasted rats, body weight was maintained until eight hours posttreatment but then declined at a rate similar to that found with the vehicle-treated group. Absolute and relative liver weight in PFDA-treated rats were increased significantly at eight hours posttreatment when compared to those receiving vehicle, and this increment was maintained throughout the rest of the 24 hours following dosing. While the activity and enzyme content of hepatic malic enzyme decreased in the vehicle-treated group, administration of PFDA to rats fasted for 48 hours prevented their decline. The specific activity of hepatic malic enzyme in 48 hours fasted rats receiving PFDA was also elevated significantly at 16 hours posttreatment. Thus, the administration of PFDA to the adult male rat in both the fed and fasted nutritional states was found to regulate hepatic malic enzyme by not only increasing enzyme quantity but also by augmenting the specific activity, (ie, catalytic state) of the enzyme.

Insulin mediates the asynchronous accumulation of hepatic albumin and malic enzyme messenger RNAs.

We have compared the rate of accumulation of hepatic albumin and malic enzyme mRNAs following insulin treatment of diabetic rats to determine whether insulin coordinately increases mRNA levels or specifically induces the accumulation of individuals mRNAs. Initially, the quantities of both albumin and malic enzyme mRNAs are reduced in diabetic rats compared to normal rats as determined by RNA blot analysis using complementary DNA probes. Following insulin administration for 12 h, albumin and malic enzyme mRNA levels increase at similar rates. However, after 12 h the rate of malic enzyme mRNA accumulation increases dramatically while albumin mRNA continues to increase at its initial rate. This accelerated rate of accumulation of malic enzyme mRNA continued through 60 h of hormone treatment and was associated with the onset of hepatic lipogenesis. Thus, our results suggest that insulin regulates the accumulation of mRNAs encoding these two inducible proteins in an asynchronous manner directly related to the metabolic requirements of the animal.

Insulin and fructose regulate malic enzyme activity by different processes.

A comparison of the regulatory processes controlling hepatic malic enzyme activity following treatment of diabetic rats with insulin or with a high fructose diet demonstrated several important differences. Insulin treatment caused a 50-fold increase in activity, due to a 12-fold increase in enzyme quantity and a 4-fold increase in specific activity(units/nmol). Dietary fructose caused a 3-fold increase in enzyme activity, due to a 3-fold increase in enzyme quantity, with no change in the specific activity of the enzyme. Thus, while fructose initiated a minor increase in malic enzyme activity, insulin was more effective, causing a substantially greater increase in enzyme activity and activating a hormone specific alteration in the catalytic activity of each enzyme molecule.

Abnormal hepatic lipid accumulation following treatment of diabetic rats with insulin and a high-carbohydrate, fat-free diet.

This study correlates the morphological and biochemical events during the accumulation of hepatic lipids in diabetic rats in response to insulin treatment and a high-carbohydrate, fat-free diet. Alloxan-diabetic rats were fed a high-carbohydrate, fat-free diet and treated with insulin for 12, 36, or 60 hr or 4.5 or 6.5 days. Samples of livers were obtained for determination of malic enzyme activity and the histochemical demonstration of lipids. An increased accumulation of hepatic lipids, although delayed, was observed following insulin treatment of diabetic rats fed the special diet. Small lipid droplets were visible after 36 hr of treatment, which later increased and coalesced into larger droplets present in all hepatocytes. Maximal accumulation was observed at 4.5 days of treatment. These changes were paralleled by an increase in the activity of hepatic malic enzyme. By 6.5 days of treatment, the lipid content of the hepatocytes had decreased and a periportal pattern was discernible. In contrast, malic enzyme activity continued to increase through 6.5 days of treatment. By comparison, no hepatic lipid accumulation occurred in regular chow-fed diabetic rats receiving insulin treatment or in diabetic rats placed on the special diet alone. These results suggest that the combination of insulin treatment and a high-carbohydrate, fat-free diet caused an imbalance in the production and mobilization of hepatic lipids.

Insulin regulation of fat cell ribosomes, protein synthesis, and lipoprotein lipase.

Ribosomes of high purity were isolated from fat cells by discontinuous sucrose gradient centrifugation. Approximately 23% of the ribosomes were membrane bound. A reproducible fraction of ribosomes was recovered as polysomes capable of incorporating [3H]leucine into peptides in a cell-free system. Insulin (0.1-1.0 mU/ml) produced an increase in polysomal activity. Linear sucrose gradient profiles also revealed an increase in the ratio of polysomes to total ribosomes. Coincident with this effect, insulin increased the lipoprotein lipase activity fraction inhibitable by cycloheximide (0.01 mg/ml), and the immunotitratable enzyme activity. Insulin also enhanced the incorporation of labeled amino acids into adipose tissue immunoprecipitable lipoprotein lipase and total fat cell proteins. Cordycepin (0.1 mg/ml) or alpha-amanitin (10 micrograms/ml) partially inhibited insulin effects on ribosomes and protein synthesis but not on lipoprotein lipase. EGTA (1mM) prevented all of the insulin effects, whereas the calcium ionophore A-23187 (2 microM) augmented the hormone actions. The ionophore alone partially mimicked the insulin effects. In adipocytes, insulin increased the size of the protein-synthesizing polysomal pool by a mechanism which, in part, requires nuclear mRNA processing. Insulin, however, increased lipoprotein lipase synthesis independent of nuclear events. Calcium ions may be important for the expression of these insulin effects.

Insulin stimulation of hepatic malic enzyme activity in normal and diabetic rats controlled by different regulatory processes.

A comparison of the processes controlling the increase in hepatic malic enzyme activity in insulin-treated normal and diabetic rats indicated the existence of two distinct regulatory mechanisms. Livers were removed at 12, 36, and 60 h after insulin treatment of normal and alloxan-diabetic rats, and the activity, quantity, and specific activity (units/nmol), of malic enzyme was determined. In normal rats, a significant increase in activity occurred 12 h after insulin, whereas 36 h of insulin treatment was required for diabetic rats to show an increase in enzyme activity. This suggested that the return of malic enzyme activity from the depleted levels measured in diabetic rats probably involved a different sequence of events. A malic enzyme specific radioimmunoassay confirmed this. The increase in activity in insulin-treated normal rats was due to an increase in the quantity of malic enzyme. In insulin-treated diabetic rats, the increase in activity resulted from increases in both enzyme quantity and the specific activity of the enzyme, which returned to levels observed in normal rats.